Srividya Balasubramanian

Targeting MYC dependence in cancer by inhibiting BET bromodomains.

The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein–DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain–promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G1 arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitts lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.

Identification of EZH2 and EZH1 Small Molecule Inhibitors with Selective Impact on Diffuse Large B Cell Lymphoma Cell Growth

The histone methyltransferase enhancer of Zeste homolog 2 (EZH2) is a candidate oncogene due to its prevalent overexpression in malignant diseases, including late stage prostate and breast cancers. The dependency of cancer cells on EZH2 activity is also predicated by recurrent missense mutations residing in the catalytic domain of EZH2 that have been identified in subtypes of diffuse large B cell lymphoma, follicular lymphoma and melanoma. Herein, we report the identification of a highly selective small molecule inhibitor series of EZH2 and EZH1. These compounds inhibit wild-type and mutant versions of EZH2 with nanomolar potency, suppress global histone H3-lysine 27 methylation, affect gene expression, and cause selective proliferation defects. These compounds represent a structurally distinct EZH2 inhibitor chemotype for the exploration of the role of Polycomb Repressive Complex 2-mediated H3K27 methylation in various biological contexts.

EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation.

The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, application across a large cell panel representing various non-Hodgkins lymphoma (NHL) subtypes, and their efficacy in EZH2mutant-containing GCB-DLBCL xenograft models. Surprisingly, our EZH2 inhibitors selectively affect the turnover of trimethylated, but not monomethylated histone H3 lysine 27 at pharmacologically relevant doses. Importantly, we find that these inhibitors are broadly efficacious also in NHL models with wild-type EZH2.

Discovery and Optimization of Tetramethylpiperidinyl Benzamides as Inhibitors of EZH2

The identification and development of a novel series of small molecule Enhancer of Zeste Homologue 2 (EZH2) inhibitors is described. A concise and modular synthesis enabled the rapid development of structure-activity relationships, which led to the identification of 44 as a potent, SAM-competitive inhibitor of EZH2 that dose-dependently decreased global H3K27me3 in KARPAS-422 lymphoma cells.

Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent and Selective Inhibitor of Histone Methyltransferase EZH2, Suitable for Phase I Clinical Trials for B-Cell Lymphomas.

Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 μM, cellular EC50 = 0.032 μM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.

Pharmacological Inhibition of the Histone Lysine Demethylase KDM1A Suppresses the Growth of Multiple Acute Myeloid Leukemia Subtypes.

Lysine-specific demethylase 1 (KDM1A) is a transcriptional coregulator that can function in both the activation and repression of gene expression, depending upon context. KDM1A plays an important role in hematopoiesis and was identified as a dependency factor in leukemia stem cell populations. Therefore, we investigated the consequences of inhibiting KDM1A in a panel of cell lines representing all acute myelogenous leukemia (AML) subtypes using selective, reversible and irreversible KDM1A small-molecule inhibitors. Cell models of AML, CML, and T-ALL were potently affected by KDM1A inhibition, and cells bearing RUNX1-RUNX1T1 (AML1-ETO) translocations were especially among the most sensitive. RNAi-mediated silencing of KDM1A also effectively suppressed growth of RUNX1-RUNX1T1-containing cell lines. Furthermore, pharmacologic inhibition of KDM1A resulted in complete abrogation of tumor growth in an AML xenograft model harboring RUNX1-RUNX1T1 translocations. We unexpectedly found that KDM1A-targeting compounds not only inhibited the catalytic activity of the enzyme, but evicted KDM1A from target genes. Accordingly, compound-mediated KDM1A eviction was associated with elevated levels of local histone H3 lysine 4 dimethylation, and increased target gene expression, which was further accompanied by cellular differentiation and induction of cell death. Finally, our finding that KDM1A inhibitors effectively synergize with multiple conventional as well as candidate anti-AML agents affords a framework for potential future clinical application. Cancer Res; 76(7); 1975-88. ©2016 AACR.

Discovery, design, and synthesis of indole-based EZH2 inhibitors.

The discovery and optimization of a series of small molecule EZH2 inhibitors is described. Starting from dimethylpyridone HTS hit (2), a series of indole-based EZH2 inhibitors were identified. Biochemical potency and microsomal stability were optimized during these studies and afforded compound 22. This compound demonstrates nanomolar levels of biochemical potency (IC50=0.002 μM), cellular potency (EC50=0.080 μM), and afforded tumor regression when dosed (200 mpk SC BID) in an EZH2 dependent tumor xenograft model.

Abstract PR09: EZH2 inhibitors reveal broad EZH2 dependencies in multiple myeloma

Histone methyl transferases (HMTs) and demethylases are chromatin modifying enzymes known to play a key role in establishing and maintaining chromatin structure and thereby contributing to the control of gene expression. The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2) is the catalytic component of the Polycomb Repressive Complex 2 and mediates trimethylation of lysine 27 on histone 3 (H3K27me3), which correlates with transcriptional repression. EZH2 has been widely implicated in cancer and inhibition of its catalytic activity recently emerged as a novel therapeutic approach to treat human cancers. Constellation has developed potent, selective and reversible EZH2 small molecule inhibitors that are currently being tested in clinical trials. We have previously reported EZH2 dependencies across non-Hodgkin Lymphoma subtypes, including models harboring both wild-type and mutant EZH2. To identify other cancer types that may rely on EZH2 for survival, we carried out long term growth assays across a 200+ cancer cell line panel. We observed that over 50% of multiple myeloma cell lines show -time and -dose dependent phenotypic response to EZH2 inhibition. Similar to lymphoma, EZH2 inhibitors induce apoptosis after continuous treatment over a longer time period. To understand the underlying molecular consequences of EZH2 inhibition in multiple myeloma, we performed RNA-sequencing and ChIP-sequencing in the absence and presence of the inhibitor. We identified an EZH2-controlled transcriptional signature across various multiple myeloma models and key downstream effectors including CDKN1A in individual models. EZH2 inhibitors such as CPI-169 achieve tumor growth inhibition in several multiple myeloma subcutaneous xenograft models at well tolerated doses, and this impact on tumor growth correlated well with target inhibition. To expand the scope of EZH2 inhibitor application in multiple myeloma, we systematically combined EZH2 inhibitors with standard of care agents, including, lenalidomide, prednisolone, bortezomib and HDAC inhibitors. We observed synergy of EZH2 inhibitors with several of these agents in vitro and in vivo and are currently exploring the molecular basis of these combinatorial effects. In conclusion, we provide ample evidence suggesting multiple myeloma as a disease indication in which EZH2 inhibitors may show clinical benefit as a single agent and in combination with approved therapeutics. Citation Format: Shilpi Arora, Kaylyn Williamson, Srividya Balasubramanian, Jennifer Busby, Shivani Garapaty-Rao, Charlie Hatton, Dhanalakshmi Sivanandhan, Barbara Bryant, Emmanuel Normant, Patrick Trojer. EZH2 inhibitors reveal broad EZH2 dependencies in multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR09.

Abstract B179: Mechanism of antitumor activity of BET inhibitors in models of lymphomas and leukemia.

Proteins of the BET family bind to acetylated histones and promote gene expression by recruiting transcriptional activators. We developed BET inhibitors and demonstrated a strong association between biochemical inhibition of BET-chromatin binding, MYC suppression and reduced proliferation in hematopoietic cancer cells. We assessed the gene loci affected by BET inhibition by integrating ChipSeq and gene expression profiling, and identified genes that are directly regulated by BRD4 binding, most notably MYC, or genes that are targets of MYC. BET inhibition resulted in robust suppression of MYC expression in tumor xenograft models. Moreover, based on the PK/PD relationships established for these compounds we designed dosing regimens that resulted in significant anti-tumor efficacy in xenograft models of Burkitt9s lymphoma and acute leukemia. Compound doses and schedules resulting in sustained MYC suppression were most effective in inhibiting tumor growth and were well tolerated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B179.