2011 Innovative Research Grant 30-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 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.
Targeting Protein Quality Control for Cancer Therapy
Estela Jacinto, Ph.D., University of Medicine & Dentistry of New Jersey - Robert Wood Johnson Medical School
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.
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 PP2Aactivity 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.
Chimeric RNAs Generated by Trans-Splicing and Their Implications in Cancer
Hui Li, Ph.D, University of Virginia
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”)
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. 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.
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. 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.
A Systems Approach to Understanding Tumor Specific Drug Response
Dana Pe’er, Ph.D., Columbia University Medical Center
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.
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) & 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.
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 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.
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 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.
Framing Therapeutic Opportunities in Tumor-Activated Gametogenic Programs
Angelique Whitehurst, Ph.D., University of North Carolina, School of Medicine
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.
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.