Emmanuel Normant

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.

Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

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.

Abstract 1697: CPI-169, a novel and potent EZH2 inhibitor, synergizes with CHOP in vivo and achieves complete regression in lymphoma xenograft models

Enhancer of Zeste Homolog 2 (EZH2) is the histone lysine methyltransferase (HKMT) component of the Polycomb Repressive Complex 2 (PRC2). In conjunction with other members of the complex, EZH2 represses gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumor progression and correlates with poor prognosis in several tumor types and enzymatic hyperactivity of EZH2 has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Recently, direct inhibition of EZH2 HKMT activity by small molecules has been shown to be effective in inhibiting the proliferation of EZH2 mutant diffuse large B-cell lymphoma (DLBCL) cell lines and the growth of tumors in EZH2 mutant DLBCL xenografts. We have identified and optimized a series of small molecule EZH2 inhibitors that is structurally distinct from previously published chemotypes. CPI-169, a representative compound from that effort, inhibits the catalytic activity of PRC2 with an IC50 of Administered subcutaneously at 200 mpk twice daily (BID), CPI-169 is well tolerated in mice with no observed toxic effect or body weight loss. In the present study we show that CPI-169 treatment led to tumor growth inhibition (TGI) of an EZH2 mutant KARPAS-422 DLBCL xenograft. The TGI is proportional to the dose administered and to the reduction of the pharmacodynamic marker H3K27me3. The highest dose, 200 mpk, BID led to complete tumor regression. Since CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) is the standard treatment of advanced DLBCL, we were interested in combining a suboptimal dose of CPI-169 (100 mpk, BID) with a single dose of CHOP in the KARPAS-422 model. After a week of combinatorial treatment the tumors rapidly regressed and became unpalpable. Four weeks after the last dose only a single mouse presented a palpable tumor. The immunohistochemical analysis of tumor samples revealed a strong correlation between the global decrease of H3K27me3, the decrease in the proliferation marker Ki-67 and the increase in cleaved-caspase 3 positive cells. In conclusion, we identified a strong synergistic anti-tumor activity between the standard of care CHOP and CPI-169, a distinct EZH2 inhibitor in an in vivo model of DLBCL. Citation Format: Vidya Balasubramanian, Priya Iyer, Shilpi Arora, Patrick Troyer, Emmanuel Normant. CPI-169, a novel and potent EZH2 inhibitor, synergizes with CHOP in vivo and achieves complete regression in lymphoma xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1697. doi:10.1158/1538-7445.AM2014-1697

Abstract 2827: Activity of the proprietary Hsp90 inhibitor IPI-493 in models of colorectal cancer correlates with RAS pathway activation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Heat shock protein 90 (Hsp90) plays a role in regulating the stability of key cancer-causing proteins through its role as a protein chaperone. Proteins chaperoned by Hsp90, known as client proteins, include cancer-causing forms of ALK, BCR-ABL, EGFR, FLT3, and HER2. Infinity is developing two drug candidates in its Hsp90 chaperone inhibitor program: IPI-504 (retaspimycin hydrochloride), an intravenously-administered small molecule, and IPI-493, which is administered orally. To investigate the activity of IPI-493 in colorectal cancer (CRC), we performed in vitro growth inhibition (GI) studies on a CRC cell line panel. IPI-493 demonstrated GI50s in the range of 10-100nM in cell lines harboring either kRAS or bRAF mutations. To explore the in vivo potency of IPI-493, several mutant bRAF, mutant kRAS, as well as a wild-type kRAS/bRAF xenograft models were developed. Administration of IPI-493 at 100 mg/kg three times weekly demonstrated dramatic effects in all of the mutant models tested, with tumor growth inhibition (TGI) values between 70 and 90% and regression in one mutant bRAF model. Importantly, when IPI-493 was evaluated in combination with irinotecan, the combination resulted in greater tumor regression than seen with either agent alone with complete regressions observed in 4 of 10 animals. Interestingly, in the wild-type kRAS/bRAF models, IPI-493 administration did not lead to tumor growth inhibition. These results suggest that activation of the MAPK pathway may predispose these cells to sensitivity to Hsp90 inhibition. To investigate the effect of IPI-493 on MAPK pathway activity, we performed pharmacodynamic analysis after a single dose of IPI-493 in multiple xenograft models differing in their RAF/RAS mutation status. In mutant bRAF models, pathway activity was high, and IPI-493 administration resulted in downregulation of the activity of both bRAF and MEK. In models containing no mutations in kRAS or bRAF, we detect low baseline levels of both p-bRAF and p-MEK and little effect of IPI-493 administration. When Ras pathway activity in all CRC xenografts was compared with IPI-493 efficacy, there was a clear correlation between pathway activation and tumor growth inhibition by IPI-493. Our finding that Ras pathway activation predisposes CRC cells to sensitivity to IPI-493 and our combination data with irinotecan provide a clear rationale for the evaluation of Hsp90 inhibitors in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2827. doi:10.1158/1538-7445.AM2011-2827

Abstract LB-237: In vitro and in vivo characterization of CPI-267203, a potent Inhibitor of bromodomain-containing proteins

The transcription factor c-Myc regulates diverse biological processes including growth, proliferation, differentiation, quiescence, and senescence or apoptosis. c-Myc proto-oncogene overexpression has been implicated in the genesis of many human malignancies. Identification of compounds that selectively and potently inhibit the expression of c-Myc hence could be beneficial for the treatment of these diseases. We and others have recently shown that inhibiting the binding of bromodomain-containing proteins such as Brd4 to their acetylated histone substrates leads to the specific suppression of c-Myc transcription. We report here the characterization of CPI-267203, a potent, selective and competitive inhibitor of BET domain proteins (Brd4 IC50 = 26 nM and CBP IC50 = 6.0 uM). In vitro, this molecule inhibits IL-6 production (EC50 = 11 nM) induced by lipopolysaccharide (LPS) in acute monocytic leukemia cells (THP1) and c-Myc mRNA production (EC50 = 27 nM) in Burkitt lymphoma cells (Raji). c-Myc mRNA and protein levels decline as early as 4hrs after treatment with CPI-267203, leading to cell cycle arrest and apoptosis. In vivo, CPI-267203 has 84% oral bioavailability and a plasma half-life of 1.9 h. In a xenograft model of human acute myelogenous leukemia (MV4:11), CPI-267203 inhibits the production of c-Myc mRNA and protein, inhibits tumor growth, and triggers tumor apoptosis. Reduction of c-Myc protein levels can also be detected in the normal skin of treated animals, suggesting that the level of c-Myc in the skin might be a useful surrogate pharmacodynamic marker to aid clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-237. doi:1538-7445.AM2012-LB-237

A novel [125I]iodinated carboxypeptidase A substrate detects a metallopeptidase activity distinct from carboxypeptidase A in brain

We have designed two radioactive substrates, hippuryl-L-[3H]phenylalanine and 3-(p-hydroxy, m-[125I]phenyl)propionic acid ([125I]Bolton reagent) derivative of L-arginyl-L-phenylalanine, i.e. [125I]BRF, for a highly sensitive assay of carboxypeptidase A (CPA) activity. After cleavage of the C-terminal phenylalanine residue by CPA, the radioactive product of the reaction was conveniently separated by polystyrene bead chromatography. Using [125I]BRF, typical CPA activity inhibited by 1 microM 2-benzyl-3-mercaptopropanoic acid was detected in extracts from rat pancreas or intestine. In brain and some other tissues, however, [125I]BRF-hydrolyzing activity was only inhibited by this compound in 1000-fold higher concentration, suggesting the participation of a metallopeptidase distinct from CPA.

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 1691: Synergistic anti-tumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) leading to the overexpression of cyclin D1. As a consequence of its poor responses to conventional chemotherapy and relatively short patient survival, new therapeutic strategies are required. Despite the promising introduction of the proteasome inhibitor bortezomib in the treatment of MCL, not all the patients respond and relapse frequently occurs. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, that was associated with plasmacytic differentiation features, like IRF4 and Blimp-1 upregulation. As the immunomoduladory drug lenalidomide has been shown to modulate IRF4 expression in various B-cell malignancies, we assessed its activity in in vitro and in vivo settings by means of flow cytometry, western blot, antibody array, real-time PCR, immunofluorescence, in vivo imaging, and immunohistochemistry. In vitro, lenalidomide as single agent was found to exert antitumor activity in 4/11 MCL cell lines, corresponding to those cells with either primary or acquired resistance to bortezomib. Lenalidomide-treated cells showed decreased IRF4 expression, increased cytosolic amounts of p27 and caspase-dependent apoptosis. Accordingly, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a lenalidomide regimen of 10-50 mg/kg/day, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated mice (p=0.04), with several hallmarks of lenalidomide activity, like downregulation of IRF4 and its target gene MYC, decreased mitotic index, p27 cytosolic accumulation and caspase-3 processing. Importantly, the inhibition of tumor growth induced by the combination of lenalidomide with bortezomib (0.15 mg/kg, twice a week) was 37% and 66% greater than that for lenalidomide alone and vehicle arms, respectively (p=0.02). Moreover, repression of MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide therapy. Accordingly, co-treatment of mice with lenalidomide plus CPI203 synergistically reduced MYC and IRF4 expression and tumor burden, and induced caspase processing. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition. Citation Format: Alexandra Moros, Vanina Rodriguez, Ifigenia Saborit-Villarroya, Arnau Montraveta, Patricia Balsas, Peter Sandy, Antonio Martinez, Emmanuel Normant, Patricia Perez-Galan, Elias Campo, Dolors Colomer, Gael Roue. Synergistic anti-tumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2014-1691

Abstract 5526: Inhibition of the histone methyl transferase EZH2 causes viability defects in multiple myeloma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Histone lysine methylation, which is dynamically regulated by methyltransferases and demethylases, plays an important role in the establishment and maintenance of chromatin structure and thereby contributes to the control of gene expression. The development of small molecule methyltransferase and demethylase inhibitors provides an approach to manipulate transcriptional programs, and thus potentially allow interference with aberrant cellular states as observed in cancer. The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2) is a component of the Polycomb Repressive Complex 2. It mediates trimethylation of lysine 27 on histone 3 (H3K27me3), which leads to transcriptional repression. EZH2 has been widely implicated in cancer and inhibition of its catalytic activity provides a novel therapeutic approach to treat human cancers. Constellation has identified, characterized and optimized potent, selective and reversible EZH2 small molecule inhibitors as well as studied the biological impact of such inhibition. We have previously shown that pharmacological inhibition of EZH2 causes selective lymphoma cell viability defects with cell lines harboring EZH2 mutations being the most sensitive. The discovery of small molecules that specifically inhibit EZH2 has enabled us to look for other disease indications that might be dependent on EZH2 for survival. We carried out a long term cell viability screen in ∼75 cell lines across several different hematological malignancies using an EZH2 inhibitor. About 30% of all tested Mutliple Myeloma and Plasmacytoma cell lines showed a time-dependent phenotypic response. In these cell lines H3K27me3 levels were effectively reduced in a dose dependent manner within 4 days of compound treatment which was followed by the induction of apoptotsis at later time points. EZH2 inhibitors also achieved tumor growth inhibition in a Multiple Myeloma subcutaneous xenograft model. To understand the underlying molecular mechanism of EZH2 inhibitor sensitivity in Multiple Myeloma, genome-wide mapping of EZH2 and H3K27me3 sites in the absence and presence of the compound have been performed in conjunction with gene expression profiling and the results will be discussed. In conclusion, we identified Multiple Myeloma as a disease modality where EZH2 inhibition leads to cell viability defects both in vitro and in vivo. Citation Format: Shilpi Arora, Vidya Balasubramanian, Kaylyn Williamson, Victor Gehling, Chris Nasveschuk, Rishi Vaswani, Jennifer Busby, Shivani Garapaty, Priya Iyer, Feng Zhao, Robert Campbell, Richard Cummings, Jim Audia, JC Harmange, Brian Albrecht, Andrew Cook, Les Dakin, Emmanuel Normant, Patrick Trojer. Inhibition of the histone methyl transferase EZH2 causes viability defects in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5526. doi:10.1158/1538-7445.AM2014-5526