Debabrata Chakravarti - US grants

Steroid hormones and vitamins including glucocorticoids and vitamin A derivatives act to induce cellular differentiation by directly modulating gene transcription and are often associated with the inhibition of cell growth. Nuclear receptors bind to hormones and vitamins and activate hormone responsive genes by recruiting an acetylase co-activation complex. The broad long- term objectives of this proposal are: (a) to decipher novel regulatory mechanisms involved in receptor and coactivator function; (b) to identify new components of the receptor coactivation-complex; and (c) to dissect the regulatory properties of the complex that translate a hormonal signal into transcriptional and physiological responses. To achieve the above goals, the following Specific Aims will be pursued: (I) To test the hypothesis that both nuclear receptor interaction domains of CBP/p300 are necessary for nuclear receptor mediated gene activation. (II) To determine the roles of the histone acetyltransferase (HAT)- associated domains of CBP/p300 in transcriptional activation and to test the hypothesis that additional proteins are necessary for HAT-mediated transactivation. (III) To test the hypothesis that constant region 2 (CR2) and constant region 3 (CR3) of adenoviral oncoprotein E1A function as the HAT-inhibitory domains and to determine the mechanism of transcription inhibition by E 1 A. A combination of in vitro binding, site directed mutagenesis, deletion analysis, and mammalian cell transfection based in vivo assays will be utilized to address the above Specific Aims. Yeast two-hybrid screens will be employed to identify and characterize novel CBP/p300 regulatory proteins. These studies are clinically significant because: (a) nuclear receptor agonists and antagonists are useful in treatment of breast, and prostate cancer, leukemia, diabetes, and cardiovascular diseases; and (b) chromosomal translocations and amplification of nuclear receptors and their cofactors have been implicated in leukemia and breast cancer suggesting abnormal targeting or regulation of the activities of receptors or their coactivators may play important roles in leukemia and oncogenesis. The accomplishment of the Specific Aims described above geared towards achieving the long- term goals should provide a better understanding of the role of nuclear receptors and coactivators in transcription and in human diseases including cancer.

DESCRIPTION (provided by applicant): Transcriptional regulation plays a fundamental role in specifying cellular and tissue differentiation, cell growth, development and susceptibility to diseases. The highly regulated process of eukaryotic gene expression is coordinated by the interplay of multiple mechanisms involving chromatin, transcription factors such as nuclear hormone receptors and critical accessory/regulatory proteins with histone/chromatin modifying activities. Histones undergo posttranslational modifications such as acetylation, phosphorylation and methylation. These modified histones subsequently led to the establishment of a "histone code" of transcription. For example, unmodified histones repress transcription while acetylated histories promote gene activation. The mechanisms by which unmodified histones "code" for transcriptional repression in higher eukaryotes are largely unknown. Post-translational modification of coregulators also influence gene transcription. The long-term goal of our research is to identify and molecularly characterize novel mechanisms regulating hormonal signaling and transcription. We have recently shown that a human cellular complex termed INHAT regulates nuclear receptor function and transcription at least in part by binding to histones and modulating histone acetyltransferase activity of coactivators, pp32 is a subunit of the INHAT complex and a nuclear phosphoprotein. We hypothesize that pp32 is a critical component in transcriptional repression and that phosphorylation of pp32 plays a critical role in its in vivo function. To test the hypotheses we will (1) determine the role ofpp32 in translating the repressive "histone code" of transcription; (2) characterize molecular mechanisms of transcriptional repression by pp32; and (3) analyze the role of phosphorylation in pp32 function in vivo. We will address the above specific aims using a combination of in vitro and in vivo analyses involving biochemical, molecular, and cell biological methodologies. Altered activities of proteins regulating histone modifications and protein phosphorylation have been linked to altered hormone signaling and human diseases including cancer, and developmental abnormalities. The information stemming from the proposed studies should not only provide a better understanding of transcription and hormone signaling but may also be useful in targeted drug development to treat human diseases.

DESCRIPTION (provided by applicant): Transcriptional regulation plays a critical role in normal cell and organ differentiation, development and physiology in humans. Not surprisingly, up or downregulation of transcription factors and their coregulators has been tightly linked to tumor development and the progression to malignant and metastatic stages. Cancer cells originate primarily from epithelial tissues. Accumulating evidence suggests that during cancer progression, cells begin to redistribute or downregulate proteins involved in maintaining cell adherens and tight junctions. These changes and others promote a loss of cell-cell and cell-matrix interactions and permit cell motility and invasion of neighboring tissues. While approximately 90% of all cancer deaths occur because of tumor metastasis, very few therapies are available that are effective in preventing metastasis. One way to overcome this limitation is to gain a complete understanding of the genetic and signaling networks that promote and modulate tumor cell motility and metastasis. The Thanatos associated protein (THAP) family is comprised of 12 members in the human, and only 3 members have been partially characterized. Recently we observed that THAP11, a member of the THAP family is differentially expressed in cancer cells. How THAP11 overexpression contributes to cancer cell function is completely unknown and thus represents an exciting and novel field of investigation. Based on our preliminary results, we hypothesize that THAP11 is a transcriptional repressor that plays a critical role in cancer cell function by directly regulating expression of key genes involved in intercellular and cell-matrix interactions. Since alteration of these interactions is a key initial event in metastasis, we propose that THAP11 represents a novel and critical transcriptional regulator of cancer cell function. We will undertake state-of-the-art molecular, cellular, physiologic and genome-wide analyses to define the impact of THAP11 overexpression and its mechanism of action in cancer cell function in three integrated specific aims. The proposed studies will greatly advance our knowledge of cancer progression and metastasis, and may provide a molecular target for therapeutic intervention.

Nuclear receptors bind to hormones and vitamins and regulate the activity of hormone inducible genes which in turn influence a cascade of events that impact almost all physiologic pathways including cell differentiation, fat metabolism, energy homeostasis and reproduction. Nucleosomes, the building blocks of chromatin are DNAhistone complexes and play pivotal roles in regulating both transcriptional activation and repression. Consequently posttranslational nucleosome/histone modification and nucleosome remodeling are proposed to be critical for gene regulation. While the roles of chromatin modifying proteins such as histone acetyltransferases and histone deacetylases in regulation of hormone inducible genes are well documented, surprisingly very little is known about the role of chromatin remodeling proteins in regulating hormone/vitamin inducible genes. Additionally if and how hormone alters nucleosome positioning of hormone inducible genes is not well understood. We recently identified and characterized human ATP-utilizing chromatin assembly and remodeling factor 1 (hAcf1) as a critical regulator of hormone action and NR function. Our preliminary data enable us to propose hormone and Acf1 dependent nucleosome repositioning as a defining event in hormone action. We hypothesize that Acf1 plays critical roles in integrating hormone action and chromatin remodeling that defines the transcriptional output of NR target genes. To test this hypothesis, we will perform experiments under three specific aims that will determine the in depth mechanisms of action of Acf1 and hormone induced chromatin remodeling in NR function. In this proposal we will first analyze the innovative concept that Acf1 integrates hormone action to chromatin remodeling at target genes, which is a crucial event in both gene activation and repression. The proposal will also test the hypothesis that a novel role of hormone is to reposition nucleosome and /or to promote histone eviction/exchange. Our genome wide screen for Acf1 regulated genes (as determined by microarray) as well as its genome wide binding (as determined by ChIPSeq) adds additional innovative concepts in NR research. We will use state of the art molecular, biochemical as well as newly developed techniques such as microarray and genome-wide deep sequencing methods to address the specific aims. Since epigenomic control of hormone action and NR function is new and rapidly evolving concept, we believe our work which are highly innovative in nature will significantly advance the field forward. By making key links between chromatin remodeling events and NR biology our work should provide a better understanding of hormone action and facilitate development of novel drugs that targets chromatin remodelers in treatment of human diseases

? DESCRIPTION (provided by applicant): Androgen and its receptor AR, which is a member of the human nuclear receptor (NRs) superfamily, play critical roles in prostate cancer (PC) and castrate resistant prostate cancer (CRPC) that kills thousands of people world-wide. We posit that a better understanding of NR signaling pathway and its integration in epigenomic signaling are keys for development of future therapeutics of this deadly disease. The long term goal of our research is to discover novel components of NR function in transcriptional and epigenomic regulation with strong translational implications in human diseases. The protein kinase termed PKN1 plays a critical role in AR dependent gene regulation by establishing an epigenomic marking (histone H3 threonine 11 phosphorylation (H3T11P)) on nucleosomes at AR target genes. We hypothesize that the PKN1-H3T11P- WDR5 signaling module plays a previously unrealized and critical role in castrate resistant prostate cancer (CRPC). The goal of this present work is to perform in depth and integrative mechanistic, genome-wide, cellular and animal based studies to dissect the role of this newly discovered signaling module in AR function in CRPC. To achieve our research goals, we will utilize interdisciplinary knowledge and complementary expertise of the co-investigators spanning biochemistry, patho-physiology, genome biology, animal and cellular models of PC and molecular biology. In Specific Aims, we will examine in extensive molecular details the role of WDR5, and PKN1 in gene regulation, and CRPC-cell function in vitro and in vivo using cell and animal based models. We will also determine the gene-specific and genome-wide localization profiles of WDR5 and H3T11P in CRPC cells. Epigenomic markings and identification of their effector proteins in hormone action are new and rapidly evolving concepts and we believe our work lies at the limit of current knowledge of hormone signaling. Our findings will thus significantly advance the field by providing a better understanding of the role of epigenomic modifications and their effector proteins in nuclear receptor action and in human diseases including CRPC.

? DESCRIPTION (provided by applicant): Uterine leiomyomas, also known as uterine fibroids, are benign, fibrotic smooth muscle tumors that affect an alarming 75-80% of women in their lifetime and constitute a major health problem worldwide. They remain shockingly understudied with few nonsurgical therapeutic options. In a major breakthrough in the field, mutations in the transcriptional mediator complex subunit 12, termed Med 12, have been identified at very high frequency in leiomyoma implying that Med12 mutations are the driver mutations. However, how Med12 and its mutations contribute to leiomyoma is completely unknown. The long term goal of our laboratory is to discover novel molecular events driving leiomyoma. The immediate goal of this R21-exploratory application is to test the hypothesis that Med12 mutations function by generating an altered Med12/mediator cistrome in leiomyoma, leading to abnormal expression of cell proliferation/apoptosis and profibrotic genes. We will determine the genome wide binding profile of wild type and mutant Med12 and other key mediator subunits in normal myometrial and leiomyoma primary cells. Using bioinformatic and ChIP qPCR analysis we will determine whether Med12 functions primarily by targeting communication between transcription factors such as nuclear receptors and Smad proteins, mediator complex and RNA polymerase II and establish that such interactions are defective/altered in leiomyoma. Together, these results will determine for the first time molecular defects in Med12 in leiomyoma thereby advancing the field in an unprecedented manner. Additionally, the proposed exploratory analyses will generate new hypothesis for future RO1-type research proposals in this highly understudied human health problem. The proposed work is scientifically, translationally, and clinically significant because i will provide a new perspective on the key driver mechanisms of Med 12 function in leiomyoma development and open up possibilities for identifying additional therapeutic targets and strategies to treat this disease. It is innovative because it represents the first systematic exploration of previously unrealized roles of Med12 in leiomyoma patho-biology.

Leiomyoma (Uterine fibroids) is a major disease that affects women but remains shockingly understudied with few nonsurgical therapeutic options. Leiomyoma is characterized by excessive deposition of extracellular matrix and enhanced proliferation and tumorigenesis of uterine smooth muscle cells. The roles of estrogen and progesterone and their nuclear receptors (NRs) and transforming growth factor ???TGF???signaling in leiomyoma are well established. However, the broader role of the nuclear receptor superfamily and their cross talk with TGF? signaling pathways in leiomyoma remain as some of the major unanswered questions. The long-term goal of this project is to establish systematically the roles of nuclear receptors (NRs) and their ligands and to understand how TGF? signaling is altered in leiomyoma. The goal of this project is to determine the novel roles of NR4A subfamily members, including NR4A1 (NGF1B), NR4A2 (NURR1), and NR4A3 (NOR1) in leiomyoma. We performed expression profiling of leiomyoma tissues and adjacent normal myometrium for all 48 human NRs and demonstrated severe deficiency of the NR4A subfamily members, including NR4A1 (NGF1B), NR4A2 (NURR1), and NR4A3 (NOR1), in leiomyoma. Our preliminary results show that NR4A functions by regulating expression of key profibrotic factors such as TGF?3 and SMAD3 and key extracellular matrix components such as collagen 1A1. Furthermore, NR4As regulate proliferation of leiomyoma primary smooth muscle cells. We hypothesize that the NR4A family of nuclear receptors plays critical and integrative roles involving TGF?? and SMAD signaling in leiomyoma development by regulating key proliferation and profibrotic genes. The specific aims are: Specific Aim 1: Determine how NR4A and TGF?? /SMAD signaling pathways interact to regulate pro-fibrotic genes in leiomyoma. Specific Aim 2: Determine the roles of NR4As and their selective modulators in leiomyoma cell proliferation in vitro and tumorigenesis in vivo. The proposed aims will advance our knowledge of this highly prevalent public health challenge for which treatment options are currently limited at best. The proposed work is scientifically, translationally, and clinically significant because it provides a new perspective on and better understanding of the mechanisms underlying leiomyoma development and opens up possibilities for identifying additional therapeutic targets and strategies. It is innovative because it represents the first systematic exploration of previously unrealized roles of the NR4A nuclear receptor family in leiomyoma biology.

Uterine leiomyomas (UL), also known as uterine fibroids, are benign smooth muscle tumors with excessive depostition of extracellular matrix proteins. UL is a major health problem worldwide, because it affects almost 70-80% of all women and disproportionally African Americans, but still remains poorly understood. The long term goal of our team is to systematically discover novel mechanisms regulating key molecular events that contribute to leiomyoma. The immediate goal of this R01 application is to test the hypothesis that altered epigenomic signatures define normal myometrial tissues and leiomyomas, and therapy treated human tissues. Using genome-wide studies integrated with bioinformatic and other analyses, we will determine the epigenomic signatures in uterine fibrosis for the first time. This unbiased study will identify epigenomic differentiating features of normal and diseased tissues and may allow for development of Epitherapy (targeting the epigenome) for leiomyomas The proposed work is scientifically, translationally, and clinically significant and highly innovative because it represents the first systematic exploration of the epigenome in leiomyomas. Results obtained from this analysis will be used to generate new hypotheses to better understand the molecular underpinning of leiomyomas.

Uterine leiomyoma (LM), also known as uterine fibroids, are benign, smooth muscle tumors of the uterus characterized by extensive cellular alteration and stiffness of the extracellular matrix (ECM). Alarmingly, almost 75% of all women will develop some form of fibroids in their lifetimes, with some experiencing significant symptoms. Current medical treatments for LM have off-target side effects that limit their long-term use; surgical options are the only definitive treatment. Consequently, LM is a major gynecologic healthcare problem, and its treatment costs billions of dollars annually. Unfortunately, the molecular mechanisms underlying LM tumorigenesis and progression are poorly understood, and this has posed a significant barrier to the development of new treatment options. The proposed studies are highly significant because they will fill a gap in knowledge about this major gynecologic disease, identifying the mechanisms that drive LM formation as well as potential targets for new LM therapies. Although estrogen and progesterone signaling and TGF? signaling have been implicated in LM, the search for potential driver mutations in LM led to the identification of genes that encode two major proteins that regulate transcription and 3D chromatin topology. Overexpression via chromosomal translocation of the gene encoding chromatin-binding protein high mobility group protein HMGA2 (HMGA2-ra) and mutations in the transcriptional mediator complex subunit Med12 gene (mut-MED12) have been identified as mutually exclusive driver mutations in LM. They together contribute to almost 85% of all LM. Previous cytogenetic, IHC, molecular and whole genome sequencing studies clearly established a role for HMGA2 overexpression in leiomyoma pathogenesis. In this proposal, utilizing these previous observations, we hypothesize that HMGA2 overexpression alters its association with chromatin, thereby changing epigenetic signatures and strongly influencing 3D chromatin topology, which alters gene expression compared to normal myometrial tissues. To test our hypothesis, the following two specific aims will be pursued. Specific Aim 1, we will define the cistrome, transcriptome, and epigenome in HMGA2-ra LM. In Specific Aim 2, we will define chromosomal 3D-topology in HMGA2-ra LM. We will use state-of-the-art genome-wide technology and bioinformatic analysis to achieve these aims. Once completed, the proposed studies will advance our knowledge of this highly prevalent public health challenge in gynecology that affects half of the world population. The proposed work is scientifically, translationally, and clinically significant because these studies will establish a rational pre-clinical framework to assess existing treatments and develop novel therapies for targeting LM, a major gynecologic disease.

ABSTRACT MYC oncoproteins (including c-MYC, L-MYC and N-MYC) play critical roles in the initiation, progression and recurrence of many human malignancies. Extensive studies indicate that MYC is required to maintain tumor cell survival and proliferation. We have recently used a novel approach that combined computer-aided modeling with a rapid in vivo screen to develop a new series of direct small molecule inhibitors (MYCi?s) that show excellent selectivity, potency and tolerability in multiple MYC-driven cancer models. These compounds demonstrate a dual mechanism of action. First, direct binding of MYCi to MYC in the basic helix-loop-helix (bHLH) region disrupts complex formation with MYC which is required for MYC transcriptional activity. Secondly, binding of MYCi enhances MYC phosphorylation on threonine-58 (pT58) which promotes MYC degradation via the ubiquitin-proteasome pathway. However the key downstream effectors of these events and how they might impact cellular function are unknown. Reduction of MYC protein and enhanced pT58MYC may be expected to have profound effects on MYC family protein interactions with each other and with chromatin. In this regard, we have observed in preliminary studies that MYCi leads to selective loss of MYC at genomic loci enriched for master chromatin regulators (CTCF and FOX), suggesting disruption of the 3D architecture of the MYC-bound genome in response to MYCi. Additionally, unfolded MYC due to MYCi binding and/or enhanced MYC degradation may provoke a cellular stress response. Using unbiased ATAC-seq and RNA-seq approaches, we found that MYCi treatment activates the ATF4/CHOP stress response pathway. Importantly, activation of ATF4/CHOP by MYCi is an on-target, MYC-dependent effect. ATF4 mediates MYCi antitumor activity as ATF4 depletion partially ameliorates the antitumor effects of MYCi. Furthermore, we propose that MYCi-induced ATF4 cytokines modulate the tumor microenvironment. Activation of the ATF4 pathway by MYCi exposes potential therapeutic vulnerabilities for rational combination approaches, such as combination of MYCi with proteasome inhibitors that activates ATF4. Based on the preliminary findings, our central hypotheses is that MYCi inhibits MYC-dependent tumorigenesis by a dual-pronged mode of action. First, MYCi affects MYC family target gene expression by disrupting MYC/MAX interaction and by promoting MYC degradation. Secondly, binding of MYCi to MYC and/or MYC degradation activates an ATF4/CHOP stress response pathway that suppresses tumor cell viability. We propose the following specific aims to test these hypotheses: Aim 1). To investigate the mechanisms by which MYC inhibitor modulates MYC transcriptional activity and the epigenetic landscape. We will investigate the consequences of MYCi treatment on the recruitment of MYC, pT58MYC, and associated factors to chromatin; changes to 3D chromatin architecture; as well as the effects on MYC-driven transcriptional output in tumor cells vitro and in vivo. Aim 2). To define the mechanisms and functional consequences of ATF4/CHOP pathway activation by MYCi. We will determine mechanism of ARF4 upregulation by MYCi; define the role of MYCi- induced ATF4 in regulating target gene expression, cell viability and tumorigenicity; and assess strategies that exploit the consequences of ATF4 activation as a means of enhancing MYCi anti-tumor efficacy. These studies are significant as MYC is implicated in the majority of human cancers. The studies advance the use of MYCi as chemical probes to unmask distinct biology that complements the knowledge derived from genetic manipulations of MYC proteins. The findings will contribute to the efforts aimed at developing small molecule MYCi as potential therapeutics. Specifically, this work indicates that small-molecule MYC inhibitors have an additional anti-tumor effect due to the activation of the ATF4 pathway beyond the antitumor effects of suppressing MYC function. Finally, understanding this on-target ATF4 response provoked by small-molecule MYCi will provide rational strategies for combination therapy to enhance MYCi efficacy.