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Investigator Address Date Body Grant Program
Investigating and Targeting Diverse Kinase Alterations Driving Systemic Histiocytic Neoplasms United States -

Histiocytic diseases are a group of blood disorders that affect both children and adults and can lead to severe disability and death. Until recently, there were no effective treatments for the more severe forms of these diseases in adults. However, it was recently discovered that about half of these patients have a mutation in a gene called BRAF and that treating these patients with a medicine that blocks this mutation results in astounding benefits for these patients. Unfortunately, half of histiocytosis patients do not have the BRAF mutation and therefore cannot benefit from this breakthrough therapy. We and others have conducted intensive genetic analyses of these “non-BRAF” patients and found that nearly all have mutations in other genes that turn on a single tumor growth pathway. Fortunately, a class of medicines (called “MEK” inhibitors) blocks this pathway and in compassionate use protocols we have provided this drug to 2 adult patients dying from a histiocytic disorder with dramatic resolution of grave symptoms.

Based on these data, we believe that MEK inhibition will provide an important new therapy for those patients without the BRAF mutation as well as an opportunity to understand the pathways that drive these disorders in more detail. In this grant we propose to test the effects of the MEK inhibitor, cobimetinib, in a clinical study in these non-BRAF patients and study the biological importance of the diverse non-BRAF mutations in histiocytic disorder patients.

Career Development Program
Targeting the stress response machinery in pediatric T cell acute lymphoblastic leukemia (T-ALL) United States -

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that results from the accumulation of genetic alterations (such as mutations) during the development of precursors of T cells, a type of white blood cells with critical role in the human immune system. Despite the improvements in patient outcomes after introduction of intensified chemotherapy, a significant percentage of patients do not respond to therapy or experience disease relapse after a transient initial response. Moreover, serious side effects, such as secondary tumors, severely compromise our therapeutic efforts especially in children. Therefore, there is an urgent need to identify more specific therapeutic targets and drug combinations.

In order to avoid therapy-induced side effects we need to identify strategies that target specifically cancer cells while sparing healthy cells. In addition, there is an urgent need to elucidate the mechanisms that result in resistance to therapy upon relapse. During carcinogenesis, cancer cells undergo various alterations in many cellular processes. This results in increased levels of biological stress. To cope with stress, cancer cells depend on pathways that relieve stress, known as stress response pathways. Importantly, healthy cells are not dependent on stress response pathways under baseline conditions, making drug targeting of this pathway a promising therapeutic approach. Understanding the biology of stress responses and its crosstalk with the cancer machinery is a major focus of our laboratory. Using novel animals models and pediatric patient samples, this proposal will focus on identifying the group of patients who will benefit from a promising drug that is currently in clinical trials for solid tumors. In addition, we will investigate whether targeting a potential Achilles’ heel for resistant T-ALL, in combination with current therapy may prevent or combat recurrent disease. In conclusion, with our proposed research we hope to therapeutically exploit a tumor supportive pathway on which both newly diagnosed and relapsed leukemia are dependent.

Translational Research Program
Role and mechanisms of enhancer deregulation in in T-ALL United States -

Acute lymphoblastic leukemia (ALL) is a cancer originating from blood cells that proliferate in the bone marrow. ALL represents the most frequent pediatric leukemia, making up about a third of all childhood cancers. Gradual improvement in treatments have led to better survival rates. However, patients with ALL who fail to achieve complete remission or those whose disease relapses have very poor outcomes. A better understanding of the molecular events underlying leukemia, will be needed to develop specific and more effective antileukemic drugs.

During leukemia development, abnormal blood cells accumulate mutations in their DNA that dysregulate important cellular processes that enhance survival and proliferation of cancer cells. In addition to mutations to the DNA, epigenetic changes, which are heritable changes that do not involve direct modifications of the DNA sequence, have a clear functional impact on cancer development. Temporal and spatial gene transcriptional regulation of cellular states during development is coordinated by clusters of regulatory enhancers organized in regulatory domains. These enhancers regulate gene transcription through changes in DNA binding activity of proteins as well as epigenetic changes. We hypothesize that oncogenic genetic drivers and mutations in T-ALL alter the regulatory logic controlling chromatin accessibility of developmental enhancers active in early thymocytes locking T-ALL lymphoblasts in a hyperproliferative and differentiation-arrested developmental state. This opens a new therapeutic window for cancer treatment.

We are studying the epigenetic dysregulation of T cell acute lymphoblastic leukemia (T-ALL). By using innovative technologies and primary patient samples, we will study the functional relevance of chromatin accessibility gene regulatory enhancers, providing a high-resolution map of the active (open) and inactive (closed) regions of the genome. The comparison of this oncogenic map with normal healthy cells will highlight the regions that change their functionality in leukemia. For the first time we will elaborate a complete map of the regulatory functions of the genome in T-ALL, defining active as well as inactive genes as well as areas with no expression that function to enhance the expression of other genes. This study will also elucidate the strategies of oncogenic activation of these DNA regions, the proteins involved in these pathways and the dependency of leukemic cells on these dysregulated processes. This may eventually lead to therapeutic strategies to target these processes.

Career Development Program
Refining Molecular Risk Prediction & Individualized Lymphoma Therapy Using Circulating Tumor DNA United States -

For patients with the most common lymphoma subtype, diffuse large B-cell lymphoma (DLBCL), curative outcomes are common. Still, nearly a third of DLBCL patients succumb to their disease. Survival has not significantly improved over the last 15 years despite many clinical trials during this period. Effective strategies to predict early treatment failures have largely been elusive.

Our overall goal is to study the relations among baseline and dynamic risk factors including genetic mutations and circulating tumor DNA (ctDNA) in DLBCL patients. Circulating tumor DNA is DNA that originates from tumor cells and is shed into the bloodstream. Our central hypothesis is that novel biomarkers of cancer risk such as detection of ctDNA, and detailed genetic profiling can be used for early detection of residual disease, to identify for the identification of dynamic changes that anticipate treatment failure, and for the provision of early surrogate endpoints for future clinical trials.

We will examine the relations among clinical variables, molecular risk factors, very early treatment responses (measured using blood samples and imaging), and clinical outcomes (including survival) in a large cohort of patients with DLBCL. This is relevant to patients with blood cancers because new strategies for cancer risk stratification and early detection of adverse outcomes have the potential to improve clinical outcomes in patients with these common and often deadly tumors.

This contribution is significant since knowledge of the molecular features associated with cancer outcomes and early detection of treatment failure may lead to novel ways to select better therapies for patients at highest risk of failures, by applying blood-based assays over the disease course for both tumor genotyping and disease monitoring. Our innovative approach, in which we will employ novel methods developed by our group, will lay the foundation for studies aimed at reducing risk of treatment failure as a means of improving clinical outcomes. Demonstrating that this approach can serve as a robust, early biomarker for patients with DLBCL would be transformative for future trial design and for rapid evaluation of novel, personalized treatment approaches in patients at highest risk for recurrence. Our study will also serve as a proof-of-principle that is applicable to other tumor types.

Career Development Program
Testing Targeted Therapy in Langerhans Cell Histiocytosis United States -

Rationale and Background: Children with Langerhans cell histiocytosis (LCH) develop destructive lesions that can arise in virtually any organ including bone, brain, liver and bone marrow. LCH occurs with similar frequency as pediatric Hodgkin lymphoma, but there has historically been fewer opportunities for patients with LCH to participate in cancer research studies due to uncertain identity. LCH was first identified over 100 years ago, but only in the past ~5 years has been recognized as a disease in the family of pediatric cancers. Outcomes for children with LCH remain suboptimal, with over 50% failing to be cured with initial chemotherapy, and the majority of patients who are cured suffer long term consequences including problems with growth, control of hormones, and some develop a devastating progressive neurodegenerative condition. Patients who relapse or do not respond to front-line therapy are typically treated with highly toxic chemotherapy or eventually bone marrow transplant. Improved therapies are clearly needed for children with LCH.

Preliminary Studies: In 2010, a mutation in the BRAF gene (called BRAF-V600E), was discovered in over half of LCH tumor samples tested. Over the past 5 years, more mutations in the same cell growth pathway as BRAF (the MAPK pathway) have been discovered, accounting for over 85% of all cases of LCH. In the MAPK pathway, a series of proteins transmit messages from the cell surface to the nucleus of the cell, where that message is translated by turning on or off certain genes. In LCH, this MAPK pathway is overactive and never turns off, resulting in uncontrolled cell growth, resistance to cell death, and formation of destructive LCH lesions. Studies in blood cells from patients with LCH and in mice demonstrated that LCH is caused by activation of the MAPK pathway at specific stages of blood cell development. In early clinical trials with adult patients, LCH lesions responded to vemurafenib, a drug that blocks BRAF-V600E activation. Studies with cells from patients with LCH and experimental mice suggest that blocking MAPK pathway activation with drugs that inhibit MEK activation may be an effective therapeutic strategy for patients with LCH.

Hypothesis and Aims: We proposed to test the hypothesis that cobimetinib, which targets MEK activation, will be a safe and effective treatment for patients with refractory LCH, LCH-neurodegenerative disease, and disorders related to LCH that are also driven by MAPK activation. Additionally, we propose to study the responses associated with certain mutations, determine if cells in blood carrying LCH mutations can be used to follow disease activity, and study new mutations in patients who relapse despite cobimetinib therapy. This study will be carried out through a consortium of LCH disease experts at 11 different institutions through the North American Consortium for Histiocytosis Research.

Translational Research Program
Molecular mechanisms of PHF6 mutations in leukemogenesis United States -

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia derived from malignant early T cell blood progenitors. It accounts for 10–15% of pediatric and 20–25% of adult cases of the total number of acute lymphoblastic leukemia (ALL) in Europe, the United States and Japan. It is the most common malignancy diagnosed in children, representing nearly one third of all pediatric cancers with a peak incidence in children aged 2-5 years. The current treatment is severely toxic. Moreover, despite intensive chemotherapy, 20% of pediatric and over 50% of adult T-ALL patients fail to achieve a complete remission which, inexorably results in fatal outcomes. These devastating effects highlight the need to determine the molecules involved in the onset and spread of T-ALL, which may lead to the development of novel targeted therapies.

PHF6 is a tumor suppressor gene commonly mutated by deactivation in adult and pediatric T-ALL. This gene plays an important role in hematopoietic stem cell (HSC) self-renewal, the process by which these blood progenitors generate new blood cells for the lifetime of an organism. PHF6 inactivation enhances HSC self-renewal in mice and drives enhanced T-ALL leukemia stem cell activity. However, the mechanisms by which loss of PHF6 contributes to leukemia remain poorly understood. By testing genetic data and analyzing the interactions of PHF6 at the protein level, I have identified a second mutated gene in T-ALL called PHIP. Notably, loss of PHF6 and PHIP show convergent cellular and developmental phenotypes supporting a common molecular and functional role. My central hypothesis is that PHF6 and PHIP mutations contribute to leukemia through common mechanisms. Specifically, here I will biochemically characterize the protein composition of the PHF6 and PHIP complex to evaluate the mechanisms by which they control gene expression. Moreover, I will assess the proposed convergent role of PHIP and PHF6 in the control of normal HSC-self renewal as well as the oncogenic transformation of healthy cells to T-ALL cells. These results will improve our understanding of the leukemia initiating potential of HSCs potentially providing new therapeutic targets to prevent T-ALL initiation and progression. Ultimately, these studies will likely contribute to patient stratification and personalized treatment based on their specific molecular profiles.

Career Development Program
FACTORS REGULATING IMMUNOGLOBULIN-PRODUCING B-CELLS IN PATIENTS WITH WALDENSTROM’S MACROGLOBULINEMIA – PART V United States - Special Grants
Modulating Immune Function in Peripheral T-cell Lymphoma (PTCL) United States -

Patients with peripheral T- cell lymphoma (PTCL) constitute approximately 12-15% of all lymphoma cases and PTCL patients typically have a poor outcome. Patients with PTCL typically respond to initial combination chemotherapy, but most patients subsequently progress and require additional therapy. Treatments such as romidepsin and belinostat have been approved for patients with PTCL, but their efficacy has been limited. There is therefore clearly a need for additional new therapies to treat patients with PTCL.

Multiple mechanisms account for the lack of durable benefit with current treatment, but the inability of tumor-specific immune cells to eradicate the lymphoma is a central issue. A novel mechanism used by cancer cells to inhibit the immune response, however, is overexpression of programmed death ligand 1 or 2 (PD-L1 or PD-L2). These proteins interact with the programmed death-1 (PD-1) receptor expressed on intratumoral T-cells and provide an inhibitory signal thereby suppressing anti-tumor immunity. In previous work, we have shown that PD-1/PD-1 ligand interactions are critically important in PTCL and have found that PD-1 is expressed both on malignant cells and in the tumor microenvironment. Similarly, we have found that PD-L1 is highly expressed by malignant T-cells and intratumoral monocytes, and have shown that tumor-associated PD-L1 suppresses T-cell immunity.

Monoclonal antibodies that block PD-1 signaling can prevent T-cell suppression and promote an anti-lymphoma immune response. Antibodies, directed against PD-1 or PD-L1, are currently being tested in clinical trials and have shown remarkable efficacy in some hematologic malignancies. Despite broad clinical use of PD-1 directed therapy, very little is known about the immunology of PD-1 signaling in hematological malignancies. In PTCL in particular, PD-1/PD-L1/2 signaling is complicated by the fact that the receptor and ligands can both be expressed on the cancer cell. Furthermore, initial clinical trials have included very few patients with PTCL; however, in 5 patients receiving nivolumab, an antibody that blocks PD-1 signaling, 2 patients had an objective response, suggesting possible efficacy of PD-1 blockade in PTCL.

In this application, therefore, we propose to 1) measure the immune consequences of inhibiting PD-1/PD-1 ligand signaling, 2) determine the clinical efficacy of PD-1 blockade alone, and in combination with romidepsin, in patients with relapsed and refractory PTCL, and 3) determine the immunological predictors of clinical response to PD-1 therapy in patients with PTCL. In the proposed aims, we will determine how well PD-1 blockade works in PTCL, the specific mechanism of action of PD-1 blockade in PTCL, and in which subgroup of PTCL patients PD-1 blockade is predicted to have clinical benefit. Successful completion of this project is likely to have a major impact on clinical practice by potentially leading to an effective new therapy for patients with PTCL.

Translational Research Program
Checkpoint-Based Immunotherapy in Follicular Lymphoma United States -

Follicular lymphoma (FL) is a subtype of non-Hodgkin lymphoma that is incurable with conventional therapy. Yet a majority of patients can be cured by stem cell transplantation (SCT), the success of which depends primarily on the donor’s immune system. This demonstrates that FL is a very immune-sensitive disease. However, the toxicity of SCT is too high to justify its routine use, and while other forms of immunotherapy have been used in FL, none have yielded sufficiently frequent and durable responses. There is tremendous interest in oncology in monoclonal antibodies (mAbs) that target various immune checkpoints. Immune checkpoints are normally used by the body to regulate immune responses but can be usurped by tumors in order to shield themselves from immune attack. Several checkpoint mAbs have already been tested in FL patients that have relapsed or that have disease that is refractory to conventional treatment (R/R FL). Though this approach showed a favorable safety profile, there were durable responses only in a minority of patients. This suggests that FL may rely on immune checkpoints to survive but has anti-immunity shields that are too complex to be effectively disabled by single checkpoint mAbs. It is likely that multiple immune checkpoints are used by FL cells to evade the immune system. We seek to identify safe and effective immunotherapy approaches in FL by simultaneously targeting multiple checkpoint pathways and therefore propose a clinical trial of combination checkpoint immunotherapy in FL. This trial will sequentially test combinations of checkpoint mAbs, targeting three different immune checkpoints (PD-1, 4-1BB, and Ox-40), in conjunction with rituximab administered for just 6 months to patients with R/R FL. Those combinations that meet pre-defined success criteria will also be tested in patients with previously untreated FL. The clinical goal of this trial is to identify, within 30 months, promising short-duration combination treatment regimens in both R/R and untreated FL that can then be further tested in larger clinical trials. In addition, we will characterize in detail the immune landscape in patients receiving these treatments, to investigate how this relates to treatment response and resistance. The understanding gained through this study may allow the identification of markers of treatment sensitivity, the design of future combinations, and the successful treatment of FL with safe and effective immunotherapy.

Career Development Program
Cardiovascular reserve capacity in survivors of hematopoietic cell transplantation United States -

Hematopoietic cell transplantation (HCT) is the treatment of choice for many hematologic malignancies such as acute leukemia and lymphoma. During the past four decades, there have been tremendous advances in HCT, contributing to a growing number of long-term survivors of living in the U.S. today. However, these survivors are at risk for severe and life-threatening complications that can significantly impact quantity and quality of survival. Among these survivors, the risk of cardiovascular diseases (CVDs) such as heart failure, stroke, or heart attacks is nearly four times greater than would be expected for the general population, and five-year overall survival in patients with CVD is <50%. As such, it is important to develop reliable accurate screening strategies for early detection of CVD, so that proper interventions can be initiated to mitigate the long-term risk and devastating consequences of CVD. We will perform a comprehensive assessment of cardiovascular health in long-term survivors of HCT, using an established and validated tool (cardiopulmonary exercise testing) that is highly prognostic for CVD and premature mortality in individuals with and without cancer. In addition, HCT survivors will undergo comprehensive organ-specific (cardiac, pulmonary, musculoskeletal) evaluations, to better understand the mechanisms which contribute to impairment of cardiovascular health after HCT, so that targeted interventions can be developed to minimize long-term CVD risk.

Career Development Program
Selective BRD4 degradation in pediatric leukemia United States -

Despite advances through cancer research that have improved outcomes for many children diagnosed with leukemia, subsets of the disease, including the high-risk MLL-rearranged acute myeloid leukemia (MLL-r AML), remain difficult to cure, establishing an area of significant unmet medical need. While many pediatric leukemias lack the multitude of mutations that are a hallmark of adult cancer, recurring abnormalities in proteins called transcription factors are quite common. Gene fusions, such as rearrangement of the MLL gene, mutation or amplification of a single transcriptional regulator often lead to the inappropriate regulation of the expression of genes in the cell that control cellular pathways involved in cell division and cell death, and thereby contribute to tumor development and cancer progression. One way to stop this abnormal gene expression program and to eradicate cancer cells would be to perturb a transcriptional regulator named BRD4. BRD4 is a member of the Bromodomain and Extra-Terminal motif (BET) family of proteins that can bind to chromatin and regulate gene expression. In leukemia, BRD4 has been shown to regulate cancer-promoting gene expression and to be an excellent target for the treatment of high-risk childhood cancers, including acute leukemia. Likewise, new drugs that inhibit BET proteins have been developed and shown to strongly downregulate expression of cancer-promoting transcription factors in acute leukemia in the laboratory. Clinical trials of BET inhibitors in adult patients are ongoing.

Unfortunately, current BET inhibitors target the entire BET family of proteins, including BRD2, 3, 4 and T, and toxicity has been reported in both preclinical and clinical research data. We hypothesize that targeting of BRD4 exclusively, may reduce this toxicity while still having a potent anti-cancer effect. As such, we have recently developed a molecule, ZXH-3-26, that can cause the selective degradation of the BRD4 protein. Not only does this new molecule discriminate between the BET family members but it also works via a new mechanism – protein degradation versus protein inhibition.

In order to better understand this new BRD4-selective degrader molecule, we propose to test ZXH-3-26 in laboratory models of pediatric acute leukemia first, to understand how it works to kill cancer cells and second, to compare it to currently available pan-BET inhibitors and degraders, which demonstrate toxicity and off-target effects. Finally, we plan to chemically optimize ZXH-3-26 so that it has improved properties of a drug that can be used to treat humans. This research will lay the foundation for a new therapy to treat children with cancer in clinical trials.

Translational Research Program
Dissecting the role of the super elongation complex and the acetyl-lysine epigenetic reader protein ENL in acute myeloid leukemia United States -

Acute myeloid leukemia (AML) is a highly aggressive cancer arising from the white blood cells that can affect both children and adults. The five-year overall survival rate for adult patients with AML in the United States remains less than 30%, and only marginal improvements in patient outcomes have been achieved in the last 30 years. The implementation of advanced DNA sequencing technology has led to a dramatic increase in our understanding of the underlying molecular basis of AML. Remarkably, we have discovered that many of the alterations that lead to the development of AML in patients are not due to mutations within genes themselves but result from abnormal expression of growth-promoting genes. Our lab and others have shown that developing treatments to target this altered gene expression is possible and represents an effective new way to treat AML. Oncogenic fusions in a gene called MLL are found in both adult and pediatric AML. We recently identified the critical importance of a protein called ENL, which maintains the altered level of gene expression observed in MLL-fusion AML. Our work will build on this discovery by further characterizing the function of the ENL protein and increasing our understanding of why AML cells are so dependent on ENL for their sustained growth. In particular, little is known of how ENL interacts with MLL fusions. It is also unknown how ENL interacts with DOT1L1 and BRD4, which are also important in the context of MLL fusions and which are targeted by drugs that are in clinical development. We will examine how treatments aimed at targeting ENL may be incorporated into existing treatment strategies. We will also evaluate the cancer cells for potential mechanisms of treatment-resistance, given that patients with AML can often develop resistance to treatments that are initially effective. This will also allow us to better predict which patients will most likely benefit from this type of treatment. The research will incorporate multiple lines of investigation and approaches, including genome editing and a number of experimental models of AML in the laboratory. With a better understanding of the role of ENL in AML, and the subsequent targeting of this protein, it is our goal that new, more effective and safer treatment strategies will be developed for patients with AML.

Career Development Program
Safer and More Effective T cells for Immunotherapy of Viral-associated Hematological Malignancies United States -

Immunotherapy is leading to dramatic changes in how cancer is treated. A particularly exciting approach to immunotherapy is to genetically engineer immune cells to target molecules produced preferentially, or in some cases solely, by the tumor cell. For example, T cells of the immune system producing chimeric antigen receptors (CARs) that recognize targets associated with certain blood cancers have been recently approved by the FDA.

A related approach to immunotherapy under development is to engineer T cells to produce T cell receptors (TCRs) that target molecules produced by tumor cells. Whereas CAR-engineered T cells combine the recognition potential of an antibody with the T cell signaling machinery, TCR-engineered T cells rely on the biological recognition mechanisms used natively by T cells. One advantage TCRs have over CARs is that they can thus target a broader array of molecules produced by cancerous cells. Moreover, for cancers such as adult T cell lymphoma and Hodgkin’s lymphoma that are caused by and associated with ongoing viral infection, TCRs can target molecules that are uniquely produced by the cancer cells.

A limitation of TCRs, however, is that in their normal biological role, they possess a high degree of cross-reactivity. This can (and in some clinical trials has) led to significant “off-target” toxicity, in which the engineered T cells attack healthy tissue. We are pursuing a novel protein engineering approach that can reduce TCR off-target toxicity; however, it can also lead to reduced T cell potency. In other work though, we have shown that T cell potency can be manipulated separately by enhancing intracellular T cell signaling.

In this collaborative proposal, we propose to combine these two approaches to generate safer and more effective T cells for treating virus-associated blood cancers. As we will be able to build on a significant amount of previous work, we will focus initially on adult T cell leukemia, caused by Human T-lymphotropic virus 1 (HTLV-1). We will combine structural biology and protein engineering with molecular and cellular immunology and test efficacy in a mouse model of HTLV-1 associated leukemia. Although we will initially study HTLV-1 and adult T cell leukemia, our approach will be generalizable to other virus-associated cancers, such as lymphomas and leukemias associated with Eptsein-Barr virus and Hepatitis B virus. Thus, our work could set the stage for generation of new immunotherapies for a wide range of viral- and non-viral associated cancers of the blood and other tissues.

Translational Research Program
Targeting Immune Checkpoint protein B7-H3 (CD276) in Acute Myeloid Leukemia United States -

Acute myeloid leukemia (AML) is the most common and aggressive acute leukemia found in adults. An estimated 13,000 people develop AML in the United States every year, and 8,800 die from it. Immunotherapy has led to important clinical advances in cancer therapy in recent years due to superior cure rates compared with standard therapy. Here, we propose targeting B7-H3 (CD276), a promising immune checkpoint protein that has been reported to inhibit immune cells function by binding its receptor on natural killer (NK) cells and T lymphocytes. It has been reproted that B7-H3 is overexpressed in several solid tumors and hematological malignancies. However, this protein has not been used as a target in immunotherapy so far, probably due to lack of tools that could inhibit its function. Our preliminary data suggest that B7-H3 is over expressed in paeripheral blood and bone marrow cells collected from AML patients (n=61) at MD Anderson cancer center compared to normal samples (n=10). Inhibition of B7-H3 expression in AML cells activated NK cells-mediated killing of AML cells. To therapeutically target B7-H3 in AML cells, we have generated four monoclonal antibodies that binds B7-H3 with unique specificity and possess immuno-modulatory function. Our preliminary data suggests that addtion of anti-B7-H3 mAbs to AML and NK cell co-cultures, blocked B7-H3 function and enhanced NK cell-mediated killing of AML cells.

In order to develop these antibodies for human trials, we need to generate human comaptible anti-B7-H3 antibodies. To this end, we identified the protein sequences of all four antibodies that binds to B7-H3 protein. We will use these sequences to generate mouse-human chimeric monoclonal antibdoies that could be tested in animal models. We will test these antibodies for their ability to block B7-H3 fucntion and activate NK cells to kill AML cells. We will use these novel anti–B7-H3 chimeric mAbs to inhibit AML growth in humanized NSG mouse models containing human NK cells. We will use AML patient-derived xenograft (PDX) models expressing B7-H3 developed in our laboratory which are ideal for the proposed experiments in animals. We will implant AML-PDX cells in humanized mice and treat them with anti–B7-H3 chimeric mAbs to determine the effect of anti–B7-H3 chimeric antibodies on NK cell–mediated AML cell killing. Next, to understand the mechanism of B7-H3 mediated immunomodulation, we aim to identify its receptor on NK cells. To this end, we have generated recombinant B7-H3 protein that is conjugated with a molecular tag. We will perform protein pull down assays to isolate B7-H3 binding proteins in natural killer cells followed by mass spectrometry to identify the B7-H3 receptor. The objective of this proposal is to develop therapeutic tools to functionally block B7-H3 and enhance immune cell–mediated AML cell killing. We anticipate that the selected recombinant chimeric antibody will be suitable for use in human trials.

Translational Research Program
Therapeutic Targeting of Oncogenic RAS Signaling in Myeloid Leukemias with Stapled SOS1 Helices United States -

The Ras protein is found in many cancers, including myeloid leukemias. Despite its important role in cancer, there are no known drugs to block it. There are multiple Ras proteins, including KRas and NRas that are found in a variety of leukemias. The unique properties of Ras preclude a clear path to drug discovery using the approaches that have worked with the drug targeting of many other proteins. Thus, we are using a new approach based on a knowledge of Ras function to target its oncogenic activity. Ras proteins cycle between on and off states, and this cycling is regulated by a protein called SOS1. The association of Ras and SOS1 is critical for placing Ras back in an activated state that the cancer uses to promote its own pathogenic survival. Our laboratory has a long standing interest in a novel approach using stapled peptides to block the function of oncogenic proteins. By mimicking only a small portion of the SOS1 protein that interacts with Ras, and using a peptide-stapling technology that reinforces the structure of such peptides, we discovered a prototype compound that directly disrupts the SOS1/KRas complex, inhibits a broad spectrum of cancer-causing KRas proteins, and kills cancer cells as a result. Surprisingly, we also found that our compound can independently block KRas activity by another unknown mechanism. I will apply analytical approaches from a variety of fields, including chemistry, structural biology, leukemia biology, and translational medicine to investigate how our prototype stapled peptides directly block the activity of KRas and to determine whether it also blocks NRas activity. I will chemically synthesize a large tool box of SOS1-based stapled peptides and characterize their functional interactions with Ras family proteins and their cancer-causing mutant forms. I will then use structural biology methods to interrogate this new mechanism of direct Ras blockade by stapled peptides, and generate blueprints of their inhibitory interactions with Ras proteins, which is a critical step for developing optimal drugs. Using a series of assays that measure cancer cell death, I will test the best inhibitors that emerge and determine the relative susceptibilities of various Ras-driven leukemia cell lines to the drugs. Importantly, I will evaluate the anti-tumor activity of our best stapled peptides in mouse models of leukemia driven by Ras proteins. I anticipate that my research will reveal a novel mechanism for targeting Ras and provide a new therapeutic strategy to induce cancer cell death in patients suffering from myeloid leukemias driven by Ras.

Career Development Program
The Who and Why of Anthracycline-related Cardiotoxicity in Childhood Cancer Survivors United States -

Anthracycline chemotherapies are critical for treating children with leukemia and lymphoma. However, one of the most widely-recognized side-effects of anthracycline chemotherapy is the risk of heart failure. Childhood cancer survivors are at a 5- to 15-fold higher risk of heart failure when compared with the general population; less than 50% survive 5 years from diagnosis of heart failure. However, the risk of heart failure is not borne equally by all; thus while some develop heart failure at low-dose exposure to anthracyclines, others escape even after receiving very high doses; this observation suggests that genetic predisposition to how an individual handles anthracyclines and how the heart responds to the anthracyclines may be at play. Indeed, we and others have shown an association between certain genes and heat failure. These observations indicate a critical need to identify children with leukemia or lymphoma at highest risk of heart failure, such that targeted interventions can be instituted. We propose to develop a risk prediction model in childhood leukemia/ lymphoma survivors; this model will include a patient’s genetic make-up, clinical characteristics and treatment exposures. We will replicate the model in independent cohorts of childhood cancer survivors and will then apply the model to newly-diagnosed children with leukemia or lymphoma. These resources present us with an unprecedented opportunity to determine the association of the risk prediction model with: a) relapse of cancer, b) survival, and of course, c) risk of heart failure. Finally, we will determine the molecular basis of anthracycline-related heart failure. Thus, a clinical+genetic risk prediction model will allow identification of children with leukemia or lymphoma at highest risk for heart failure. This risk prediction model, when applied to newly-diagnosed patients with hematologic malignancies will allow for personalized interventions, thus reducing the burden of morbidity due to heart failure, while optimizing cancer-free survival. Finally, an understanding of the mechanism of anthracycline-related cardiotoxicity will set the stage for future interventions targeting molecular mechanisms of heart failure. The necessary infrastructure for the proposed research will be leveraged to test our hypotheses and take the field of personalized medicine to the clinic in children with hematologic malignancies, by minimizing long-term morbidity, while preserving survival.

Translational Research Program
Deciphering the role of disordered methylation in chronic lymphocytic leukemia development United States -

This project will explore the differences between DNA methylation, a process by which the DNA building blocks are modified, in cancer cells and in healthy cells. DNA methylation affects gene expression and plays a key role in tumorigenesis. By analyzing many chronic lymphocytic leukemia (CLL) patient samples, our group has uncovered pervasive disordered methylation: high variability of DNA methylation across diverse regions of DNA. This is not seen in cells from healthy people. We have previously shown that higher methylation variability in CLL cells is associated with poorer clinical outcomes, suggesting that a better understanding of this phenomenon may lead to new clinical interventions. Interestingly, we also observed that these changes are present early in cancer development, suggesting that disordered methylation may be a central driver leading to cancer development. These findings provide the foundation for my research, which aims to decipher the impact of disordered methylation on the development, onset, and severity of CLL.

To pursue these aims, I will employ new mouse models of CLL reflecting key CLL driver mutations first identified in our lab. I will also utilize state-of-the-art technologies such as single-cell RNA-sequencing and single-cell methylation profiling, to determine the chronologic origin of disordered methylation with respect to cancer diagnosis and progression. I have demonstrated that mouse CLL cells, like their human counterpart, present with disordered methylation. This provides an exciting and amenable disease context in which I can monitor mice over the course of disease. Furthermore, I aim to elucidate the cellular pathways affecting disordered methylation. Using CRISPR-mediated gene-editing tools, I will target genes that might affect disordered methylation, such as the DNMT3 and TET families, and monitor the development of CLL. Results from these studies will enhance our growing understanding of CLL development as well as the development of other cancers that demonstrate disordered methylation. Ultimately, our findings may inform the development of new tools to predict cancer development in healthy individuals and identify new therapeutic targets for blood cancer patients.

Career Development Program
The oncogene eIF4E coordinates extracellular signalling in AML Canada -

Relative to normal cells, cancer cells are often characterized by substantial changes to their surface in order to mediate their oncogenic properties. Factors on these surfaces can include multidrug resistance transporters which pump drugs out of the cell, enzymes that are involved in breaking down the surrounding tissue to enable cancer cells to migrate and invade other parts of the body as well as features that help the cells survive in the tumour microenviroment. In this way, changes to the cell surface architecture can lead to dramatic alterations in how cells respond to growth signals, drugs and affect their mobility. We identified the oncoprotein eIF4E as a factor that could substantially alter the cell surface and identified this ability as critical for its cancer causing properties. eIF4E alters the surface by increasing the levels of a large sugar molecular known as hyaluronic acid (HA) and also of key factors that bind HA such as CD44. In fact, upon elevation of eIF4E, the cells become coated in HA and further, the HA forms protrusions radiating outward from the cell surface. If the HA is removed using a clinically available enzyme called hyaluronidase, the cells lose their cancer-causing properties in the lab even though they still have elevated eIF4E. This suggests that the HA is important for the activity of eIF4E. eIF4E is elevated in acute myeloid leukemia (AML), lymphomas and other hematological malignancies. In this renewal, we will further examine the functional effects of these HA mediated alterations to the cell surface and investigate the use of hyaluronidase in patients in AML in a Phase I trial as a first step in determining the safety and preliminary clinical utility of this approach.

Translational Research Program
Randomized Phase II Study of Autologous Stem Cell Transplantation With Tadalafil and Lenalidomide Maintenance With or Without Activated Marrow Infiltrating Lymphocytes (MILs) in High Risk Myeloma United States - Therapy Acceleration Program
Understanding sex differences in myeloid and dendritic differentiation and function to target high-risk leukemias including BPDCN United States -

This project will explore the genetic causes as to why blastic plasmacytoid dendritic cell neoplasm (BPDCN) is vastly more common in men, with an aim to determine whether a gender-specific treatment approach would be effective. There are important differences between male and female patients with cancer that we can’t explain. Men develop cancer more often than women and are more likely to die from cancer. This holds true even when adjusting for risks such as smoking or exposure to toxic chemicals. It is not because of a difference in cancer types (prostate versus breast, for example), but rather because within nearly every type of cancer, men are more affected than women, including for blood cancers. In particular, BPDCN occurs over 3 times more frequently in men than women. BPDCN is a very aggressive subtype of leukemia, and has a dire prognosis.

We believe that studying the influence of gender on the biology of BPDCN is critical to understanding the mechanisms driving this cancer and to treating it more efficiently. Despite the observed gender bias in leukemia, there is currently a lack of research investigating its causes. Our lab recently identified a genetic mechanism of cancer gender differences tied to the X chromosome. We found cancer-protective genes, called tumor suppressor genes, located on the X chromosome that are expressed more highly in female cells compared to male cells. Because women have two copies of the X chromosome compared to only one in men, they would need mutations on both chromosomes to knock out the tumor suppressor whereas a man would only need one mutation on their only X chromosome. This makes women statistically less likely to get these cancer-associated mutations.

We plan to determine whether such mechanisms are at play in BPDCN, and specifically which genes are involved. Normal pDCs have a finite lifespan and naturally die through a normal process called apoptosis. There is evidence that pDCs in men are less likely to die when exposed to cell death signals, which might increase their potential to acquire cancer-causing mutations. We will directly test if altered survival regulation is more often seen in normal pDCs in males using our mouse models as well as in a male-specific BPDCN cell line. Understanding these mechanisms in the normal cells that may give rise to BPDCN will better our understanding of BPDCN and may help identify therapeutic strategies. Furthermore, our findings might also provide new therapeutic strategies to test in other blood cancers, including the possibility that males and females with the same cancer might benefit from different treatments.

Career Development Program
Loss of the non-canonical BAF complex as a driver and therapeutic target in SF3B1-mutant MDS and leukemia United States -

Myelodysplastic syndromes (MDS) are a heterogenous group of clonal blood disorders characterized by ineffective differentiation of stem cells to produce blood. MDS usually has a poor prognosis and patients are at elevated risk for disease transformation to acute myeloid leukemias. Because the genetic and molecular aberrations that give rise to MDS were unknown until relatively recently, there are few treatment options for MDS.

The most common cause of MDS is a genetic mutation occurring in blood cells that affects a process called “RNA splicing.” RNA splicing is a molecular process that is critical to the means by which genetic information encoded in DNA is used to make proteins. The most commonly mutated RNA splicing factor gene is called SF3B1. We now know that many patients with MDS carry mutations in SF3B1, but we do not have a good understanding of why these mutations cause disease.

We propose to determine how mutations in SF3B1 cause MDS and potentially create new opportunities for treating this disease. In particular, we will study how SF3B1 mutations disrupt a biochemical complex called the non-canonical BAF complex, which is involved in chromatin remodeling, and plays an important but incompletely understood role in hematologic malignancies. We will also determine whether it is possible to restore normal function of the non-canonical BAF complex as a potential therapeutic for treating hematologic malignancies with SF3B1 mutations.

Blood Cancer Discoveries Grant Program
The biological and therapeutic consequences of SF3B1 mutations in myelodysplastic syndromes United States -

Myelodysplastic syndromes (MDS) are a group of blood disorders characterized by impaired differentiation of hematopoietic stem cells into functional blood cells. MDS frequently has a poor prognosis and is associated with a high risk of transformation into acute myeloid leukemia. There are few treatment options for MDS, largely because the underlying molecular changes that drove MDS were not known until recently.

Recent genome sequencing studies revealed that MDS and related diseases are associated with specific mutations (genetic changes) in hematopoietic stem cells. These mutations most commonly affect genes that control a molecular process termed "RNA splicing." RNA splicing is critical to the process by which genetic information in DNA is "read" to make proteins. We now know that MDS-associated mutations that affect RNA splicing cause mistakes during the transfer of genetic information from DNA to protein. However, we do not yet know precisely which mistakes ultimately give rise to MDS.

We plan to use both experimental and computational methods to determine how mutations that affect RNA splicing give rise to MDS. Understanding the specific molecular changes that occur in MDS cells carrying these mutations will enable us to identify potential new therapeutic opportunities for treating MDS. Because the same mutations affecting RNA splicing are found in other blood diseases as well, such as chronic lymphocytic leukemia, we hope that our discoveries will improve the treatment of many different blood diseases.

Career Development Program
Novel Immune Therapy of Lymphoma United States -

Patients with relapsed diffuse large B cell lymphoma (DLBCL) have limited curative options, once their tumor fails to respond to standard chemotherapy regimens. Our group and others have recently demonstrated that activating the patients’ own immune cells can induce clinical responses, even in chemotherapy-refractory DLBCL patients. The central goal of this SCOR is to establish a collaborative team-science approach aiming at the development of new immune therapeutic strategies for DLBCL. Our ultimate goal is to translate our findings into novel clinical trials to improve the cure rate of patients with DLBCL.

Specialized Center of Research Program
The Immunobiology of Blinatumomab Response and Resistance in Relapsed Pediatric B-ALL United States -

Pediatric pre-B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. With current therapies, long-term survivorship reaches nearly 90%. However, despite these advances in therapy, nearly 20% of patients will relapse. In addition, survivors are at higher risk for other medical problems in the future, as a result of the years of intensive chemotherapy needed to effectively treat B-ALL. These issues underscore the need to develop better and more targeted therapies for B-ALL. Blinatumomab is an antibody that targets two types of cells simultaneously – B-ALL leukemia cells and normal T-cells. By bringing T-cells in proximity to B-ALL cells, T-cells are able to activate pathways to result in killing of the leukemia cells. This occurs without significant toxicity to other cells in the body. In order for blinatumomab to be effective, the B-ALL cells must express a protein called CD19 on their cells. In addition, there has to be a sufficient quantity of T-cells, which express the protein CD3. However, even when these criteria are met, some patients have no response to blinatumomab at all. We hypothesize that there are differences in other cells in the bone marrow of responders and nonresponders that lead to this difference.

I am the principal investigator for a current study run through the Children’s Oncology Group (COG) for children and young adults with relapsed B-ALL. In this study, patients are randomized to receive either blinatumomab or standard chemotherapy. Prior to this randomization, bone marrow is collected from all patients. For patients who receive blinatumomab, blood samples are collected at various times prior to and during the blinatumomab infusion. All of these samples from across the world are sent to my laboratory, where they undergo processing prior to freezing. We have samples from nearly 500 patients, approximately half of whom go on to receive blinatumomab. This provides us with a robust number of patient samples with which we can test our hypothesis and perform the necessary experiments for validation as well. We also have the knowledge of 1) which patients received blinatumomab and 2) which patients are classified as responders versus nonresponders after blinatumomab exposure. We will investigate the bone marrow of known blinatumomab responders and nonresponders to see if there are any changes in the types of cells that are present. We will also test the peripheral blood that was collected during blinatumomab treatment to see if there are differences in the proteins that are found in blood when we compare responders and nonresponders.

We expect that this will allow us to identify patients who are likely to respond to blinatumomab therapy, thereby personalizing therapy for B-ALL patients. Patients who are predicted to respond to blinatumomab may be able to avoid significant toxicity from exposure to chemotherapy.

Translational Research Program
RUNX1-ETO Targeted Small Molecule Therapy for t(8;21) Acute Myeloid Leukemia United States - Special Grants