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2011 IRG 24 Month Progress Reports

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2011 Innovative Research Grant 18-Month Progress Reports

2009 Innovative Research Grant 1 Year Progress Reports

2011 Innovative Research Grant 6-Month Progress Reports

2011 36 Month - Coupled Genetic and Functional Dissection of Chronic Lymphocytic Leukemia

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In this reporting period, Dr. Wu has continued to focus on understanding the impact of mutations in the gene for an RNA splicing factor called SF3B1 on CLL, and is optimizing methods to better understand the impact of these mutations. She has continued to investigate the hypothesis that epigenetic (methylation) variability also shapes CLL clonal evolution through interrelation with genetic variability. She identified random methylation as the primary cause of methylation changes in CLL, and cancer in general, and determined that this phenomenon influences gene transcription, genetic evolution, and clinical outcome. She has also analyzed how resistance to the drug ibrutinib arises, which is generally thought to result from mutations in a gene called Burton tyrosine kinase (BTK) or related genes. Dr. Wu has found, however, that that ibrutinib resistance does not uniformly involve mutations in BTK or related genes, but involves possible cancer-driving mutations that can bypass the need for BTK-dependent survival signaling. She has 8 publications further detailing this work.


2011 36 Month - Framing Therapeutic Opportunities in Tumor-Activated Gametogenic Programs

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Previously, Dr. Whitehurst used a panel of tumor derived cell lines representing six different tumor types to assess the specific contribution of certain proteins to the hallmarks of cancer cell biology. She identified a number of individual cancer-germline proteins that make essential contributions to cellular survival. In the current reporting period, Dr. Whitehurst made progress in understanding how each of these proteins contributes to tumor cell survival. In particular, her studies have identified proteins referred to as Cancer Testes Antigens (CTAs) that can directly activate normal cell suicide programs in response to stress. She has uncovered a CTA (ZNF165) that promotes the expression of pro-tumor oncogenes and oncogenic signaling pathways and a CTA (FATE1) that induces the activation of genes required for cell growth.


2011 36 Month - Developing New Therapeutic Strategies for Soft-Tissue Sarcoma

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During the past 6 months, Dr. Wagers has made important progress towards identify new candidate drug targets for sarcoma-induced genes. She has performed additional studies to assess the anti-sarcoma activity of 8 chemical compounds she previously identified. One drug in particular, Asparaginase (an FDA-approved drug to treat blood cell cancers), works exceedingly well in humanized mouse models of alveolar rhabdomyosarcoma. Given that Asparaginase is a drug that already is used routinely in the treatment of blood cell cancers, Dr. Wager’s is hopeful that this data may translate rapidly into clinical trials to test the efficacy of this FDA-approved drug in patients with sarcoma.


2011 36 Month - Inhibiting Innate Resistance to Chemotherapy in Lung Cancer Stem Cells

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Previously, Dr. Sweet-Cordero established a 3D culture method and analyzed the genes expressed in 3D cell culture in response to the cancer therapy drug cisplatin. In this reporting period, Dr. Sweet-Cordero identified 3 cell surface markers that are associated with tumor re-initiation, and identified a population of cells associated with drug resistance. During the no-cost-extension period of this grant, Dr. Sweet-Cordero will work on further validation of these results.


2011 36 Month - Targeting Sleeping Cancer Cells

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During the term of this grant, Dr. Ramaswamy has outlined a complete signaling pathway that governs sleeping cancer cells using cell lines grown in the laboratory. He has also generated preliminary evidence that suggests slowly dividing cells may actually promote tumor growth in certain contexts – a surprising and exciting possibility that may change the way people think about cancer progression, dormancy, and treatment resistance. He is currently finishing analysis of other important cellular regulatory mechanisms (the epigenetic state) of these slow proliferators, which is providing novel insight into potential ways to therapeutically target these cells.

Taken together, his work raises the possibility that new drugs to specifically target the pathway associated with slow proliferators (AKT1low) may also prove clinically useful. The molecular details, framework, and rationale that he provided through the SU2C-IRG grant mechanism may aid in developing such drugs. Dr. Ramaswamy believes that these findings will be of broad interest to those interested in cancer biology and therapeutics.


2011 36 Month - A Systems Approach to Understanding Tumor Specific Drug Response

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During the past 3 years, Dr. Pe’er’s computational work resulted in the identification of a novel synergy between two FDA approved drugs to treat melanoma. This observation supported the use of a combination approach to treatment that is currently being tested in clinical trials. Furthermore, a sub-type of melanoma, which currently does not have a personalized care approach (NRAS melanoma), may benefit from this combination approach. In addition, the Pe’er group have found a genetic marker that may help predict which patients will respond to a certain drugs. Her work on this grant has resulted in new technology to better investigate differences in the tumor.


2011 36 Month - Identification and Targeting of Novel Rearrangements in High-Risk ALL

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Dr. Mullighan has sequenced leukemic cells from 156 individuals with Ph-like ALL and identified the genetic basis of 91% of cases, which includes 31 different fusions. He has also developed experimental models to examine how these fusions affect the cells. Furthermore, he has tested drugs in animal models, which showed profound inhibition of leukemia growth. Dr. Mullighan has exceeded the stated aims of the project in terms of the size of the sample cohorts, the extent of genomic characterization, and the range of experimental models established. Importantly, the studies funded by this grant have provided preliminary data justifying clinical trials of a therapy (tyrosine-kinase inhibitor) in ALL.


2011 36 Month - Exome Sequencing of Melanomas with Acquired Resistance to BRAF Inhibitors

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Previously, Dr. Lo made the unexpected observation that certain mutations, proposed previously as a mechanism of drug resistance, cannot account for acquired resistance since these mutations are found prior to therapy. He has identified genetic alterations in key resistance pathways for further study.

He has identified a pathway (PI3K-AKT) that contributes to BRAF inhibitor resistance and has proposed Phase I/II clinical trial using combined AKT inhibitor and BRAF inhibitor drugs, which have been approved. In his final progress report, he states that he exceeded his goals in both the number of patient-matched tumor samples analyzed and the types of analysis performed as a result of additional funding for this work. He also reported that several publications and manuscripts have resulted from the work sponsored by this grant.


2011 36 Month - Targeting PP2A and the Glutamine-Sensing Pathway as Cancer Treatment

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Previously, Dr. Kong demonstrated that among 16 different PP2A regulatory proteins, only one, called the B55α subunit, was specifically upregulated upon glutamine deprivation. She also identified the molecular pathway that is critical for mediating B55α’s pro-survival effect on cancer cells. In this reporting period, Dr. Kong again showed that B55α is important for tumor growth in mice and decreased levels of B55α sensitizes tumor to a drug that inhibits glutamine metabolism. In addition, she identified a mechanism, mediated by a protein named IKK, that leads to B55α induction. During her 6 month no cost extension of the grant, she will continue to examine the effect of targeting glutamine metabolism in tumors.


2011 36 Month - Targeting Protein Quality Control for Cancer Therapy

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Dr. Jacinto has continued her work on the mTORC2 signaling pathway that plays an important role in protein production and quality control in the cell. Dr. Jacinto focused on two enzymes related to mTORC2; one a cellular receptor called CD147 and the other,called GFAT1, which she identified as a target of mTORC2. During this reporting period, she analyzed how mTORC2 can directly or indirectly regulate GFAT1. Since GFAT1 responds directly to cellular nutrients such as glucose and glutamine, she examined how these nutrients can regulate mTORC2. She also studied CD147 processing and found that this cellular receptor needs mTORC2 to function. During the 6 month no cost extension of the grant, Dr. Jacinto will continue to analyze how mTORC2 regulates GFAT1 and how blocking mTORC2 and GFAT1 activity can prevent mammary tumorigenesis in mice.


2011 36 Month - Targeting Genetic and Metabolic Networks in T-ALL

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For the past three years, Dr. Ferrando has worked towards identifying new drugs for the treatment of acute lymphoblastic leukemia (ALL). Towards this goal, he has analyzed a highly representative panel of human T-cell leukemia samples to catalog their genetic alterations, genetic programs, and metabolic signatures. He focused on cellular pathways that were specific to leukemia to identify new drugs. His work has resulted in the identification of two molecularly distinct groups of T-cell leukemia. He has identified numerous new genes mutated in T-ALL which have resulted in new biomarkers to help identify high-risk patients in the clinic.

His studies also looked at drug resistance in leukemia patients who have relapsed. These studies have identified new recurrent mutations that activate NT5C2, which is a metabolic gene responsible for the inactivation of mercaptopurine, an essential drug in the treatment of T-ALL. This result highlights the importance of drug metabolism in the response to therapy. In addition, he has uncovered the mechanistic role of two major genes driving T-cell leukemia (TLX1 and TLX3) and identified the PI3K-AKT1 cell signaling pathway as a new therapeutic target in this disease. He has also uncovered new mechanisms of resistance to therapies directed against a target called NOTCH1. Finally, Dr. Ferrando has profiled T-cell leukemias at the cellular level to better understand their metabolism and showed that targeted therapies result in dramatic changes in cell metabolism.

Overall, Dr. Ferrando’s SU2C-work has pointed to new drugs and drug combinations for the treatment of T-ALL and shown that perturbed cell metabolism represents an important Achilles heel in leukemia cells. This work demonstrate the role of high throughput technologies and network analyses to identify key regulators of leukemic cell growth and survival, and to identify novel and highly effective targeted therapies in high risk human leukemias.


2011 36 Month - Chimeric RNAs Generated by Trans-Splicing and their Implications in Cancer

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To better understand gene fusions and their chimeric fusion products, Dr. Li focused on a known fusion: PAX3-FOXO1. This gene fusion is common in the pediatric cancer alveolar rhabdomyosarcoma (aRMS) and detection of PAX3-FOXO1 fusion RNA by RT-PCR is a standard diagnostic procedure. Dr. Li found the PAX3-FOXO1 fusion in normal cells, which raises concern for tests coming back positive for aRMS when in fact they are negative. Furthermore, therapies targeting this fusion protein may have side effects due to the disruption of functions performed by PAX3-FOXO1 in normal developing muscle.

Another gene fusion Dr. Li studied was SLC45A3-ELK4, which is expressed at much higher level in malignant prostate cancer. His preliminary studies suggest that expression of SLC45A3-ELK4 correlate with prostate cancer progression. He plans to continue to study the SLC45A3-ELK4 fusion, specifically, how it occurs (the mechanisms) in order to enhance the understanding of disease and develop better strategies to fight cancer.

Dr. Li’s work during the 3-year grant term has not only enhanced the understanding of specific RNA and protein fusions, but has also provided valuable insight to understand the mechanism for generating chimeric RNAs in the absence of DNA rearrangement. His work has also resulted in the identification of other RNA fusions that are not the result of DNA rearrangement and plans to continue to study their importance in the development of cancer.


2011 36 Month - Targeting MLL in Acute Leukemia

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In the past three years, Dr. Dou has made significant progress toward the goal of developing therapeutics that specifically target Mixed Lineage Leukemia 1 (MLL1), which functions as an enzyme called histone methyltransferase. Her results show that blocking a specific interaction between MLL1 and a protein called WDR5 hinders MLL leukemia but not normal blood formation (hematopoiesis). In addition, while working on these proteins, she identified the molecular mechanism for the role of MLL1 activity in disease progression and has used this information to better understand other therapeutic agents for MLL leukemia. The work supported by SU2C was instrumental in translating basic knowledge of histone methyltransferase into potential clinical application. In the future, Dr. Dou will keep working in this area to examine the potential for cooperative or synergistic actions with other therapies used to treat leukemia. She will also try to identify and confirm new compounds that target MLL methyltransferase activity that are more suitable for clinical development.


2011 30 Month - Coupled Genetic and Functional Dissection of Chronic Lymphocytic Leukemia

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The treatment of chronic lymphocytic leukemia (CLL) poses two main challenges: 1) predicting the clinical course in a disease that shows many differences across patients, and 2) overcoming the insensitivity of some patient tumors to chemotherapy. At this time, genetic abnormalities are the best predictors of disease progression, based on gross chromosomal changes. However, an urgent need remains for improved understanding of how disease starts and progresses, which would lead to better predictive markers and potentially more effective (and non-toxic) therapies. Recent advances in genomic technologies provide a unique opportunity to find the genes and molecular circuits that make tumors grow in CLL. We have collected tumor and normal cells from 200 CLL patients and are almost done with sequencing all their genes. We are also looking at how genes are expressed in the same patient tumors using gene microarrays. Most importantly for enabling this project, our laboratory has pioneered the use of silicon-coated nanowires as a method of delivering DNA, RNA to primary CLL and normal B cells, which allows us to genetically manipulate CLL cells for the first time in a high-throughput fashion. Analysis of the first sixty patients has already identified genes that are important for CLL (called ‘driver mutations and pathways’). We have used our nanowires to verify the importance of some of these genes in CLL tumors cells. We now propose to find all the major genes and pathways that control CLL tumor formation. We will use a combination of sequencing technologies with statistical analyses to find the key genes that are important in creating tumors in CLL patients. In addition, we will find out which genes are good predictors of disease progression. Then, we will use our nanowires to place the mutant genes from CLL tumors into normal B cells and see how they affect their behavior. By taking this unique approach of combining different kinds of data collected from patient samples and using nanowires to manipulate the tumor cells in culture, we hope to understand the basic reasons why CLL patients develop cancer. This information will help us predict the progression of disease and provide new strategies for therapy. Finally, our approach can be extended to other tumors, especially leukemias and lymphomas.


2011 30 Month - Framing Therapeutic Opportunities in Tumor-Activated Gametogenic Programs

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Ideal anti-cancer treatments are those that selectively target tumor cells, while leaving normal tissues unharmed. Thus, there is a strong demand for the identification for molecular targets that are uniquely required for tumor cells to grow, divide and survive, but innocuous when perturbed in the normal setting. One potential untapped discovery space for these tumor-selective vulnerabilities is the cohort of genes whose expression is typically biased to the testes but frequently re-expressed in a wide range of cancers. Expression of these Cancer/Testes Antigens (CT-Antigens, CTAs) has been correlated with tumorigenesis for over 20 years, however, their functional roles in supporting neoplastic behaviors and any efficacy as therapeutic intervention points have not been extensively investigated. The goal of this grant is to begin to determine if these re-activated testes proteins do contribute to behaviors that make tumors so deadly. If so, these testes proteins may represent a novel set of therapeutic targets that could spare impacts on normal tissues. To do this, we inhibit each of these proteins one by one and ask whether tumor cells require these proteins to survive. To date, the screening platform we have developed has found that inhibition of many of these testes proteins can reduce the ability of tumor cells to remain viable. Thus, this work has demonstrated that testes proteins found in tumors are not merely epiphenomenon, but can directly contribute to a number of behaviors that tumor cells rely on to
survive. Importantly, these proteins, which have not previously been implicated in cancer, are now potential candidates for therapeutic intervention. Thus, our immediate goal is to further evaluate how these proteins function in cells to promote cancer. This work is essential to expand target space for therapeutic intervention. Additionally, understanding how these proteins interface with tumorigenic regulatory process in cells, will provide a mechanism to assess the effectiveness of intervention agents. Finally, we will be assessing how inhibition of these proteins impacts tumor growth in animal models of human cancer.


2011 30 Month - Developing New Therapeutic Strategies for Soft-Tissue Sarcoma

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Sarcomas are highly aggressive cancers that arise in connective tissues such as bone, fat and cartilage, as well as in muscles and blood vessels embedded within these tissues. Approximately 12,000 Americans are diagnosed with sarcoma each year, and current treatment strategies, especially for advanced forms of the disease, are often ineffective, leading to high rates of mortality among sarcoma patients. To advance sarcoma treatment and develop new approaches to cure these tumors, my lab established a new mouse model of soft-tissue sarcoma in skeletal muscle that introduces disease-relevant genetic modifications into tissue stem cells found normally in the skeletal muscle. We used this model to identify a small group of 141 genes present at increased levels in both mouse and human sarcomas. Our goal in this SU2C Innovative Research Grant is to test this novel set of sarcoma-induced genes to identify new candidate drug targets for these poorly-treatable tumors.

In the past 6 months, we have made important progress towards this goal. First, we confirmed the anti-sarcoma activity of 8 chemical compounds with predicted activity against high priority targets identified in the custom genetic “knock down” screen we completed earlier in this project. This knock down screen was thus highly effective in prioritizing the most promising candidates for follow up from among the 141 genes we initially identified. The 8 chemicals we have identified with anti-sarcoma activity include Asparaginase (an FDA-approved drug), Amino Sulfoximine 5, Bortezomib, Latrunculin A, UA62784, 4-Methylumbelliferone and Aldehyde Erastin derivates. We speculate that these chemicals may be useful as therapeutic agents for soft-tissue sarcoma, and have begun testing them in vivo. We are excited to report that in preliminary experiments Asparaginase had significant growth-inhibitory effects on human alveolar rhabdomyosarcoma xenografts.

In summary, our studies have employed a highly integrated strategy to identify and validate novel targets for sarcoma therapy. We believe that this work will help to uncover the root causes of sarcoma formation and identify new strategies to cure these aggressive cancers.


2011 30 Month - Inhibiting Innate Resistance to Chemotherapy in Lung Cancer Stem Cells

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Lung cancer is the leading cause of cancer fatalities worldwide. The most common form is non-small cell lung cancer (NSCLC). Platinum-based chemotherapy drugs (such as cisplatin) are commonly used to treat NSCLC, but only marginally increase survival due to the innate resistance of some tumor cells to chemotherapy. There is an urgent need to develop new ways to increase the effectiveness of chemotherapy for this disease. Past strategies for developing new drug targets have relied almost exclusively on testing cell lines grown directly on plastic culture dishes in “2D”. However, the biology of these cells is very different from that of tumor cells, which survive in a “3D” environment. To address this problem, we have developed methods for growing primary tumor cells in “3D” cultures (suspended in a gel-like material that mimics the tumor environment, rather than attached to plastic).

In our studies, we use tumor cells isolated from a well-characterized mouse model of NSCLC in which tumors carry one of the most frequent genetic mutations found in human lung cancer (a gene called K-ras). We have identified a population of tumor cells from these mice that form spheres in a 3D culture system and can reinitiate tumor growth if transplanted into the lungs of an immunocompromised mouse. We are testing whether we can make tumor spheres growing in 3D more sensitive to chemotherapy by inhibiting potential drug targets using shRNAs (short hairpin RNAs) that target and inhibit individual genes.

The goal of Aim 1 of our grant is to identify novel regulators of chemoresistance using shRNAs screens in 3D culture. In previous research periods, we optimized the design of a pooled shRNA screen to find genes required for chemoresistance in tumor spheres, and performed mock screens that showed we could identify genes required for self-renewal or proliferation of spheres in 3D. We used genomic and computational approaches to create a list of candidate genes relevant to chemotherapy resistance and thrapy response. The final set of genes includes genes associated with chemoresistance in cisplatin-resistant tumors, pathways related to DNA damage repair, a gene signature associated with tumor propagating sphere cells and poor prognosis, and genes that are up-regulated in response to cisplatin in “3D” tumor spheres.

We are now actively performing the full shRNA screen using this shRNA library. As this is being done in
primary tumors derived from mice in triplicate, this is a time-consuming process as it requires breeding of mice, isolating of tumor cells, infecting with pools of shRNAs and assessing relative frequency of shRNAs over time. To date, over 40% of our gene list (~600 genes/2,500 shRNAs) has been screened and the initial bioinformatics have been performed to analyze the earliest results. We expect to have the entire library screened in the next 4- 6 weeks. Preliminary results from shRNAs targeting major DNA damage repair pathways indicate that translesion synthesis and mismatch repair genes could play a dominant role in regulating chemoresistance in the tumor propagating cell population.

The goal of Aims 2-3 is to validate the findings of Aim 1 using in vivo tumors derived from mice (Aim 2) or humans (Aim 3). Methods to carry out these aims are actively being developed. The final experiments are awaiting the completion of Aim 1.


2011 30 Month - Targeting Sleeping Cancer Cells

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Cancer cells of different types have the very strange ability to go to sleep and then eventually wake up. While cancer cells sleep they are highly resistant to virtually all currently available forms of treatment. However, we do not understand how highly aggressive cancer cells can become dormant. It has proven extremely difficult to study these cells directly in patients and we have lacked suitable model systems to study them in the laboratory. We recently made a remarkable observation, however, that has the potential to open this important area for new investigation. We found that highly aggressive cancer cell lines of various types occasionally produce dormant cells. We went on to develop reliable methods for the prospective identification, isolation, molecular tracking, and experimental study of these “G0-like” dormant cancer cells in human cancer cell lines. Our preliminary results raised the possibility that epigenetic or signaling networks regulate these spontaneously dormant cancer cells. With a SU2C-AACR Innovative Grant Award, we have been using cutting edge molecular and cellular biology and genomic (next-generation sequencing (ChIP-seq / RNA-seq)), proteomic (reverse-phase protein microarrays), and computational technologies to identify and validate 1) genetic and 2) protein signaling networks that might trigger and maintain cancer cell dormancy.

Since the start of the award, we have made tremendous progress (see Dey-Guha, PNAS 108:12845 (2011) & Dey-Guha, Submitted (2013)). In brief, we have now delineated the complete signaling pathway that governs sleeping cancer cells in vitro. We have also generated preliminary evidence that these slowly proliferating cells may actually promote tumor growth in certain contexts – a surprising and exciting possibility that may change the way we think about cancer progression, dormancy, and treatment resistance. We are currently finishing analysis of the epigenetic state of these slow proliferators which is providing novel insight into potential ways to therapeutically target these cells.


2011 30 Month - A Systems Approach to Understanding Tumor Specific Drug Response

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Our research project focuses on understanding the heterogeneity underlying tumor response to drug. Some patients respond to therapy, at times achieving a full remission, while others are less lucky. The focus of our research is to understand these differences in patient response. We sought out to collect data using modern high throughput technologies and then to develop cutting-edge computational “machine learning” algorithms it interpret this complex data. Early in the project we realized that understanding the heterogeneity within a patient is no less important. Why do some of the cancer cells die, while other remain dormant, to recur at a later date. By studying heterogeneity both between and within tumors we can begin to piece together general principles and patterns in response to drug. These studies should teach us what drives cancers and what part of the networks we should target. For each individual patient, we wish to determine the best drug regime for that individual, informed by a model that can predict tumor response to drugs and their combinations. Treatment that is based not only on understanding which components go wrong, but also how these go wrong in each individual patient, will improve cancer therapeutics.

Our research progress includes the following:

  • Our computational method identified a novel synergy between two previously approved drugs for melanoma. MAPK inhibition (e.g. PLX, a BRAF inhibitor currently) and interferon, the combinatorial therapy is now being tested in a number of trials.
  • Most exciting, this combination might also offer possible therapeutics for NRAS melanoma patients (20% of malignant melanoma), who currently have no course of personalized care.
  • We detected a genetic marker that can help predict response to MAPK inhibition (e.g. PLX) and help determine which patients are likely to have a sustained response to this therapy.
  • We developed a new technology to better investigate intra-tumor heterogeneity and found surprising insights regarding which cells continue to grow following therapy.

 


2011 30 Month - Identification and Targeting of Novel Rearrangements in High-Risk ALL

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Acute lymphoblastic leukemia (ALL) is the commonest childhood cancer, and the leading cause of non-traumatic death in children and young adults. This project has focused on a recently described subtype of ALL termed “BCR-ABL1-like” or “Ph-like” ALL characterized by a range of previously unknown chromosomal changes and mutations that result in activation of cellular growth signals called kinases. Ph-like ALL is common, comprising up to15% of childhood ALL and up to one third of ALL in adolescents and young adults, and associated with a high risk of treatment failure, hence new therapeutic approaches to improve treatment outcomes are required. Work supported by the Stand Up to Cancer Innovative Research Grant has supported genetic analysis of leukemia cells from patients with Ph-like ALL in order to identify the range of genetic alterations in this disorder, to examine the frequency of these changes in large cohorts of ALL patients, and to examine their role in the development of leukemia, and potential responsiveness to therapy.

The first aim of this project is to use genomic sequencing and recurrence testing analysis to determine the nature and frequency of kinase activating genetic alterations in children and young adults with ALL. An initial pilot study that used mRNA-sequencing and whole genome sequencing of leukemia cells from 15 children with Ph-like ALL and identified a range of genetic changes activating kinases including CRLF2, ABL1, JAK2, PDGFRB, IL7R, and SH2B3 (LNK). At the time of the last report I described how the scope of analysis, including the number of cases, and the extent of sequencing, has expanded to over 1500 cases. This analysis is now mature. We have completed analyses of 2013 children, adolescents and young adults with ALL. 1725 had genomic data sufficient to enable identification of Ph-like ALL, the frequency of which rose from 11% in children with standard risk ALL, to 26% in young adult ALL. We have used multiple types of genome-wide sequencing in 156 cases of Ph-like ALL to identify the driver genetic changes, and have identified the genetic basis of 91% of cases. At the time of the last report sequencing and analysis was ongoing. This is now complete. We have identified 31 different fusion involving kinases or cytokine receptors that fall into a limited number of cell signaling pathways. The majority of these are potentially amenable to treatment with currently available tyrosine kinase inhibitors.

The second aim of this project was to develop experimental models to examine the way in which the alterations identified in aim 1 contribute to the development of leukemia, and to develop experimental systems to test the potential effectiveness of TKIs. At the time of the last report, I reported initial results of modeling of a small number of fusions. This has now been expanded to include testing of multiple representative fusions of each class of kinase signaling alteration. These cell lines have been successfully used to show that the fusions trigger cell growth and activation of signaling pathways, and that this activation is inhibited by use of the logical kinase inhibitor. These data provide important support for the rationale of treating patients with these alterations. We have also expanded the number of xenografts models of Ph-like ALL, in which human leukemia cells are propagated in immunodeficient mice, to 25, and have performed preclinical testing of tyrosine kinase inhibitors in 5 of these, all of which showed profound inhibition of leukemia growth. Finally, in conjunction with colleagues in the Children’s Oncology Group, we have collected data on 30 children from around the US referred due to features suggestive of Ph-like ALL. Remarkably, the majority was confirmed to have Ph-like ALL, and 19 had kinase rearrangements confirmed. Four were treated with appropriate TKIs with evidence of response.

The project has achieved the stated goals: to comprehensively define the genetic basis of Ph-like ALL, to show that the kinase-activating alterations transform blood cells, and that these alterations are amenable to treatment with tyrosine kinase inhibitors. These findings have formed the basis of a trial of tyrosine kinase inhibitor therapy being established by the Children’s Oncology Group. Ongoing work supported by this grant includes (1) performing whole genome sequencing of the remaining 9% of Ph-like cases that lack a kinase-activating alteration on existing analyses; (2) examining the frequency and nature of Ph-like ALL in older adults with ALL (for whom the prognosis of treatment is poor); (3) performing more detailed mouse modeling of kinase alterations and associated alterations in leukemogenesis; and (4) expanding the scope of preclinical testing of tyrosine kinase inhibitors in xenografts.


2011 30 Month - Exome Sequencing of Melanomas with Acquired Resistance to BRAF Inhibitors

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A small molecule (PLX4032/vemurafenib/Zelboraf) targeting a common melanoma mutation, V600EB-RAF, has shown unprecedented promise in advanced clinical trials (80% of patients respond if their tumors harbor the V600EB-RAF mutation) and confers survival benefit, prompting FDA approval. However, its ultimate success is challenged by so-called acquired drug resistance, which leads to clinical relapse. This type of drug resistance that develops over time occurs within months to years of drug initiation and cuts short the “sudden reprieve” that awakens patients’ hope for a cure (see NY Times stories by Amy Harmon on December 22-24, 2010). Earlier, we reported in Nature the discovery of two means by which melanomas escape from vemurafenib, which suggest new treatment strategies that are testable in clinical trials. This study along with others gave us another insight, that is, melanomas likely use a variety of different ways to escape from B-RAF inhibitors. Discovering other mechanisms of acquired resistance is logically the first step in constructing a therapeutic strategy closer to a cure.

We set forth three research aims centered on this group of V600EB-RAF-positive melanomas treated with B-RAF inhibitors (vemurafenib as well as another competing B-RAF inhibitor, GSK2118436). These aims are based on several premises. First, we need to directly study precious tissues derived from clinical trial patients. Second, we need to enlarge this tissue collection by collaborating among distinct clinical sites. Third, because finding a specific mechanism among the myriad of cancer-related changes is akin to finding a needle in a haystack, we should capitalize on the latest, “high-throughput” genomic technologies. Here, we report assembling a collaboration of multiple clinical sites to study acquired resistance directly in tissue samples from patients. For each patient that participates in this study, we are obtaining a set of normal tissue (e.g., blood), melanoma tissue before drug treatment, and melanoma tissue after an initial shrinkage followed by re-growth. Each set of tumor samples is first studied for the existence of known mechanisms which we have already discovered and characterized with in-depth molecular details in laboratory models. Works along this line have been published recently (Poulikakos et al, Nature, Nov 2011; Shi et al, Nature Communications, March 2012; Shi et al, Cancer Discovery, April 2012; Shi, Hugo,…, Lo, Cancer Discovery 2013; Shi, Hong,…, Lo, Cancer Discovery 2013). This workflow culls out tumor sample sets or patients for detailed genetic analysis. By harnessing the speed of “next-generation” DNA sequencing technology, we are examining the whole exome or the protein-coding, “business end” of the melanoma genomes for key genetic alterations that account for acquired resistance to B-RAF inhibitors in melanoma. From a patient’s perspective, we can now claim we know how melanomas escape from BRAF inhibitors in over 70% of patients. This knowledge has generated additional hypotheses to improve therapeutic response (both number of responders and duration of response) which are being tested in a clinical trial (1) or will be tested in two additional clinical trials (2-3) currently under review.

1. Safety and efficacy of the AKT inhibitor GSK2141795 in combination with the BRAF inhibitor dabrafenib in patients with BRAF mutant metastatic melanoma and study of the non-MAPK pathway resistance. Phase I/II.
2. A randomized phase II trial of intermittent versus continuous dosing of dabrafenib and trametinib in BRAFV600E/K mutant melanoma (SWOG-sponsored; study chairs: A. P. Algazi, A. I. Daud, & R. S. Lo)
3. Biomarkers of durable response with intermittent therapy with LGX818 and MEK162 combined therapy in patients with BRAF mutant metastatic melanoma (UCLA investigator-initated; PIs: A. Ribas & R. S. Lo)

Going forward, as we recruit patients for these next-generation clinical trials, we will need to iteratively sample tumor tissues donated by these patients in order to derive further knowledge and improve upon therapeutic outcomes. The SU2C Bud and Sue Selig Innovative Research Grant (IRG) has thus allowed us to take one large step forward in the treatment of 50-60% of all melanoma patients. The success of this research funding and the urgency of the next-step questions should allow us to compete for additional funding to accelerate the next big step forward.


2011 30 Month - Chimeric RNAs Generated by Trans-Splicing and their Implications in Cancer

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Aim1: identification of additional trans-splicing events in both normal and cancer cells. One approach we are taking is the candidate fusion approach. As cancer cells often hijack processes involved in normal development, we hypothesized that at least some of the well-known gene fusions associated with cancer may not be unique to cancer cells. We chose a particular fusion PAX3-FOXO1, which is associated with alveolar rhabdomyosarcoma, as a model and test whether the fusion RNA is also present during normal muscle development. Using a stem differentiation approach, we detected the fusion RNA transiently in the myogenesis. Both the fusion RNA and protein can be detected in the fetal muscle biopsies, confirming the results. Contradictory to the common paradigm, the fusion products are generated in the absence of the t(2;13) chromosomal translocation as seen in the case of alveolar rhabdomyosarcoma, suggesting a mechanism of RNA trans-splicing. W also found that the time points the PAX3-FOXO1 were detected preside other myogenic factors. If the cells are forced to express PAX3-FOXO1 continuously, they will remain at a stage of muscle precursor while terminal differentiation is inhibited. These findings further challenge the traditional dogma that gene fusions are unique to cancer. Together with the loss-of-function evidence, we have come to the conclusion that such chimeric RNA is not unique to the tumor, it is expressed in normal muscle development process and serves important physiological function. The original manuscript was rejected by Nature. We have since submitted half of the story (that PAX3-FOXO1 exists in normal myogensis) to Cancer Discovery and it has been published on the December issue. We plan to carry other additional functional study of the chimera in normal muscle development and submit this half to a stem cell journal.

The other approach that we used to identify more trans-splicing events is RNA-sequencing. We used this approach to investigate whether other trans-spliced chimeras are also present in normal myogenesis. Towards this goal, we did 100bp paired-end transcriptome sequencing with 50 millions read-depth. The samples we sequenced are various time points of muscle differentiation from mesenchymal stem cells and RH30, an alveolar rhabdomyosarcoma cell line. We compared four different packages of software to identify candidate fusion RNAs and selected SoapFuse as the most reliable and informative one to use. A total of 133 fusions were identified that at least in one sample. 18 fusions were present in at least two samples. Interestingly, the muscle differentiation time point that PAX3-FOXO1 is detected shared the highest number of fusions with RH30, supporting the idea that the cells express PAX3-FOXO1 may be the cell of origin for alveolar rhabdomyosarcoma. Consistently, we picked 6 fusions identified in RH30 cells and checked their expression pattern during normal myogenesis. All 6 are seen in the same time points that PAX3-FOXO1 is expressed.

The PAX3-FOXO1 gene fusion is a prominent marker of ARMS and detection of PAX3-FOXO1 fusion RNA by RT-PCR is a standard diagnostic procedure. Our findings of the presence of PAX3-FOXO1 RNA in normal cells raise concerns for false positive diagnoses. Additionally, therapies targeted at the fusion protein may have side effects due to disruption of functions performed by PAX3-FOXO1 in normal developing muscle. Knowledge about the mechanism and the cells that express the fusion products could lead to more specific diagnostic methods with fewer false positives and treatment strategies with less side effects. In addition, knowing the temporal and kinetic expression characteristic and pattern of PAX3-FOXO1 in normal cells will shed light on the etiology of the tumors (ARMS maybe the result of continuous expression of PAX3-FOXO1 in combination with other oncogenic “hits”)


2011 30 Month - Targeting PP2A and the Glutamine-Sensing Pathway as Cancer Treatment

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Fast-growing cancer cells rely on enhanced nutrient uptake to grow and divide. However, as tumors grow, increased uptake of nutrients and poor vascularization often lead to nutrient deprivation in tumor cells. Understanding the molecular mechanisms that promote cancer cell survival under poor nutrient conditions is important for developing new drugs that could starve tumor cells and block cancer progression. The amino acid glutamine is a major nutrient that supports cell growth and survival. Solid tumors consume glutamine at a rate that outstrips its supply and inevitably end up facing low glutamine conditions. The goal of this project is to determine the molecular basis for tumor cell survival under conditions of glutamine deprivation in order to develop novel drugs targeting this pathway. We have shown that the enzyme PP2A (protein phosphatase 2A) plays a critical role in mediating cell survival upon glutamine deprivation. However, PP2A is a member of a large family of protein complexes that regulate many different cellular functions. In this study, we worked to identify the specific PP2A complex that regulates cancer cell survival upon glutamine deprivation. Our aims are to determine: (1) whether PP2A complexes are regulated by glutamine levels; (2) the mechanism by which PP2A exerts a cell survival effect during glutamine deprivation; (3) whether PP2A contributes to tumor cell survival and whether impairment of PP2Aactivity combined with inhibition of glutamine metabolism can alter cancer cell viability.

During the first 2 years, we have demonstrated that only the regulatory subunit B55 is strongly and selectively upregulated in response to glutamine withdrawal, thereby triggering the formation of an active PP2A complex consisting of catalytic C, scaffolding A subunits, and the specifically induced B55 subunit via a ROS-dependent mechansim. This B55α-containing PP2A complex is critical for cancer cell survival upon glutamine deprivation. We further demonstrated that glutamine deprivation results in activation of p53, an important sensor of metabolic stress, and that B55α-mediated cell survival is p53-dependent. In these six months following the previous progress report in June of 2013, we have successfully fufilled the milestone defined in “Milestones and Deliverables Timeline”, which is to identify the specific substrate of the B55α complex upon glutamine deprivation. We demonstrated a protein named EDD directly assoicates with B55α and negatively regulates p53 function. In the next funding period, we will continue experiments outlined in the aims proposed for this funding period in “milestones and deliverables,” to determine the effect of concomitant impairment of B55α activity and glutamine metabolism on cancer cell viability in mouse xenograft model.


2011 30 Month - Targeting Protein Quality Control for Cancer Therapy

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Normal growth and proliferation of cells is orchestrated by a cascade of events that is initiated by binding of a stimulus to a receptor at the cell membrane. The receptor communicates to the rest of the cell via recruitment of a number of signaling molecules. Depending on the quality and quantity of signals from the receptor, the cellular output can be modified, for example proliferation versus death. Signals from growth receptors on the cell surface can become altered in cancer due to either increased expression of these receptors or mutations that lead to increased activity. In our project, we are addressing how inhibition of the expression critical growth receptors can be exploited for cancer therapy. Our lab had initial findings that a protein complex called mTORC2 is involved in protein production and quality control. When mTORC2 is inhibited by pharmacological agents or by genetic manipulation, proteins that are known to become deregulated in cancer such as Akt and growth receptor have defects in their synthesis. In the past months, we have shown that mTORC2 functions in protein quality control by controlling enzymes that play a role in cellular metabolism. Cells take up nutrients such as glucose and process (or metabolize) these nutrients in order to provide building blocks for synthesis of cellular macromolecules such as proteins, lipids and nucleic acids. Quality control of proteins involves addition of modifications such as carbohydrate moieties that alter protein conformation, stability and activity. We found that mTORC2 functions in regulating enzymes involved in the synthesis of these carbohydrate moieties. Since cancer cells are known to have defective metabolism and are addicted to nutrients such as glucose, our findings provide cellular mechanisms that become deregulated in cancer. Thus, identification of the mTORC2 targets in metabolism would provide new insights how we can develop more effective therapy to exploit the metabolic defects in cancer cells.


2011 30 Month - Targeting Genetic and Metabolic Networks in T-ALL

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Acute lymphoblastic leukemia is the most frequent cancer in children. Despite much progress in the treatment of this disease, leukemia still represents a clinical challenge, particularly in cases diagnosed with T-cell disease. In this project, we aim to elucidate the malignant mechanisms that control T-cell acute lymphoblastic leukemia. Our ultimate objective is to identify effective new drugs and drug combinations for the treatment of this disease.

Towards this goal we have analyzed a highly representative panel of human T-cell leukemia samples to catalog their genetic alterations, genetic programs and metabolic signatures and exploited leukemia specific regulatory circuitries to identify new active drugs and drug combinations. Our results have identified and cataloged two molecular groups of T-cell leukemia characterized by different gene expression programs; identified numerous new genes mutated in T-ALL including ETV6, RUNX1, SH2B3, EZH2 and SUZ12. Most notably these genetic alterations provide new biomarkers for the identification of high risk patients in the clinic.

Following on these results and to gain better understanding of the mechanisms of drug resistance we have extended our mutation analyses to relapsed leukemias. These studies have identified new recurrent mutations that activate NT5C2, a metabolic gene responsible for the inactivation of mercaptopurine, an essential drug in the treatment of T-ALL. This result highlights the importance of drug metabolism in the response to therapy. Ongoing analyses have extended these studies to a broader panel of relapsed tumors uncovering over 200 new mutations associated with leukemia relapse.

A central component of this research is the analysis of genetic and metabolic networks. Using this approach we have uncovered the mechanistic role of two major genes driving T-cell leukemia (TLX1 and TLX3) and identified the PI3K-AKT1 pathway as a new therapeutic target for the reversal of resistance to glucocorticoids, a key drug in the treatment of T-ALL. Applying these principles and approaches to the study of NOTCH1, the most critical factor in T-ALL development, we have uncovered new mechanisms of resistance to anti-NOTCH1 therapy in this disease. Strikingly, these results pointed to new drugs and drug combinations for the treatment of T-ALL.

Finally, and along this line, we have performed global metabolic profiling of T-cell leukemias and shown that targeted therapies result in dramatic changes in cell metabolism. Most notably these analyses have uncovered cell metabolism as an important Achilles heel in leukemia.

Overall, we have made significant progress towards our goal of using high throughput technologies and network analyses to identify key regulators of leukemic cell growth, and survival and to develop novel and highly effective targeted therapies in this disease.


2011 30 Month - Targeting MLL in Acute Leukemia

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Our broad objective in the proposed research is to develop novel chemotherapeutic agents that target the activity of a regulator of a subtype of acute myeloid leukemia, namely the Mixed Lineage Leukemia (MLL) protein. MLL was originally cloned by its direct involvement in a group of distinct human acute leukemia with extremely poor prognosis. MLL gene abnormalities account for 5% to 10% of the disease, and at least 70% of the cases in infants under 1 year old. It is general consensus that MLL mutations disrupt expression of specific genes that are important in early blood cell development. MLL is an enzyme and its activity is essential for leukemia development. Biochemical analyses have shown that MLL activity is tightly regulated by several interacting proteins. Therefore, it is conceivable that disrupting these protein-protein interactions involving MLL will compromise MLL enzymatic activity, which in turn leads to inhibition of leukemogenesis. Using the biochemistry and medicinal chemistry approaches, we have designed a series of inhibitors that target the MLL activity. In the past several months, we have made significant progress in improving our lead compounds in both in vitro and in vivo assays. These results suggest that our approach is valid and is likely to provide new therapeutics for MLL mediated leukemia.


2011 24 Month - Framing Therapeutic Opportunities in Tumor-Activated Gametogenic Programs

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The overarching goal of this grant is to identify and nominate new targets for cancer therapy. The focus is on a set (119) of proteins that are only expressed in the male testes but also reactivated in a diverse set of (male and female) tumors. Our work here aims to determine which of these proteins are required for tumor to grow, thus presenting new targets for anti-cancer therapy,whose inhibition would be less detrimental to normal tissues than current therapies. Over the previous 2 years, we have constructed a platform for investigating which of the 119 testes proteins are critical for tumor cells. This study has preliminarily revealed that 75 % of these testes proteins are essential for tumor cells to survive, creating a fresh set of potential targets for therapeutic intervention. In the last phase of this project, we will be elaborating how theses testes protein contribute to tumorigenesis as follows: 1) FATE1 is essential for the survival of nearly every tumor we have tested. Over the last reporting period, we have found that this protein can prevent cell suicide programs from being activated in tumor cells. In the next phase of the project, we will assess if targeting this protein in whole animals also kills tumor cells. If these experiments succeed, FATE1 would be an idea target for therapeutic intervention in a broad range of tumors. 2) This work has revealed that tumor cells required the function of CSAG1 to promote the deflection of growth arrest signals. Continued studies on this protein, will determine if direct targeting would be an entry point to prevent the growth of tumor cells. 3) One common characteristic to all tumors is the ability to grow in low oxygen conditions and we have identified 4 testes proteins that permit survival under these low oxygen conditions. In the next reporting period, we are extending our studies to whole animal analysis to the in vivo roles of these proteins. 4) One of the most aggressive forms of breast cancer is the highly metastatic claudin-low subtype. Our work here has revealed that this type of breast tumor requires a re-expressed testes protein, ZNF165, for survival. We have found that ZNF165 supports the Transforming Growth Factor β signaling pathway, which allows tumors cells to take on the characteristics of migratory cells and move to distant sites. We are currently working to identify which genes ZNF165 regulates in this process. 5) We have also discovered that tumor cells require a testes protein called MAGEA4 to divide. In particular, lung tumor cells express this testes protein and use it to regulate processes involved in both DNA replication and the segregation of genomic material. We have initiated animal studies to determine whether MAGEA4 enhances the growth of tumor cells in vivo. In addition, we have identified a potential binding pocket on MAGEA4 for a small molecule inhibitor, and we are developing assays amenable for screening to identify therapeutics that could inhibit the function of MAGEA4.

Our work here has demonstrated that tumor cells frequently employ testes proteins to support a range of different tumorigenic features. Given that these proteins are not expressed in other adult tissues, they are ideal targets for therapies that may have limited impacts on normal tissues. In fact, mice lacking a number of these proteins are perfectly viable and healthy. These studies have have isolated those testes proteins that are most potently required for tumor cell survival and may present ideal entry points for therapeutic intervention.


2011 24 Month - Coupled Genetic and Functional Dissection of Chronic Lymphocytic Leukemia

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The treatment of chronic lymphocytic leukemia (CLL) poses two main challenges: 1) predicting the clinical course in a disease that shows many differences across patients, and 2) overcoming the insensitivity of some patient tumors to chemotherapy. At this time, genetic abnormalities are the best predictors of disease progression, based on gross chromosomal changes. However, an urgent need remains for improved understanding of how disease starts and progresses, which would lead to better predictive markers and potentially more effective (and non-toxic) therapies. Recent advances in genomic technologies provide a unique opportunity to find the genes and molecular circuits that make tumors grow in CLL. We have collected tumor and normal cells from 200 CLL patients and are almost done with sequencing all their genes. We are also looking at how genes are expressed in the same patient tumors using gene microarrays. Most importantly for enabling this project, our laboratory has pioneered the use of silicon-coated nanowires as a method of delivering DNA, RNA to primary CLL and normal B cells, which allows us to genetically manipulate CLL cells for the first time in a high-throughput fashion. Analysis of the first sixty patients has already identified genes that are important for CLL (called ‘driver mutations and pathways’). We have used our nanowires to verify the importance of some of these genes in CLL tumors cells. We now propose to find all the major genes and pathways that control CLL tumor formation. We will use a combination of sequencing technologies with statistical analyses to find the key genes that are important in creating tumors in CLL patients. In addition, we will find out which genes are good predictors of disease progression. Then, we will use our nanowires to place the mutant genes from CLL tumors into normal B cells and see how they affect their behavior. By taking this unique approach of combining different kinds of data collected from patient samples and using nanowires to manipulate the tumor cells in culture, we hope to understand the basic reasons why CLL patients develop cancer. This information will help us predict the progression of disease and provide new strategies for therapy. Finally, our approach can be extended to other tumors, especially leukemias and lymphomas.


2011 24 Month - Developing New Therapeutic Strategies for Soft-Tissue Sarcoma

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Sarcomas are highly aggressive cancers that arise in connective tissues such as bone, fat and cartilage, as well as in muscles and blood vessels embedded within these tissues. Approximately 12,000 Americans are diagnosed with sarcoma each year, and current treatment strategies, especially for advanced forms of the disease, are often ineffective, leading to high rates of mortality among sarcoma patients. To advance sarcoma treatment and develop new approaches to cure these tumors, my lab established a new mouse model of soft-tissue sarcoma in skeletal muscle that introduces disease-relevant genetic modifications into tissue stem cells found normally in the skeletal muscle. We used this model to identify a small group of 141 genes present at increased levels in both mouse and human sarcomas. Our goal in this SU2C Innovative Research Grant is to test this novel set of sarcoma-induced genes to identify new candidate drug targets for these poorly-treatable tumors.

In the past 6 months, we have made substantial progress towards this goal. First, we identified 11 chemical compounds with predicted activity against high priority targets from the custom genetic “knock down” screen we complete earlier in this project. This screen allowed us to select the most promising candidates for follow up from the 141 genes we initially identified. We analyzed the ability of these 11 compounds to inhibit sarcoma cell growth in cell culture, using a panel of cells derived from different mouse and human sarcoma tumors, and confirmed that 8 of the 11 compounds substantially reduced tumor cell growth in culture. These 8 chemicals include Asparaginase (an FDA-approved drug), Amino Sulfoximine 5, Bortezomib, Latrunculin A, UA62784, Aldehyde Erastin, MKE and AKE. We speculate that these chemicals may be useful as anti-sarcoma therapeutic agents, and have begun in vivo testing of these compounds. We are particularly excited about asparaginase, which is already in clinical use for the treatment of some leukemias. Unfortunately, our initial studies have not shown a detectable response of established sarcomas to systemic asparaginase therapy; however, additional dose and delivery optimization is needed before we draw conclusions from this study.

Finally, we have advanced in our analysis of metastatic disease in soft-tissue sarcoma, confirming that our mouse sarcoma model faithfully recapitulates differences in metastatic potential seen in myogenic vs. non-myogenic human pleomorphic sarcomas. This result, together with our ongoing biomarker analyses, further establishes the relevance of our model to discover new candidate therapeutics for human sarcoma.

In summary, our studies pursue a highly integrated strategy to identify novel targets for sarcoma therapy. Ultimately, we believe that this work will help to uncover the root causes of sarcoma formation and identify new strategies to cure these aggressive cancers.


2011 24 Month - Inhibiting Innate Resistance to Chemotherapy in Lung Cancer Stem Cells

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Lung cancer is the leading cause of cancer fatalities worldwide. The most common form is non-small cell lung cancer (NSCLC). Platinum-based chemotherapy drugs (such as cisplatin) are commonly used to treat NSCLC, but they only marginally increase survival due to the innate resistance of some tumor cells to chemotherapy. There is an urgent need to develop new ways to increase the effectiveness of chemotherapy for this disease. Past strategies for developing new drug targets have relied almost exclusively on testing cell lines grown directly on plastic culture dishes in “2D”. However, the biology of these cells is very different from that of tumor cells, which survive in a “3D” environment. To address this problem, we have developed methods for growing primary tumor cells in “3D” cultures (suspended in a gel-like material that mimics the tumor environment, rather than attached to plastic).

In our studies, we use tumor cells isolated from a well-characterized mouse model of NSCLC in which tumors carry one of the most frequent genetic mutations found in human lung cancer (a gene called K-ras). We have identified a population of tumor cells from these mice that form spheres in a 3D culture system and can re-initiate tumor growth if transplanted into the lung of an immunocompromised mouse. We call these cells “tumor propagating cells” or “TPCs” We have also determined that these TPC cells are resistant to chemotherapy treatment. In our study, we are trying to determine what genes make these TPCs chemoresistant. To do this, we have used a technique called “gene expression analysis” to find genes that change in their expression when TPCs are treated with chemotherapy either in vivo (in mice) or in vitro (in 3D culture). We have also used gene expression analysis to identify genes that have different gene exrpression in TPCs compared to non-TPC tumor cells. Importantly, we find that human patients that have a more “TPC like” gene expression signature have a worse prognosis. So we feel if we can find ways to target these “TPC and chemoresistanct genes” we may find better ways to treat lung cancer. We have come up with a list of genes that we plan to test for relevance to chemoresistance using a technique called “shRNA knock-down” where we test the effects of inhibiting expression of each of these genes on chemotherapy response. We have carried out preliminary experiments to make sure we have the right conditions to do the “shRNA screen” of chemoresistance factors in TPCs. During the first 24 month funding period, we have completed the preliminary work needed to identify genes most likely to be involved in chemoresistane and tested the conditions for the shRNA screen. Over the next 6 months we plan to carry out the screen and begin to validate the results.


2011 24 Month - Targeting Sleeping Cancer Cells

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Cancer cells of different types have the very strange ability to go to sleep and then eventually wake up. While cancer cells sleep they are highly resistant to virtually all currently available forms of treatment. However, we do not understand how highly aggressive cancer cells can become dormant. It has proven extremely difficult to study these cells directly in patients and we have lacked suitable model systems to study them in the laboratory. We recently made a remarkable observation, however, that has the potential to open this important area for new investigation. We found that highly aggressive cancer cell lines of various types occasionally produce dormant cells. We went on to develop reliable methods for the prospective identification, isolation, molecular tracking, and experimental study of these “G0-like” dormant cancer cells in human cancer cell lines. Our preliminary results raised the possibility that epigenetic or signaling networks regulate these spontaneously dormant cancer cells. With a SU2C-AACR Innovative Grant Award, we have been using cutting edge molecular and cellular biology and genomic (next-generation sequencing (ChIP-seq / RNA-seq)), proteomic (reverse-phase protein microarrays), and computational technologies to identify and validate 1) genetic and 2) protein signaling networks that might trigger and maintain cancer cell dormancy.

Since the start of the award, we have made tremendous progress (see Dey-Guha, PNAS 108:12845 (2011)). Importantly, we have found that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating “G0-like” progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT1 kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with allosteric small-molecule inhibitors can induce asymmetric cancer cell division and the production of slow proliferators.

Most recently, we have discovered that AKT1 (rather than AKT2 or AKT3) is both necessary and sufficient for entry into the G0-like cell state. Moreover, AKT1 signaling is suppressed by suppression of AKT1 total protein levels via an mTORC2-induced, TTC3 / proteasome-mediated degradation pathway. In addition, RNA-seq studies suggest that G0-like cells actually assume a unique “stem-like” state with activation of the CTTNB1, FOXO1, and NOTCH1 pathways and global alterations in chromatin state. Furthermore, we have found that RNAi-mediated disruption of mTORC2 signaling does not alter the bulk proliferative properties of multiple human cancer cell lines, but completely abrogates the production of “G0-like” cancer cells, which in turn profoundly alters the tumorigeneity of these cell lines as xenografts in nude mice. We have submitted these exciting new mechanistic results for publication (2nd manuscript in preparation).

Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the triggering of a previously unappreciated mTORC2-AKT1-TTC3-proteasome signaling pathway during cancer cell mitosis, and the G0-like cancer cells arising through this mechanism play an important but previously unappreciated role in driving tumorigenesis. This model promises significant implications for understanding how tumors grow, evade treatment, and recur.


2011 24 Month - A Systems Approach to Understanding Tumor Specific Drug Response

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We propose using genomic technologies to track tumor response to potent drug inhibition of critical pathways across a diverse tumor panel. We will develop cutting-edge computational machine learning algorithms to piece these data together and illuminate how a cell’s regulatory network processes signals, and how this signal processing goes awry in cancer. By studying a large panel of diverse tumors we can begin to piece together general principles and patterns in response to drug. These studies should teach us what drives cancers and what part of the networks we should target. For each individual patient, we wish to determine the best drug regime for that individual, informed by a model that can predict tumor response to drugs and their combinations. Treatment that is based not only on understanding which components go wrong, but also how these go wrong in each individual patient, will improve cancer therapeutics.

At the end of the second year we are have made some major progress in our research which include:
1. A potential new combination therapy for melanoma, currently being tested in mice, that can potentially offer response to many patients who are resistant to PLX alone (the current BRAF inhibitor in use), most exciting combination might also offer possible therapeutics for NRAS melanoma patients, who currently have no course of personalized care.
2. We have gained a much better understanding of why and which melanoma cells either die or not follow treatment. This understanding suggests a number of potential avenues to target with drugs.
3. We have made first steps towards understanding a mechanism through which some melanoma cells evade drugs and continue to grow.
4. Importantly, the 3 findings above were discovered using new tools that could be applied to additional cancers and drugs.


2011 24 Month - Identification and Targeting of Novel Rearrangements in High-Risk ALL

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Acute lymphoblastic leukemia (ALL) is the commonest childhood cancer, and the leading cause of non-traumatic death in children and young adults. This project has focused on a recently described subtype of ALL termed “BCR-ABL1-like” or “Ph-like” ALL characterized by a range of previously unknown chromosomal changes and mutations that result in activation of cellular growth signals called kinases. Ph-like ALL is common, comprising up to15% of childhood ALL and up to one third of ALL in adolescents and young adults, and associated with a high risk of treatment failure, hence new therapeutic approaches to improve treatment outcomes are required. Work supported by the Stand Up to Cancer Innovative Research Grant has supported genetic analysis of leukemia cells from patients with Ph-like ALL in order to identify the range of genetic alterations in this disorder, to examine the frequency of these changes in large cohorts of ALL patients, and to examine their role in the development of leukemia, and potential responsiveness to therapy

The first aim of this project is to use genomic sequencing and recurrence testing analysis to determine the nature and frequency of kinase activating genetic alterations in children and young adults with ALL. An initial pilot study that used mRNA-sequencing and whole genome sequencing of leukemia cells from 15 children with Ph-like ALL and identified a range of genetic changes activating kinases including CRLF2, ABL1, JAK2, PDGFRB, IL7R, and SH2B3 (LNK). We have now expanded the profiling of cases to define the frequency of Ph-like ALL, and have tested the frequency of each of these changes in cohorts of childhood and adolescent and young adult (AYA) ALL, with current numbers of these cohorts exceeding 1500. The changes identified by sequencing of the first 15 cases were present in 80% of the recurrence cohorts. To identify the kinase-activating alterations in the remaining cases, we are performing mRNA-sequencing, exome sequencing and whole genome sequencing in all cases with suitable genetic material lacking one of the kinase-activating alterations identified in the pilot project (mRNA-seq and whole genome sequencing in almost 100 patients). This second phase of sequencing is complete, and analysis is nearing completion. This has already identified new fusion partners of the known kinases (e.g. ABL1, JAK2 and PDGFRB) and importantly, has identified new kinases as targets for rearrangement, (EPOR, ABL2, AKT2, STAT5B). The next 6 months of the project will witness completion of this sequencing analysis and testing for recurrence of each new alteration.

The second aim of this project was to develop experimental models to examine the way in which the alterations identified in aim 1 contribute to the development of leukemia, and to develop experimental systems to test the potential effectiveness of TKIs. Using laboratory cell lines, I have shown that several of these alterations accelerate cell growth and activate downstream signaling pathways. In addition, alterations such as EBF1-PDGFRB induce leukemia when expressed in mouse bone marrow cells. We have also developed xenograft models in which human leukemia cells are grown in immunodeficient mice. Importantly, growth of these cell lines is inhibited by several TKIs including include imatinib (Gleevec), dasatinib (Sprycel) and ruxolitinib (Jakafi). We are establishing xenografts of additional tumors to test the activity of other targeted agents.

Together, these studies continue to identify the range of lesions underlying BCR-ABL1-like ALL, and show that these alterations directly contribute to the development of leukemia. Importantly, the experimental models show that these alterations are targetable with TKIs. These results have generated tremendous excitement in the ALL field, and efforts to identify patients harboring these lesions at diagnosis and to treat them with these drugs are already underway.


2011 24 Month - Exome Sequencing of Melanomas with Acquired Resistance to BRAF Inhibitors

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A small molecule (PLX4032/vemurafenib/Zelboraf) targeting a common melanoma mutation, V600EB-RAF, has shown unprecedented promise in advanced clinical trials (80% of patients respond if their tumors harbor the V600EB-RAF mutation) and confers survival benefit, prompting FDA approval. However, its ultimate success is challenged by so-called acquired drug resistance, which leads to clinical relapse. This type of drug resistance that develops over time occurs within months to years of drug initiation and cuts short the “sudden reprieve” that awakens patients’ hope for a cure (see NY Times stories by Amy Harmon on December 22-24, 2010). Earlier, we reported in Nature the discovery of two means by which melanomas escape from vemurafenib, which suggest new treatment strategies that are testable in clinical trials. This study along with others gave us another insight, that is, melanomas likely use a variety of different ways to escape from B-RAF inhibitors. Discovering other mechanisms of acquired resistance is logically the first step in constructing a therapeutic strategy closer to a cure.

We set forth three research aims centered on this group of V600EB-RAF-positive melanomas treated with B-RAF inhibitors (vemurafenib as well as another competing B-RAF inhibitor, GSK2118436). These aims are based on several premises. First, we need to directly study precious tissues derived from clinical trial patients. Second, we need to enlarge this tissue collection by collaborating among distinct clinical sites. Third, because finding a specific mechanism among the myriad of cancer-related changes is akin to finding a needle in a haystack, we should capitalize on the latest, “high-throughput” genomic technologies. Here, we report assembling a collaboration of multiple clinical sites to study acquired resistance directly in tissue samples from patients. For each patient that participates in this study, we are obtaining a set of normal tissue (e.g., blood), melanoma tissue before drug treatment, and melanoma tissue after an initial shrinkage followed by re-growth. Each set of tumor samples is first studied for the existence of known mechanisms which we have already discovered and characterized with in-depth molecular details in laboratory models. Works along this line have been published recently (Poulikakos et al, Nature, Nov 2011; Shi et al, Nature Communications, March 2012; Shi et al, Cancer Discovery, April 2012) or are currently under peer review for publication likely early in 2014 (Shi, Hugo,…, Lo, 2013; Shi, Hong,…, Lo, 2013). This workflow culls out tumor sample sets or patients for detailed genetic analysis. By harnessing the speed of “next-generation” DNA sequencing technology, we are examining the whole exome or the protein-coding, “business end” of the melanoma genomes for key genetic alterations that account for acquired resistance to B-RAF inhibitors in melanoma. From a patient’s perspective, we can now claim we know how melanomas escape from BRAF inhibitors in over 70% of patients. This knowledge has generated additional hypotheses to improve therapeutic response (both number of responders and duration of response) which are being tested in a clinical trial (1) or will be tested in two additional clinical trials (2-3) currently under review.

1. Safety and efficacy of the AKT inhibitor GSK2141795 in combination with the BRAF inhibitor dabrafenib in patients with BRAF mutant metastatic melanoma and study of the non-MAPK pathway resistance. Phase I/II.
2. A randomized phase II trial of intermittent versus continuous dosing of dabrafenib and trametinib in BRAFV600E/K mutant melanoma (SWOG-sponsored; study chairs: A. P. Algazi, A. I. Daud, & R. S. Lo)
3. Biomarkers of durable response with intermittent therapy with LGX818 and MEK162 combined therapy in patients with BRAF mutant metastatic melanoma (UCLA investigator-initiated; PIs: A. Ribas & R. S. Lo)

Going forward, as we recruit patients for these next-generation clinical trials, we will need to iteratively sample tumor tissues donated by these patients in order to derive further knowledge and improve upon therapeutic outcomes. The SU2C Bud and Sue Selig Innovative Research Grant (IRG) has thus allowed us to take one large step forward in the treatment of 50-60% of all melanoma patients. The success of this research funding and the urgency of the next-step questions should allow us to compete for additional funding to accelerate the next big step forward.


2011 24 Month - Chimeric RNAs Generated by Trans-Splicing and their Implications in Cancer

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Substantial progress we made in the last 6 months are summarized in the following:

For aim1: identification of additional trans-splicing events in both normal and cancer cells, we have found the presence of PAX3-FOXO1 (FKHR) during stem cell differentiation process. The fusion has been thought to be a unique feature of alveolar rhabdomyosarcoma, a common childhood cancer. We found the same fusion product in fetal muscle samples as well. These findings further challenge the traditional dogma that gene fusions are unique to cancer. In addition to some functional evidence, we have come to the conclusion that such chimeric RNA is not unique to the tumor, it is expressed in normal muscle development process and serves important physiological function, and that its generating mechanism in the normal cells is independent of chromosomal translocation, which is the mechanism for the fusion production in alveolar rhabdomyosarcoma. Unfortunately, the manuscript was not accepted by Nature. We have since submitted half of the story (that PAX3-FOXO1 exists in normal myogensis) to Cancer Discovery and it is under review now. We plan to carry other additional functional study of the chimera in normal muscle development and submit this half to a stem cell journal.

It turns out that PAX3-FOXO1 chimera is uniquely expressed during the myogensis process, as no signal of BCR-ABL fusion (associated with CML) or EWS-FLI1 (associated with Ewing sarcoma) was detected in these samples.

The PAX3-FOXO1 gene fusion is a prominent marker of ARMS and detection of PAX3-FOXO1 fusion RNA by RT-PCR is a standard diagnostic procedure. Our findings of the presence of PAX3-FOXO1 RNA in normal cells raise concerns for false positive diagnoses. Additionally, therapies targeted at the fusion protein may have side effects due to disruption of functions performed by PAX3-FOXO1 in normal developing muscle. Knowledge about the mechanism and the cells that express the fusion products could lead to more specific diagnostic methods with fewer false positives and treatment strategies with less side effects. In addition, knowing the temporal and kinetic expression characteristic and pattern of PAX3-FOXO1 in normal cells will shed light on the etiology of the tumors (ARMS maybe the result of continuous expression of PAX3-FOXO1 in combination with other oncogenic “hits”)

Aim2 is designed to study the implications of chimeric gene fusions in cancer. In the field of endometrial cancer research, a big caveat of using mouse as a model is that mouse as well as most mammals do not have menstrual cycle. By accident, we found that the fusion JAZF1-JJAZ1 we have been studying is unique in species that have menstrual cycle. We have found that the fusion is necessary for the process. To test whether it is sufficient to trigger the process, we used human foreskin fibroblast cells and found that the fusion can induce at least two markers associated with decidualization, a trigger for menstruation. We also found that when combined with EZH2 and EED, two core components of Polycomb Repressive Complex, we can enhance the effect. These surprising findings have led us to hypothesize that by inducing the fusion at right time, we may be able to generate a mouse model that go through menstrual cycle. Such a tool will be extremely useful not only for endometrial cancer or breast cancer research, but also for any research fields related to menstrual cycle. We are now testing the hypothesis in cell culture models. If successful, we will generate a transgenic mouse model. Such a model will allow us to study the fusion’s oncogenic effect when expressed continuously, and test whether the animal will menstruate if the fusion is induced at the right time.


2011 24 Month - Targeting PP2A and the Glutamine-Sensing Pathway as Cancer Treatment

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Fast-growing cancer cells rely on enhanced nutrient uptake to grow and divide. However, as tumors grow, increased uptake of nutrients and poor vascularization often lead to nutrient deprivation in tumor cells. Understanding the molecular mechanisms that promote cancer cell survival under poor nutrient conditions is important for developing new drugs that could starve tumor cells and block cancer progression. The amino acid glutamine is a major nutrient that supports cell growth and survival. Solid tumors consume glutamine at a rate that outstrips its supply and inevitably end up facing low glutamine conditions. The goal of this project is to determine the molecular basis for tumor cell survival under conditions of glutamine deprivation in order to develop novel drugs targeting this pathway. We have shown that the enzyme PP2A (protein phosphatase 2A) plays a critical role in mediating cell survival upon glutamine deprivation. However, PP2A is a member of a large family of protein complexes that regulate many different cellular functions. In this study, we worked to identify the specific PP2A complex that regulates cancer cell survival upon glutamine deprivation. Our aims are to determine: (1) whether PP2A complexes are regulated by glutamine levels; (2) the mechanism by which PP2A exerts a cell survival effect during glutamine deprivation; (3) whether PP2A contributes to tumor cell survival and whether impairment of PP2Aactivity combined with inhibition of glutamine metabolism can alter cancer cell viability.

During the first eighteen months, we have demonstrated that only the regulatory subunit B55α is strongly and selectively upregulated in response to glutamine withdrawal, thereby triggering the formation of an active PP2A complex consisting of catalytic C, scaffolding A subunits, and the specifically induced B55α subunit. This B55α-containing PP2A complex is critical for cancer cell survival upon glutamine deprivation. We further demonstrated that glutamine deprivation results in activation of p53, an important sensor of metabolic stress, and that B55α-mediated cell survival is p53-dependent. In these six months following the previous progress report in December of 2012, we have successfully fufilled two pre-defined milestones. First, we determined if α4 and B55α promote cell survival upon glutamine deprivation via inhibition of c-Myc activity. Our data shows that B55α functions through regulation of p53, but not c-Myc activity. Second, we identified that B55α enhances cell survival upon glutamine deprivation via a ROS-dependent mechanism. In the next funding period, we will continue experiments outlined in the aims proposed for this funding period in “milestones and deliverables,” which is to identify the specific substrate of the B55α complex upon glutamine deprivation.


2011 24 Month - Targeting Protein Quality Control for Cancer Therapy

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The normal growth and proliferation of cells is orchestrated by a cascade of events that is initiated by binding of a stimulus to a receptor at the membrane. Once triggered, the receptor communicates to the rest of the cell via recruitment of a number of signaling molecules. Depending on the quality and quantity of signals from the receptor, the cellular output can be modified, for example proliferation versus death. Signals from growth receptors on the cell surface can become altered in cancer due to either increased expression of these receptors or mutations that lead to increased activity. In our project, we are addressing how inhibition of the expression of the epidermal growth factor receptors (referred in here as ErbB) can be exploited for cancer therapy. Our lab had initial findings that a protein complex called mTORC2 is involved in protein production and quality control. When mTORC2 is inhibited by pharmacological agents or by genetic manipulation, proteins that are known to become deregulated in cancer such as Akt and ErbB have defects in their synthesis. During the first year of this grant, we have established a role for mTORC2 in controlling the amount and quality of ErbB1 that is expressed in the surface of breast cancer cells. In the second year (July 2012-June 2013), we have identified a possible mediator of the mTORC2 function in ErbB1 quality control. Using protein purification and mass spectrometry, we identified a protein called GFAT1. This protein has been previously characterized in the field of diabetes since it is involved in cellular metabolism. Not much is known about how this protein becomes regulated by nutrients. Our findings now provide a connection between cellular proliferation (via ErbB1 signaling) and metabolism (GFAT1) and that these two pathways could be coupled by mTORC2. In the last six months, we have analyzed how mTORC2 can regulate GFAT1. We found that GFAT1 expression is diminished upon mTORC2 disruption and that GFAT1 is likely phosphorylated by mTORC2 during translation in order to stabilize this protein. We have identified a possible target site in GFAT1 by mTORC2. In the coming year, we will analyze how the regulation of GFAT1 by mTORC2 at this site plays a role in the control of ErbB1 expression. We have also identified another receptor, CD147, that is controlled by mTORC2 in a similar way. CD147 has been indentified in the past as a marker of tumor metastasis and is required for lactate transport in the cell. Therefore, this receptor could play a crucial role in the changes in metabolism that occurs in cancer cells. Since we found that CD147 is highly dependent on mTORC2 for proper expression, we will alternatively examine this receptor, along with ErbB1, to elucidate the function of mTORC2 in protein quality control.


2011 24 Month - Targeting Genetic and Metabolic Networks in T-ALL

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Acute lymphoblastic leukemia is the most frequent cancer in children. Despite much progress in the treatment of this disease, leukemia still represents a clinical challenge, particularly in cases diagnosed with T-cell disease. In this project, we aim to elucidate the oncogenic circuitries that control T-cell acute lymphoblastic leukemia. Our ultimate objective is to identify effective new drugs and drug combinations for the treatment of this disease.

In the first two years of funding we have analyzed a highly representative panel of human T-cell leukemia samples to catalog their genetic alterations, genetic programs and metabolic signatures and exploited leukemia specific regulatory circuitries to identify new active drugs and drug combinations. Our results have identified and cataloged two molecular groups of T-cell leukemia characterized by different gene expression programs; identified numerous new genes mutated in T-ALL including ETV6, RUNX1, EZH2 and SUZ12. Most notably these genetic alterations provide new biomarkers for the identification of high risk patients in the clinic. We are currently extending these studies to the analysis of epigenetic lesions and testing the effects of DNA methylation in leukemia development and therapy response.

Following on these results and to gain better understanding of the mechanisms of drug resistance we have extended our mutation analyses to relapsed leukemias. These studies have identified new recurrent mutations that activate NT5C2, a metabolic gene responsible for the inactivation of mercaptopurime, an essential drug in the treatment of T-ALL. This result highlights the importance of cell metabolism in the response to therapy. Ongoing analyses have extended these studies to a broader panel of relapsed tumors uncovering over 180 new mutations associated with leukemia relapse.

A central component of this research is the analysis of genetic and metabolic networks. Using this approach we have uncovered the mechanistic role of TLX1 and TLX3, two major genes driving T-cell leukemia. Moreover, analysis of the circuitries involved in resistance to chemotherapy with glucocorticoids has identified the PI3K-AKT1 pathway as a new therapeutic target for the reversal of resistance to glucocorticoids, a key drug in the treatment of T-ALL. Applying these principles and approaches to the study of NOTCH1 and PTEN, the two most critical factors in T-ALL development, we have uncovered the mechanisms mediating resistance to anti-NOTCH1 therapy in this disease. Strikingly, these results pointed to new drugs and drug combinations for the treatment of leukemia. Ongoing testing of these drugs in highly aggressive leukemias shows strong synergism with anti NOTCH1 therapies, opening the way towards the development of new combinations in the clinic.

Finally, and along this line, we have performed global metabolic profiling of T-cell leukemias and shown that targeted drugs that inactivate NOTCH1 result in dramatic changes in cell metabolism. Strikingly inactivation of PTEN induces reprogramming of cell metabolism and effectively reverses the metabolic shutdown resulting from NOTCH1 inhibition. Most notably these analyses have uncovered cell metabolism as an important Achilles heel in leukemia.

Overall, we have made significant progress towards our goal of using high throughput technologies and network analyses to identify key regulators of leukemic cell growth, and survival and to develop novel and highly effective targeted in this disease.


2011 24 Month - Targeting MLL in Acute Leukemia

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Our broad objective in the proposed research is to develop novel chemotherapeutic agents that target the activity of a regulator of a subtype of acute myeloid leukemia, namely the Mixed Lineage Leukemia (MLL) protein. MLL was originally cloned by its direct involvement in a group of distinct human acute leukemia with extremely poor prognosis. MLL gene abnormalities account for 5% to 10% of the disease, and at least 70% of the cases in infants under 1 year old. It is general consensus that MLL mutations disrupt expression of specific genes that are important in early blood cell development. MLL is an enzyme and its activity is essential for leukemia development. Biochemical analyses have shown that MLL activity is tightly regulated by several interacting proteins. Therefore, it is conceivable that disrupting these protein-protein interactions involving MLL will compromise MLL enzymatic activity, which in turn leads to inhibition of leukemogenesis. Using the biochemistry and medicinal chemistry approaches, we have designed a series of inhibitors that target the MLL activity. In the past several months, we have made significant progress in improving our lead compounds in both in vitro and in vivo assays. These results suggest that our approach is valid and is likely to provide new therapeutics for MLL mediated leukemia.


2009 42 Month - Probing EBV-LMP-1’s Transmembrane Activation Domain with Synthetic Peptide

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The human herpesvirus Epstein-Barr virus (EBV) infects and immortalizes naïve B lymphocytes and is
associated with a number of human malignancies. Understanding the signaling mechanisms by which EBV gene products hijack existing B cell signaling pathways to gain access to B cell memory is crucial for designing rational strategies for the prevention and treatment of EBV-dependent malignancies. One of the key players for EBV activation is LMP-1, a viral oncoprotein that activates B cell signaling pathways promoting survival and proliferation. LMP-1 functions as a ligand-independent Tumor Necrosis Factor Receptor (TNFR) superfamily “analog”, most closely resembling the TNFR family member CD40. Unlike CD40, LMP-1 is constitutively active. LMP-1’s activity depends on the homo-oligomerization of its hydrophobic multi-spanning transmembrane domain. We have developed antagonistic peptides (anti-TMD-5) specific for LMP1’s fifth transmembrane domain (TMD-5) to use as probes to study LMP-1 signaling. Currently, we attempt to optimize the anti-TMD-5 sequences by combining rational design and the directed evolution. Next, we report a small molecule agent, NSC 259242,to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of NSC 259242 are essential for its inhibitory activity. Two dimensional heteronuclear multiple quantum coherence (2D-HMQC) NMR titrations showed that NSC 259242 recognizes the TMD-5 helix and disrupts TMD-5 homo-trimerization. Furthermore, cell-based assays revealed that NSC 259242 inhibits full-length LMP-1 signaling in EBV infected B cells. Most recently, we engineered an EBV-infected 721 B cell line that responds to LMP-1 activation by expressing green fluorescent protein (GFP) using the commercially available pGreenFireTM NF-κB reporter lentivectors. Reiterative screening by this method produced novel traptamer sequences that potently inhibit LMP-1 signaling. These studies demonstrated a new strategy for identifying novel anti-TMD peptide/small molecule inhibitors for investigating transmembrane protein-protein interactions and ultimately as therapeutic agents in the treatment of EBV-dependent lymphoproliferative disease.


2009 42 Month - Identifying Solid Tumor Kinase Fusions Via Exon Capture and 454 Sequencing

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Cells rely upon molecular switches to carry out normal functions. One class of switches is called kinases. In a highly simplified model, when kinases are ‘on’, cells divide; when kinases are ‘off’, cells stop dividing. In some cancers, kinases have become altered, leading to abnormal signaling. One major type of alteration is called a ‘fusion’. A fusion ‘fuses’ together a part of a cellular protein (that normally has another function in the cell) with the signaling portion of the kinase molecule. Instead of turning ‘off’ and ‘on’ in a tightly regulated manner, kinase fusions are ‘on’ all the time, tricking cells into constantly dividing. If one blocks the abnormal signals from a kinase fusion with a drug, cancer cells can die, and patients can enormously benefit. When we started this project, only a limited number of TK fusion proteins had been identified in solid tumors, because experimental procedures for their identification were inadequate.

As part of this grant, we developed a novel method to identify kinase fusion proteins using DNA from any type of tumor. We demonstrated the feasibility of this platform on tumor cell lines (Chmielecki et al., Nucleic Acids Res, 2010) and then used the results of the pilot study to create an improved platform with streamlined workflow and computational analysis. Using the newer kinase fusion design, we screened multiple tumor samples and identified a novel PDGFRbeta fusion from one patient sample (Chmielecki et al., Genes Chromosomes Cancer, 2011). Since then, commercial entities like Foundation Medicine have incorporated a similar approach to detecting kinase fusions from formalin-fixed paraffin-embedded tumor samples; their FoundationOne test is now used routinely for patient care.

During the course of this grant, next-generation sequencing platforms were constantly improving. Thus, we subsequently used multiple different sequencing platforms to identify novel actionable targets for therapy. These included projects in non-small cell lung cancer, small cell lung cancer, and melanoma.


2009 42 Month - Cancer Cell-Specific Self-delivering Prodrugs

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We sought to develop a novel system for targeted delivery of drugs that localize specifically to tumor cells in whole animals. To do this, we made use of an alternate class of targeting molecules composed of nucleic acids called aptamers. These cell type specific aptamers were composed of or linked to cytotoxic nucleoside analog drugs. Following internalization within cancer cells, breakdown of the aptamers should result in release of the drug molecules. In this way, drugs will preferentially accumulate in cancer cells, which we believe will translate into minimization of off-target adverse side effects on healthy tissue while maintaining or even enhancing the efficacy of these drugs on diseased cells.


2009 42 Month - Modeling Ewing Tumor Initiation in Human Neural Crest Stem Cells

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Many pediatric tumors develop when stem cells or other fetal cells develop alterations in their DNA leading them to differentiate into cancerous rather than normal tissues. Alterations can be induced by mutations that disrupt the DNA or by so-called epigenetic processes that alter gene expression without changing DNA sequence. In this project we used novel stem cell models and innovative technologies to study how changes in DNA methylation contribute to the origin and progression of Ewing sarcoma (ES).

ES is believed to arise from neural crest stem cells (NCSC). In Aim 1 we defined what DNA methylation should look like before, during and after these stem cells differentiate. Although changes in methylation occurred during differentiation, most did not occur in the regulatory regions of genes, so-called gene promoters. Instead, most changes to DNA methylation occurred in the regions that lie between genes. In contrast, the pattern of DNA methylation in ES cells differed dramatically and gene promoters were found to be selectively hypermethylated. Thus, DNA methylation in ES deviates from both normal NCSC as well as their normal progeny.

ES arises from cells that acquire a genetic mutation, termed EWS-FLI1. Although EWS-FLI1 can initiate the process of malignant transformation it is not sufficient to create a tumor. In Aim 2 we investigated whether NCSC that express EWS-FLI1 acquire changes to DNA methylation. Our studies showed that the methylation profiles of EWS-FLI1+ stem cells were abnormal and evolved over time. In particular, the promoters of genes that control embryonic development became progressively more methylated over time and the global profiles of EWS-FLI1+ cells evolved to resemble those of fully malignant tumor cells. Evaluation of DNA methylation in ES tumor biopsies confirmed that the patterns of abnormal methylation that we detected in EWS-FLI1+ stem cells and ES cell lines were present in patient tumors and were not simply a consequence of cell culture. Analysis of gene expression data confirmed that abnormal methylation was associated with abnormal gene expression. In particular, the normal regulation of genes involved in early embryonic development was disrupted in EWS-FLI1+ stem cells and tumor cells. Thus, our studies in Aim 2 confirm that abnormal epigenetic regulation of gene expression, via abnormal DNA methylation, is a critical event in the creation of ES and is driven by the EWS-FLI1 oncogene.

In Aim 3 we tested whether the growth of ES tumors in mice could be blocked by a drug that inhibits DNA methylation or by turning off the EWS-FLI1 oncogene. Significantly, both interventions slowed tumor growth demonstrating that the oncogene and abnormal DNA methylation both serve as key drivers of ES tumor progression.

In summary, our studies together confirm that abnormal DNA methylation and disrupted epigenetic regulation of embryonic gene expression is a key driver of ES initiation and progression. These studies support introduction of epigenetic modifying agents into ES treatment protocols.


2009 36 Month - Functional Oncogene Identification

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Despite advances in diagnosis and treatment, more than one-half of adults with cancers of the blood (i.e., leukemia, lymphoma and multiple myeloma) will die from their disease. One of the limitations in our current approach is that most cancer chemotherapy does not target abnormalities unique to the tumor cells, but instead kills all growing cells. Thus, the identification of specific cancer-associated abnormalities is an essential first step toward newer and more effective therapies. We developed a system to identify new targets for therapy directly from leukemia and lymphoma samples. Briefly, we isolate the many millions of pieces of genetic material from a tumor sample and then individually insert each into cells that can only grow in a special kind of culture. If one of the pieces of genetic material has a cancer-promoting effect, it allows the cells to grow in normal culture. Thus, any cell that survives in the normal culture must contain a piece of genetic material from the tumor that has a cancer-promoting effect. We can easily identify that piece of genetic material and then confirm that it is important for the tumor’s growth. The system we developed is efficient and can be scaled up to analyze a large number of individual specimens. Using this approach, we have already discovered a new cancer protein called CRLF2 in some cases of acute lymphocytic leukemia. The overall goal of our Stand Up To Cancer Innovative Research Grant proposal is to identify important alterations that promote the growth of other types of blood cancer. During the funding period, we utilized our approach to screen 16 types of human leukemia and lymphoma samples for new mutations. From this screen and from sequencing large amounts of DNA in the specimens, we identified multiple mutations that have never been described from tumor specimens. Of particular interest, we identified mutated versions of proteins that can be targeted with available drugs. We are in the process of confirming that the mutations we identified contribute to tumor growth. We are also defining the frequency of these mutations in other leukemia and lymphoma specimens. These studies have led directly to additional funding from the Leukemia and Lymphoma Society, Claudia Adams Barr Program in Cancer Research and Dana-Farber/Novartis Drug Discovery Program, with the goal of identifying new treatment approaches that target these alterations.


2009 36 Month - A Transformative Technology to Capture and Drug New Cancer Targets

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The goal of my SU2C project was to create a powerful, new, and versatile approach to identifying
and drugging new cancer targets. To that end, we combined a chemical technology termed
“hydrocarbon stapling” that restores natural shape to bioactive peptide alpha-helices with a protein
capture technology in order to trap and characterize critical targets involved in defective signaling in
cancer. With the first year of SU2C funding, we achieved our goal of chemically synthesizing a pilot
panel of “photoreactive stabilized alpha-helices” or pSAHs, which are the chemical tools designed to
capture and characterize new cancer targets. We examined and optimized the sensitivity and specificity of these new tool compounds for crosslinking to discrete physiologic targets of the BCL-2 pathway, a key signaling network implicated in cancer pathogenesis and chemoresistance. This SU2Csponsored
proof-of-concept study was published as a cover article in Cell’s Chemistry and Biology in December of 2010. In this publication, we demonstrated our capacity to successfully and reproducibly generate pSAHs that recapitulate the structure of distinct bioactive domains and deploy them to trap, purify, and identify their natural cellular targets with high fidelity. In addition, we reported a rapid and reliable method for inputing our crosslinking data into a binding site algorithm that employs mass spectrometry and computational docking analysis to calculate model structures of the key-in-lock binding interfaces we discovered. This critical information provided the basis for validating new proteinprotein interactions for drug development and therapeutic targeting. Having defined and published the “rules” for successful production and application of pSAHs by the end of year 1, we dedicated year 2
support to expanding our arsenal of pSAH constructs in order to home in on key protein interactions
that drive cancer. In doing so, we ultimately synthesized a library of over 80 photoreactive helices
spanning the death domains of 10 seminal BCL-2 family apoptosis proteins implicated in oncogenesis
and the response to chemotherapy. In applying these unique reagents during the final year of the grant, we characterized novel interaction sites at the atomic level, identified unanticipated interactors, and developed a versatile chemical toolbox for validating and modulating these critical protein
interactions in cancer cells. In addition to the development and deployment of the technology itself, our flagship achievement from the grant period was the discovery and characterization of novel interaction surfaces to “inhibit the inhibitors” and “activate the activators” of cell death in resistant human cancers. These blueprints provided fundamental new directions for targeted drug development in cancer, and have already catalyzed the advancement of novel stapled peptide and small molecule modulators that reactivate cancer cell death.


2009 36 Month - Noninvasive Molecular Profiling of Cancer via Tumor-Derived Microparticles

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Obtaining molecular information about an individual cancer patient’s tumor would allow for the development of patient-specific treatment plans.  However, obtaining tumor biopsy samples in many cases requires invasive surgery which can be painful, disfiguring and potentially dangerous.  Consequently, alternative non-invasive sampling methods are needed which could serve as a reliable surrogate for actual tumor tissue.  The approach we are pursuing takes advantage of the fact that cancer cells release information into the bloodstream.  This information is packaged into what are called tumor-derived microparticles; essentially small parcels derived from the contents of the tumor cells.  The goal of this project is to develop methods to efficiently capture these particles from patient blood samples and decode the information within them in order to gain molecular information about the cancer cells from which they originated.  Over the course of this project we developed methods for the capture and evaluation of a number of different particles types.  In doing so, we were able to focus our efforts on a specific type of microparticle known as as an exosome.  We have developed methods to specifically and efficiently capture an purify exosomes from clinical samples.  Molecular characterization of the contents of these capture particles is in progress. 

Furthermore, we also recently discovered that although exosomes do contain some of the information we seek, much of this information is in fact present is a larger, mid-sized class of particles that we are currently characterizing.  Consequently, we are now engaged in investigating the nature of these particles using different techniques that will allow us to determine their size, as well as to identify molecules on their surface that will permit us to capture them from blood samples.  Taken together, we believe that these various particle types will provide molecular information derived from the tumor and serve as a tumor surrogate.  Our ultimate goal is to obtain key information using a blood sample that can inform critical clinical decisions such as choosing the most effective drug for an individual patient.


2009 36 Month - Modulating Transcription Factor Abnormalities in Pediatric Cancer

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There is an urgent unmet need for more efficacious therapies for childhood cancers. Many of the cancerpromoting, tumor-specific proteins in these pediatric cancers, however, have been considered “undruggable” by traditional drug discovery approaches. One class of challenging cancer-promoting proteins is proteins that bind to DNA called transcription factors. Our laboratory developed new chemical genomic approaches to target these elusive proteins. We applied these approaches to two pediatric malignancies: Ewing sarcoma, the second most common cause of bone cancer in children, and neuroblastoma, the most common extracranial solid tumor of childhood, both diseases characterized by the expression of cancer-promoting transcription factors, and diseases where treatment for patients with high-risk tumors remains poor.

In Ewing sarcoma, the majority of tumors express the cancer-promoting transcription factor, EWS/FLI. We developed an alternative to traditional drug discovery using DNA microarrays (“gene chips”) to characterize the genes that are turned on or off in the presence or the absence of the Ewing sarcoma protein (Gene Expressionbed High-throughput Screening (GE-HTS)). We completed a screen of over 10,000 chemicals, prioritized 160 top scorers, and focused our attention on several molecules already FDA-approved in humans or in clinical development. These compounds now serve as tools to further our understanding of Ewing sarcoma development and as leads toward clinical trial development. Moreover, we have “platformized” GE-HTS at the Broad Institute to enable access to many investigators. In parallel to these efforts, we also conducted a screen to identify more druggable targets in this disease. We identified the protein focal adhesion kinase (FAK) as highly activated in Ewing sarcoma tumors. Treatment of Ewing sarcoma cells with a FAK-inhibitory drug, PF-562271, impaired viability and induced cell death. Additionally, PF-562271 attenuated Ewing sarcoma growth in mouse
xenograft models. With FAK inhibitors currently in clinical trials for adult malignancies, these findings may bear immediate relevance to patients with Ewing sarcoma.

A second approach taken by my laboratory is large-scale screening of genetically-defined cancer cell lines for response to specific small molecules of interest. We collaboratively screened a panel of over 600 genetically characterized cancer cell lines for response to a new class of epigenetic-modifying drugs called BET bromodomain inhibitors. BET bromodomain inhibitors have been studied in a handful of discrete malignancies, but genomic biomarkers to direct clinical translation have been lacking. In our study, integration of genetic features with chemosensitivity data demonstrated a robust correlation between amplification of MYCN, a gene encoding for a transcription factor, and sensitivity to bromodomain inhibition. We have characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent MYCN amplification. Genome-wide expression analysis demonstrated down regulation of the MYCN transcriptional program and the expression of the MYCN transcript. Functionally, bromodomain-mediated inhibition of MYCN induced cell death in neuroblastoma cells. BET inhibition conferred a significant survival advantage in three mouse models of neuroblastoma. A clinical trial to test BET bromodomain inhibitors in patients with neuroblastoma is now in development.


2009 36 Month - Endogenous Small Molecules that Regulate Signaling Pathways in Cancer Cells

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A major goal in cancer biology is the comprehensive understanding of signals that drive the growth and spread of cancer cells. My long-term goal is to develop methods to isolate new small molecules that play a role in cancer signaling and then to identify the proteins that interact with these small molecules. Such small molecule-protein pairs are likely to be particularly good drug targets in oncology. To develop tools for this endeavor, we are focusing on the identification of small molecules that regulate the “Hedgehog” signaling circuit. Damage to this circuit has been shown to drive the development of a large number of adult and childhood cancers. Major progress over the course of this grant, now published in Nature Chemical Biology, has been the unexpected discovery that cholesterol-like small molecules called oxysterols can directly influence the protein Smoothened, a cancer-driving protein that is the major drug target in this pathway. This discovery of the mechanism by which these cholesterol-like molecules influence a cancersignaling pathway was a major goal in our initial proposal. Importantly, we have shown that this mechanism can be exploited to develop a new class of anti-Hedgehog drugs based on a cholesterol scaffold. We believe that this discovery supports our initial IRG hypothesis that endogenous small molecules can have dramatic effects in the activities of cancer-relevant proteins, and we are now actively engaged in discovering such regulatory interactions and establishing their therapeutic relevance.


2009 36 Month - Genetic Approaches for the Next Generation of Breast Cancer Tailored Therapies

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Cancer therapy has radically changed during the last decade. Novel therapies based on the specific molecular changes that drive tumorigenesis in every patient are emerging as low toxicity and more efficient alternatives to classical treatments. An alternative promising approach for the design of these personalized therapies is the use of genetic synthetic lethal interactions. These occur when two genetic alterations that are individually innocuous appear in the same cell, causing growth inhibition. This concept can be exploited to identify genes that, when inhibited, exclusively reduce the viability of tumor cells that carry a preexisting genetic lesion.

Recently, RNA interference (RNAi) technology has emerged as a very powerful approach to attenuate the expression of any chosen gene. Thus, we envision using RNAi to identify genes that, when attenuated, exclusively reduce the viability of tumor cells carrying specific genetic lesions without affecting normal cells. During the last years, my group has pioneered the development of RNAi based genetic tools for studies in mammalian cells. This technology represents a unique opportunity to identify synthetic lethal effects with major cancer alterations. In this project we proposed to apply our state-of-the-art technology to uncover synthetic lethal interactions with the major breast cancer genes.

Our proposal is divided into three specific aims that represent the transition from target discovery and validation to mechanistic characterization.

-Specific Aim 1. Identify genes that interact with the major breast cancer alterations to produce synthetic lethality in vitro (1st year): During the first year of this project, we proposed to complete four genome-wide RNAi screens in vitro to identify target genes that, upon inhibition, reduce the cell viability in breast cancer cells with any of the major breast cancer alterations; ErbB2, c-Myc, Cyclin-D or RB. In our pre-defined milestones we estimated that two of the four RNAi screens would be completed during the first six months and the rest during the second semester of the first year. At this point, the four genome-wide RNAi screens have been completed and analyzed.

-Specific Aim 2. Model selected lethal interactions in vivo (1st and 2nd year): Completion of the above mentioned screens has provided us with a list of candidates that were further validated in vivo by candidate driven RNAi screens in mouse models during the second year of the award.

-Specific Aim 3.Initial characterization of the molecular mechanism of the genetic lethality (2nd and 3nd year): Upon completion of Aim 2, we selected the most promising (2-3) target (inhibiton of STAT3 in ErbB2+ tumors) to investigate the biology of the lethal phenotype in more detail.


2009 36 Month - Therapeutically Targeting the Epigenome in Aggressive Pediatric Cancers

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Recently there has been a growing realization that some of the critical changes in gene expression required for the development of cancer do not arise via genetic mutations in DNA but rather are ‘epigenetic’ changes that affect gene expression indirectly by affecting DNA packaging. The SWI/SNF complex controls the protein support structure that surrounds key growth genes and is thus at the heart of this epigenetic regulation. Mutations in SNF5, a core subunit of the SWI/SNF complex, are present in the large majority of malignant rhabdoid tumors (MRT), a highly lethal cancer that occurs in kidney, brain, and soft tissues of young children. Inactivating mutations in SNF5 have recently been found to occur in a variety of other cancers as well including epithelioid sarcomas, small cell hepatoblastomas, undifferentiated sarcomas, chondrosarcomas, familial schwannomatosis, and renal medullary carcinomas. Mutation in SNF5 is also the basis of an inherited cancer predisposition syndrome. As the cancers that arise following Snf5 loss appear to be largely driven by the epigenetic consequences, we hypothesize that these cancers will be particularly susceptible to drugs that interfere with epigenetic mechanisms of gene regulation. The experiments in this proposal were designed to reveal the underlying mechanisms by which SNF5 loss affects gene expression and thereby causes cancer with a goal of identifying improved therapies that can be rapidly translated into patients. Of note, during the time of SU2C grant support, cancer genome sequencing studies have revealed remarkable findings: in addition to SNF5, at least 6 other SWI/SNF subunits are frequently and recurrently mutated in a wide variety of cancers, both pediatric and adult, including cancers of lung, breast, stomach, liver, ovary, uterus, kidney, bladder, brain and melanoma. Consequently, the work performed during this IRG now has broad implications for many types of cancer.

Over the course of the IRG support, we exceeded our expectations. While not all of our hypotheses proved correct, we made outstanding progress on others. Indeed, the first clinical trial based upon our work is now open at 6 centers nationwide, and several in Europe. In addition, we anticipate opening of a second clinical trial in 2014 directly based upon work that was accomplished via IRG support. With respect to the specific aims of this proposal, based upon identifying over-expression of miR-21 in SNF5
deficient cancers, we had hypothesized that inactivation of this micro RNA might reverse the growth of Snf5-deficient cancers. This proved incorrect – it had no effect. With respect to changes in histone modifications caused by Snf5 inactivation, we identified the existence of epigenetic antagonism between SNF5 and EZH2, a member of a different chromatin modifying complex. This suggested that inhibiting EZH2 might stop the growth of SNF5 mutant cancers and, indeed, that is precisely what we found in our pre-clinical models, and we’re hopeful that this will be headed into the clinics for patients with rhabdoid tumors in 2014. With respect to or DNA methylation hypothesis, we’re still working on obtaining data to determine the extent to which Snf5 loss affects DNA methylation, and whether this will be a good target. However, we have performed trials of DNA methylation in mice bearing Snf5-deficient tumors. Bottom line: work from my lab has now led to a new, first-time mechanistically targeted clinical trial that is now open, and a new trial directly derived from the SU2C work slated to open in 2014.


2009 36 Month - Targeted Inhibition of BCL6 for Leukemia Stem Cell Eradication

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Despite significant advances in the treatment of leukemia over the past four decades, the rate of long-term survival has reached a plateau and still large numbers of leukemia patients die, mostly because of relapse and drug-resistance. These two clinical problems were recently attributed to the persistence of leukemia stem cells. If a therapy succeeds in eradicating leukemia stem cells, renewed initiation of the disease (relapse) is no longer possible. Therapeutic progress in recent clinical trials has likely been stalled, partly because current chemotherapy approaches target proliferating bulk leukemia cells rather than non-dividing leukemia stem cells. We now discovered that BCL6, a factor known to play a central role in lymphomas, also plays a key role in the maintenance of leukemia stem cells. Since leukemia stem cells represent the origin of relapse and drug-resistance in leukemia in many cases, the identification of BCL6 as a target for leukemia stem cell eradication holds great promise. BCL6 is a master regulatory factor that controls the production of many different important genes. BCL6 was not previously known to be involved in leukemia. In preliminary studies for this proposal, we have discovered aberrant expression of BCL6 as a central component of a fundamentally novel pathway of leukemia stem cell self-renewal and drug-resistance in a wide array of human leukemias, some of which are still difficult to treat. In these leukemias, drug-treatment results in aberrant production of BCL6 by the leukemia cells, which appears to allow leukemia stem cell to self-renew and become resistant against chemotherapy. Recently a drug has been developed that can attach to BCL6 and block its cancer-causing activities. We found that this BCL6 inhibitor, called RI-BPI, has strong cooperative activity when combined with conventional chemotherapy. This opens up a powerful new therapeutic strategy for leukemia stem cell eradication through targeted inhibition of BCL6. Based on the discovery of BCL6 as a key component of a novel pathway of drug-resistance and stem cell self-renewal in a wide array of leukemias, we propose three Aims to develop these findings towards application in patient care: (1) To test the hypothesis that BCL6 is critical for leukemia-initiation and relapse of leukemia, (2) To determine the frequency and appearance of BCL6-dependent leukemia stem cells in human leukemia samples and (3) To validate the role of the BCL6 inhibitor RI-BPI as a novel therapeutic agent for targeted eradication of leukemia stem cells. Since RI-BPI is currently going through the process of approval for use in clinical trials, we expect to be able to test the power of this approach in clinical trials by the end of the funding period.


2009 36 Month - An Emerging Tumor Suppressor Pathway in Human Cancer

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Hippo is a novel biochemical pathway that can regulate organ growth. It seems to work by preventing further cell division once organs have reached their proper size. Our group believes that Hippo signals participate in a novel and powerful “checkpoint” that restricts cell proliferation and activates cell death. Such a checkpoint would normally enable an organ to “know” its size and stop growing when the appropriate dimensions are achieved. A checkpoint mechanism would also serve to suppress the unchecked cell proliferation that characterizes tumor growth. Our proposal aims to study the existence of this checkpoint and to examine its role in tumor growth. In Aim 1, we proposed to test this hypothesis by using genetically engineered mice in which Hippo signaling can be turned on or off. For Aim 2, we proposed to identify novel proteins and small molecules that can modulate Hippo signaling in cultured cells and that could eventually be used to develop therapies for cancer.

For Specific Aim 1, we have found that inactivation of the Hippo pathway in the skin of mice leads to an expansion of the stem cells that normally form and maintain the skin, and this eventually leads to the development of invasive squamous cell carcinomas. We have also found that a specific sub-class of human squamous cell carcinomas might be driven by alterations in this pathway, indicating that these tumors might benefit directly from therapies targeting components of this pathway. Along these same lines, we have made similar observations in skeletal muscle, where inactivation of the Hippo pathway leads to the amplification of the muscle-specific stem cells, and the subsequent development of invasive rhabdomyosarcomas. We have validated these observations in human cells and tumors suggesting that Hippo might be an important pathway to target in soft-tissue targets. Additional results indicate that the Hippo pathway is an important suppressor of tumor growth in the small intestine and the colon. Our discoveries to date highlight a previously unappreciated global role for Hippo as an important cancer preventing checkpoint in mammals.

We have also demonstrated using mouse models that re-activation of the pathway can lead to an important suppression of growth in a model of basal cell carcinoma and hepatocellular carcinoma. Conversely, in the intestine over-activation of the pathway might not be beneficial and might even be harmful.

For Specific Aim 2, we have finalized a genetic screen to identify kinases or phosphatases (specific subset of proteins in the human genome) that can regulate Hippo signaling. We have identified approximately 40 molecules that can have important effects on Hippo activity. Among these, we have discovered an important connection between Lkb1, a very common gene altered in human cancer, and the Hippo pathway. This means that human cancers with mutations in Lkb1 could be amenable to treatment by manipulating the Hippo pathway. Additionally, another molecule identified in this screen, alpha-catenin, is a known tumor suppressor in the skin and other epithelial tissues. How alpha-catenin prevented tumor growth was unclear, our results suggest that alpha-catenin inhibits growth by directly controlling the activity of the Hippo pathway. Thus, our results would argue that these alpha-catenin mutant tumors would benefit from therapies inhibiting Hippo signaling.


2011 18 Month - Coupled Genetic and Functional Dissection of Chronic Lymphocytic Leukemia

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The treatment of chronic lymphocytic leukemia (CLL) poses two main challenges: 1) predicting the clinical course in a disease that shows many differences across patients, and 2) overcoming the insensitivity of some patient tumors to chemotherapy. At this time, genetic abnormalities are the best predictors of disease progression, based on gross chromosomal changes. However, an urgent need remains for improved understanding of how disease starts and progresses, which would lead to better predictive markers and potentially more effective (and non-toxic) therapies. Recent advances in genomic technologies provide a unique opportunity to find the genes and molecular circuits that make tumors grow in CLL. We have collected tumor and normal cells from 200 CLL patients and are almost done with sequencing all their genes. We are also looking at how genes are expressed in the same patient tumors using gene microarrays. Most importantly for enabling this project, our laboratory has pioneered the use of silicon-coated nanowires as a method of delivering DNA, RNA to primary CLL and normal B cells, which allows us to genetically manipulate CLL cells for the first time in a high-throughput fashion. Analysis of the first sixty patients has already identified genes that are important for CLL (called ‘driver mutations and pathways’). We have used our nanowires to verify the importance of some of these genes in CLL tumors cells. We now propose to find all the major genes and pathways that control CLL tumor formation. We will use a combination of sequencing technologies with statistical analyses to find the key genes that are important in creating tumors in CLL patients. In addition, we will find out which genes are good predictors of disease progression. Then, we will use our nanowires to place the mutant genes from CLL tumors into normal B cells and see how they affect their behavior. By taking this unique approach of combining different kinds of data collected from patient samples and using nanowires to manipulate the tumor cells in culture, we hope to understand the basic reasons why CLL patients develop cancer. This information will help us predict the progression of disease and provide new strategies for therapy. Finally, our approach can be extended to other tumors, especially leukemias and lymphomas.


2011 18 Month - Framing Therapeutic Opportunities in Tumor-activated Gametogenic Programs

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1. The overarching goal of this grant is to identify and nominate new targets for cancer therapy. The focus is on a set of 119 proteins, which are typically expressed during the growth and maturation of sperm, oocytes and during fetal development. Abundant evidence indicates that these proteins, known as CT-antigens (CTA’s), are frequently re-activated in tumor cells, but not expressed in normal adult tissue. Furthermore, a number of studies have indicated that high expression of CTA’s can correlate with poor prognosis. However, the tumorigenic function of this reactivated gametogenic program is largely unknown. Given their restricted expression pattern, these proteins may represent ideal therapeutic entry points, if they are essential for tumor cell growth and survival. Thus, our proposal seeks to: 1) establish a tumor cellbased platform to study the function of CTAs, 2) employ a systematic screening strategy to identify those CTA’s that are essential to tumorigenic behaviors and 3)elaborate how lead candidates function at the molecular level and in vivo.

During the initial phase of this work, a discovery platform to interrogate CTA function in a range of neoplastic processes was devised and deployed in a diverse set of tumor-derived cell-lines (Aim 1 and 2). This effort has revealed that distinct CTA’s can support many of the hallmarks of cancer, including: 1) enhanced energy production 2) hijacking developmental programs that promote self-renewal and metastases 3) modulation of cell-fate programs that support proliferation 4) adaption to low oxygen conditions inherent in the tumor microenvironment and 5) activation of survival and inflammatory signaling. FATE1 and COX6B2 were found to support tumor cell survival, potentially by enhancing mitochondrial energy production. IGSF11, FTHL17, and ZNF165 each make individualized contributions to TGFβ signal propagation. All three proteins are also required for tumor cell viability, potentially by promoting cell adhesion, iron homeostasis and the transcription of genes that regulate epithelial to mesenchymal transition, respectively. The CTA XAGE2 modulates the WNT developmental pathway by supporting the activation of a key signaling component, β-catenin. PIWIL2, IGF2BP3, TDRD1 and MAGEA3/6 proteins mediate adaptation to hypoxia by enhancing the stabilization of the HIF-1α protein. Finally, the CTA, MAGEA1, supports activation of the NF-κB pathway, which promotes tumor cell survival. Over the next 6 months, our goal is to validate these high-priority candidates and continue to elaborate their mechanisms of action at the molecular level as well as in animal tumor models. Thus far, our work suggests that CTA’s may confer traits that promote growth and survival in the tumorigenic environment. Further investigation of the mechanisms of action of these CTA’s may identify new anti-tumor therapeutic strategies.


2011 18 Month - Developing New Therapeutic Strategies for Soft-tissue Sarcoma

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Sarcomas are highly aggressive cancers that arise in connective tissues such as bone, fat and cartilage, as well as in muscles and blood vessels embedded within these tissues. Approximately 12,000 Americans are diagnosed with sarcoma each year, and current treatment strategies, especially for advanced forms of the disease, are often ineffective, leading to high rates of mortality among sarcoma patients. To advance sarcoma treatment and develop new approaches to cure these tumors, my lab established a new mouse model of soft- tissue sarcoma in skeletal muscle that introduces disease-relevant genetic modifications into tissue stem cells found normally in the skeletal muscle. We further used this model to identify a small group of 141 genes present at increased levels in both mouse and human sarcomas.

Our goal in this SU2C Innovative Research Grant is to test this novel set of sarcoma-induced genes to identify new candidate drug targets for these poorly-treatable tumors.

In the past 6 months, we have made substantial progress towards this goal, using genetic and pharmacological approaches to test the importance of candidate genes and pathways in sarcoma biology. We first found that one high-priority target, Gremlin1, acts as an extracellular growth factor for sarcomas and positively regulates tumor malignancy. Further studies are now underway to evaluate the impact of Gremlin1 inhibition on tumor growth and metastasis in mouse tissues. Our analysisof sarcoma-associated genes also implicated the avoidance of a novel form of iron-dependent cell death (called “ferroptosis”) in the continued growth of sarcoma cells. In collaboration with Brent Stockwell’s lab at Columbia University, we tested the effects of 10 chemical compounds that trigger ferroptosis on mouse and human sarcoma cells, and found that a subset of these significantly inhibit sarcoma cell growth. Our future studies will assess the ability of these compounds to serve as drugs that can be used to slow or shrink established tumors in animal models. Finally, we completed and analyzed a custom screen to systematically evaluate the effects of genetic “knock down” of each of the 141 candidate genes we identified. This comprehensive study revealed 17 high-priority targets whose inhibition resulted in substantial reduction in sarcoma cell growth in culture. Silencing of one of these targets, an enzyme that stimulates production of the amino acid asparagine, resulted in dramatic blockade of tumor cell growth in culture for both rhabdomhyosarcoma and non- myogenic sarcoma. Likewise, exposure to asparaginase, which enhances the degradation of cellular asparagine stores, also blocked sarcoma cell growth in culture. As asparaginase is already in clinical use for the treatment of some leukemias, we are excited to test its impact on established sarcomas in animal models. Positive results in these studies could lead rapidly to the repurposing of this anti-leukemia drug as an anti-sarcoma therapeutic.

In summary, our studies pursue a highly integrated strategy to identify novel targets for sarcoma therapy. Ultimately, we believe that this work will help to uncover the root causes of sarcoma formation and identify new strategies to cure these aggressive cancers.


2011 18 Month - Inhibiting Innate Resistance to Chemotherapy in Lung Cancer Stem Cells

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Lung cancer is the leading cause of cancer fatalities worldwide. The most common form is non- small cell lung cancer (NSCLC). Platinum-based chemotherapy drugs (such as cisplatin) are commonly used to treat NSCLC, but they only marginally increase survival due to the innate resistance of some tumor cells to chemotherapy. There is an urgent need to develop new ways to increase the effectiveness of chemotherapy for this disease. Past strategies for developing new drug targets have relied almost exclusively on testing cell lines grown directly on plastic culture dishes in “2D”. However, the biology of these cells is very different from that of tumor cells, which survive in a “3D” environment. To address this problem, we have developed methods for growing primary tumor cells in “3D” cultures (suspended in a gel-like material that mimics the tumor environment, rather than attached to plastic). Our approach combines the advantage of rapidly testing new drug targets in a 3D culture system with the ability to validate our findings in vivo using a mouse model of NSCLC. We will use this approach as a platform to identify new ways to make chemotherapy more effective at killing lung tumor cells.

In our studies, we use tumor cells isolated from a well-characterized mouse model of NSCLC in which tumors carry one of the most frequent genetic mutations found in human lung cancer (a gene called K-ras). We have identified a way to isolate a population of tumor cells from these mice that form spheres in a 3D culture system. We are testing whether inhibiting specific genes makes cells growing in 3D more sensitive to chemotherapy using shRNAs (short hairpin RNAs) that target individual genes and inhibit their action. We are using this approach to identify new ways to make chemotherapy more effective at killing lung tumor cells by (1) identifying novel regulators of chemoresistance in lung cancer cells, (2) determining how targeting regulators of chemoresistance can increase tumor clearance in combination with chemotherapy, and (3) finding novel regulators of chemoresistance in human NSCLC that could be targeted to improve patient outcomes with chemotherapy.

In previous research periods, we optimized the design of screens to test which shRNAs inhibit chemoresistance. During this period, we have completed a mock screen and are in the process of determining how many shRNAs can be tested in the full screen. The completion of the mock screen will allow us to move forward and complete the entire shRNA screen in the next research period. In addition, we have also analyzed the genes expressed in 3D spheres in response to cisplatin using a novel approach called “RNAseq.”. We will utilize this information to tailor which shRNAs we test to the targets most relevant to chemoresistance in the 3D system and create a custom library of shRNAs that is also composed of clinically relevant genes. In the next research period, we will analyze and validate the results of the screen in vitro and begin testing our results in vivo.

In our initial proposal, we have also outlined plans for transferring the studies done in mice to primary human lung cancer to confirm that our studies are of relevance to human disease. We are continuing our efforts to establish a “tumor bank” of human samples that are established by direct grafting of human tumor samples into immunocompromised mice.


2011 18 Month - Targeting Sleeping Cancer Cells

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Cancer cells of different types have the very strange ability to go to sleep and then eventually wake up. While cancer cells sleep they are highly resistant to virtually all currently available forms of treatment. However, we do not understand how highly aggressive cancer cells can become dormant. It has proven extremely difficult to study these cells directly in patients and we have lacked suitable model systems to study them in the laboratory. We recently made a remarkable observation, however, that has the potential to open this important area for new investigation. We found that highly aggressive cancer cell lines of various types occasionally produce dormant cells. We went on to develop reliable methods for the prospective identification, isolation, molecular tracking, and experimental study of these “G0-like” dormant cancer cells in human cancer cell lines. Our preliminary results raised the possibility that epigenetic or signaling networks regulate these spontaneously dormant cancer cells.
With a SU2C-AACR Innovative Grant Award, we have been using cutting edge molecular and cellular biology and genomic (next-generation sequencing (ChIP-seq / RNA-seq)), proteomic (reverse-phase protein microarrays), and computational technologies to identify and validate 1) genetic and 2) protein signaling networks that might trigger and maintain cancer cell dormancy.

Since the start of the award, we have made tremendous progress (see Dey-Guha, PNAS 108:12845 (2011)). Importantly, we have found that rapidly proliferating cancer cells can divide asymmetrically to produce slowlyproliferating “G0-like” progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT1 kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with allosteric small-molecule inhibitors can induce asymmetric cancer cell division and the production of slow proliferators.

Most recently, we have discovered that AKT1 (rather than AKT2 or AKT3) is both necessary and sufficient for entry into the G0-like cell state. Moreover, AKT1 signaling is suppressed by suppression of AKT1 total protein levels via an mTORC2-induced, TTC3 / proteasome- mediated degradation pathway. In addition, RNA-seq studies suggest that G0-like cells actually assume a unique “stem-like” state with activation of the CTTNB1, FOXO1, and NOTCH1 pathways and global alterations in chromatin state. Furthermore, we have found that RNAi- mediated disruption of mTORC2 signaling does not alter the bulk proliferative properties of multiple human cancer cell lines, but completely abrogates the production of “G0-like” cancer cells, which in turn profoundly alters the tumorigeneity of these cell lines as xenografts in nude mice. We have submitted these exciting new mechanistic results for publication (2nd manuscript in preparation).

Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the triggering of a previously unappreciated mTORC2-AKT1-TTC3-proteasome signaling pathway during cancer cell mitosis, and the G0-like cancer cells arising through this mechanism play an important but previously unappreciated role in driving tumorigenesis. This model promises significant implications for understanding how tumors grow, evade treatment, and recur.


2011 18 Month - A Systems Approach to Understanding Tumor Specific Drug Response

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We propose using genomic technologies to track tumor response to potent drug inhibition of critical pathways across a diverse tumor panel. We will develop cutting-edge computational machine learning algorithms to piece these data together and illuminate how a cell’s regulatory network processes signals, and how this signal processing goes awry in cancer. By studying a large panel of diverse tumors we can begin to piece together general principles and patterns in response to drug. These studies should teach us what drives cancers and what part of the networks we should target. For each individual patient, we wish to determine the best drug regime for that individual, informed by a model that can predict tumor response to drugs and their combinations. Treatment that is based not only on understanding which components go wrong, but also how these go wrong in each individual patient, will improve cancer therapeutics.

The in the middle of the second year we are focused on better understanding differences between patients and their response to drug:

1. We learned that most of the differences in response to a prevalent targeted treatment of melanoma (PLX) is likely not due to differences related to the drug target, but more global differences in other pathways and their cross-talk with the drug target. We are developing systematic ways to analyze this. Both to help pinpoint the patients that will most benefit from PLX and combinatorial treatment to expand this cohort of patients.

2. We have learned that there is heterogeneity not only between patients, but also with subpopulations of a single patient. We are developing methods to characterize, identify and understand drug resistant subpopulations.


2011 18 Month - Identification and Targeting of Novel Rearrangements in High-Risk ALL

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Acute lymphoblastic leukemia (ALL) is the commonest childhood cancer, and the leading cause of non-traumatic death in children and young adults. This project has focused on a recently described subtype of ALL termed “BCR-ABL1-like” or “Ph-like” ALL characterized by a range of previously unknown chromosomal changes and mutations that result in activation of cellular growth signals called kinases. These cases of ALL are common, comprising up to15% of childhood ALL and over one third of ALL in adolescents and young adults, and associated with a high risk of treatment failure, hence new therapeutic approaches to improve treatment outcomes are required. Work supported by the Stand Up to Cancer Innovative Research Grant has supported genetic analysis of leukemia cells from patients with BCR-ABL1-like ALL in order to identify the range of genetic alterations in this disorder, to examine the frequency of these changes in large cohorts of ALL patients, and to examine their role in the development of leukemia, and potential responsiveness to therapy.

The first aim of this project is to use genomic sequencing and recurrence testing analysis to determine the nature and frequency of kinase activating genetic alterations in children and young adults with ALL. An initial pilot study that used mRNA-sequencing and whole genome sequencing of 15 children with BCR-ABL1-like ALL identified a range of genetic changes activating kinase signaling in each case. These included large chromosomal alterations, or rearrangements, changes in DNA sequence, and loss of DNA material (deletions), in genes involved include CRLF2, ABL1, JAK2, PDGFRB, IL7R, and SH2B3 (LNK). We have then tested the frequency of each of these changes in cohorts of childhood and adolescent and young adult (AYA) ALL, with current numbers of these cohorts exceeding 800. The changes identified by sequencing of the first 15 cases were present in 80% of the recurrence cohorts. To identify the kinase-activating alterations in the remaining cases, we are performing mRNA- sequencing, exome sequencing and whole genome sequencing in all cases with suitable genetic material lacking one of the kinase-activating alterations identified in the pilot project (mRNA-seq and whole genome sequencing N=50). This second phase of sequencing is complete, and analysis is underway. This has already identified new fusion partners of the known kinases (e.g. ABL1, JAK2 and PDGFRB) and importantly, has identified new kinases as targets for rearrangement, notably CSF1R. The next 6 months of the project will witness completion of this sequencing analysis and testing for recurrence of each new alteration.

The second aim of this project was to develop experimental models to examine the way in which the alterations identified in aim 1 contribute to the development of leukemia, and to develop experimental systems to test the potential effectiveness of TKIs. Using laboratory cell lines, I have shown that several of these alterations accelerate cell growth and activate downstream signaling pathways. In addition, alterations such as EBF1-PDGFRB induce leukemia when expressed in mouse bone marrow cells. We have also developed xenograft models in which human leukemia cells are grown in immunodeficient mice. Importantly, growth of these cell lines is inhibited by several TKIs including include imatinib (Gleevec), dasatinib (Sprycel) and ruxolitinib (Jakafi). We are establishing xenografts of additional tumors to test the activity of other targeted agents.

Together, these studies continue to identify the range of lesions underlying BCR-ABL1-like ALL, and show that these alterations directly contribute to the development of leukemia. Importantly, the experimental models show that these alterations are targetable with TKIs. These results have generated tremendous excitement in the ALL field, and efforts to identify patients harboring these lesions at diagnosis, and to treat them with these drugs are already underway.


2011 18 Month - Exome Sequencing of Melanomas with Acquired Resistance to BRAF Inhibitors

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A small molecule (PLX4032/vemurafenib/Zelboraf) targeting a common melanoma mutation, V600EB-RAF, has shown unprecedented promise in advanced clinical trials (80% of patients respond if their tumors harbor the V600EB-RAF mutation) and confers survival benefit, prompting FDA approval. However, its ultimate success is challenged by so-called acquired drug resistance, which leads to clinical relapse. This type of drug resistance that develops over time occurs within months to years of drug initiation and cuts short the “sudden reprieve” that awakens patients’ hope for a cure (see NY Times stories by Amy Harmon on December 22-24, 2010). Earlier, we reported in Nature the discovery of two means by which melanomas escape from vemurafenib, which suggest new treatment strategies that are testable in clinical trials. This study along with others gave us another insight, that is, melanomas likely use a variety of different ways to escape from B-RAF inhibitors. Discovering other mechanisms of acquired resistance is logically the first step in constructing a therapeutic strategy closer to a cure.

We set forth three research aims centered on this group of V600EB-RAF-positive melanomas treated with B-RAF inhibitors (vemurafenib as well as another competing B-RAF inhibitor, GSK2118436). These aims are based on several premises. First, we need to directly study precious tissues derived from clinical trial patients. Second, we need to enlarge this tissue collection by collaborating among distinct clinical sites. Third, because finding a specific mechanism among the myriad of cancer-related changes is akin to finding a needle in a haystack, we should capitalize on the latest, “high-throughput” genomic technologies. Here, we report assembling a collaboration of multiple clinical sites to study acquired resistance directly in tissue samples from patients. For each patient that participates in this study, we are obtaining a set of normal tissue (e.g., blood), melanoma tissue before drug treatment, and melanoma tissue after an initial shrinkage followed by re-growth. Each set of tumor samples is first studied for the existence of known mechanisms which we have already discovered and characterized with in-depth molecular details in laboratory models. Work along this line has been published recently (Poulikakos et al, Nature, Nov 2011; Shi et al, Nature Communications, March 2012; Shi et al, Cancer Discovery, April 2012), under peer review (one manuscript) or under preparation (one manuscript). This workflow culls out tumor sample sets or patients for detailed genetic analysis. By harnessing the speed of “next-generation” DNA sequencing technology, we are examining the whole exome or the protein-coding, “business end” of the melanoma genomes for key genetic alterations that account for acquired resistance to B-RAF inhibitors in melanoma. From a patient’s perspective, we can now claim we know how melanomas escape from BRAF inhibitors in over 70% of patients. This knowledge will undoubtedly inform clinical trials for in-human hypotheses testing and rationally guide patient care.


2011 18 Month - Chimeric RNAs Generated by Trans-Splicing and their Implications in Cancer

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Substantial progress we made in the last 6 months are summarized in the following:

For aim1: identification of additional trans-splicing events in both normal and cancer cells, we have found the presence of PAX3-FKHR during stem cell differentiation process. The fusion has been thought to be a unique feature of alveolar rhabdomyosarcoma, a common childhood cancer. We found the same fusion product in fetal muscle samples as well. These findings further challenge the traditional dogma that gene fusions are unique to cancer. In addition to some functional evidence, we have come to the conclusion that such chimeric RNA is not unique to the tumor, it is expressed in normal muscle development process and serves important physiological function, and that its generating mechanism in the normal cells is independent of chromosomal translocation, which is the mechanism for the fusion production in alveolar rhabdomyosarcoma. The manuscript was sent to nature, and the reviewers raised a few concerns. We now have gathered further evidence to demonstrate the presence of the fusion at protein level, at RNA level by non-RT-PCR based methods, and its key role in muscle differentiation by loss-of-function approach. In addition, we repeated the previous experiment in a new embryonic-stem cellderived mesenchymal stem cell system. We hope to resubmit our revised manuscript soon. In addition to PAX3-FKHR fusion, we attempted to study EWS-FLI1 fusion associated with Ewing sarcoma. Collaborating with another IRG lab, Elizabeth Lawlor at the University of Michigan, we tested whether the fusion is expressed at a few time points along the stem differentiation to neural crest cells. The initial results turned out negative. Is the expression of EWS-FLi1 too transient to be detected by a few scarce time points? Is neural crest cells no the cell of origin for Ewing sarcoma? Or are we just wrong about the assumption that we will see the fusion in normal development? These are the questions we will try to address in the future.

Because of the broadness of chimeric gene fusions in cancer, our discovery has already raised concerns for false positive cancer diagnoses with current diagnostic methods based solely on the detection of chimeric fusion RNA, as well as for potential side effects in normal tissues caused by therapies targeting these fusion protein products. Through this study, we hope to better characterize the trans-splicing process, and translate our knowledge into better diagnostic and therapeutic approaches.

Aim2 is designed to study the implications of chimeric gene fusions in cancer. In the field of endometrial cancer research, a big caveat of using mouse as a model is that mouse as well as most mammals do not have menstrual cycle. By accident, we found that the fusion JAZF1- JJAZ1 we have been studying is unique in species that have menstrual cycle, and that the fusion is necessary and maybe sufficient to induce menstrual cycle. These surprising findings have led us to hypothesize that by inducing the fusion at right time, we may be able to generate a mouse model that go through menstrual cycle. Such a tool will be extremely useful not only for endometrial cancer or breast cancer research, but also for any research fields related to menstrual cycle. We are now testing the hypothesis in cell culture models. If successful, we will generate a transgenic mouse model. Such a model will allow us to study the fusion’s oncogenic effect when expressed continuously, and test whether the animal will menstruate if the fusion is induced at the right time.


2011 18 Month - Targeting PP2A and the Glutamine-Sensing Pathway as Cancer Treatment

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Fast-growing cancer cells rely on enhanced nutrient uptake to grow and divide. However, as tumors grow, increased uptake of nutrients and poor vascularization often lead to nutrient deprivation in tumor cells. Understanding the molecular mechanisms that promote cancer cell survival under poor nutrient conditions is important for developing new drugs that could starve tumor cells and block cancer progression. The amino acid glutamine is a major nutrient that supports cell growth and survival. Solid tumors consume glutamine at a rate that outstrips its supply and inevitably end up facing low glutamine conditions. The goal of this project is to determine the molecular basis for tumor cell survival under conditions of glutamine deprivation in order to develop novel drugs targeting this pathway. We have shown that the enzyme PP2A (protein phosphatase 2A) plays a critical role in mediating cell survival upon glutamine deprivation. However, PP2A is a member of a large family of protein complexes that regulate many different cellular functions. In this study, we worked to identify the specific PP2A complex that regulates cancer cell survival upon glutamine deprivation. Our aims are to determine: (1) whether PP2A complexes are regulated by glutamine levels; (2) the mechanism by which PP2A exerts a cell survival effect during glutamine deprivation; (3) whether PP2A contributes to tumor cell survival and whether impairment of PP2A activity combined with inhibition of glutamine metabolism can alter cancer cell viability.

During the first year of the grant period, we demonstrated that among 16 different PP2A regulatory proteins, only one, the B55 subunit, was specifically upregulated upon glutamine deprivation. We also demonstrated that suppression of B55 expression impairs cancer cell survival in the presence of low glutamine in a p53-dependent manner. In these last six months, we have successfully met two of our proposed milestones. First, we demonstrated that glutamine deprivation induces assembly of a specific B55 containing complex, including B55, PP2A catalytic subunit (C subunit) and scaffolding subunit (A subunit). Second, we demonstrated that the PP2A 4 subunit, which is often elevated in cancer cells, is required for the assembly of the B55 containing complex via providing the C subunit. Thus, we idenfied a precise complex in cancer cells that mediates cell survival in responding to glutamine deprivation. In the next funding period, we will continue experiments outlined in our “milestones and diverables,” which are (1) to determine if 4 and B55 promote cell survival upon glutamine deprivation via inhibition of c-Myc activity, and (2) to determine the mechanism by which ROS induce B55 and enhance cell survival upon glutamine deprivation.


2011 18 Month - Targeting Protein Quality Control for Cancer Therapy

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The normal growth and proliferation of cells is orchestrated by a cascade of events that is initiated by binding of a stimulus to a receptor at the membrane. Once triggered, the receptor communicates to the rest of the cell via recruitment of a number of signaling molecules. Depending on the quality and quantity of signals from the receptor, the cellular output can be modified, for example proliferation versus death. Signals from growth receptors on the cell surface can become altered in cancer due to either increased expression of these receptors or mutations that lead to increased activity. In our project, we are addressing how inhibition of the expression of the epidermal growth factor receptors (referred in here as ErbB) can be exploited for cancer therapy. Our lab had initial findings that a protein complex called mTORC2 is involved in protein production and quality control. When mTORC2 is inhibited by pharmacological agents or by genetic manipulation, proteins that are known to become deregulated in cancer such as Akt and ErbB have defects in their synthesis. During the first year of this grant, we have established a role for mTORC2 in controlling the amount and quality of ErbB1 that is expressed in the surface of breast cancer cells. In the past six months (July 2012-Dec 2012), we have identified a possible mediator of the mTORC2 function in ErbB1 quality control. Using protein purification and mass spectrometry, we identified a protein called GFAT1. This protein has been previously characterized in the field of diabetes since it is involved in cellular metabolism. Not much is known about how this protein becomes regulated by nutrients. Our findings now provide a connection between cellular proliferation (via ErbB1 signaling) and metabolism (GFAT1) and that these two pathways could be coupled by mTORC2. We have begun to characterize how GFAT1 could be involved in the regulation of ErbB1 expression. Ourfindings would now suggest that combined inhibition of mTOR and GFAT1 could serve as a more effective therapy in breast cancers wherein the activities of these proteins become deregulated.


2011 18 Month - Targeting Genetic and Metabolic Networks in T-ALL

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Acute lymphoblastic leukemia is the most frequent cancer in children. Despite much progress in the treatment of this disease, leukemia still represents a clinical challenge, particularly in cases diagnosed with T-cell disease. In this project, we aim to elucidate the oncogenic circuitries that control T-cell acute lymphoblastic leukemia. Our ultimate objective is to identify effective new drugs and drug combinations for the treatment of this disease.

In the first year and a half of funding we have analyzed a highly representative panel of human T-cell leukemia samples to catalog their genetic alterations, genetic programs and metabolic signatures. Our results have identified and cataloged two molecular groups of T-cell leukemia characterized by different gene expression programs; identified numerous new genes mutated in T-ALL including ETV6, RUNX1, EZH2 and SUZ12. In addition over the last months we have completed the analysis of global DNA methylation of T-cell leukemias and analyzed the impact of these changes in the activity of leukemia genes.

In addition we have used network analysis to uncover the mechanistic role of TLX1 and TLX3, two major genes driving T-cell leukemia growth and proliferation. Moreover, analysis of the circuitries involved in resistance to chemotherapy with glucocorticoids has identified the PI3K- AKT1 pathway as a new therapeutic target for the reversal of resistance to glucocorticoids, a key drug in the treatment of T-ALL.

Following on these results and to gain better understanding of the mechanisms of drug resistance we have extended our mutation analyses to relapsed leukemias. These studies have identified new recurrent mutations that activate NT5C2, a metabolic gene responsible for the inactivation of mercaptopurime, an essential drug in the treatment of T-ALL. This result highlights the importance of cell metabolism in the response to therapy.

Finally, and along this line, we have performed global metabolic profiling of T-cell leukemias and shown that targeted drugs that inactivate NOTCH1, a central gene activated by mutations in T-ALL, results in dramatic changes in cell metabolism. Strikingly activation of PI3K-AKT, a second cancer pathway, effectively reverses this metabolic shutdown and induces leukemia resistance to anti NOTCH1 therapies. Most notably these analyses have uncovered new important drugs and synergistic drug interactions for the treatment of T-ALL.

Overall, we have made significant progress towards our goal of analyzing a broad panel of primary T-cell leukemia samples using high throughput technologies to build a network that integrates the information obtained from different platforms to identify key regulators of leukemic cell growth, proliferation and survival for the development of targeted therapies against T-cell leukemia.


2011 18 Month - Targeting MLL in Acute Myeloid Leukemia

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Our broad objective in the proposed research is to develop novel chemotherapeutic agents that target the activity of a regulator of a subtype of acute myeloid leukemia, namely the Mixed Lineage Leukemia (MLL) protein. MLL was originally cloned by its direct involvement in a group of distinct human acute leukemia with extremely poor prognosis. MLL gene abnormalities account for 5% to 10% of the disease, and at least 70% of the cases in infants under 1 year old. It is general consensus that MLL mutations disrupt expression of specific genes that are important in early blood cell development. MLL is an enzyme and its activity is essential for leukemia development. Biochemical analyses have shown that MLL activity is tightly regulated by several interacting proteins. Therefore, it is conceivable that disrupting these protein-protein interactions involving MLL will compromise MLL enzymatic activity, which in turn leads to inhibition of leukemogenesis. Using the biochemistry and medicinal chemistry approaches, we have designed a series of inhibitors that target the MLL activity. In the past several months, we have made significant progress in improving our lead compounds in both in vitro and in vivo assays. These results suggest that our approach is valid and is likely to provide new therapeutics for MLL mediated leukemia.


2009 24 Month - Probing EBV-LMP-1’s Transmembrane Activation Domain with Synthetic Peptide

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Dr. Yin developed a new technique to identify transmembrane proteins that are part of the signaling pathways that drive cancer cell growth. Dr. Yin is currently using CHAMP-designed peptide antagonists to study the contribution of LMP-1 activation. This could lead to the development of new therapies that target transmembrane proteins like LMP-1. Dr. Yin’s laboratory has:

  • Begun developing a second-generation CHAMP algorithm design tool.
  • Completed the training database for the supervised-machine learning process.
    • Preliminary testing showed an 85% success rate to predict/recognize protein flexible regions.
    • The flexibility-prediction element has been integrated into CHAMP design package developing the second generation design tools.
  • Established a method to synthesize transmembrane peptide sequences on Tentagel beads.
    • A second approach to optimize these sequences that makes it possible to screen a larger peptide library has now been developed.
  • Established a biophysical assay to evaluate peptide/membrane protein insertion/binding. This assay has been validated.
    • This work led to the development of a novel reporter assay that the laboratory is now using to study the peptide/membrane protein interactions in E coli membranes.
  • Based on his hypothesis that the fifth transmembrane domain of LMP-1 mediates LMP-1 signaling, Dr. Yin successfully designed anti-transmembrane domain 5 (TMD5) that has been shown to be able to disrupt the LMP-1 TMD5 oligomerization in cell membranes.
  • Using a previously established assay to test for compounds from the NCI Diversity Set II library, Dr. Yin identified four potential compounds that may disrupt TMD5 oligomerization.
    • One of the four compounds is being characterized and preliminary data shows that this compound specifically inhibits LMP-1 signaling in EBV-infected B cells.

 


2009 24 Month - Functional Oncogene Identification

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Dr. Weinstock is using a novel molecular profiling approach to identify abnormalities that promote the growth of cancers of the blood. Dr. Weinstock’s laboratory has:

  • Screened 12 types of human leukemia and lymphoma samples for new mutations.
    • This screen and additional DNA sequencing led to the identification of multiple mutations that have never been described from tumor specimens.
      • Mutated versions of four additional proteins in three cancer types, including proteins called kinases that can be targeted with available drugs.
    • The laboratory is also defining the frequency of these mutations in other leukemia and lymphoma specimens.
      • Multiple mutations are present in several lymphomas of the same type.
    • Once further confirmation is completed, the laboratory will test leukemias and lymphomas dependent on these new mutations with a panel of targeted agents to define drugs for further testing in patients.
    • The laboratory will then apply the same screening approach to identify targetable mutations in other blood cancers, including subtypes of leukemia, non-Hodgkin lymphoma and Hodgkin’s disease.
  • Identified a donor-recipient bone marrow transplantation (BMT) pair who both developed follicular lymphoma nine years after transplantation (published in Cancer Discovery 2012;2:47-55). Sequencing identified 15 mutations that were shared between the two lymphomas as well as of mutations unique to each lymphoma.


2009 24 Month - A Transformative Technology to Capture and Drug New Cancer Targets

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Dr. Walensky is using modified natural peptides that bind to and capture target proteins to identify cancer-causing interactions and to develop therapies that interfere with these interactions. Dr. Walensky’s laboratory has:

  • Chemically synthesized a pilot panel of “photoreactive stabilized alpha-helices” or pSAHs, which are the chemical tools designed to capture and characterize new cancer targets.
  • Assessed and optimized these pSAHs so that they could be cross-linked to specific targets in the BCL-2 pathway, which regulate the cell’s life-death decision and has been implicated in the development of cancer and chemo-resistance.
  • Developed a rapid method for localizing helix-target binding sites in order to identify protein interfaces, which will help in the discovery of potential new targeted therapies.
  • Demonstrated the ability to successfully and reproducibly generate pSAHs that recapitulate the structure of distinct bioactive domains and deploy them to trap, purify, and identify their natural cellular targets with high fidelity.
    • The laboratory is now putting these design principles into practice so that new pSAH reagents can be effectively deployed to discover and target cancer-causing protein interactions.
    • This work was published in Cell’s Chemistry and Biology journal in December 2010.
  • Developed and published a rapid method for localizing helix-target binding sites that will expedite the discovery of protein interfaces for targeted drug development and clinical translation.
  • Expanded its panel of pSAHs for target discovery.
    • The laboratory has successfully expanded to a library of 60 PSAHs that encompass 10 BCL-2 family subdomains.
    • This library has been used to identify several new protein targets that are currently being confirmed.

     


    2009 24 Month - Noninvasive Molecular Profiling of Cancer via Tumor-Derived Microparticles

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    Dr. Tewari is trying to develop a blood test that can efficiently capture and decode the data about a tumor that can be obtained from the tumor-derived microparticles that circulate in the patient’s bloodstream. Dr. Tewari’s laboratory has:

    • Improved its procedures for concentrating the microparticles, making it easier to see them using electron microscopy.
    • Obtained a Nanosight nanoparticle tracking system and analyzed its reproducibility and accuracy in sizing exosomes and microvesicles.
    • Used the Nanosight system to compare plasma exosome abundance in ovarian cancer cases and controls, as well as pre- and post-surgery for ovarian cancer.
      • No significant difference in bulk exosome abundance in patients with cancer vs. healthy controls was found, which differs from what others have reported.
      • This indicates that capture of the tumor-derived subpopulation of exosomes will be important for successful development of an exosome-based tumor transcriptome profiling.
    • Based on the above data, Dr. Tewari has optimized a protocol capturing exosomes using antibodies that recognize EpCAM, a protein which is found on ovarian cancer cells.
      • This will be used to capture and elute ovarian cancer cell line-secreted exosomes and their associated microRNAs for analysis.
      • Used the Nanosight system to quantitatively characterize the performance of our exosome isolation protocol.
    • Found in a set of matched fresh and frozen plasma specimens from advanced prostate cancer patients, that freezing leads to a dramatic (10-100 fold) decrease in miRNA biomarker abundance in plasma
      • These results suggest: (i) frozen specimens may not be suitable for exosome-based tumor transcriptome analysis, and (ii) the potential for this approach is enhanced if working with fresh samples is able to increase signal by 10-100 fold.


    2009 24 Month - Modulating Transcription Factor Abnormalities in Pediatric Cancer

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    Rather than using standard approaches to chemical screening, Dr. Stegmaier’s laboratory developed a genomic approach that uses DNA microarrays (gene chips) to characterize genes that are turned on or off in the presence of or absence of the Ewing sarcoma protein. Over the past 18 months Dr. Stegmaier’s laboratory has:

    • Developed a 123-gene signature that distinguishes Ewing sarcoma cells with the active versus inactive EWS-FLI protein.
    • Adapted the signature to a robust assay that can be measured in a high-throughput format.
    • Confirmed that the signature identifies the active versus inactive EWS-FLI protein across a number of different Ewing sarcoma cell lines.
    • Performed a successful pilot screen of 1600 bioactive chemicals and FDA-approved drugs.
      • Two chemical hits that induce the EWS-FLI inactive expression signature were identified.
    • Completed a large-scale screen of more than 10,000 small molecules. This chemical collection consists of bioactive molecules (including many FDA-approved drugs), natural products, and novel chemicals created by a new approach called diversity oriented synthesis.
      • Diversity oriented synthesis is expected to yield chemicals with greater similarity to these products in nature.
      • These chemicals may also have greater biological activity than compounds produced by standard approaches to chemical synthesis.
    • Prioritized 160 top-scoring compounds to retest in a secondary screen across multiple doses.
      • Based on these findings, 36 of these compounds have been prioritized for additional testing.
      • Attention is being focused on several molecules that are already FDA-approved or in clinical trials, as well as 15 diversity oriented synthesis molecules.
    • Identified two classes of molecules to target: a compound which activates the protein retinoic acid receptor γ (RARγ) and a compound which inhibits a protein called focal adhesion kinase (FAK).
      • Dr. Stegmaier has demonstrated that the RARγ agonist molecule, CD437, leads to a decrease of the ID2 protein, one of the downstream target proteins of EWS/FLI.
      • It was shown that both CD437 treatment and genetic decrease of ID2 prevent growth of Ewing sarcoma cells
      • Dr. Stegmaier has demonstrated that FAK is activated in most Ewing sarcoma tumors from patients and that inhibiting FAK with a genetic tool called shRNA prevents the growth of Ewing sarcoma cells cell culture and in mice and
      • Evaluation of the activity of a FAK inhibitor in a mouse model of Ewing sarcoma is currently in progress.


    Genetic Approaches for Next Generation of Breast Cancer Tailored Programs

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    Dr. Silva’s laboratory has pioneered the development of RNAi based genetic tools for studies in mammalian cells. This technology represents a unique opportunity to identify genetic synthetic lethal interactions. Dr. Silva’s laboratory has:

    • Completed and analyzed four genome-wide RNAi screens in vitro to identify target genes that upon inhibition can stop the growth of breast cancer cells that have one of the four major breast cancer alterations: ErbB2(HER2), c-Myc, Cyclin-D or RB.
    • Selected 30-50 genes as synthetic lethal candidates for each of the four major breast cancer alterations.
    • Developed a procedure to see whether the lethal interactions found in vitro are reproduced in vivo in mouse models.
    • Identified several components involved in lipid processing that were shown to be highly significant in c-MYC alterations and have been targeted for further study
    • Identified the Jak/Stat pathway as differentially activated in cells that are ErbB2(HER2)-positive. This pathway has previously been identified as a key pathway in other tumor types, including non-small cell lung cancer.
      • Several small molecule inhibitors already exist that potentially could be used for translational clinical trials.
      • Studies are now underway that will advance the research on this genetic interaction.
      • Studies identified upregulation of multiple molecules and, importantly, their receptors. The most upregulated interleukin was interleukin-6 (IL-6), which is known to activate STAT-3.
      • This suggests that cells that are HER2-postiive produce high levels of IL-6 and upregulate its receptor, which activates the Jak/Stat pathway.
      • Has identified 37 new potential inhibitor molecules of STAT-3 from a screen of more than 20,000 compounds, which will be tested for their potential use as clinically relevant drugs.


    2009 24 Month - Endogenous Small Molecules that Regulate Signaling Pathways in Cancer Cells

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    Dr. Rohatgi’s laboratory is attempting to identify the undiscovered small molecule-protein pair that is believed to regulate the Hedgehog pathway. Studies suggest that Hedgehog plays a role in a diverse range of cancers and it has become an important drug target in oncology. Dr. Rohatgi’s laboratory has:

    • Continued to conduct studies designed to isolate small molecule lipids that activate the Hedgehog pathway and a method to measure the activation of the pathway in a cell free system.
    • Conducted studies designed to build upon their previous observation that molecules related to cholesterol, called oxysterols, can activate the Hedgehog pathway.
      • Their studies demonstrated that radio-labeled directly interact with a cancer-driving protein called Smoothened that is the major drug target in the hedgehog pathway.
      • This work, which has been submitted for publication, suggests both novel ways in which this pathway can be targeted by drugs and novel avenues for cross-talk between cancer signaling and lipid levels in cells.
    • Performed screens to identify Oxysterol Binding Proteins (OSBPs) that activate and inactivate the Hedgehog Pathway.
      • Identified several natural compounds that are toxic to several cancer cell lines.


    2009 24 Month - Therapeutically Targeting the Epigenome in Aggressive Pediatric Cancers

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    Dr. Roberts’ laboratory is conducting studies designed to determine the role SNF5 loss plays in the development of cancer and to identify therapies that can block or reverse the epigenetic changes that result from the loss of SNF5. Dr. Roberts’ laboratory has:

    • Generated an animal model that it used to test whether inactivation of a small RNA molecule, called miR-21, could block the growth of cancers driven by SNF5 loss.
      • These studies determined that the absence of miR-21 has no effect upon the formation of lymphomas driven by SNF5 loss.
    • Initiated experiments to evaluate whether SNF5-deficient cancer cells are susceptible to treatment with histone deacetylase inhibitors.
      • The trials of DNA methylation inhibitors used SNF5 deficient cancer cells transplanted into recipient mice.
      • The data suggests that SNF5-loss does indeed affect histone acetylation. But that it leads to increased, rather than decreased, acetylation.
      • Pre-clinical development trials of a drug targeting EZH2, a protein that stops the expression of genes by adding a chemical modification to the DNA, have led to decreased tumor burden, however cell growth was not completely blocked and led to decreased bone marrow activity.
      • Further studies are being conducted before deciding to pursue other pre-clinical trials.


    2009 24 Month - Identifying Solid Tumor Kinase Fusions via Exon Capture and 454 Sequencing

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    Dr. Pao’s laboratory is screening 120 tumor samples and cell lines from multiple cancer types, including lung, breast, and head and neck cancers, angioscarcomas, and acute T-cell leukemias/lymphomas for TK fusions. To date Dr. Pao’s laboratory has:

    • Fully analyzed 72 of these samples. The other samples are in various phases of the of process, which involves quality control, DNA capture, sequencing, and computational analysis.
    • Characterized a novel TK fusion in a patient with recurrent T-cell acute lymphoblastic leukemia and eosinophilia (a higher than normal level of eosinophilis, one of five major types of white blood cells.) Their studies suggested that this fusion was likely the ‘driver’ of the patient’s eosinophilia.
    • Begun to use orthogonal methods to identify driver mutations in cancers that may not contain kinase fusions but may have other genetic alterations.
      • Whole exome sequencing has been completed on18 pairs of tumor/normal samples from lung cancer patients, including five never smokers. Whole exome sequencing focuses on the part of the genome formed by exons, the portion of the gene that codes for proteins.
      • Conventional sequencing analysis had not identified any known mutations in EGFR, KRAS, BRAF, PIK3CA, and ALK.
      • Two tumors were found to have mutations in a growth factor called HGF, which binds to the TK MET both of which have been implicated in cancer progression. Studies are ongoing to further understand the role of HGF in lung cancer.
    • Sequenced the expressed genes in 29 small-cell lung carcinoma tumors, which accounts for 15% of lung tumors, and found a high mutation rate and support for the role a biologic process in this cancer that is being investigated.

     


    2009 24 Month - Targeting Inhibition of BCL6 for Leukemia Stem Cell Eradication

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    Dr. Müschen’s laboratory is investigating the hypothesis that BCL6 is critical for leukemia-initiation and relapse. They are examining the frequency and appearance of BCL6-dependent leukemia stem cells in human leukemia samples, and studying whether the BCL6 inhibitor RI-BPI is effective in targeting and eradicating leukemia stem cells. Dr. Müschen’s laboratory has:

    • Conducted studies that showed that BCL6 enables Ph+ acute lymphoblastic leukemia (ALL) cells to survive after treatment with a BCR-ABL1 kinase inhibitor.
      • BCR-ABL1tyrosine kinase inhibitors are widely used to treat patients with Ph+ ALL and chronic myeloid leukemia (CML).
      • These findings identify a novel mechanism of drug resistance, and identify BCL6 as a central component of this drug resistance pathway.
      • These findings demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukemia-initiating clones.
      • This suggests that combining a tyrosine kinase inhibitor with a BCL6 inhibitor might potentially overcome drug resistance in Ph+ ALL.
      • This led the laboratory to question which function BCL6 might have (if any) in the regulation of normal B cell development and in the regulation of cell cycle progression of differentiating pre-B cells, which represent the target cell for malignant transformation toward ALL.
      • Studies to pursue this question showed that pre-B cell receptor signaling activates BCL6 and BLC6-mediated repression of proteins called MYC and CCND2.
    • Conducted studies that showed that BCL6 is required for the maintenance of leukemia-initiating cells in chronic myeloid leukemia (CML).
      • CML can be treated for many years with tyrosine kinase inhibitors. But unless patients use these drugs throughout their lives, the CML will eventually recur.
      • This research showed that BCL6 plays a role in this process by repressing Arf and p53 in CML cells.
      • It also showed that BCL6 is required to form leukemia.
      • This suggests that a BCL6 inhibitor might be able to eradicate leukemia-initiating cells in CML and, in turn, limit how long CML patients must stay on a tyrosine kinase inhibitor.
      • Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in CML. Dr. Müschen’s laboratory identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. These findings identify inhibition of BCL6 as a novel strategy to eradicate leukemia-initiating cells in CML.
        • This suggests the potential for a dual strategy in which a tyrosine kinase inhibitor is used along with BCL6 inhibition to treat CML.
    • Assessed gene expression data in 207 pediatric patients, in addition to an adult patient study, which demonstrated that high BCL6 expression associated with lower overall survival and relapse free survival.

     


    2009 24 Month - Modeling Ewing Tumor Initiation in Human Neural Crest Stem Cells

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    Tumors often look like tissues that have not developed normally. In fact, some tumors arise when abnormalities in the DNA of normal stem cells create changes that lead to the formation of malignant rather than normal tissues. We are studying whether changes in DNA methylation contribute to the genesis and growth of Ewing sarcoma family tumors (ET), highly aggressive tumors that primarily affect children and young adults. Precise control of DNA methylation is essential for the creation of normal adult tissues. We believe that ET arises from normal neural crest stem cells (NCSC) in which the expression of an abnormal gene, EWS-FLI1, induces changes to DNA methylation that result in cancer formation. To date, Dr. Lawlor’s laboratory has:

    • Developed a way to generate and then differentiate neural crest stem cells in the laboratory.
      • The cells are being studied to determine both their normal differentiation process and the changes that normally occur in their methylation over time.
    • Analyzed gene expression data and identified a list of approximately 350 genes they suspect to be abnormally methylated in Ewing sarcomas.

    • Performed experiments to determine whether abnormal DNA methylation in Ewing sarcomas can be corrected by treatment with decitabine, a targeted therapy that inhibits DNA methylation.

      • Dr. Lawlor’s laboratory tested the efficacy of decitabine in Ewing sarcoma cells that are grown as tumors in mice and found that tumor growth was inhibited.
      • Several genes were identified that were turned back after exposure to decitabine and will be studied further.


    2009 24 Month - An Emerging Tumor Suppressor Pathway in Human Cancer

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    Dr. Camargo’s laboratory is conducting research that will expand our understanding of the Hippo pathway and the role it plays in cancer development. In the last 6 months Dr. Camargo’s laboratory has:

    • Discovered that when Hippo is turned off, the amount of Yap1, the main molecule responsible for Hippo’s expression, is increased. Studies were conducted which revealed that inactivation of the Hippo pathway in the skin of mice leads to the development of invasive squamous cell carcinoma. Similarly, inactivation of the Hippo pathway in the muscle of mice leads to the formation of rhabdomyosarcomas.
    • Recent studies demonstrate that over-activation of this pathway can lead to the suppression of growth in the epidermis, but may not be helpful in the intestine.
    • Finalized a genetic screen to identify which proteins can control Hippo signaling.
         
      • Results suggest that alpha-catenin, a protein known to suppress tumors in the skin, and a different signaling pathway called JNK, are able to regulate the Hippo pathway.
      • Future experiments will investigate how these molecules control Hippo signaling.


    2009 12 Month - Probing EBV-LMP-1’s Transmembrane Activation Domain with Synthetic Peptide

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    Although many therapeutic strategies exist for molecular targets accessible from the outside of the cell (e.g. therapeutic antibodies) or within the cytoplasm (e.g. small molecule inhibitors), they are not applicable to molecular targets that lie within the membrane bilayer. The hydrophobic core of the phospholipid bilayer imposes an impenetrable barrier to water-soluble polar therapeutic agents. The Yin lab recently developed a computational method, Computed Helical Anti-Membrane Protein (CHAMP), to rationally design peptide probes that recognize protein transmembrane domains with high affinity and selectivity. This study utilizes this cutting edge technology to study the activation mechanism of oncogenic Latent Membrane Protein 1 (LMP-1) of Epstein-Barr virus (EBV). EBV is a human tumor virus associated with a number of malignancies and lymphoproliferative syndromes. EBV’s ability to infect and immortalize B-lymphocytes underlies its contribution to cancers.

    EBV’s transforming activity depends on the expression and activity of LMP-1, the viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 functions as a constitutively active Tumor Necrosis Factor Receptor (TNFR) whose activity requires the function of its hydrophobic transmembrane domain. LMP-1 most resembles the TNFR CD40 in its signaling. Unlike CD40, whose activity requires activation by ligand, LMP-1’s activity is constitutive and ligand-independent. Constitutive homo-oligomerization and lipid raft association, activities of LMP-1’s transmembrane domain, play a key role in activation of downstream signaling. This proposal focuses on LMP-1 as a model membrane protein target for the design of peptide inhibitors because of LMP-1’s essential role in EBV-dependent B cell transformation, LMP-1’scontribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and EBV’s dependence on LMP- 1’s hydrophobic transmembrane domain for activity.

    This study aims to develop novel prevention and treatment methods to target LMP-1’s transmembrane domain, using CHAMP-designed anti-TMD peptide antagonists as probes to study the contribution of oligomerization to LMP-1 activation, with the goal of inhibiting downstream signaling. Results of this research will provide insight into the molecular interactions within the membrane environment and the mechanisms underlying constitutive/oncogenic receptor signal transduction across membranes; will reveal the mechanism of LMP-1’s constitutive activation of signaling; and will be applicable to the future development of novel therapeutics targeting cancers dependent on critical transmembrane proteins.

    Specifically, this proposal addresses the following Aims:

    1) Develop specific peptide probes targeting the TMD-mediated homo-oligomerization.

    2) Answer the question as to what the role of TMD- mediated oligomerization in LMP-1 activation is.


    2009 12 Month - Functional Oncogene Identification

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    Despite advances in diagnosis and treatment, more than half of adults with cancers of the blood (i.e., leukemia, lymphoma and multiple myeloma) will die from their disease. One of the limitations in our current approach is that most cancer chemotherapy nonspecifically kills all growing cells, and does not target abnormalities unique to the tumor cells. Thus, the identification of specific cancer-associated abnormalities is an essential first step toward newer and more effective therapies. We developed a system to identify new targets for therapy directly from leukemia and lymphoma samples. Briefly, we isolate the many millions of pieces of genetic material from a tumor sample and then individually insert each into cells that can only grow in a special kind of culture. If one of the pieces of genetic material has a cancer-promoting effect, it allows the cells to grow in a normal culture. Thus, any cell that survives in the normal culture must contain a piece of genetic material from the tumor that has a cancer-promoting effect. We can easily identify that piece of genetic material and then confirm that it is important for the tumor’s growth. The system we developed is efficient and can be scaled up to analyze a large number of individual specimens. Using this approach, we have already discovered a new cancer protein called CRLF2 in some cases of acute lymphocytic leukemia.

    The overall goal of our Stand Up To Cancer Innovative Research Grant proposal is to identify important alterations that promote the growth of other types of blood cancer. During the initial 12 months of funding, we utilized our approach to screen six types of human leukemia and lymphoma samples for new mutations. From this screen and from sequencing large amounts of DNA in the specimens, we identified multiple mutations that have never been described from tumor specimens. Of particular interest, we identified mutated versions of two proteins called kinases that can be targeted with available drugs. We are in the process of confirming that the mutations we identified contribute to tumor growth. We are also defining the frequency of these mutations in other leukemia and lymphoma specimens. At least one of the mutations is present in multiple different lymphomas of the same type. Once further confirmation is completed, we will test leukemias and lymphomas dependent on these new mutations with a panel of targeted agents to define drugs for further testing in patients. Over the next two years, we will apply the same screening approach to identify targetable aberrations in other blood cancers, including subtypes of leukemia, non-Hodgkin lymphoma and Hodgkin’s disease.


    2009 12 Month - A Transformative Technology to Capture and Drug New Cancer Targets

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    The goal of our SU2C project is to create a powerful, new, and versatile approach to identifying and drugging new cancer targets. To that end, we are combining a chemical technology termed “hydrocarbon stapling” that restores natural shape to bioactive peptide alpha-helices with a protein capture technology in order to trap and characterize critical targets involved in defective signaling in cancer. For the first year of funding, our goal was to chemically synthesize a pilot panel of “photoreactive stabilized alpha-helices” or pSAHs, which are the chemical tools designed to capture and characterize new cancer targets. With our first series of pSAHs in hand, we examined and optimized the sensitivity and specificity of these new tool compounds for cross-linking to discrete physiologic targets of the BCL-2 pathway.

    In an SU2C-sponsored manuscript published on December 22nd in Cell’s Chemistry and Biology journal, we demonstrate our capacity to successfully and reproducibly generate pSAHs that recapitulate the structure of distinct bioactive domains and deploy them to trap, purify, and identify their natural cellular targets with high fidelity. In addition, we developed and reported a reliable method for inputiing our cross-linking data into a binding site algorithm that employs mass spectrometry and computational docking analysis to calculate model structures of the key-in-Iock binding interfaces we discover. This critical information will provide the basis for validating new protein-protein interactions for drug development and therapeutic targeting. Having defined and published the “rules” for successful production and application of pSAHs based on our year one work, we are currently expanding our arsenal of pSAH constructs to home in on key protein interactions that drive cancer. Indeed, as we enter year two of the funding cycle, the implementation phase of our technology for cancer target discovery is well underway, setting the stage for an exciting year ahead. From a laboratory development standpoint, SU2C funds have enabled us to dramatically expand our pSAH production capacity through the purchase of a Liberty microwave-enhanced peptide synthesizer, which is now installed and fully operational. The success of our proteomic approach has further motivated me to deploy laboratory funds to purchase our own Thermo L TO Orbitrap mass spectrometer, which will enable us to build a state-of-the-art infrastructure for advancing our cancer protein target discovery platform.


    2009 12 Month - Noninvasive Molecular Profiling of Cancer via Tumor-Derived Microparticles

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    Obtaining molecular information about an individual cancer patient’s tumor would allow for the development of patient-specific treatment plans. However, obtaining tumor biopsy samples in many cases requires invasive surgery, which can be painful, disfiguring and potentially dangerous. Consequently, alternative non-invasive sampling methods are needed, and could serve as a reliable surrogate for actual tumor tissue. The approach we are pursuing takes advantage of the known fact that cancer cells release “information” into the bloodstream in the form of specific types of RNA and DNA molecules. This information is packaged into what we call tumor-derived microparticles; essentially, small parcels derived from and representative of the contents of tumor cells. The goal of this project is to develop methods to efficiently capture these particles from patient blood samples and decode the information within them in order to gain molecular information about the cancer cells from which they originated. In the recent period, our major progress includes: (i) improvement in our methods for concentrating microparticles that yielded a 400-fold improvement in our ability to see them using electron microscopy, (ii) improvement in methods to detect RNA associated with one class of microparticles, and (iii) use of the improved methods to detect the presence of a specific class of microparticles in ovarian cancer tissue samples. Moving forward we plan to continue to develop the methods needed to capture and decipher the molecular information contained within these particles. Our ultimate goal is to use a blood sample to obtain key information that will inform critical clinical decisions, such as identifying the most effective therapy for an individual patient.


    2009 12 Month - Modulating Transcription Factor Abnormalities in Pediatric Cancer

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    The identification of cancer-promoting proteins has advanced at a rapid pace with the development of new genomic technologies. However, the majority of these proteins have been considered “undruggable” by traditional pharmacological approaches. One class of “undruggable” proteins critically important in both pediatric and adult malignancies is proteins that bind to DNA, the so-called transcription factors. One such protein historically considered “undruggable” is the cancer-promoting protein in the pediatric solid tumor Ewing sarcoma, EWS/FLI. Ewing sarcoma is the second most common cause of bone cancer in children and young adults, and the EWS/FLI protein can be identified in over 80% of tumor samples. While progress has been made in treating patients with localized Ewing sarcoma, little advancement has been made for those patients with metastatic or recurrent disease. Current regimens employ cytotoxic chemotherapy, and targeted treatments are only beginning to emerge. New approaches to treating this disease are needed.

    One approach to tackling this challenging class of proteins is to develop alternatives to traditional drug discovery. Instead of using standard approaches to chemical screening, we developed a genomic approach using DNA microarrays (“gene chips”) to characterize the genes that are turned on or off in the presence or the absence of the Ewing sarcoma protein. Over the past 12 months, we have developed a 123-gene signature that distinguishes Ewing sarcoma cells with the active versus inactive EWS/FLI protein. We next adapted this signature to a robust assay, which can be measured in 384-well high-throughput format. We confirmed that the signature identifies the active versus inactive EWS/FLI protein across a number of different Ewing sarcoma cell lines with genetic knockdown of EWS/FLI. We next performed a successful pilot screen of 1600 bioactive chemicals and FDA-approved drugs. We identified two chemical hits, which induce the EWS/FLI inactive expression signature. We have just completed a large-scale screen of over 10,000 small molecules. This chemical collection consists of bioactive molecules (including many FDA- approved drugs), natural products, and novel chemicals created by an approach called diversity oriented synthesis. This new approach to chemical synthesis is expected to yield chemicals with greater similarity to those produced in nature, with the hope that they will also have greater biological activity than compounds produced by standard approaches to chemical synthesis. We have prioritized 150 top scoring compounds, which we are actively testing in a secondary screen across multiple doses.

    Over the next funding period, we will identify those chemicals which reproducibly induce the EWS/FLI inactive gene signature. For those chemicals confirmed to inactivate the EWS/FLI signature we will next measure their effects on various aspects of Ewing sarcoma cells, including the effects on cell growth/death and the cancerous properties of the tumor cells. Chemicals that emerge from this screen will be used in the laboratory as tools to identify new drug targets for Ewing sarcoma and as lead compounds for clinical trial development. Moreover, if this work is successful, it can be extended to many more tumor types both in adults and in children (e.g. prostate cancer and leukemia) where proteins similar to EWS/FLI promote the development of the tumor.


    2009 12 Month - Genetic Approaches for Next Generation of Breast Cancer Tailored Therapies

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    Cancer therapy has radically changed during the last decade. Novel therapies based on the specific molecular changes that drive tumorigenesis in every patient are emerging as low toxicity and more efficient alternatives to classical treatments. An alternative promising approach for the design of these personalized therapies is the use of genetic synthetic lethal interactions. These occur when two genetic alterations that are individually innocuous appear in the same cell, causing growth inhibition. This concept can be exploited to identify genes that, when inhibited, exclusively reduce the viability of tumor cells that carry a preexisting genetic lesion.

    Recently, RNA interference (RNAi) technology has emerged as a very powerful approach to attenuate the expression of any chosen gene. Thus, we envision using RNAi to identify genes that, when attenuated, exclusively reduce the viability of tumor cells carrying specific genetic lesions without affecting normal cells. During the last years, my group has pioneered the development of RNAi based genetic tools for studies in mammalian cells. This technology represents a unique opportunity to identify synthetic lethal effects with major cancer alterations. In this project we propose to apply our state- of-the-art technology to uncover synthetic lethal interactions with the major breast cancer genes.

    Our proposal is divided into three specific aims that represent the transition from target discovery and validation to mechanistic characterization.

    Specific Aim 1. Identify genes that interact with the major breast cancer alterations to produce synthetic lethality in vitro (1st year):

    During the first year of this project, we proposed to complete four genome-wide RNAi screens in vitro to identify target genes that, upon inhibition, reduce the cell viability in breast cancer cells with any of the major breast cancer alterations; ErbB2, c-Myc, Cyclin-D or RB. In our pre-defined milestones we estimated that two of the four RNAi screens would be completed during the first six months and the rest during the second semester of the first year. At this point, the four genome-wide RNAi screens have been completed and analyzed.

    Specific Aim 2. Model selected lethal interactions in vivo (1st and 2nd year):

    Completion of the above mentioned screens has provided us with a list of candidates that will be further validated in vivo by candidate driven RNAi screens in mouse models during the second year of the award.

    Specific Aim.3- Initial characterization of the molecular mechanism of the genetic lethality (2nd and 3nd year):

    Upon completion of Aim 2, we will select the most promising (2-3) target genes to investigate the biology of the lethal phenotype in more detail.


    2009 12 Month - Endogenous Small Molecules that Regulate Signaling Pathways in Cancer Cells

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    A major goal in cancer biology is the comprehensive understanding of signals that drive the growth and spread of cancer cells. My long-term goal is to develop methods to isolate new small molecules that play a role in cancer signaling and then to identify the proteins that interact with these small molecules. Such small molecule- protein pairs are likely to be particularly good drug targets in oncology. To develop tools for this endeavor, we are focusing on the identification of small molecules that regulate the “Hedgehog” signaling circuit. Damage to this circuit has been shown to drive the development of a large number of adult and childhood cancers. We have taken three complementary trajectories to tackle this project. First, we are searching for new small molecules present in crude homogenates of cells and tissues that can modulate the Hedgehog pathway. We are also taking advantage of our previous observation that molecules related to cholesterol, called oxysterols, can activate the Hedgehog pathway. To understand how these enigmatic molecules work, we are attempting to find the proteins through which oxysterols function. In addition to continuing to develop the approaches described in the last progress report, we have made substantial progress on understanding how oxysterols function at a molecular level in the Hedgehog pathway. This work will be submitted for publication shortly.


    2009 12 Month - Therapeutically Targeting the Epigenome in Aggressive Pediatric Cancers

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    Recently there has been a growing realization that some of the critical changes in gene expression required for the development of cancer do not arise via genetic mutations in DNA but rather are ‘epigenetic’ changes that affect gene expression indirectly by affecting DNA packaging. The SWI/SNF complex controls the protein support structure that surrounds key growth genes and is thus at the heart of this epigenetic regulation. Mutations in SNF5, a core subunit of the SWI/SNF complex, are present in the large majority of malignant rhabdoid tumors (MRT), a highly lethal cancer that occurs in kidney, brain, and soft tissues of young children. Inactivating mutations in SNF5 have recently been found to occur in a variety of other cancers as well including epithelioid sarcomas, small cell hepatoblastomas, undifferentiated sarcomas, chondrosarcomas, familial schwannomatosis, and renal medullary carcinomas. Mutation in SNF5 is also the basis of an inherited cancer predisposition syndrome and mutation of another SWI/SNF sub-unit that is frequently found in adult lung cancers. As the cancers that arise following Snf5 loss appear to be largely driven by the epigenetic consequences, we hypothesize that these cancers will be particularly susceptible to drugs that interfere with epigenetic mechanisms of gene regulation. The experiments in this proposal were designed to reveal the underlying mechanisms by which SNF5 loss affects gene expression and thereby causes cancer with a goal of identifying improved therapies that can be rapidly translated into patients.

    We have continued to make substantial advances during months seven through twelve of funding from Stand Up 2 Cancer and have met and exceeded our milestones. We have generated an animal model with which we are now specifically testing whether inactivation of a small RNA molecule miR-21 can block the growth of cancers driven by Snf5 loss. Unfortunately, we now have some mice that have developed cancers despite absence of miR-21, suggesting that this may not be sufficient to block tumor formation. We are awaiting further data to determine whether the absence of miR-21 has any effect on the rate of tumor formation caused by Snf5 inactivation. We are in the process of collecting systematic data on the effects of Snf5 loss upon other histone acetylation and DNA methylation so that we can determine whether drugs that target these related modifications may be therapeutically beneficial. Related to this work, we have identified an antagonistic relationship between Snf5 and another epigenetic regulator, Polycomb and have now shown that inactivation of Ezh2 can completely block tumor formation driven by Snf5 loss. We published this finding in Cancer Cell in October and were subsequently contacted by multiple pharmaceutical companies that wish to work with us on this, a collaboration that we are now pursuing.


    2009 12 Month - Identifying Solid Tumor Kinase Fusions via Exon Capture and 454 Sequencing

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    Cells rely upon molecular switches to carry out normal functions. One class of switches is called tyrosine kinases (TKs). In a highly simplified model, when TKs are ‘on’, cells divide; when TKs are ‘off’, cells stop dividing. In some cancers, TKs have become altered, leading to abnormal signaling. One major type of alteration is called a ‘fusion’. A fusion ‘fuses’ together a part of a cellular protein (that normally has another function in the cell) with the signaling portion of the TK molecule. Instead of turning ‘off’ and ‘on’ in a tightly regulated manner, TKs fusions are ‘on’ all the time, tricking cells into constantly dividing. If one blocks the abnormal signals from a TK fusion with a drug, cancer cells can die, and patients can enormously benefit. The best example of this idea involves the drug Gleevec, which targets a TK fusion (called BCR-ABL) in patients with a type of leukemia.

    Thus far, only a limited number of TK fusion proteins have been identified because experimental procedures for their identification are inadequate, especially in solid tumors that contain cells from multiple tissue types. As part of this grant, we developed a novel method to identify TK fusion proteins using DNA from any type of tumor. After demonstrating the feasibility of this platform on tumor cell lines, we used the results of the pilot study to create an improved more powerful platform with streamlined workflow and computational analysis. Using the newer kinase fusion design, we have screened multiple tumor samples and have identified a novel fusion from one patient sample thus far. We are currently exploring how this TK fusion contributes to the development of cancer and can be treated with existing targeted therapies. The screening of multiple other tumor samples with no known targets for therapy is ongoing. Finally, we have mapped the genomic sequences of multiple known kinase fusions in order to gain further insights into the DNA structures of these cancer-specific alterations.


    2009 12 Month - Targeted Inhibition of BCL6 for Leukemia Stem Cell Eradication

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    Despite significant advances in the treatment of leukemia over the past four decades, the rate of long-term survival has reached a plateau. Large numbers of leukemia patients still die, mostly because of relapse and drug- resistance. These two clinical problems were recently attributed to the persistence of leukemia stem cells. If a therapy succeeds in eradicating leukemia stem cells, renewed initiation of the disease (relapse) is no longer possible. Therapeutic progress in recent clinical trials has likely been stalled, partly because current chemotherapy approaches target proliferating bulk leukemia cells rather than non-dividing leukemia stem cells.

    We have now discovered that BCL6, a factor known to play a central role in lymphomas, also plays a key role in the maintenance of leukemia stem cells. Since leukemia stem cells represent the origin of relapse and drug-resistance in leukemia in many cases, the identification of BCL6 as a target for leukemia stem cell eradication holds great promise. BCL6 is a master regulatory factor that controls the production of many different important genes. BCL6 was not previously known to be involved in leukemia. In preliminary studies for this proposal, we have discovered aberrant expression of BCL6 as a central component of a fundamentally novel pathway of leukemia stem cell self-renewal and drug-resistance in a wide array of human leukemias, some of which are still difficult to treat. In these leukemias, drug-treatment results in aberrant production of BCL6 by the leukemia cells, which appears to allow leukemia stem cells to self-renew and become resistant against chemotherapy.

    Recently, a drug has been developed that can attach to BCL6 and block its cancer- causing activities. We found that this BCL6 inhibitor, called RI-BPI, has strong cooperative activity when combined with conventional chemotherapy. This opens up a powerful new therapeutic strategy for leukemia stem cell eradication through targeted inhibition of BCL6. Based on the discovery of BCL6 as a key component of a novel pathway of drug-resistance and stem cell self-renewal in a wide array of leukemias, we propose three Aims to develop these findings towards application in patient care: (1) To test the hypothesis that BCL6 is critical for leukemia-initiation and relapse of leukemia, (2) To determine the frequency and appearance of BCL6-dependent leukemia stem cells in human leukemia samples and (3) To validate the role of the BCL6 inhibitor RI-BPI as a novel therapeutic agent for targeted eradication of leukemia stem cells. Since RI-BPI is currently going through the process of approval for use in clinical trials, we expect to be able to test the power of this approach in clinical trials by the end of the funding period.


    2009 12 Month - Modeling Ewing Tumor Initiation in Human Neural Crest Stem Cells

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    Tumors often look like tissues that have not developed normally. In fact, some tumors arise when abnormalities in the DNA of normal stem cells creates changes that lead to the formation of malignant rather than normal tissues. We are studying whether changes in DNA methylation contribute to the genesis and growth of Ewing sarcoma family tumors (ET), highly aggressive tumors that primarily affect children and young adults. Precise control of DNA methylation is essential for the creation of normal adult tissues. We believe that ET arises from normal neural crest stem cells (NCSC) in which the expression of an abnormal gene, EWS-FLI1, induces changes to DNA methylation that result in cancer formation.

    Because NCSC are very rare cells very little is known about their DNA methylation profiles. In Aim 1 of our proposal we are studying normal human NCSC before, during and after differentiation. In the first year we developed the means to reproducibly generate high quality NCSC and sufficient numbers of terminally differentiated cells for whole genome DNA profiling. The first set of DNA samples from these studies is currently being processed at the USC Epigenome Center. Replicate studies will be performed in year two and data from these assays used to define what methylation looks like during normal neural crest development.

    In our second aim we will assess how EWS-FLI1 alters the normal pattern of DNA methylation. DNA samples from NCSC that were grown for up to 30 weeks in the presence of EWS-FLI1 have been sent to USC for analysis. We are also profiling the whole-genome methylation status of five established ET cell lines along side these NCSC samples. In year two and three of the proposal we will repeat these studies and also test cells that have been grown in low oxygen concentrations, a condition that better represents the environment of tumor cells in the patient. These studies will help us to determine which disruptions to DNA methylation are mediated by EWS-FLI1 and important for the creation of ET cells.

    In Aim 3 we are testing whether abnormal DNA methylation in ET cells can be corrected by treatment with decitabine, a drug that inhibits DNA methylation and/or by turning off EWS-FLI1. In year one we performed experiments to determine if decitabine treatment in vitro would lead to re-expression of three abnormally silenced genes. Our data show that, in some cells, decitabine is effective at achieving this. In the final months of year one we initiated in vivo studies in which mice harboring ET tumors were being treated with decitabine. It is our hope that the drug will reverse the abnormal methylation patterns and inhibit tumor growth. Over the next year we will also to test to what extent turning off EWS-FLI1 mimics the effects of decitabine. These studies will determine if how DNA methylation contributes to continued growth of established tumors and whether targeting DNA methylation might be an effective therapeutic option.


    2011 6 Month - Coupled Genetic and Functional Dissection of Chronic Lymphocytic Leukemia

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    To discover genetic alterations important in chronic lymphocytic leukemia formation, Dr. Wu’s Laboratory has:

    • Sequenced the DNA of 91 CCL samples. She detected approximately 2,000 mutations that changed protein sequences. Nine of these genes were mutated significantly more often than normal. Four of the nine genes have previously been described occurring in CLL, but five have not been implicated in CLL before. Dr. Wu’s laboratory has begun work on confirming these results through other methods.
    • Found strong associations between common mutations and key chromosomal deletions in CLL.
    • Defined the effects of mutations in the Wnt pathway, which is often mutated in cancer. Using innovative nanowire delivery, Dr. Wu decreased expression of Wnt pathway proteins and found this decrease increased cell death in cancer cells compared to normal B cells. This indicates that mutational profiling may be able to identify patients more susceptible to Wnt pathway inhibition.


    2011 6 Month - Framing Therapeutic Opportunities in Tumor-activated Gametogenic Programs

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    During this initial reporting period, Dr. Whitehurst’s laboratory has:

    • Focused on a set of 22 tumor cell lines from a range of cancers and evaluated the level of expression of the 120 gametogenic genes previously demonstrated to be present in tumors.
    • Identifying 12 cell lines that cover 90% of the original data set.
    • Initiated the screening phase of the project, which consists of the systematic inhibition the function of each of these gametogenic proteins in the 12 cell lines selected. From these experiments, they can determine which of these proteins are required for tumor cells to grow, divide and survive. They have completed roughly half of the cell lines.
    • Found one protein, FATE1, expressed in Ewing’s Sarcoma cells and essential for survival of those tumor cells.
    • This work has revealed that many, but not all, of these proteins are indeed required for tumor cells to survive and divide and is now the basis for their further research into how these proteins are being exploited by tumor cells.


    2011 6 Month - Developing New Therapeutic Strategies for Soft-tissue Sarcoma

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    The ultimate goal of these studies is to identify new, more effective anti-sarcoma therapies, based on a better understanding of how these cancers arise and grow in skeletal muscle. So far, Dr. Wagers’ laboratory has:

    • Confirmed that 104 out of 141 candidate sarcoma-relevant genes identified previously are indeed upregulated in their mouse model.
    • Identified the gene “Gremlin” as a high-priority candidate gene, because it is upregulated 1326-fold in their mouse model. They also have in vitro evidence for decreased cell growth in sarcoma lacking Gremlin.
    • Shown that their mouse model for rhabdomyosarcomas (RMS) metastasized to the lungs of tumor-bearing animals.
    • Established protocols to induce and phenotype human sarcoma xenografts in mouse skeletal muscle.
    • Found that the mTOR inhibitor prolongs survival both in their mouse model and in human HT1080 fibrosarcoma xenografts, thereby validating that their sarcoma platform in skeletal muscle can be used to identify targets that modulate the progression of sarcomas in vivo.

     


    2011 6 Month - Inhibiting Innate Resistance to Chemotherapy in Lung Cancer Stem Cells

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    The approach that Dr. Sweet-Cordero has chosen to identify novel regulators of chemoresistance in lung cancer cells involves inhibiting a set of genes whose expression is altered in response to chemotherapy treatment in vivo in a mouse model of lung cancer.
    To date, Dr. Sweet-Cordero’s laboratory has:

    • Established the methods to effectively infect primary cells and induce silencing and functional knockdown with lentiviral shRNAs in the 3D culture system.
    • Established the sensitivity of primary spheres to cisplatin in the 3D culture system.
    • Optimized the conditions of the 3D culture model and tested alternatives to Matrigel to determine if another matrix could be more effective at producing sphere-forming cells and, therefore, more cost-effective. None of the alternatives have been capable of substituting for Matrigel.

    Dr. Cordero has also continued collecting human NSCLC primary tumor samples with the goal of establishing a tumor sample bank of frozen single cells. His laboratory is currently obtaining 2-3 samples a month and these are being processed into single cell suspensions and frozen for later use.


    2011 6 Month - Targeting Sleeping Cancer Cells

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    The goals of Dr. Ramaswamy’s project are to identify the dormant cancer cell epigenomic and signaling networks, and to validate candidate transcriptional and signaling targets: During this initial project period, Dr. Ramaswamy’s laboratory has:

    • Found that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating progeny that resemble dormant cells.
    • Found that this population of “quasi dormant” cells is enriched following chemotherapy in breast cancer patients.
    • Determined that asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during division.


    2011 6 Month - A Systems Approach to Understanding Tumor Specific Drug Response

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    The first 6 months of the project was primarily focused on collecting the first phase of necessary data described in the proposal. Dr. Pe’er’s laboratory has:

    • Characterized the transcriptional and phenotypic response following pathway inhibition of MAPK, AKT and their combination in 12 melanoma cell lines of different genetic backgrounds.
    • Built two mathematical models that both consists of four different cell cycle states, between which cells can transition. Dr. Pe’er and her group are modeling how the drugs influence these transition rates, which allows them to compute how many cells are undergoing apoptosis, senescence and proliferation after treatment.
    • Collected proteomic data from three different melanoma cell lines at multiple time points following treatment with a B-RAF inhibitor (PLX4032/vemurafenib) in order to determine the relevant time points for further studies.

     


    2011 6 Month - Identification and Targeting of Novel Rearrangements in High-Risk ALL

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    Dr. Mullighan is using genomic profiling to identify novel targets of rearrangement in high- risk ALL, and to determine the frequency and spectrum of these alterations.

    • They have used a variety of complementary approaches to screen over 500 cases of ALL and have identified the genetic bases of approximately 70% of childhood cases of BCR-ABL1-like ALL.
    • They are continuing to identify the genetic basis of additional BCR-ABL1-like ALL cases using advanced (next-generation) sequencing technology, including transcriptome sequencing and exome sequencing.

    Dr. Mullighan is also developing experimental models of B-progenitor ALL that recapitulate the genetic alterations identified above and enable testing of targeted therapeutic agents.

    • Using laboratory cell lines, Dr. Mullighan’s laboratory has shown that several of these alterations accelerate cell growth and activate downstream signaling pathways. In addition, alterations such as EBF1-PDGFRB induce leukemia when expressed in mouse bone marrow cells.
    • They have also developed xenograft models in which human leukemia cells are grown in immunodeficient mice. Importantly, growth of these cell lines is inhibited by several TKIs including include imatinib (Gleevec), dasatinib (Sprycel) and ruxolitinib (Jakafi).


    2011 6 Month - Exome Sequencing of Melanomas with Acquired Resistance to BRAF Inhibitors

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    To discover genetic alterations that cause disease progression of initially B-RAF inhibitor- sensitive human metastatic melanomas, Dr. Lo’s Laboratory has:

    • Made the unexpected observation that MEK1 mutations, proposed previously as a mechanism of acquired B-RAF inhibitor (B-RAFi) resistance, cannot account for acquired resistance since these mutations are found prior to B-RAFi therapy. This discovery of B-RAF/MEK1 double mutant melanomas and its implications for B-RAF targeted therapy has been submitted for publication.
    • Published work describing splicing isoforms of BRAF (V600E) that dimerize in a RAS independent manner, and showing that these may be mediating acquired resistance to RAF inhibitors.
    • Discovered, using a new type of analysis called ExomCNV, a gene amplification that mediates acquired resistance in melanoma. This discovery has also been submitted for publication.
    • Completed 20 whole exome sequences from six patients (each set consists of a normal tissue, a baseline pretreatment tumor, and a disease progression (DP), on- therapy tumor or tumors in some patients), with one patient contributing 3 DP tumors.

     


    2011 6 Month - Chimeric RNAs Generated by Trans-Splicing and their Implications in Cancer

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    To better characterize the trans-splicing process, and translate our knowledge into better diagnostic and therapeutic approaches, Dr. Li’s Laboratory has:

    • Detected a gene fusion product, PAX3-FKHR, which is characteristic for alveolar rhabdomyosarcoma, which is produced transiently during the muscle differentiation by using an adult stem cell differentiation system to induce cells into to many distinguishable lineages at many time points.
    • Found that the expression of the newly identified fusion, SLC45A3-ELK4, correlates with prostate cancer progression and it plays important roles in prostate cancer cell growth.
    • Generated a specific antibody to JAZF1-JJAZ1 fusion that only recognizes the fusion product.
    • Using this method, Dr. Li found that the fusion protein is localized to a specific compartment inside the cellular nucleus called PML nuclear body that is known to suppress tumor growth by inducing cell death.


    2011 6 Month - Targeting PP2A and the Glutamine-Sensing Pathway as Cancer Treatment

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    To determine the molecular basis of tumor cell survival under glutamine deprivation in order to develop novel drugs targeting this pathway, Dr. Kong’s Laboratory has:

    • Demonstrated that, among 16 different PP2A regulatory proteins, only the B55α subunit is induced upon glutamine deprivation.
      • This induction is specific to glutamine deprivation.
      • Initial results indicate that increased reactive oxygen species due to glutamine deprivation contribute to B55α induction.
    • Demonstrated that suppression of B55α expression impairs cancer cell viability in the presence of low glutamine.

     


    2011 6 Month - Targeting Protein Quality Control for Cancer Therapy

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    Dr. Jacinto’s laboratory is working to confirm the hypothesis that mTORC2 plays a role in translation and processing of ErbB1 and ErbB2 in cell models.

    • Models that genetically disrupt mTORC2:
      • Using murine embryonic fibroblasts (MEFs) that have genetically disrupted mTORC2, the lab has demonstrated that TORC2 is critical for synthesizing normal amounts of ErbB1.
      • Using another MEF cell line that inhibits the formation of the mTORC2 complex (SIN1 knockout cells), the lab also confirmed that mTORC2 is critical for ErbB expression and normal levels of glycosylation.
    • Wild-type MEFs and breast cancer cell lines treated with mTOR inhibitors:

      • ErbB2 expression (translation) is not altered in wild-type MEFs treated with an mTOR inhibitor (Torin 1).
      • ErbB1 and ErbB2 expression is not altered upon mTOR inhibition in the breast cancer cell lines tested. The lab is expanding to test other cell lines.
      • It is hypothesized that other signaling pathways might compensate for the mTOR inhibition in these models.


    2009 24 Month - Cancer Cell Specific, Self-delivering Pro-Drugs

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    Dr. Levy’s research efforts are focused on the development of a new type of targeted therapy that can home to and deliver anti-cancer drugs specifically to the cancer cells. His initial research is focused on prostate cancer, but the technologies developed will be broadly applicable to most other cancers. His laboratory has:

    • Synthesized a series of drug-laden oligonucleotides (small molecules) and aptamers with different types of modifications and/or different chemotherapy agents incorporated inside them.
    • Performed tests to find the optimal drug/vehicle combination that will enable them to use two core aptamers as the basis for targeting: one that binds the human transferrin receptor (a receptor commonly overexpressed on different types of cancer cells) and one that binds the prostate specific membrane antigen (PSMA; a marker of prostate cancer).
      • Both sets of minimized aptamers were confirmed for their ability to bind to and internalize into the appropriate receptor-bearing cells. The ability for the aptamers to enhance cell death have been modest. New drug “cargoes” are being developed in order to enhance the ability to increase cell death.
    • Explored a number of different approaches for the targeted delivery of nucleoside analog drugs using nucleic acid aptamers.
    • Initiated the production of liposomes that could bear up to 2000 drug molecules per liposome and are decorated with targeting aptamers on the outside.

      • Experiments testing different and more potent drugs to be delivered by the liposomes are being performed.
    • Begun development of a new transferrin specific aptamer that is optimized to work in conditions with higher transferrin.
    • Begun collaboration with two different SU2C Dream Teams—Dr. Haber’s group and Dr. Baylin’s group.