2011 Innovative Research Grant 18-Month Progress Reports
Targeting MLL in Acute Myeloid Leukemia
Yali Dou, Ph.D., University of Michigan
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.
Targeting Genetic and Metabolic Networks in T-ALL
Adolfo A. Ferrando, M.D., Ph.D., Columbia University
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.
Targeting Protein Quality Control for Cancer Therapy
Estela Jacinto, Ph.D., University of Medicine & Dentistry of New Jersey - Robert Wood Johnson Medical School
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.
Targeting PP2A and the Glutamine-Sensing Pathway as Cancer Treatment
Mei Kong, Ph.D., City of Hope
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.
Chimeric RNAs Generated by Trans-Splicing and Their Implications in Cancer
Hui Li, Ph.D, University of Virginia
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.
Exome sequencing of melanomas with acquired resistance to BRAF inhibitors
Roger Lo, M.D., Ph.D., Santa Monica-UCLA Medical Center and Orthopaedic Hospital
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.
Identification and Targeting of Novel Rearrangements in High-Risk ALL
Charles G. Mullighan, MBBS(Hons), MSc, MD., St. Jude Children’s Research Hospital
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.
A Systems Approach to Understanding Tumor Specific Drug Response
Dana Pe’er, Ph.D., Columbia University Medical Center
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.
Targeting Sleeping Cancer Cells
Sridhar Ramaswamy, M.D., Harvard Medical School
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.
Inhibiting Innate Resistance to Chemotherapy in Lung Cancer Stem Cells
E. Alejandro Sweet-Cordero, M.D., Stanford University School of Medicine
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.
Developing New Therapeutic Strategies for Soft-Tissue Sarcoma
Amy Wagers, Ph.D., Harvard Medical School and Joslin Diabetes Center
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.
Framing Therapeutic Opportunities in Tumor-Activated Gametogenic Programs
Angelique Whitehurst, Ph.D., University of North Carolina, School of Medicine
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.
Coupled Genetic and Functional Dissection of Chronic Lymphocytic Leukemia
Catherine J. Wu, M.D., Dana-Farber Cancer Institute
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.