Iannis Aifantis - US grants

DESCRIPTION (provided by applicant): The pre-T Cell Receptor (pre-TCR) is a receptor expressed early during the development of T cells in the thymus. It's signaling is essential for the maturation of T cells as it induces proliferation and differentiation through downstream signaling pathways that involve the activation of key transcription factors. One of the main functions of the pre-TCR is the induction of cell survival as cells unable to express the receptor die by programmed cell death. However, the mechanisms of both apoptotic death of pre-TCR non-expressing thymocytes and the pre-TCR mediated survival are poorly understood. In this proposal we attempt to identify the molecular mechanisms responsible for the induction of survival and study the role of well-characterized death inducers in the apoptotic cascades of pre-T cells. To achieve that we a) analyze biochemically the constituents of the apoptotic pathway in these cells, b) study thymic phenotypes of mice deficient for several genes - key components of the apoptotic machinery, and c) characterize the nature of pre-TCR derived antiapoptotic signals and in particular the expression pattern and the function of the pre-TCR-induced prosurvival factor A1 and the pre-TCR-suppressed pro-apoptotic Bim. We are also linking constitutive pre-TCR signaling, that leads to deregulated survival and proliferation, to the induction of T cell acute lymphoblastic leukemia (T-ALL). To study the role of the pre-TCR in thymocytes transformation we will a) address the nature of interactions of oncogenes (Notch) with pre-TCR signaling, b) study the significance of pre-TCR gene-targets in the triggering of transformation and c) attempt to identify additional genetic events contributing in the establisment of the disease. The proposed experiments will help to identify the molecular mechanisms regulating pre-T cell survival and death and pinpoint pre-TCR-induced signaling pathways responsible for the malignant transformation of thymocytes.

The generation of mature and functional T cells is the endpoint of a long process that starts in the bone marrow or the fetal liver where hematopoietic stem cells (HSCs) reside. It requires orchestrated inputs from several signaling pathways. The identification of signaling cascades which affect the differentiation of lymphoid progenitors and HSCs is of unique importance for the understanding of the molecular mechanisms of hematopoiesis but also for the development of novel methods to culture, expand, manipulate and transplant adult HSCs to immunodeficient patients. Here, we identify a novel signaling pathway, the Hedgehog (Hh) signaling network, as a master positive regulator of T cell development and hematopoiesis in mammals. Hh is a secreted protein family that was originally described as a major regulator of cell fate decision and body segment polarity. In this proposal we demonstrate that inhibition of Hh signaling (using a conditional knock-out of Smoothened, the Hh signal transducer) abrogates the development of lymphoid progenitors in the thymus. We also show both in vitro and in vivo that Hh signaling is essential for the homeostasis and the differentiation of bone marrow HSCs. These data lead us to hypothesize that Hh signaling controls differentiation of intra- and extra-thymic hematopoietic progenitors and that the Hh signaling network is a novel master regulator of mammalian hematopoiesis. To test this hypothesis we will study the mechanisms of Hh function in the thymus and in the bone marrow where stem cells are generated and develop. In the thymus we will study the mechanisms of Smoothened (Smo) mediated signal transduction and the role of the Gli transcription factors (targets of Hh signaling) in thymocyte development. We will identify genes, targets of the Gli proteins that can control T cell development and we will address the role of Hh in mature T cell differentiation and function. In the bone marrow we will study in detail the effect of Smoothened deletion in the differentiation of adult hematopoietic stem cells and committed lymphoid and myeloid progenitors. We will complement these experiments with the study of novel Hh activating mouse mutants in which we will define the exact role of the Gli factors in the regulation of mammalian lymphopoiesis and hematopoiesis.

DESCRIPTION (provided by applicant): T cell acute lymphoblastic leukemia (T-ALL) is a disease induced by transformation of hematopoietic stem cells/progenitors. It afflicts mainly children and adolescents. Although treatment outcome in T-ALL has improved in recent years, patients with relapsed disease continue to have dismal prognosis. It is thus very important to identify and study the molecular pathways that control both induction of transformation and treatment resistance in this particular type of leukemia. Recent evidence demonstrated that activating mutations in the Notch1 oncogene are the trigger for cell transformation in the majority of T-ALL patients. The majority of the T-ALL Notch1 mutations are truncating a portion of the Notch1 protein called the PEST domain. Although this domain has been previously suggested to be important for Notch1 proteasome-mediated degradation, the exact mechanism of regulation of Notch1 stability and its role in T cell transformation and human T-ALL is currently unknown. We present here a large amount of experimental evidence that identifies the E3 ubiquitin ligase Fbw7 as an important regulator of Notch1 protein stability, through its interaction with a Threonine-centered degron sequence situated in the Notch1 PEST domain. We also demonstrate that T-ALL- inducing Notch1 mutations target this Fbw7 degron and that the Fbw7 gene itself is mutated and inactivated in a significant portion of human T-ALL. These and other preliminary observations presented in our application make us hypothesize that Fbw7 is an important novel tumor suppressor in T cell leukemia and its inactivation can trigger T cell transformation due to the stabilization of essential Fbw7 substrates. In this application we initially test the importance of the Notch1: Fbw7 interaction in leukemia by generating "knock-in" mice that lack the essential degron on the Notch1 PEST domain. Moreover, to directly prove that Fbw7 is a tumor suppressor in T-ALL we generate conditional, T-cell specific Fbw7 knock-out mice and study the effect of the deficiency in T cell transformation and development. Moreover, using these genetic tools we identify the essential downstream targets of Fbw7 in T cell transformation. Finally, we study both in vitro and in vivo the effect of Fbw7 mutations on gamma-secretase inhibitor treatment of T-ALL. PUBLIC HEALTH RELEVANCE: T cell acute lymphoblastic leukemia (T-ALL) is a disease induced by malignant transformation of T lymphocytes. Although treatment outcome of T-ALL has improved in recent years, patients with relapsed disease continue to have dismal prognosis. In an attempt to identify and study the molecular pathways that control T cell transformation we study here the interplay between Notch1, an oncogene mutated in the majority of T- ALL patients, and Fbw7, a ubiquitin ligase that can ubiquitinate and degrade nuclear, oncogenic Notch1.

DESCRIPTION (provided by applicant): All mature blood cells arise from a rare and specialized population, the hematopoietic stem cells (HSCs), which exist mostly in a quiescent state. Cell division of HSCs results in both their proliferation and progressive differentiation into increasingly lineage-restricted mature blood cells, as well as maintenance of a small pool of HSCs that do not differentiate, but rather carry out hematopoiesis throughout the life of an organism. Due to the significance of HSC function, the elucidation of the signals that govern the balance between HSC self-renewal and differentiation is a paramount task. Interestingly, several studies have suggested an intimate balance between physiological hematopoiesis and induction of hematopoietic malignancy (leukemia) controlled by aberrant signaling that is able to transform HSC and progenitor cells. In agreement with this notion, we have recently identified the E3 ubiquitin ligase Fbw7 as an important tumor suppressor in acute lymphocytic leukemia (ALL). Fbw7 inactivating mutations are found in a large fraction of ALL patients and induce transformation due to the aberrant stability of several important oncogenes, including Notch1 and c-Myc. We have addressed the role of Fbw7 in HSC function using a novel conditional knock-out mouse model. We have found that deletion of Fbw7 specifically and rapidly affected the HSC compartment in a cell-autonomous manner. Fbw7-/- HSCs showed defective maintenance of quiescence, leading to impaired self-renewal and a severe loss of competitive repopulating capacity. Furthermore, genome-wide transcriptome studies of Fbw7-/- HSC indicated that Fbw7 regulates a global transcriptional signature associated with the quiescent, self-renewing HSC phenotype. In this application we: a) Identify HSC-specific protein substrates targeted by Fbw7 and playing important roles in HSC differentiation and function, b) address the universal function of Fbw7 in stem cell self-renewal by studying its role in embryonic stem cell function and c) study the effects of ALL Fbw7 missense mutations in hematopoiesis and HSC self- renewal.

DESCRIPTION (provided by applicant): Acute lymphoblastic leukemia (ALL) is a disease induced by the transformation of blood (hematopoietic) stem cells and progenitors. It mainly afflicts children and adolescents. Although treatment outcome in ALL has improved in recent years, patients with relapsed disease continue to have dismal prognosis. It is thus very important to understand the molecular pathways that control both induction of transformation and treatment resistance in this cancer type. Recent studies demonstrated that the majority of T-cell ALL (T-ALL) patients show signs of hyperactivation of the Notch signaling pathway. Also, the leukemic cells appear to be addicted to Notch signaling. Excessive Notch activity is either due to gain-of-function Notch1 mutations or loss-of-function mutations of the ubiquitin ligase Fbw7 (SEL- 10) which is able to bind and induce the degradation of nuclear oncogenic Notch. Identical, inactivating, Fbw7 mutations were also recently found in a significant fraction (approximately 10%) of several types of solid tumors including breast, colon and gastric carcinomas suggesting that Fbw7 is a key tumor suppressor. To prove this hypothesis, we have conditionally deleted the Fbw7 locus and shown that hematopoietic progenitor-specific loss of Fbw7 predisposes the animals to T cell leukemia and lymphoma due to the stabilization of Notch1 and pathway hyperactivation. All these observations underline the importance of the Notch-Fbw7 interaction in cancer, and more specifically in leukemia. They also suggest that regulation of this interaction could prove to be a powerful therapeutic tool for the treatment of the disease. In this application we propose experiments that will identify novel regulators of the Notch-Fbw7 regulatory interaction and test their ability to modulate Notch pathway activation in leukemia using animal models of the disease. To achieve that, we will use a powerful RNAi-based in vivo screen using the C.elegans as our model organism. Genes, putative regulators of the Notch:Fbw7 interaction will be then tested in mammalian in vitro and in vivo screens as well as mouse models of T-ALL. PUBLIC HEALTH RELEVANCE: The interaction between the oncogene Notch1 and the tumor suppressor Fbw7 is crucial for the induction of blood malignancies and solid tumors. In this application we are identifying genes that can regulate the Notch:Fbw7 interaction using a powerful C.elegans RNAi-based screen. Genes identified in this screen are further tested in both human cancer and animal models of Notch-induced disease.

DESCRIPTION (provided by applicant): T cell acute lymphoblastic leukemia (T-ALL) is a disease induced by the transformation of T cell progenitors. It mainly afflicts children and adolescents. Although treatment outcome in T-ALL has improved in recent years, patients with relapsed disease continue to have dismal prognosis despite the use of protocols involving hematopoietic stem cell transplantation. T-ALL patients present at diagnosis with elevated white cell counts, hepatosplenomegaly, and are at elevated risk for central nervous system (CNS) relapse. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The dramatic increase in survival is thought to be worth the significant side effects associated with this therapy. Such complications include secondary tumors, neurocognitive deficits, endocrine disorders and growth impairment. Little is known about the mechanism of leukemic cell infiltration of the CNS despite its clinical significance. Here, we show using T-ALL animal modeling and gene-expression profiling that the chemokine receptor CCR7 is the essential adhesion signal required for the targeting of leukemic T-cells into the CNS. CCR7 gene expression is controlled by the activity of the T- ALL oncogene Notch1, the most central oncogene in this disease, and is expressed in human tumors carrying Notch1 activating mutations. Silencing of either CCR7 or its chemokine ligands in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, CNS targeting by human T-ALL cells depends on their ability to express CCR7. All these observations made us hypothesize that the CCR7 chemokine receptor and its ligands are potent regulator of leukemic cell migration/tissue infiltration in response to oncogenic Notch1 signaling. This hypothesis is tested in this application. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS targeted therapy, thus reducing short- and long-term complications of therapy. PUBLIC HEALTH RELEVANCE: T cell acute lymphoblastic leukemia (T-ALL) is a deadly blood tumor that afflicts mainly children. One of the devastating manifestations of the disease is infiltration of leukemic T cells in to the central nervous system (CNS) that can cause neural tissue damage and paralysis. In this application we study the mechanism of CNS entry and propose novel targets for future therapy.

DESCRIPTION (provided by applicant): Currently, the standard treatment of T cell acute lymphoblastic leukemia (T-ALL) patients is intensive chemotherapy with almost 70% 5-year event free survival for pediatric and 50% for adult T-ALL patients. Moreover, such non-targeted therapies fail to address high-risk T-ALL subtypes. Unfortunately, recent attempts to introduce targeted therapies were plagued by both toxicity and resistance, further underlining the urgency for new treatments that also deal with leukemia-initiating cells (LIC) leading to complete disease remission. During the last funding period we were able to make important steps towards the understanding of the role of the ubiquitin system in acute lymphoblastic leukemia (ALL). We were able to identify somatic mutations on Fbw7, a ubiquitin ligase, in a large fraction of T cell ALL patients. We were able to show that Fbw7 can ubiquitinate and degrade NOTCH1 and MYC, two of the most important oncogenes in T-ALL. Using conditional Fbw7 alleles and were able to prove in vivo its role as a tumor suppressor. More recently we generated animals that expressed the exact Fbw7 mutations found in T-ALL patients and we were able to show that such missense mutations are able to directly expand leukemia initiating cells (LIC) but at the same time space normal hematopoiesis, providing an explanation on the selection of such mutations in this disease. Further analysis has shown that Fbw7 mutations expand LIC by aberrantly increasing the half-life of MYC. Moreover, conditional MYC deletions lead to LIC targeting and complete disease eradication opening the way for therapeutic targeting of T-ALL stem cells. Indeed, preliminary studies described here show that bromodomain and extra-terminal (BET) inhibitors can efficiently target MYC transcription and lead to suppression of growth in human T-ALL lines and primary cells. In this renewal application we would like to take advantage of all these significant developments and take our studies all the way from basic understanding of leukemia stem cell biology and the impact of FBW7 mutations to the identification of novel drugs that can target initiation and progression of this devastating tumor that frequently afflicts children and adolescents.

DESCRIPTION (provided by applicant): AML is a devastating blood tumor, the most common type of leukemia in adults, a disease that continues to have the lowest survival rate within leukemia. Nearly 45,000 people are diagnosed each year in the US, and the current 5-year survival frequency is only 24% with an almost 50% relapse rate after treatment. AML is a part of a wider family of myeloid neoplasms that include diverse but related diseases like myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and chronic myelomonocytic leukemia (CMML), a disease that frequently develops into AML. Currently, there are no targeted therapies for most of these diseases, including AML, making the study of the molecular mechanisms of their induction and progression of great significance. We have recently identified novel somatically acquired mutations inactivating the Notch signaling pathway in patients with CMML. Interestingly, these Notch mutations do not act in isolation but they co-occur with additional genomic hits, including mutations on the TET2, ASXL1, KRAS and JAK2 genes. Our published and preliminary data presented here demonstrate that: a) The Notch pathway is switched-off in myelo-monocytic leukemias, including AML, b) Inhibition of the pathway is achieved both by inactivating mutations and epigenetic silencing, c) Re-activation of the pathway can inhibit the growth of mouse CMML in vivo and human AML in vitro. In this application we: a) Assess the biological effects of Notch pathway re- activation in AML, b) Identify the molecular and epigenetic mode of pathway inhibition in human AML and c) Study co-operation of Notch pathway mutations with additional genomic lesions and their effect in AML initiation and progression. We strongly believe that these studies can lead to future targeted therapies of AML and related myeloid neoplasms, as re-activation of the Notch pathway can be achieved using both peptide and antibody agonist approaches.

? DESCRIPTION (provided by applicant): Recent efforts to functionally annotate the human genome have revealed that up to 75% of our DNA is transcriptionally active. Since a very small portion of our genome encodes proteins many have hypothesized that a portion of this pervasive transcription results in production of long non-coding RNA. By generating high depth RNA-Seq datasets integrated with chromatin features, our lab and others have revealed the presence of many thousands of previously un-annotated lncRNAs, which are dynamically expressed in response to various stimuli in diverse cellular contexts. Despite these compelling advances, the vast majority of putative lncRNAs have not been proven to be functionally important, although a small portion have clearly been shown to play key regulatory roles. Most importantly, very little is known on the biological role of lncRNAs in human cancer. We provide here, using a combination of deep transcriptome sequencing, high-resolution transcription factor occupancy mapping and chromatin interaction data, the first comprehensive identification and characterization of lncRNA expression and function in T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematologic malignancy driven by oncogenic NOTCH1 transcriptional activity. Moreover, we provide the first map of oncogene (NOTCH1)-targeted lncRNAs in this tumor and identify an individual lncRNA (called LUNAR1) that appears to be essential for tumor growth as it controls cytokine (IGF1) signaling. These studies suggest that: a) lncRNAs could be used as both biomarkers and therapy targets in human cancer (see AIM1), b) more efficient methods for large-scale inference and validation of lncRNA function are needed to fully understand their biological significance (see AIM2) and, c) LUNAR1 is one of the first lncRNAs that can control growth of acute leukemia and a potential therapeutic target (see AIM3). In this application we address in detail all these important issues and attempt to directly connect lncRNA deregulation to leukemia initiation and progression.

Melanoma is one of the deadliest forms of skin cancer and affects tens of thousands of people each year. Although novel targeted and immune therapies have been approved, they often work transiently with resistance eventually ensuing, or are accompanied by significant toxicities. The transcriptional program organized by melanoma drivers is poorly understood. Here we propose to dissect the melanoma metastasis-supportive transcriptional program organized by the transcription factor Heat Shock Factor 1 (HSF1). Although this pathway has been evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. Our preliminary data indicate that the ubiquitin ligase FBXW7 interacts with HSF1 through a conserved motif phosphorylated by GSK3? and ERK1. FBXW7 is either mutated or transcriptionally down-regulated in melanoma and HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. We proposed to investigate the molecular basis for FBXW7 silencing in melanoma (Aim 1), and dissect the role of HSF1 in melanoma initiation and progression and the effects of FBXW7 loss or silencing on HSF1 stability by using novel in vivo models (Aim 2). In addition, we will identify the HSF1-regulated transcriptome and examine the in vivo effects of a subset of HSF1 direct targets (Aim 3). Our studies will elucidate the metastasis-supportive transcriptional program orchestrated by HSF1 and its regulation by a tumor suppressor (FBXW7) frequently mutated or silenced and will provide us with novel therapeutic targets for melanoma.

While much is known about the cell-intrinsic factors that support leukemia progression, little is understood about the role of the microenvironment. T cell acute lymphoblastic leukemia (T-ALL) cells have commonly acquired mutations in pathways downstream of surface receptors that regulate differentiation, survival and proliferation in response to environmental cues, and it is unknown whether such genetic events free T-ALL from dependence on a putative leukemic niche. We have found that T- ALL cells are in direct, stable contact with CXCL12-producing bone marrow stroma. Moreover, both genetic targeting of the CXCL12 receptor CXCR4 in murine disease models and pharmacologic CXCR4 antagonism in human T-ALL xenografts led to rapid and sustained disease remission. Here, we propose to further map and functionally dissect the bone marrow niche for T-ALL, and test the potential of targeting T-ALL:niche interactions as a novel therapeutic avenue for this aggressive blood malignancy. We will use a combination of novel genetic reporters marking distinct niche elements and targeted alleles of factors that have been implicated in T-ALL progression ? CXCL12, Notch ligands, and the cytokine IL-7 ? to identify the key cells that maintain T-ALL. We will also use a potent CXCR4 antagonist and a panel of well-characterized primary human xenografts to test the therapeutic potential of dislodging T-ALL cells from their niches.

? DESCRIPTION (provided by applicant): Recent efforts to functionally annotate the human genome have revealed that up to 75% of our DNA is transcriptionally active. Since a very small portion of our genome encodes proteins many have hypothesized that a portion of this pervasive transcription results in production of long non-coding RNA. By generating high depth RNA-Seq datasets integrated with chromatin features, our lab and others have revealed the presence of many thousands of previously un-annotated lncRNAs, which are dynamically expressed in response to various stimuli in diverse cellular contexts. Despite these compelling advances, the vast majority of putative lncRNAs have not been proven to be functionally important, although a small portion have clearly been shown to play key regulatory roles. Most importantly, very little is known on the biological role of lncRNAs in human cancer. We provide here, using a combination of deep transcriptome sequencing, high-resolution transcription factor occupancy mapping and chromatin interaction data, the first comprehensive identification and characterization of lncRNA expression and function in T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematologic malignancy driven by oncogenic NOTCH1 transcriptional activity. Moreover, we provide the first map of oncogene (NOTCH1)-targeted lncRNAs in this tumor and identify an individual lncRNA (called LUNAR1) that appears to be essential for tumor growth as it controls cytokine (IGF1) signaling. These studies suggest that: a) lncRNAs could be used as both biomarkers and therapy targets in human cancer (see AIM1), b) more efficient methods for large-scale inference and validation of lncRNA function are needed to fully understand their biological significance (see AIM2) and, c) LUNAR1 is one of the first lncRNAs that can control growth of acute leukemia and a potential therapeutic target (see AIM3). In this application we address in detail all these important issues and attempt to directly connect lncRNA deregulation to leukemia initiation and progression.

ABSTRACT Acute myeloid leukemia (AML) is the most common adult leukemia characterized by excessive proliferation of abnormal myeloid progenitors. AML continues to have a dismal survival rate amongst all subtypes of leukemia (<50% five-year overall survival rate), which can largely be attributed to limited advances in treatment regimens that, for the last decades, have relied on the use of two non-targeted cytotoxic drugs: cytarabine and anthracycline. Large-scale sequencing efforts have shed new light on genetic and epigenetic determinants of AML. Interestingly, these studies identified a frequent co-occurrence of somatic mutation between genes encoding cohesin complex subunits (such as STAG2, SMC1A, RAD21 and SMC3) and well-characterized AML oncogenic triggers, such as FLT3-ITD, TET2, and NPM1. Recent work has demonstrated an important role for the cohesin complex in normal stem/progenitor self-renewal and differentiation, gene regulation, and suppression of myeloproliferative neoplasms and AML, despite the precise mechanisms underlying these functions remaining poorly understood. It is believed that cohesin may suppress tumor formation by regulating chromatin looping at loci critical for self-renewal and myeloid progenitor differentiation. Utilizing established models of murine and human AML, this application focuses on the molecular mechanisms of cohesin- dependent myeloid tumor-suppression, with an emphasis on understanding novel treatment approaches that can exploit these functions. Using established protocols for identifying genome-wide changes in chromatin topology and gene expression, we propose to undertake an extensive characterization of cohesin-regulated chromatin changes driving AML. Furthermore, recent studies have identified inhibition of HDAC8 and poly-ADP ribose polymerase (PARP) as an attractive targeted treatment approach for cohesin-mutated AML patients. Here we investigate the application of targeted agents in cohesin-deficient AML whilst extensively mapping the mechanisms-of-action underlying these specific treatments. Ultimately, this project aims to generate novel, pre- clinical disease models of cohesin-mutated AML with strong mechanistic insights into the tumor-suppressive function of this complex.

ABSTRACT Transcription factors require coactivators to communicate with the general transcription machinery and, thereby, ensure that biological inputs are translated into specific gene-expression programs. The Mediator complex is such a coactivator and acts as a ?molecular bridge? between transcription factor at enhancers and RNA polymerase II (Pol II) at promoters. It is a large macromolecular complex further arranged in four modules that confer high flexibility: the head, the middle, the tail and the kinase module. The member of the kinase module Mediator 12 (MED12) has been found frequently mutated in both solid (endometrial, lung, cervical, colon carcinomas) and blood (DLBCL, CLL, ALL, AML) cancers. However, the underlying mechanisms of MED12 mutations and its role in disease initiation and progression remain elusive. We have recently focused on the function of the kinase module and specifically MED12 in hematopoietic stem cell (HSC) differentiation and transformation. We found that MED12 protein expression is controlled post-translationally by the ubiquitin ligase FBXW7, a frequently mutated tumor suppressor. We also found that MED12 is an essential regulator of HSC function, as in vivo deletion of MED12 compromises HSC survival and leads to mouse lethality. Together with essential hematopoietic transcription factors, MED12 co-occupies HSC-specific enhancers. MED12 depletion destabilizes P300 binding thus leading to rapid enhancer ?inactivation?, and loss of expression of key HSC-specific genes. These data suggest that MED12 expression and function can be altered due to multiple mechanisms, including somatic mutations targeting the gene itself or its regulators (FBXW7), and that this aberrant function can lead to malignant transformation. This proposal aims to shed light on the molecular mechanisms altered upon deregulation of a crucial regulator of enhancer activity, such as MED12. While it has been suggested that MED12 mutations confer a ?gain-of-function?, no mechanistic studies have been performed up to date. To address this key question, we are studying chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world. To dissect how disruption of Mediator function contributes to this heterogeneous and complex disease, we use a combination of: a) transcriptional/epigenetic characterization of human patient samples harboring MED12 mutations, b) CRISPR-modified and ES targeted transgenic mouse models to investigate the ability of MED12 lesions to initiate and maintain disease, and, c) in vitro transcriptome, epigenetic and 3D-chromosome topology in CRISPR-modified cell lines with MED12 mutations. Defining the mechanisms by which Mediator and enhancer regulation contribute to malignant transformation will be beneficial for the development of novel therapies targeting blood malignancies and solid tumors. The recent identification of small molecules targeting Mediator pharmacologically suggests that such therapies are within reach.

SUMMARY ? PROJECT 3 (AIFANTIS) Recent studies have offered the first comprehensive maps of three-dimensional (3D) chromosomal interactions. The 3D structure of chromatin is defined in part by the organization of chromatin into highly conserved topologically associating domains (TADs). Studies presented in Project 1 and 2 directly address the role of key ?structural? elements of TAD boundaries (CTCF/Cohesin) in human cancer, including B cell and T cell malignancy (leukemia and lymphoma). In Project 3, we will test the hypothesis that 3D chromatin structure is not only affected by CTCF/Cohesin alterations but also by mutations that affect specific epigenetic regulators and by oncogenic transcription factors. For these studies, we will use T cell leukemia (T-ALL), as a model of study. We will test whether T-ALL oncogenes (the transcription factor NOTCH1, the main driver in this disease, mutated or activated in 90% of human T-ALL) use 3D DNA looping events to induce expression of gene- targets and non-protein coding RNAs that control the function of leukemia cells, including cells that can initiate the disease (leukemia initiating cells), that are characterized by the overexpression of the NOTCH1 transcriptional target MYC. In addition to oncogenic (NOTCH1, MYC) activation, chromosomal topology can also be influenced by epigenetic regulators, and it was shown that T-ALL is a disease characterized by recurrent inactivating mutations in genes that can affect DNA and histone modifications, including genes that affect DNA methylation (DNMT3A), promoter (EZH2) and enhancer (EP300) activity. We thus hypothesize that in human leukemia oncogenes (NOTCH1) and tumor suppressors (DNMT3A, EZH2, EP300) cause aberrant 3D chromatin organization changes and that targeted drug treatments are able to correct these defects. We test this hypothesis in three Aims. Aim 1 assesses the ability of oncogenes like NOTCH1 to directly alter CTCF/Cohesin distribution leading to aberrant chromosomal architecture. Aim 2 tests the hypothesis that drugs that target either oncogenic signaling pathways (NOTCH pathway inhibitors) or altered epigenetic states (BET inhibitors, targeting active H3K27ac-marked areas) can correct 3D chromosomal structure. Finally, Aim 3 focuses on potential effects of selected T-ALL somatic mutations targeting epigenetic regulators and examines their impact on chromosomal topology. We believe that these studies will complement Projects 1 and 2 that focus on the impact of CTCF/Cohesin alterations in blood cancer and generate new paradigms of gene expression regulation in human leukemia.

PROJECT SUMMARY The overall goal of the NYU Melanoma SPORE Developmental Research Program (DRP) is to support pilot projects that take maximum advantage of new research opportunities in melanoma and aid in recruiting established cancer investigators at the level of Assistant Professor or higher to the translational study of melanoma. The DRP focuses on and will select innovative and promising translational research projects in melanoma, and will monitor their progress to ensure that goals are met. The Director of the DRP is Dr. Iannis Aifantis, the Chair of the NYU Department of Pathology and an internationally regarded investigator who brings an accomplished track record of mentorship and emerging translational melanoma research success. He will work together with an internal evaluation panel and the SPORE PD/PIs to identify and support selected pilot studies with exceptional translational potential. The NYU Melanoma SPORE DRP will support projects for up to 2 years, enabling awardees to adequately explore the potential of novel topics in translational melanoma research. Focusing on innovation, the DRP will support the development of ?high-risk, high-reward? investigations in melanoma and encourage proposals for novel approaches to screening, diagnosis, prognosis, prediction, and treatment of melanoma. The DRP will monitor the progress of all projects and awardees chosen for funding to ensure that goals are met and that DRP projects may be strongly positioned to replace any SPORE project either not meeting its translational research goals or completed prior to the close of the award period. The DRP is highly integrated thematically and programmatically with the larger NYU Melanoma SPORE and recognizes that a key component to the growth of the SPORE is the continued support and development of novel investigations in melanoma, specifically in the area of improved personalized management of melanoma patients. Both NYU Langone Health (NYULH) and the NYU Perlmutter Cancer Center (PCC) have consistently used various pilot funding mechanisms to support promising novel approaches to melanoma prognosis, clinical management, and treatment.

Project Summary The response to systemic infection and tissue injury requires the rapid adaptation of hematopoietic stem cells (HSCs) in the bone marrow, which proliferate and divert their differentiation towards the myeloid lineage. Significant interest has emerged in understanding the signals that trigger this emergency hematopoietic program. However, the mechanisms that terminate this response of the HSCs and restore tissue homeostasis remain unknown. The clinical success of proteasome inhibitors, bortezomib, and E3 ubiquitin ligase glues for the treatment of hematologic diseases has made the Ubiquitin pathway a bona fide target for cancer therapeutics. Thus, defining how novel E3 ligases function in the bone marrow and investigating their specific roles in normal and emergency hematopoiesis can lead to novel therapeutic interventions. We have demonstrated that the E3 ubiquitin ligase Spop restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeds in an unresolved manner and the sustained response in the HSCs results in a lethal phenotype reminiscent of hyper-inflammatory syndrome. Our proteomic/biochemical studies demonstrated that Spop restricts inflammation by targeting the signal transducer Myd88 for proteasome-dependent degradation. Myd88 accumulation in conjunction with an inflammatory stimulus leads to Myddosome formation, the hyper-phosphorylation of the Irak4 kinase and activation of a number of transcription factor pathways (NF-kB, Jun, Pu.1, Cebpb). This proposal defines: (a) the transcriptional and chromatin landscape changes imposed during initiation and termination of emergency hematopoiesis in the bone marrow HSC and progenitor cells, (b) the role of the myddosome assembly, signaling and termination in emergency hematopoiesis and gene regulation and (c) the structural details of myddosome assembly and termination. The findings of this grant proposal will uncover HSC-intrinsic mechanisms essential for reestablishing homeostasis following emergency hematopoiesis.