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Investigator Address Date Body Grant Program
Modulating Signaling Pathways in Endothelial Cells to Abate Leukemic Progression United States -

The aging process leads to an increased susceptibility of acquiring a variety of life-threatening diseases. The growth of the elderly population around the globe has resulted in an increase in age-related blood disorders, for which there are really no effective treatments. Most reports describing age-related blood alterations have focused exclusively on changes within blood stem cells. However, an increasing body of evidence has demonstrated that the blood vessels surrounding blood stem cells in the bone marrow can nurture and instruct their ability to function properly. Understanding the intimate relationship between blood vessels and blood stem cells during aging may provide a novel opportunity to slow down and potentially reverse the age-related functional decline observed in the blood system.
The goal of my research is to determine how aging of the blood vessels leads to the disruption of healthy blood stem cell function and if these age-related deficiencies of the blood vessels can enhance the progression of blood cancers such as acute myeloid leukemia (AML). Our group has shown that blood vessels are absolutely essential for supporting blood stem cell function. We have recently demonstrated that aged blood vessels can train young blood stem cells to function as aged stem cells, whereas young blood vessels can rejuvenate and increase the functionality of aged blood stem cells. Cells within the blood vessels interact with the AML cells, particularly through the secretion of soluble factors that influence the leukemia cells. We have also shown that AML cells can hijack the ability of blood vessels to support healthy blood stem cells and induce their own expansion leading to aggressive disease. We have devised novel experimental models of blood vessel cells grown in the laboratory that will allow us to determine if we can manipulate blood vessels to safeguard healthy blood stem cells while also increasing the susceptibility of AML cells to anti-cancer regimens. We will utilize our model systems to test if aged blood vessels support aggressive leukemic cells and if altering the instructive capacity of these blood vessels can reverse age-related blood disorders and give a competitive advantage to healthy blood stem cells. The success of these studies may lead to the development of therapeutic strategies designed to augment the sensitivity of leukemic cells to chemotherapy thereby reducing AML-associated side effects. Furthermore, these studies will begin to unravel the mechanisms by which aged blood vessels can lose their natural capacity to support normal blood production and become accessories to the development of blood cancer.

Career Development Program
Synergism of cell-intrinsic and cell-extrinsic factors in the clonal evolution of pre-malignant HSCs United States -

Hematopoietic stem cells (HSCs) live in the bone marrow and are responsible for life-long regeneration of the blood system and are characterized by the ability to self-renew. As we age, HSCs acquire genetic mutations, some of which provide a growth advantage. Many mutations which provide HSCs with this abnormal growth are also driving forces for development of blood cancers, including those in the genes DNMT3A and TET2. These proteins regulate the epigenetic mark of DNA methylation, modifying its properties, resulting in altered gene regulation. Mutations in these genes enhance self-renewal in HSCs, leading to an over-production of these cells in the bone marrow, a condition called clonal hematopoiesis of indeterminate potential (CHIP). Approximately 80% of people over the age of 50 have at least one HSC with a DNMT3A or TET2 mutation. People with CHIP have a slightly increased incidence of developing blood cancer. But this observation presents a curious paradox; if CHIP is near ubiquitous in the aging population, and DNMT3A and TET2 are associated with blood cancer, why does the overall incidence of blood cancer remain relatively low? As the average age of the population continues to increase, this dilemma will become increasingly important, and it presents two major unresolved questions in hematology research which form the basis of my research;
A) Are there specific genes and pathways that are required for the expansion of the mutant HSCs?
B) What factors promote cancer progression in individuals with CHIP?
We will identify other genes specifically required for clonal expansion of HSCs with mutations in DNMT3A and TET2. We will use genome engineering technology to identify genes which sustain the growth of the mutant HSCs, but not normal HSCs. The goal is to find targets we can inhibit to prevent the mutant HSCs from accumulating in the bone marrow, while leaving the function of normal HSCs unharmed. We will also investigate how environmental changes in the aging bone marrow preferentially support the growth of mutant HSCs. We will determine how different molecules secreted from cells in to the bone marrow environment stimulate mutant HSCs and analyze the effects in individual HSCs using a variety of cutting-edge techniques. The long-term goal of this work is to define how these mutations allow the HSCs to grow better, such that we can identify methods to impede their expansion. This would act as a method of cancer prevention by reducing the chance of one of these mutant cells ever evolving to blood cancer.

Career Development Program
Mechanisms of aberrant DNA double-strand break repair leading to lymphoid malignancy United States -

An essential function of the immune system is the ability to identify and react to substances foreign to the body, called antigens. Antigens are recognized by antibodies and antigen receptors, which resemble antibodies but are anchored to the cell. These are intricate multi-protein complexes that are generated by a process of gene recombination. Recombination happens by breaking the DNA and then repairing it in new configurations. When these repair processes do not work properly, they can cause mutations, including translocations of segments of chromosomes, which may lead to lymphoma. This can happen during early lymphocyte development when DNA breaks are generated by the RAG protein. The DNA breaks provide lymphocytes an opportunity to introduce various modifications in antigen receptor genes via the DNA repair machinery, which greatly increases the diversity of immune repertoire. Developing lymphocytes preferentially use a DNA repair pathway called canonical non-homologous end joining (c-NHEJ), instead of a more error-prone pathway called alternative end joining (alt-EJ) that is more likely to cause mutations. It remains unclear how developing lymphocytes normally utilize c-NHEJ while suppressing alt-EJ in response to DNA breaks.

To study how developing lymphocytes maintain DNA repair fidelity, we use B cell lines derived from mouse bone marrow that mimic characteristics of early-stage B cells. One possibility of suppressing mutagenic alt-EJ repair at RAG-induced DNA breaks is through an antagonism with factors of c-NHEJ repair, namely the DNA end-binding protein Ku. We will perform genetic analyses to identify the roles of Ku and related proteins in protecting DNA ends from improper processing that could allow for alt-EJ and translocations. In addition, we plan to take an unbiased approach by performing a genome-wide screening using state-of-the-art CRISPR genome editing tools to identify novel factors involved in preventing mutagenic alt-EJ in lymphocytes.

These results will help elucidate how lymphocytes regulate the choice of DNA repair pathways to resolve physiological DNA breaks correctly. Defects in these protective mechanisms contribute to genome instability, such as mutations and translocations, that can drive lymphoma progression. This study will help expand our fundamental knowledge about the regulation of antigen receptor gene recombination and the impact of mutagenic DNA repair activity during lymphocyte development.

Career Development Program
The oncogenic role and underlying mechanism of TET1 in acute myeloid leukemia United States -

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. Thus, it is urgent to better understand the mechanisms underlying the pathogenesis of AML, and on the basis of such understanding, to develop novel therapies with higher efficacy and minimal side effects to treat AML. The properties of cancer are often determined by the proteins that are expressed from information provided by the genes in the cell. Expression of information from genes is regulated in part by chemical modifications of the DNA in the gene, in a process called “epigenetic regulation.” One such modification is called methylation. A family of proteins involved in epigenetic regulation are the 3 TET proteins, which ultimately affect the methylation status of critical genes. TET proteins are traditionally thought to be negative regulators of tumor growth. However, in contrast to this tumor suppressive role, we recently reported that TET1 is highly expressed in certain subtypes of AML, suggesting an opposite role for TET1, the promotion of tumor growth.

We are currently studying the mechanisms by which TET1 promotes tumorigenesis and how we might use this information to develop a novel approach to treat AML. Some central questions are to understand how tumors develop, how they are maintained after they develop, and how the leukemia stem cells provide a reservoir for continued tumor development in a patient. Therefore, we seek to understand the role of TET1 in both the development and maintenance of the AML types that overexpress TET1, and the role of TET1 in the leukemic stem cells. In addition, we are studying the critical target genes that are affected by TET1, which will provide further insight into the role of TET1 in AML. Lastly, we are examining ways to therapeutically target TET1 using mouse models of AML. The success of our studies will provide novel insights into our understanding of the critical role of TET1 in AML and may also lead to the development of novel and more effective therapeutic approaches to treat the AML.

Career Development Program
Longitudinal functional genomics in mantle cell lymphoma therapy and drug resistance United States -

Despite recent therapeutic advances, mantle cell lymphoma (MCL) remains incurable due to its development of drug resistance. With each successive treatment failure comes a more rapidly proliferating disease and fewer treatment options. Our long-term goal is to develop superior therapies for MCL that are effective, well tolerated, durable, and can be individualized for patient treatment. The immediate objectives of our study are (1) to define the genomic basis and molecular mechanisms for drug resistance in MCL via clinical trials of targeted agents and (2) to develop strategies that overcome drug resistance. Our approach goes from the laboratory bench to the patient bedside and back, leveraging our unique blend of scientific and clinical expertise, state-of-the-art technology and reagents developed at Weill Cornell Medicine and Ohio State University.

Before a healthy cell divides, it undergoes an orderly series of events that are governed by special regulatory proteins. In MCL cells, these proteins malfunction, leading to uncontrolled cell proliferation, which ultimately underlies drug resistance and disease progression. Our previous efforts to target one of these regulatory proteins in blood cancers paved the way for FDA approval of palbociclib, a novel drug for cancer treatment. In a separate effort to overcome drug resistance in MCL, we created a novel inhibitor of an enzyme that is dysregulated in many cancers, including MCL. Inhibiting this enzyme restores regulatory pathways and kills some resistant primary MCL cells. The planned project will build on these novel findings and employ three different approaches to develop new therapeutic strategies.

Project 1: To develop rational treatment strategies for new and recurrent MCL (Peter Martin, Kami Maddocks, Jia Ruan, John Leonard). We aim to develop regimens that are well tolerated and suited to patient stratification. To accomplish this goal, we will conduct a multicenter phase II clinical trial (the PALIBR trial) to define subgroups most likely to benefit from a combination of palocicilib with another key drug, ibrutinib. Results from this study, along with those from Projects 2 and 3, should reveal the mechanisms of drug resistance. We will also explore whether additional strategies can overcome immune-mediated resistance to certain drug combinations.

Project 2: To discover the genomic basis and mechanisms for drug resistance in MCL (Selina Chen-Kiang, Lewis Cantley, Olivier Elemento). Results from our phase I PALIBR trial support our hypothesis that altering the cell cycle leads to reprogramming MCL for a deeper, more durable clinical response. By studying the MCL genomes of individual patients in the phase II PALIBR trial over time together with Project 1, we will identify the mutations associated with drug-resistance.

Project 3: To target the epigenome in MCL (Jihye Paik and Robert Baiocchi). Our preliminary evidence suggests that non-genomic chromosomal changes (alterations of the “epigenome”) in MCL promote MCL proliferation and survival. Targeting epigenomic activities using our novel inhibitor may therefore circumvent drug resistance and reveal new therapeutic targets. We will thus characterize epigenomic mechanisms in MCL cells, in collaboration with investigators from Projects 1 and 2.

These innovative and timely projects will be supported by the Administrative Core and the extensive expertise of our Pathology Core (Giorgio Inghirami) and Genomics, Bioinformatics & Biostatistics Core (Olivier Elemento, Christopher Mason and Karla Ballman). We expect that the proposed study will shed new light on the genomic basis and mechanisms for drug resistance in MCL, discover novel resistance biomarkers and develop individualized therapies that overcome drug resistance in MCL – all of which have profound implications for the treatment of other blood cancers.

Mantle Cell Lymphoma Research Initiative
Oncogenic RAS induced mitochondrial fission is requisite to cellular transformation, and reveals a novel chemotherapeutic target to treat leukemia. United States -

Figuring out the causes of leukemia and identifying new treatments for this tragic disease are major struggles for researchers and clinicians worldwide. While decades of research have revealed the complexities of leukemia, our understanding of the fundamental driving forces behind this disease and its treatment remain essentially the same. Basically, cancers of the blood arise when healthy cells acquire changes to their essential building blocks (like DNA & protein), which result in enhanced lifespan and tremendous difficulty in killing these cancer cells with current drugs. Over the years, my lab discovered several new and unexpected events within cancer cells that directly control their survival and responses to almost all chemotherapeutic drugs prescribed by clinicians. In this LLS Scholar’s Application, we propose to expand upon our recent breakthrough discoveries and directly apply our unexpected insights to gain a new and powerful perspective on how leukemia develops and may be treated. At the center of our study is a protein named DRP1, which was identified in my lab as a new driving force in cancer. Here, our strategy is to investigate how DRP1 governs leukemia cell survival (Aim 1) and chemotherapeutic responses (Aim 2) using the most advanced experimental design and model systems, including DNA and cells kindly provided to us by leukemia patients in our cancer institute. We will also generate clear evidence that DRP1 is a valid target to kill leukemia cells.

Career Development Program
Development Of Pharmacologic Agents To Target Leukemia Stem Cells United States -

Most patients with acute myeloid leukemia (aml) die from the disease despite achieving initial remission upon treatment. emerging evidence shows that recurrence of the disease results from the activity of leukemia stem cells (lscs), which are capable of self-renewal, proliferation and differentiation into malignant blasts. lscs are resistant to current treatments and novel drugs killing leukemia stem cells are urgently needed. the polycomb group (pcg) proteins play an essential role in maintaining the self-renewing capacity of leukemic stem cells. bmi1 is a stem cell gene, which is a central component of the polycomb repressive complex 1 (prc1). in this project we propose to developing inhibitors of the prc1 activity as new pharmacologic agents targeting leukemia stem cells. To identify inhibitors of prc1 we have employed fragment-based drug discovery approach. subsequently, we improved potency of our inhibitors using medicinal chemistry. we developed compounds which block the activity of prc1 complex to ubiquitinate h2a in cells with low micromolar activities and block stem cell properties of leukemia cells. in this project we propose to develop more potent inhibitors of prc1 activity with optimized drug-like properties for testing in animal models of leukemia. our studies will explore a new approach to target leukemia stem cells and may lead to development of novel pharmacologic agents for acute leukemia.

Career Development Program
Validation of Critical 1q21 Vulnerabilities in multiple myeloma United States -

Multiple myeloma (MM) is a cancer of a type of immune cells called plasma cells, which are mature B cells that constitute the second most commonly diagnosed blood-related tumor. New drugs introduced in recent years have doubled survival in standard-risk patients; however, high-risk patients survive only 2-3 years. While 20% of newly diagnosed patients are high-risk, this number increases dramatically as patients relapse. The vast majority of patients relapse, and average survival is a dismal 9 months.

Genes are contained within large structures of proteins and DNA called chromosomes. Chromosomes often undergo changes that are associated with cancer. One change is the amplification of chromosomal regions, which increases the levels of certain oncogenic proteins, thereby contributing to the formation and maintenance of the cancer. A region of one chromosome called 1q21 is amplified in some MM patients, and it defines one of the most high-risk MM subtypes associated with resistance to therapy and a poor prognosis. Using a genetic screen, I have identified 5 genes within 1q21 that are likely the most essential genes for MM pathophysiology. One gene, called interleukin enhancer binding factor 2 (ILF2), makes MM cells less responsive to chemotherapy when it is present in extra copies, which may explain why 1q21 MM patients benefit less from chemotherapy than non–1q21 MM patients. I am currently extending these studies to the other 4 candidate genes by reducing their expression in MM cells to see if that reduces MM survival, and I will further see if any effect can be enhanced by the simultaneous application of MM drugs. I will also overexpress these genes in mouse models to determine their role in tumor formation and to see if they collaborate with other oncogenes to form MM. Lastly, I am partnering with IONIS Pharmaceuticals to develop a novel therapeutic approach using molecules called antisense oligonucleotides. These molecules are expected to reduce the level of ILF2, and I will functionally validate their effectiveness, both alone and in combination with chemotherapy, in inhibiting the growth of MM cells in preclinical mouse models of high-risk MM.

This research will expand our understanding of the contribution of 1q21 genes to MM development and will inform the development of new approaches to improve the outcomes of MM patients who have high-risk disease that is not responsive to current therapeutic agents.

Career Development Program
Constellation Pharmaceuticals United States - Therapy Acceleration Program
Overcoming ibrutinib resistance in mantle cell lymphoma United States -

Mantle cell lymphoma (MCL) is a deadly blood cancer that has no cure. About 4,000 patients in the U.S. are diagnosed with MCL each year. Normal B cells are white blood cells that help fight infection, but in MCL, B cells no longer perform their function but grow unchecked. Chemotherapy is the main treatment for MCL, but is not curative. Moreover, because most patients are older, they are often unable to tolerate the harsh side effects of chemotherapy drugs. Our team’s goal is to develop new targeted drugs for MCL that are more tolerable and effective than currently available treatments.
We recently learned that lymphoma B cells rely on certain proteins they carry on their surface, most importantly the B-cell receptor (BCR), to survive. Ibrutinib is a targeted drug that blocks the BCR and shows promise as a new therapy for MCL. While ibrutinib can prolong survival, a third of patients do not respond to ibrutinib while most others relapse within two years. This is because a subset of the lymphoma cells may have mutations in them that allow them to survive even in the presence of ibrutinib. While ibrutinib resistance in other cancers is often due to specific mutations in the BCR, the mechanism of resistance in MCL appears different and more complicated. There is evidence that one potential mode of ibrutinib resistance in MCL is through the signaling protein called NFB. NFB is of particular interest because there are drugs in development that can degrade NFB. Using such drugs would deprive ibrutinib-resistant MCL cells from this escape mechanism.
The goal of this research is to explore how MCL cells become resistant to ibrutinib and other similar drugs, and to seek ways to overcome this resistance. We have access to a unique combination of research tools, systems and expertise that allow us to study these questions, including laboratory models that mimic the tumor environment and samples donated by MCL patients. We will test lymphoma cells from patients obtained at different stages of ibrutinib therapy, including when MCL relapses, to determine the factors that ensure tumor survival. We will also collect patients’ plasma and use new tools to study important tumor genetics to better understand the disease and its response to therapy. Using a panel of drugs, we will explore whether new approaches we developed to target NFB will overcome ibrutinib resistance in the laboratory and in a clinical trial of MCL patients. Ultimately, this project will help us understand how ibrutinib resistance develops in MCL and may lead to new targeted therapies for MCL that are more tolerable and effective than chemotherapy.

Career Development Program
Novel approach to thwart MYC in B-cell neoplasia by selective targeting cyclin-dependent kinase 9 United States -

Diffuse large B-cell lymphoma (DLBCL), a disease of the lymph nodes, is the most common subtype of non-Hodgkin lymphoma accounting for >10,000 deaths in the United States annually. While “targeted therapy” has made significant progress in treatment of blood cancers, we continue to treat DLBCL with standard chemotherapy regimens, which are associated with significant side effects and high rate of failure. When DLBCL recurs after initial therapy, it often becomes incurable with chemotherapy. The novel class of agents called inhibitors of cyclin-dependent kinases (or “CDK inhibitors”) has shown promise in therapy of cancer. Cyclin-dependent kinases are proteins which exist in cells under normal conditions. A whole range of them exist and ensure that cells can make all the necessary components for their survival, as well as reproduce. Cancer cells co-opt CDKs in their unlimited growth, and thus CDKs are attractive targets in cancer therapy. However, drugs which inhibit multiple CDKs are toxic, probably because they inhibit he function of multiple CDKs in normal (non-cancerous) cells. By contrast, emergent selective CDKs which target only one-two specific proteins hold promise to be more efficacious and have fewer adverse events. Here we propose to study how selective CDK inhibitors work in cancer, specifically focusing on inhibition of CDK9. Our preliminary experiments suggest that selective CDK9 inhibition stems lymphoma growth by disrupting the function of another protein, called MYC. MYC regulates synthesis of many cellular constituents which ultimately ensure tumor survival and growth. MYC levels are high in DLBCL and it contributes to therapy resistance. Here we will study the effect of CDK inhibitors on MYC function. We will determine how MYC function is affected, and whether MYC disruption is the dominant mechanism of how cells die in response to CDK inhibitors. We will also search for drug partners which will make CDK inhibitors more toxic specifically to tumor cells. Together, this study will help develop novel targeted therapies with the goal of eradicating DLBCL.

Translational Research Program
Novel Combination Immunotherapies for High Risk Hodgkin's Lymphoma United States -

Hodgkin’s lymphoma (HL), a type of blood cancer is largely curable but with significant long-term side effects. Moreover 10-20% of patients are resistant to treatment and difficult to cure. HL is unique that the tumor cells are surrounded by an inhibitory environment that makes the immune system dysfunctional and allows evasion from an effective anti-tumor response. Understanding this environment may provide insight into how we can spur the immune system to attack HL cells effectively. Like many cancer cells, HL cells escape the immune attack by expressing a protein called PD1 or PDL-1, which is is normally expressed by healthy T cells to prevent them from attacking healthy cells. PD1-inhbitors are drugs that can bind to the PD-1/PDL-1 on lymphoma cells thereby releasing the “brakes” on the immune system to mount a strong attack on the HL cells. The lymphoma cells also secrete certain chemicals like TGFβ which incapacitates the immune cells to kill the lymphoma cells effectively. The goal of this project is to understand how we can change the tumor microenvironment sufficiently to unleash pre-existing anti-tumor immune responses and allow more successful incorporation of killer- T cells. We will determine if PD1 inhibitors when given in combination with the administration of a novel cancer killing T-cell therapy will produce long-lasting cures in patients with high risk HL with less side effects than conventional chemotherapy. Finally, we will continue to improve the efficacy of our combination immunotherapy approach by also engineering the cancer killing T cells to become resistant to the inhibitory chemical TGFβ which is released by the HL tumor cells and has devastating effects on T cell function in vivo. In the laboratory we will determine if these engineered T cells have enhanced killing ability compared to the non engineered T cells.

Translational Research Program
Optimizing ibrutinib combination strategies to achieve minimal residual disease negativity in CLL United States -

Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable for most patients. Only about 20% of previously untreated patients will achieve the deepest possible remission with the conventional chemoimmunotherapy regimen Fludarbine, Cyclophosphamide, Rituximab (FCR). Remission rates are even lower in patients whose CLL has come back after completing initial therapy when they are treated with a second therapy such as the immunotherapy drug obinutuzumab.

Ibrutinib was recently approved to treat CLL. Although ibrutinib can be effective on its own, it rarely leads to deep remissions, thereby requiring indefinite therapy. We hypothesized that combining ibrutinib with either FCR (for previously untreated patients) or obinutuzumab (for patients whose CLL has come back) would be an effective treatment strategy. We are evaluating this in two clinical trials. In the first trial, previously untreated CLL patients will be treated for up to 6 months with ibrutinib plus FCR followed by 2 years of ibrutinib alone, at which point patients who are in a deep remission stop ibrutinib and are observed. In the second trial, patients with relapsed CLL will receive 6 months of a combination of ibrutinib with obinuzutumab followed by ongoing treatment with ibrutinib alone.

Complementing the clinical trials will be laboratory studies to evaluate the effectiveness of the treatments. Though tumor cells are often thought of as sturdy, many have molecular attributes that makes them quite vulnerable to drug-induced cell death. Thus, we will use a functional test to determine how close a patient’s tumor cells are to dying. We believe that patients whose CLL cells are prone to die more easily will do better on these trials, whereas patients whose CLL cells are resistant to dying are less likely to do well. We are also using a new technology to sequence tumor DNA even in samples with very few cells. We will explore whether analyzing such samples from patients in remission may detect early mutations that would allow us to switch to a more effective therapy before a patient even has symptoms of a relapse.

Through these two clinical trials and the associated laboratory studies, we hope to optimize the combination of ibrutinib with chemotherapy and immunotherapy, with a goal of curing patients with drugs given for a finite period of time. Our findings may help oncologists to test patients prior to starting treatment to understand which treatment will likely be the most effective for that particular patient, which will bring us closer to our goal of personalizing treatment to maximize the chance of cure for patients with CLL.

Career Development Program
Improving targeted adoptive cell therapy of myeloma United States - Specialized Center of Research Program
Targeting the interaction of leukemia stem cells with their niche to treat myelofibrosis United States -

This project explores the connection between the niche – the area in the bone marrow where blood cells are formed – and the development of leukemia stem cells (LSCs) that give way to primary myelofibrosis (PMF). PMF is a stem cell-derived blood malignancy with the characteristics of too many cells in the blood and a large amount of scar tissue formation (fibrosis) in the bone marrow. PMF often progresses to an aggressive acute myeloid leukemia, causing high mortality. Strategies aimed at eradicating disease-causing LSCs are the key to cure the disease. Current treatment options for PMF are limited, and the only potential cure, stem cell transplantation, is often prohibitively toxic for most patients. Scientists reported in 2005 that recurrent mutations resulting in abnormal activation of the JAK-STAT pathway are drivers of PMF and other related diseases. This ground-breaking discovery led to the development of FDA-approved drugs targeting JAK2, such as ruxolitinib. However, these inhibitors only reduce some symptoms without significant impact on reducing LSCs or mutant blood cells. Thus, a deeper understanding of the pathogenesis of PMF will offer new opportunity to better treat the disease.

Blood-forming stem cells called hematopoietic stem cells (HSC), give rise to all mature blood cells. These cells are found in the bone marrow in a region called their “niche,” which is near the bone marrow vascular system. In the PMF diseased state, the fibrotic bone marrow niche is a critical component to PMF pathogenesis. We hypothesize that the abnormal bone marrow niche in PMF provides protection to disease-causing LSCs at the cost of the normal blood-forming HSCs. We further hypothesize that targeting the abnormal niche may help eliminate LSCs, preserve normal HSCs, and provide a potential cure to PMF. Our recent data show that bone marrow cells in the niche that express the Leptin receptor protein are the source of fibrosis via activation of a signaling network mediated by a protein called PDGFRa. We are elucidating the cellular and molecular mechanisms of how LSCs interact with the fibrotic niche in mouse models and how this negatively impacts normal HSCs. To explore the potential of translating our findings to the clinic, we will test whether targeting the PDGFRa pathway with a specific blocking antibody will lead to efficient elimination of LSCs. By having a deeper understanding of the interaction between LSCs and the niche, our strategy of targeting the diseased niche may provide novel therapeutics for PMF.

Career Development Program
Molecular Mechanisms Mediating the Role of Wild-type vs. Mutant IRF4 in Multiple Myeloma United States -

Plasma cells (PCs) are specialized blood cells that produce antibodies to fight infections. Multiple myeloma (MM) is a malignancy of PCs and it is estimated that over 30,000 new people will be diagnosed with this disease this year in the United States alone. Fortunately, over the last two decades, major progress has been made in MM treatment leading to improved outcomes. However, the disease is still considered to be incurable for the overwhelming majority of MM patients. Thus far, therapeutic progress achieved in MM has been attributed to medications that keep MM cells at bay by targeting molecular circuits critical to both normal and malignant PCs, but spare the overwhelming majority of normal cells from other tissues, allowing these medications to have generally manageable profile of side effects. This experience led us to hypothesize that the clinical benefit provided to MM patients by these recently developed PC-targeting medications may be extended if we identify additional molecular targets and circuits that are critical for MM cells, because of their PC biology. We have focused our efforts on gaining a deeper understanding of IRF4, a transcription factor that is preferentially critical for MM cells, compared to other non-PC malignancies or most other normal cell types. We are seeking to gain a more systematic characterization of the biology of IRF4. We will study how this gene interacts with other critical genes for PCs/MM cells to define the functional circuits that drive MM cells and their malignant behavior. We also seek to define the functional significance of a mutation that is observed in MM cells in a subgroup of patients who derive major clinical benefit from the drug pomalidomide. In addition, we seek to better understand how the interaction of the MM cells with the nonmalignant cells of the bone marrow may alter the role of wild-type or mutant IRF4 and their interactions with other MM-preferential essential genes. We anticipate that these studies will improve our understanding of the biology of MM and further our ability to create new therapies that can specifically target malignant cells from MM. We also hope that our work will establish a framework to dissect the biology and facilitate the therapeutic targeting of other transcription factors critical for blood cancers.

Career Development Program
Personalizing Medicine for AML Based on Functional Genomics United States - Therapy Acceleration Program
KDM6A mutation as an epigenetic driver of multiple myeloma United States -

DNA in the nucleus of cells is wrapped around proteins called histones to compact the DNA into a structure termed chromatin. Chromatin is malleable and can be opened or closed to regulate the expression of genes, and this occurs through the action of chromatin-modifying proteins. Chromatin structure is altered in cancer cells, and these alterations cause gene expression changes that contribute to the pathological properties of tumors. One chromatin-modifying protein that is frequently disrupted in cancer is KDM6A. KDM6A functions to open chromatin, providing other proteins that regulate gene expression with access. About 10% of multiple myeloma (MM) patients carry harmful mutations affecting KDM6A function, and my goal is to understand how loss of the KDM6A contributes to the development of MM.

KDM6A opens chromatin through the modification of chemical tags on the chromatin through a process called demethylation, which is the loss of methyl groups. Mutations that affect KDM6A demethylase activity are likely important in the pathology of MM. However, KDM6A is part of a large protein complex that interacts with and affects the behavior of the chromatin. Therefore, KDM6A may also function as a scaffolding protein that either recruits and/or retains other proteins in the complex, and this scaffolding function may also important for the pathology of MM. My research seeks to characterize the role of KDM6A in MM and to clarify the roles that the demethylating and scaffolding activities play in MM.

I am using DNA and RNA sequencing technologies to determine how KDM6A loss changes the configuration of chromatin and how this correlates with changes in gene expression. In addition, I am using an advanced gene editing technique known as CRISPR to eliminate KDM6A expression in MM cells in order to identify changes in gene expression associated with KDM6A loss. I will also use this technique to eliminate the demethylase activity of KDM6A to isolate the scaffolding activity in order to determine its role. I am developing a novel mouse model that mimics MM in patients and testing how loss of KDM6A may accelerate MM development and change the characteristics of the disease. The resources I am generating will provide important new tools for understanding how MM develops. Importantly, the completion of this study will yield new insights that are expected to lead to the development of more effective MM therapies that directly target mechanisms of chromatin structure regulation.

Career Development Program
Interaction of RUNX1 and the cohesin complex in megakaryocyte development and myeloid disease United States -

Inherited mutations in the RUNX1 gene cause abnormal platelets and a predisposition to the development of acute myeloid leukemia. Similarly, patients with RUNX1 mutations that are acquired during adult life have an analogous platelet abnormality and predisposition to acute myeloid leukemia. In order for patients with RUNX1 mutations to progress to acute leukemia, additional mutations need to be acquired. We have found that mutations in the STAG2 gene can cooperate with RUNX1 mutations, likely leading both to the development of worsening platelet abnormalities and progression to overt malignancy.
Our goals are to answer two questions: (1) Does loss of RUNX1 and STAG2 lead to abnormal platelet precursor cell formation, with resultant platelet dysfunction and increased risk of bleeding? If so, we would like to pro-actively identify these patients so that appropriate measures can be taken to mitigate their risk of bleeding, particularly when undergoing diagnostic procedures. (2) Can we model abnormal platelet precursor cell function and AML disease progression in human and mouse bone marrow stem and progenitor cells? If so, this would provide functional evidence that loss of normal function of STAG2 and RUNX1 leads to abnormal platelet precursor cell formation and/or development of leukemia. In addition, these models would facilitate studies of novel therapeutic agents, which are desperately needed for this patient population.
Treatment options for patients with RUNX1 mutations are limited, and better understanding of the mechanisms by which leukemia causing genes contribute to leukemia development in patients will inform the design of urgently needed new treatments. We propose to develop models and investigate therapeutic approaches that have the potential to benefit individuals with RUNX1 mutations.

Translational Research Program
A JAK2 V617F -directed T cell receptor transgenic T cell immunotherapy for the treatment of myeloproliferative neoplasms United States - Special Grants
Protection of proliferating B lymphocytes from transformation by a c-MYC-induced tumor suppressive program United States -

Lymphomas and leukemias are caused by uncontrolled proliferation of lymphocytes due to accumulating errors in the genome. However, cell proliferation is also an important biological activity across many different tissues and cell types. Specifically, proliferation of lymphocytes is essential for the immune responses that protect individuals from invading pathogens. Normal lymphocytes are able to proliferate even quicker than cancer cells in response to infection for extended periods of time. In the course of normal immune system development, lymphocytes also mutate their own genome to establish better immunity to infection. However, it is unknown how lymphocytes undergo normal proliferation and mutation events without increasing the risk of cancers. We hypothesize that lymphocytes engage an unidentified, unique mechanism to minimize the risk of cancers while facing the demand to proliferate and mutate their genome during their normal functional activities. To explore this hypothesis, we have studied sets of genes that are activated by a protein named c-Myc, which has been causally linked to many cancers in humans and other organisms. Since c-Myc is also essential for lymphocyte proliferation in response to infection, c-Myc may activate an unidentified pathway that is required to protect normally proliferating lymphocytes from becoming cancerous. Accordingly, mutations of genes in the pathway may increase the risk of lymphomas or leukemias. Indeed, we have identified a gene that is activated by c-Myc in proliferating lymphocytes and is necessary to suppress blood cancer development in an animal model. Moreover, a few mutations of this gene have been found in leukemic cells from human patients. Starting with these discoveries, we will define the molecular mechanisms by which this c-Myc initiated regulatory pathway suppresses leukemia and lymphoma. The knowledge from this study will contribute to the development of improved strategies for the risk assessment and diagnosis of leukemias and lymphomas and also to inventing new therapeutic approaches.

Career Development Program
Randomized Trial of a Sexual Dysfunction Intervention for Hematopoietic Stem Cell Transplant Survivors United States -

The use of Hematopoietic stem cell transplantation (HCT) as a potentially curative treatment strategy for blood cancer has increased over the last decade with more than 20,000 transplants performed in the United States each year. Sexual health problems are common among the rapidly increasing number of HCT survivors, and such problems negatively impact their quality of life, mood, and intimate relationships. Over half of HCT survivors are under 45 years of age – much younger on average than survivors of most other cancers. Therefore, this group experiences more severe sexual health concerns, resulting in a wider range of biologic, interpersonal, psychological, and social problems. Despite this, no interventions have been developed to address sexual health problems in this population. Therefore, there is a critical need to develop and test innovative interventions that comprehensively address the multiple factors affecting sexual function in HCT survivors.

We recently completed a small study of a multimodal intervention to address sexual health problems in HCT survivors. Study participants attended monthly visits with trained study nurse practitioners who 1) performed in-depth assessments of the causes of the patients’ sexual health problems; 2) educated, normalized, and empowered patients to address their sexual health issues; and 3) implemented therapeutic interventions targeting the patients’ individual sexual health needs. We demonstrated that the intervention was feasible and led to significant improvements in patients’ satisfaction and interest in sex and sexual health and function. Notably, 6 of 10 patients who were not sexually active prior to the study became sexually active afterwards. Patients also reported clinically significant improvement in their quality of life.

The goal of this project is to conduct a randomized clinical trial of 230 HCT survivors to rigorously demonstrate the efficacy of the multimodal intervention to address sexual health problems in HCT survivors, enhancing patients’ sexual function and their interest in sexual activity. We will also test whether the intervention improves patients’ quality of life and reduces their depression and anxiety symptoms. In addition to its potential to improve HCT survivorship, this intervention can easily be adapted and utilized to address the sexual health concerns of other cancer survivors. Therefore, this work represents an important opportunity to improve the quality of life and survivorship care of patients with blood cancer.

Career Development Program
Epigenetic Regulation of WM Biology United States - Special Grants
Biomarker-based strategies for personalized therapy of ALK-negative anaplastic large cell lymphoma United States -

T-cell lymphomas are a group of cancers that invade lymph nodes and other organs throughout the body, and are often fatal. The goal of this proposal is to improve the survival and quality of life for patients with ALK-negative anaplastic large cell lymphoma (ALCL), a common subtype of T-cell lymphoma with generally aggressive clinical behavior. We have set out to determine in the laboratory the differences that exist between the ALK-negative ALCL tumors in most patients, who often die soon after diagnosis, and the tumors in occasional patients that survive for many years. Specifically, our team discovered that two-thirds of patients died within 5 years if their tumor had a protein called DUSP22, whereas 9 out 10 patients survived if DUSP22 was absent. By understanding how these two types of tumor differ, we believe we can develop treatments that convert aggressive tumors into ones that grow more slowly, or possibly not at all. Our findings so far suggest that the main difference is that the patient’s immune system can efficiently recognize and attack ALCL tumors when the DUSP22 protein is absent. However, three significant areas of understanding are lacking before we can use these findings to benefit patients: (1) The way that the DUSP22 protein affects the immune response against the tumor remains unknown; (2) The best combination of drugs to improve this immune response has not been identified; and (3) There are no current tests that can be performed on tumor tissue to personalize the use of these drugs; that is, to choose the correct combination of drugs for each individual patient. We propose to address these issues in three Specific Aims. In Aim 1, we will determine the way the DUSP22 protein contributes to the development of ALK-negative ALCL tumors. We believe that DUSP22 is critical in whether or not the tumor displays antigens that are signals to the immune system to attack. We will determine this role in mice, which are ideal models for evaluating the immune response against tumors. In Aim 2, we will determine which combinations of drugs treat aggressive ALCLs most effectively. We believe the best combinations will be those that both enhance the immune response against the tumor and simultaneously block key growth-promoting proteins. We will perform these experiments in mice, which will allow us to safely test multiple combinations of drugs in different types of ALCL tumors. In Aim 3, we will develop a test that can be performed on tumor tissue to determine which drugs are most likely to be effective in a specific tumor. We believe that a combined test that separately evaluates four key features of the tumor will predict response to treatment in mice as well as identify which patients with ALK-negative ALCL have the highest risk of death. We expect that these studies will lead directly to a clinical trial, where patients will be treated based on which drugs are most likely to be effective against their individual tumor.

Translational Research Program
TOP2 Poisons: Old Drugs, New Mechanisms and Rational MLL-R AML Epigenetic Targeting Combinations United States -

Pediatric leukemias with a DNA abnormality called a MLL translocation in which the MLL gene breaks and fuses to a gene on a different chromosome have a poor prognosis. The resulting fusion gene creates a fusion protein that causes disturbances in gene transcription, the process whereby a gene is read into a message. This research will develop a novel combination treatment for MLL-R AML to target the abnormal gene transcription. The combination includes an old well-known class of chemotherapy drugs that we discovered have a new mechanism of action, and a new molecularly targeted agent. The chemotherapy drugs called “TOP2 poisons” have been used for decades. They are highly effective at killing cancer cells because they cause disturbances in DNA cutting proteins (topoisomerase II enzymes) and create damage to the DNA by converting the cell’s own cutting proteins into cellular toxins. However, TOP2 poison chemotherapies were first developed before a technology was available to know where in the DNA in cells the cutting proteins make their cuts, or how the TOP2 poisons affect locations of the cut sites. We invented a dynamic high throughput sequencing technology to detect exactly where in the bases in the DNA all along all of the genes in the genome cuts are actively being made by the TOP2 enzymes. What we discovered using this technology is that: 1) the cut sites that are made by the one of two types of these DNA cutting proteins (TOP2A) start in the middle of the gene and increase progressively along its length to relieve twisting and turning of the DNA as it is being transcribed into a message; 2) not unexpectedly, long genes have more cuts; 3) all cut genes whether long or short have higher levels of transcription; 4) the cut sites co-localize with various signals along the gene of active gene transcription; 5) TOP2 poisons cause the TOP2 cut sites to shift to the starting end of the gene where a modification to a protein called a histone is also located that causes activation of transcription. The abnormal transcription in MLL-R leukemias depends on abnormalities in the patterns of this histone modification. The modification is created by an enzyme called DOT1L. Others recently discovered that DOT1L is a therapeutic target and that a drug that inhibits DOT1L has potent activity against MLL-R AML. Therefore, our discovery that TOP2 poisons have a new mechanism of action to alter the location of the TOP2 cut sites, and the interrelated discovery made by others that the DOT1L inhibitor has anti-leukemia activity together give strong rationale to develop new TOP2 poison/DOT1L inhibitor combinations to target abnormal gene transcription in MLL-R AML. This very novel strategy has great likelihood to advance rapidly to clinical translation because, as molecularly targeted agents find their roles in treatment, they are combined with chemotherapies and the DOT1L inhibitor as a single agent is already in the clinic.

Translational Research Program