Small-molecule Inhibitors Of EZH2 Research Articles

DDDR-48. EZH2 INHIBITION AS A POTENTIAL THERAPEUTIC AVENUE IN THE TREATMENT OF MENINGIOMA

Abstract INTRODUCTION High grade meningiomas provide challenges to treatment as these neoplasms are more likely to recur, exhibit invasion of brain parenchyma, and result in decreased long term survival. Recent works demonstrate the epigenetic modifier, EZH2, to be increased in higher grade meningiomas, and to correlate with poor survival. The selective targeting of EZH2 has shown promise in other malignancies with overexpression of or gain-of-function mutations in EZH2, leading to promising findings. Here we sought to investigate the effect of EZH2 inhibition on meningioma cell growth and viability, using a panel of in vitro models. METHODS Human immortalized meningioma cell lines, mouse meningioma cell lines and a panel of human primary cell cultures with varying baseline protein expression of NF2, SMARCB1, and EZH2, were selected for investigation. Cells were treated with several selective small molecule inhibitors of EZH2. Cell proliferation, cell cycle and cell death processes were assessed. RNAseq was used to assess transcriptional changes in response to EZH2 inhibition. Western blots were used to assess protein changes in response to EZH2 inhibition. RESULTS EZH2 inhibition significantly impairs meningioma cell proliferation, at varying IC50s, in all cell lines and primary cultures tested, utilizing a panel of EZH2 inhibitors. EZH2 inhibition resulted in significantly decreased colony formation in a high-grade cell line. Furthermore, EZH2 inhibition results in a reduction of Ki67+ cells, induction of cell cycle arrest, and apoptosis. Similar grouping of differential gene expression in immortalized cell lines and primary cell cultures is observed across cell lines and primary cell cultures, after EZH2 inhibition. CONCLUSION Our work demonstrates that EZH2 inhibition results in growth inhibition, cell cycle arrest, and cell death, across all cell lines, and cell cultures tested. Futhermore, modulation of protein levels of the EZH2 target H3K27 is observed upon treatment. RNA changes are broad and involve cell proliferative and migratory processes. This work provides the framework for further investigation of EZH2 inhibition in meningioma.

BZW2 Inhibition Reduces Colorectal Cancer Growth and Metastasis.

FDA-approved small-molecule inhibitors of EZH2 can indirectly target BZW2 and because BZW2 functions as an oncogene, these inhibitors could serve as therapeutic agents for colorectal cancer.

Emerin interacts with histone methyltransferases to regulate repressive chromatin at the nuclear periphery.

X-Linked Emery-Dreifuss muscular dystrophy is caused by mutations in the gene encoding emerin. Emerin is an inner nuclear membrane protein important for repressive chromatin organization at the nuclear periphery. Myogenic differentiation is a tightly regulated process characterized by genomic reorganization leading to coordinated temporal expression of key transcription factors, including MyoD, Pax7, and Myf5. Emerin was shown to interact with repressive histone modification machinery, including HDAC3 and EZH2. Using emerin-null myogenic progenitor cells we established several EDMD-causing emerin mutant lines in the effort to understand how the functional interaction of emerin with HDAC3 regulates histone methyltransferase localization or function to organize repressive chromatin at the nuclear periphery. We found that, in addition to its interaction with HDAC3, emerin interacts with the histone methyltransferases EZH2 and G9a in myogenic progenitor cells. Further, we show enhanced binding of emerin HDAC3-binding mutants S54F and Q133H to EZH2 and G9a. Treatment with small molecule inhibitors of EZH2 and G9a reduced H3K9me2 or H3K27me3 throughout differentiation. EZH2 and G9a inhibitors impaired cell cycle withdrawal, differentiation commitment, and myotube formation in wildtype progenitors, while they had no effect on emerin-null progenitors. Interestingly, these inhibitors exacerbated the impaired differentiation of emerin S54F and Q133H mutant progenitors. Collectively, these results suggest the functional interaction between emerin and HDAC3, EZH2, and G9a are important for myogenic differentiation.

The Synergistic Anti-Tumor Activity of EZH2 Inhibitor SHR2554 and HDAC Inhibitor Chidamide through ORC1 Reduction of DNA Replication Process in Diffuse Large B Cell Lymphoma.

Simple SummaryThe EZH2-targeted drugs have demonstrated notable therapeutic effects in EZH2 mutant B-cell lymphoma patients. In this study, we demonstrated that the combination of EZH2 inhibitor SHR2554 and HDAC inhibitor HBI8000 exert synergistic anti-proliferative activity in both EZH2 wide-type and mutation B-cell lymphoma. More importantly, gene expression profile analysis revealed simultaneous treatment with these agents led to dramatic inhibition of DNA replication initiator protein ORC1, which might contribute to great efficacy of combination strategy. The combination of EZH2 inhibitor and HDAC inhibitor could provide a potential therapeutic treatment for both EZH2 wide-type and mutation B-cell lymphoma patients.Background: Upregulation of H3K27me3 induced by EZH2 overexpression or somatic heterozygous mutations were implicated in lymphomagenesis. It has been demonstrated that several EZH2-target agents have notable therapeutic effects in EZH2-mutant B-cell lymphoma patients. Here we present a novel highly selective EZH2 inhibitor SHR2554 and possible combination strategy in diffuse large B-cell lymphoma (DLBCL). Methods: Cell proliferation, cell cycle and apoptosis were analyzed by CellTiter-Glo Luminescent Cell Viability Assay and flow cytometry. Western Blot was used to detect the expression of related proteins. The gene expression profiling post combination treatment was analyzed by RNA-Seq. Finally, CDX and PDX models were used to evaluate the synergistic anti-tumor effects of the combination treatment in vivo. Results: The novel EZH2 inhibitor SHR2554 inhibited proliferation and induced G1 phase arrest in EZH2-mutant DLBCL cell lines. The combination of EZH2 inhibitor SHR2554 with histone deacetylase (HDAC) inhibitor chidamide (hereafter referred to as HBI8000) exerted synergistic anti-proliferative activity in vitro and in vivo. Gene expression profile analysis revealed dramatic inhibition of the DNA replication process in combined treatment. Conclusions: SHR2554, a potent, highly selective small molecule inhibitor of EZH2, inhibited EZH2-mutant DLBCL more significantly in vitro and in vivo. The combination of HDAC inhibitor HBI8000 with EZH2 inhibitor SHR2554 exhibited dramatic anti-tumor activity in both mutant and wild-type DLBCL, which may become a potential therapeutic modality for the treatment of DLBCL patients.

Reversing Epigenetic Gene Silencing to Overcome Immune Evasion in CNS Malignancies.

Glioblastoma (GBM) is an aggressive brain malignancy with a dismal prognosis. With emerging evidence to disprove brain-immune privilege, there has been much interest in examining immunotherapy strategies to treat central nervous system (CNS) cancers. Unfortunately, the limited success of clinical studies investigating immunotherapy regimens, has led to questions about the suitability of immunotherapy for these cancers. Inadequate inherent populations of tumor infiltrating lymphocytes (TILs) and limited trafficking of systemic, circulating T cells into the CNS likely contribute to the poor response to immunotherapy. This paucity of TILs is in concert with the finding of epigenetic silencing of genes that promote immune cell movement (chemotaxis) to the tumor. In this study we evaluated the ability of GSK126, a blood-brain barrier (BBB) permeable small molecule inhibitor of EZH2, to reverse GBM immune evasion by epigenetic suppression of T cell chemotaxis. We also evaluated the in vivo efficacy of this drug in combination with anti-PD-1 treatment on tumor growth, survival and T cell infiltration in syngeneic mouse models. GSK126 reversed H3K27me3 in murine and human GBM cell lines. When combined with anti-PD-1 treatment, a significant increase in activated T cell infiltration into the tumor was observed. This resulted in decreased tumor growth and enhanced survival both in sub-cutaneous and intracranial tumors of immunocompetent, syngeneic murine models of GBM. Additionally, a significant increase in CXCR3+ T cells was also seen in the draining lymph nodes, suggesting their readiness to migrate to the tumor. Closer examination of the mechanism of action of GSK126 revealed its ability to promote the expression of IFN-γ driven chemokines CXCL9 and CXCL10 from the tumor cells, that work to traffic T cells without directly affecting T maturation and/or proliferation. The loss of survival benefit either with single agent or combination in immunocompromised SCID mice, suggest that the therapeutic efficacy of GSK126 in GBM is primarily driven by lymphocytes. Taken together, our data suggests that in glioblastoma, epigenetic modulation using GSK126 could improve current immunotherapy strategies by reversing the epigenetic changes that enable immune cell evasion leading to enhanced immune cell trafficking to the tumor.

Abstract 1161: EZH2 inhibitor tazemetostat demonstrates activity in preclinical models of Bruton's tyrosine kinase inhibitor-resistant relapsed/refractory mantle cell lymphoma

Abstract Tazemetostat (EZM6438) is a potent, orally bioavailable small molecule inhibitor of EZH2, the enzymatic subunit of the polycomb repressive complex 2, which has been approved for treatment of epithelioid sarcoma and relapsed/refractory follicular lymphoma. EZH2 has been shown to play a key role in B-cell maturation and multiple B-cell malignancies are dependent on EZH2 for survival. Mantle cell lymphoma (MCL) is a rare subtype of mature B cell non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32) translocation leading to overexpression of cyclin D1, which plays a significant role in tumor cell proliferation via cell cycle dysregulation, chromosomal instability, and epigenetic regulation. This disease most often presents at an advanced stage and while initial responses occur, most cases relapse to frontline therapy, including Bruton's Tyrosine Kinase (BTK) inhibitors. The rate of intrinsic and acquired resistance to these treatments results in a high unmet medical need. We previously reported that treatment with EZH2 inhibitors, as monotherapy or in combination with BTK inhibitors, elicited in vitro anti-proliferative activity and in vivo tumor growth inhibition in MCL models, demonstrating that EZH2 may be a promising therapeutic target in this indication. To extend our previous in vitro combination studies, we demonstrated that the recently approved BTK inhibitor, zanubrutinib synergized with tazemetostat and demonstrated that tazemetostat resensitized a subset of cell lines intrinsically resistant to BTK inhibitors. In vivo studies confirmed these findings, showing a significant tumor growth delay when tazemetostat was combined with zanubrutinib in the MCL MINO cell line-derived murine xenograft model compared to the single agent treatments. To understand if EZH2 inhibition offered potential therapeutic benefit to the MCL BTK inhibitor-resistant population, we generated in vitro MCL models of acquired resistance to the BTK inhibitors ibrutinib and zanubrutinib. These cell lines retained the in vitro sensitivity to tazemetostat observed in the parental cell line, suggesting a potential therapeutic option for tazemetostat in the setting of acquired resistance to BTK inhibitors. Additionally, we showed that tazemetostat induced anti-proliferative effects ex vivo in samples derived from MCL patients that were relapsed or refractory to one or more current standard of care agents, including the BTK inhibitor ibrutinib. Subsequent studies aim to identify the mechanisms of growth inhibition driven by tazemetostat in MCL models sensitive and resistant to BTK inhibitors. In summary, these data suggest that tazemetostat treatment (alone or in combination with a BTK inhibitor) could be a potential therapeutic option in the treatment of the MCL patient population that is relapsed/refractory to BTK inhibitor therapy. Citation Format: Jeffrey A. Keats, Arleide Lee, Jeremy C. Cunniff, Weiqing Chen, Revonda Mehovic, Vania Estanek, Crag Markwood, Cuyue Tang, Daniel T. Dransfield, Veronica Gibaja, Alejandra Raimondi. EZH2 inhibitor tazemetostat demonstrates activity in preclinical models of Bruton's tyrosine kinase inhibitor-resistant relapsed/refractory mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1161.

EZH2 Regulates the Correlation between Skin Regeneration and the Duration of Mechanical Stretch

It has been widely recognized that mechanical stretch can regulate the fate of stem cells (SCs). Previous research has shown that short-term mechanical stretch induces SC proliferation by activating the predominant transcription factor YAP, and YAP is a critical modulator in controlling epidermal proliferation. However, our study finds that after this phase, cell growth arrest appears, which is induced by long-term mechanical stretch. In the interfollicular epidermal SCs undergoing long-term mechanical stretch invivo and invitro, the level of H3K27me3 and its histone methyltransferase EZH2 are significantly elevated with suppressed expression of the target genes of YAP. EZH2 forms repressive H3K27me3 that suppresses gene transcription. Small-molecule inhibitor of EZH2 rescues significantly the cell growth arrest in interfollicular epidermal SCs induced by long-term mechanical stretch, thus promoting epidermal proliferation invivo again. These findings reveal that there is an unexpected correlation between SC proliferation and the duration of mechanical stretch regulated by EZH2. This study of long-term mechanical stretch that induces cell growth arrest provides a strategy for clinical translation to promote skin regeneration.

Salinomycin inhibits epigenetic modulator EZH2 to enhance death receptors in colon cancer stem cells

ABSTRACT Drug resistance is one of the trademark features of Cancer Stem Cells (CSCs). We and others have recently shown that paucity of functional death receptors (DR4/5) on the cell surface of tumour cells is one of the major reasons for drug resistance, but their involvement in the context of in CSCs is poorly understood. By harnessing CSC specific cytotoxic function of salinomycin, we discovered a critical role of epigenetic modulator EZH2 in regulating the expression of DRs in colon CSCs. Our unbiased proteome profiler array approach followed by ChIP analysis of salinomycin treated cells indicated that the expression of DRs, especially DR4 is epigenetically repressed in colon CSCs. Concurrently, EZH2 knockdown demonstrated increased expression of DR4/DR5, significant reduction of CSC phenotypes such as spheroid formation in-vitro and tumorigenic potential in-vivo in colon cancer. TCGA data analysis of human colon cancer clinical samples shows strong inverse correlation between EZH2 and DR4. Taken together, this study provides an insight about epigenetic regulation of DR4 in colon CSCs and advocates that drug-resistant colon cancer can be therapeutically targeted by combining TRAIL and small molecule EZH2 inhibitors.

Abstract B5: Reversing epigenetic gene silencing to overcome immune evasion in CNS malignancies

Abstract Glioblastoma (GBM) is a lethal brain malignancy, and standard of care only offers a modest survival benefit. Emerging evidence of immune surveillance in the brain has prompted investigation of immunotherapy as a potential treatment strategy. However, preliminary studies have demonstrated impaired immune cell trafficking to the tumor, making GBMs immunologically “cold.” One mechanism of immune evasion by the tumor is to epigenetically silence the expression of chemoattractant cytokines. Previous studies in ovarian and prostate cancer have successfully established the ability of GSK126, a small-molecule inhibitor of histone methyl transferase EZH2, to promote tumor T-cell infiltration and subsequent antitumor response. Mechanistically, GSK126 was shown to promote expression of interferon gamma (IFN gamma)-induced chemokines CXCL9 and CXCL10. In the present study we evaluated the therapeutic efficacy of GSK126 in preclinical models of GBM. Our studies determined that treatment of murine and human glioblastoma cell lines with GSK126 increased IFN gamma-mediated gene expression of CXCL9 and CXCL10. Transwell migration assays performed using human T cells demonstrated increased T-cell migration when exposed to conditioned medium from tumor cells treated with GSK126 and IFN gamma. In vivo, efficacy of GSK126 was tested in combination with anti-PD-1 antibody in C57Bl/6 immunocompetent mice subcutaneously implanted with the syngeneic glioma cell line CT2A. Mice treated with the combination of GSK126 and anti-PD-1 antibody showed a significant decrease in tumor volume and improved overall survival. Tumors treated with combination therapy showed increased infiltration of CD8+ IFN gamma expressing T cells and increased CXCR3+ migratory T cells in the tumor-draining lymph node. Interestingly, treatment of mice with intracranial CT2A tumors with GSK126 alone resulted in small tumors with better prognosis. Treatment-related survival benefit was lost in immune-compromised mice, suggesting that efficacy depended upon drug-related increased lymphocyte trafficking and subsequent T cell-mediated antitumor response. Analysis of post-treatment tumor by LC-MS confirmed that GSK126 crosses the blood-brain barrier. Taken together, our data suggest a potential synergistic therapeutic role for GSK126 in glioblastoma by enhancing current immunotherapy regimens with reversing the epigenetic changes that enable immune cell evasion with resultant increases in immune cell trafficking to the tumor. Citation Format: Nivedita M. Ratnam, Heather M. Sonnemann, Mark R. Gilbert, Amber J. Giles. Reversing epigenetic gene silencing to overcome immune evasion in CNS malignancies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B5.

Abstract LB-A09: EED targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex

Abstract Deregulation of the PRC2 components EZH2, SUZ12, and EED plays critical roles in driving aberrant hypermethylation of H3K27 and tumorigenicity of many solid and hematological malignancies. Although SAM competitive small molecule inhibitors of EZH2 show promising clinical activity in PRC2-dependent cancers, preclinical data suggests that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (Proteolysis Targeting Chimera) to efficiently target EED for elimination. Our EED-targeting PROTACs bind to EED (pKD= 9.02-9.27) and promote stable ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50= 8.11-8.17) that results in a decrease in H3K27me3 levels in cells. Interestingly, EED-targeting PROTACs induce rapid degradation of EED, as well as its associated proteins, including EZH2 and SUZ12 in the PRC2 complex. Inhibition of the ubiquitin proteasome pathway abrogates PROTAC-mediated degradation of EED and its associated proteins. Furthermore, the EED targeting PROTACs selectively inhibit proliferation and survival of PRC2-dependent cancer cells (GI50= 49-58 nM). In summary, our data demonstrate a novel therapeutic modality in treating PRC2 dependent cancer through PROTAC mediated platform. Citation Format: Jessie Hao-Ru Hsu, Timothy Rasmusson, James Robinson, Fiona Pachl, Jon Read, Sameer Kawatkar, Daniel H O'Donovan, Sharan Bagal, Erin Code, Philip Rawlins, Argyrides Argyrou, Ronald Tomlinson, Ning Gao, Xiahui Zhu, Elisabetta Chiarparin, Kelly Jacques, Minhui Shen, Haley Woods, Emma Bednarski, David M. Wilson, Lisa Drew, M. Paola Castaldi, Stephen Fawell, Andrew Bloecher. EED targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A09. doi:10.1158/1535-7163.TARG-19-LB-A09

Identification and Assessments of Novel and Potent Small-Molecule Inhibitors of EED-EZH2 Interaction of Polycomb Repressive Complex 2 by Computational Methods and Biological Evaluations.

Polycomb repressive complex 2 (PRC2) is an attractive drug target for anti-cancer treatment. Among the three core subunits (EZH2, EED and SUZ12) of PRC2, EZH2 is the catalytic subunit that methylates histone H3 lysine 27 (H3K27), while EED is the regulatory subunit. Besides the small-molecule inhibitors of EZH2, those targeting the protein-protein interaction (PPI) between EZH2 and EED have also been reported. Here, for the first time, we have identified the key residues that contributed most to the EED-EZH2 binding affinity by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations based on the 200 ns molecular dynamics simulation. Moreover, we report the identification of two novel and potent small-molecule inhibitors (35 and 49) of EZH2-EED interaction (bottom interaction surface) by virtual screening and biological evaluations. Binding modes of the two identified molecules with EED were probed by molecular docking. Additionally, 35 and 49 displayed cellular antiproliferative activity against diffuse large B-cell lymphoma (DLBCL) cancer cell line Toledo whose cell growth was driven by aberrant PRC2 activity. Our findings have provided structural insights for the design of novel EZH2-EED interaction inhibitors to regulate the activity of PRC2 complex.

Inhibition of Polycomb Repressive Complex 2 activity reduces trimethylation of H3K27 and affects development in Arabidopsis seedlings

BackgroundPolycomb repressive complex 2 (PRC2) is an epigenetic transcriptional repression system, whose catalytic subunit (ENHANCER OF ZESTE HOMOLOG 2, EZH2 in animals) is responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In mammals, gain-of-function mutations as well as overexpression of EZH2 have been associated with several tumors, therefore making this subunit a suitable target for the development of selective inhibitors. Indeed, highly specific small-molecule inhibitors of EZH2 have been reported. In plants, mutations in some PRC2 components lead to embryonic lethality, but no trial with any inhibitor has ever been reported.ResultsWe show here that the 1,5-bis (3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one compound (RDS 3434), previously reported as an EZH2 inhibitor in human leukemia cells, is active on the Arabidopsis catalytic subunit of PRC2, since treatment with the drug reduces the total amount of H3K27me3 in a dose-dependent fashion. Consistently, we show that the expression level of two PRC2 targets is significantly increased following treatment with the RDS 3434 compound. Finally, we show that impairment of H3K27 trimethylation in Arabidopsis seeds and seedlings affects both seed germination and root growth.ConclusionsOur results provide a useful tool for the plant community in investigating how PRC2 affects transcriptional control in plant development.

Abstract 5190: EZH2 epigenetically silences anti-tumor immunity in melanoma via dual histone and DNA methylation

Abstract Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remain key obstacles. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution. EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Additionally, EZH2 has been shown to interact with DNA methyltransferases (DNMTs). Aberrant EZH2 in cancer results in the silencing of hundreds of tumor suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2. Using ChIP-seq, microarrays and DNA methylation sequencing, we identified a subset of EZH2 target genes that are silenced by both histone and DNA methylation in melanoma. Interestingly many of these genes play a role in the anti-tumor immune response, including RASSF5 and ITGB2 that facilitate lymphocyte infiltration into tumors and correlate with better survival outcomes in melanoma patients when their expression is high. We show that such genes can be significantly up-regulated by combined treatment with inhibitors of both EZH2 and DNMT in melanoma cells in vitro. Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the anti-tumor immune response. The progression of several preclinical, small molecule inhibitors of EZH2 and DNMTs remains promising. Citation Format: Jessamy Tiffen, Dilini Gunatilake, Stuart Gallagher, Peter Hersey. EZH2 epigenetically silences anti-tumor immunity in melanoma via dual histone and DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5190.

Abstract CT094: Phase Ib results of ProSTAR: CPI-1205, EZH2 inhibitor, combined with enzalutamide (E) or abiraterone/prednisone (A/P) in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Increased expression and neomorphic mutations of the histone methyltransferase EZH2 are often observed in cancer, leading to repression of genes associated with apoptosis and differentiation. High EZH2 expression and low expression of EZH2 target genes in mCRPC correlate with poor prognosis. CPI-1205 is a potent, reversible, cofactor-competitive small molecule inhibitor of EZH2. Preclinical studies revealed that prostate cancer models dependent on androgen receptor signaling (ARS) are sensitive to EZH2 inhibition, and that CPI-1205, when combined with novel ARS inhibitors, results in synergistic cell growth inhibition (Bradley 2018). Here we report preliminary results from the Phase Ib component. Methods: In this multicenter Phase Ib/II study, patients (pts) with mCRPC previously treated with a novel ARS inhibitor were enrolled in different cohorts exploring two regimens of oral CPI-1205 on a continuous 28-day cycle: 800 mg TID or 400 mg BID with cobicistat, a CYP3A4 inhibitor, combined with the standard dose of E (160 mg QD) or standard dose of A/P (1000 mg QD/5 mg BID). Prior chemotherapy was allowed. The primary objective in Phase Ib is to determine safety, a recommended Phase II dose, pharmacokinetics (PK), and pharmacodynamics (PD). Results: As of 11/16/2018, 35 pts were enrolled in four cohorts (two cohorts of CPI-1205 + E; two cohorts of CPI-1205 + A/P) and were treated for ≥1 cycle. Age ranged from 55 to 90 years (median 72). 63% were ECOG 0 and 37% ECOG 1. 13 pts (37%) tested positive for ARV7. 21 pts (60%) had unfavorable circulating tumor cells (CTC) counts at baseline. 6 pts (17%) had prior taxane-based chemotherapy for mCRPC. 16 pts received CPI-1205 + E and 19 pts received CPI-1205 + A/P. Only 1 DLT (asymptomatic reversible ALT increase, Grade 4) was reported in the CPI-1205 + A/P + cobicistat. No serious AE related to the study drug was reported in any cohort. Overall, the commonly reported drug-related treatment-emergent adverse events (TEAEs ≥10%) were low-grade diarrhea (n= 11, 31%), fatigue and nausea (n= 9, 26% each), and decreased appetite (n=5, 14%). Grade ≥3 TEAEs were fatigue and elevated ALT (n=2, 6% each), and nausea, pruritus, hypokalemia, and muscular weakness (n=1, 3% each). 16 pts remain on treatment. Cases of PSA >80% reduction, CTC ≥30% reduction, and RECIST response were observed. Both CPI-1205 regimens resulted in significant target engagement (reduced H3K27me3 in PBMCs and CTCs). Patient sample analysis to maximize biomarker identification is ongoing. PK data will be presented. Conclusions: Both CPI-1205 800 mg PO TID and 400 mg PO BID plus cobicistat, combined with full doses of either E or A/P, are well tolerated, with acceptable safety. Encouraging clinical activity has been observed. CPI-1205 800 mg PO TID + E or A/P has been selected for phase 2 expansion, and patient enrollment is ongoing Citation Format: Mary-Ellen Taplin, Arif Hussain, Satish Shah, Neal D. Shore, William Jeffery Edenfield, Oliver A. Sartor, Luke T. Nordquist, Manish Agrawal, William Clark, David R. Wise, William K. Oh, Mark T. Fleming, James E. Butrynski, Gurkamal S. Chatta, Manojkumar Bupathi, Claudia Lebedinsky, Adrian Senderowicz, Jian Li, Gozde Colak, David Nash, Patrick Trojer, William D. Bradley, Jessica Piel, Emmanuel S. Antonarakis. Phase Ib results of ProSTAR: CPI-1205, EZH2 inhibitor, combined with enzalutamide (E) or abiraterone/prednisone (A/P) in patients with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT094.

ProSTAR: A phase Ib/II study of CPI-1205, a small molecule inhibitor of EZH2, combined with enzalutamide (E) or abiraterone/prednisone (A/P) in patients with metastatic castration-resistant prostate cancer (mCRPC).

TPS335 Background: EZH2 is frequently mutated and/or overexpressed in numerous cancers. High EZH2 expression is correlated with poor outcomes in prostate cancer patients (Varambally, 2002). EZH2 inhibition combines synergistically with anti-androgen therapies in preclinical models of advanced prostate cancer, suggesting epigenetic reprogramming as a pathophysiologic mechanism to enhance the combination therapy (Ku, 2017; Xiao 2018; Constellation, unpublished). CPI-1205 is a potent, selective, and cofactor-competitive inhibitor of wild type and mutant EZH2 catalytic activity, which demonstrates anti-proliferative effects in prostate and other cancer cell models. Methods: We present a Phase 1b/2 multicenter study of CPI-1205 combined with either E or A/P in patients with mCRPC, which includes phase 1 dose escalation, an expansion cohort in heavily pretreated patients (HPEC), and a randomized phase 2 study. Key eligibility criteria include progressive mCRPC in patients previously treated with a second-generation androgen inhibitor, ECOG 0-1, and measurable or non-measurable disease. During the phase 1b, patients will receive CPI-1205 continuously in 28-day cycles combined with the standard dose of either E (160mg PO once daily) or A/P (1000mg PO once daily/5 mg BID). The primary objective in Phase 1b is to determine the safety, tolerability, and recommended phase 2 dose (RP2D) of CPI-1205 combined with either E or A/P. Secondary objectives include safety, pharmacokinetic and pharmacodynamic profiles, and anti-tumor activity. The HPEC arm, with Simon’s 2-stage design, may begin with the regimen that is deemed safe by the study safety committee. The primary endpoint of the HPEC arm is objective response rate (ORR) per PCWG3, with a requirement ≥ 1 measurable lymph node at baseline. Once RP2D is established, we will start a randomized Phase 2 trial of E or A/P combined with CPI-1205 vs. E or A/P alone. The co-primary endpoint includes PSA50 and composite response rate (either CTC 30% reduction or ORR per PCWG3). Patient accrual in US sites began in December 2017. Clinical trial information: NCT03480646.

Epigenetic Targeting of the Myeloma-Bone Microenvironment in 3D

EZH2, the methyltransferase subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes H3K27me3 histone modifications and epigenetically regulates genes involved in cellular pluripotency and differentiation. EZH2 plays a role in myeloma (MM) cell proliferation, survival, stemness and its elevated expression correlates with poor prognosis in MM patients. We have previously shown that EZH2 plays a critical role in preventing osteoblast differentiation of myeloma-exposed bone marrow stromal cells (BMSCs). Here we show that GSK126 blocks MM-induced hyperactivation of osteoclast precursors (OCLp). RNA-seq profiling revealed that inhibition of EZH2 prevented RANKL-induced repression of genes associated with bivalent and/or H3K27me3 promoter signatures including OCL inhibitory factors MafB, Irf8, Bcl6b and Arg1. In contrast, we found that OCLp expansion in MM1.S-conditioned media induced significant gene expression changes, which correlated with TNF and IKK signaling, inflammatory responses and CXC-chemokine receptor pathways.Several classes of small molecule EZH2 inhibitors exhibited anti-MM effects, but their efficacy has been primarily studied in 2-dimensional (2D) cell culture systems or subcutaneous MM-tumor models in vivo. Therefore, we evaluated the effectiveness of GSK126 in the context of the bone microenvironment in a novel 3D model of MM co-cultures (3D-MM). We combined basement membrane extract (BME) hydrogels with devitalized bone slices to mimic the 3D setting of MM with the OCL-resorbing endosteal surface. This enabled us to test GSK126 alone or in combination with bortezomib simultaneously on MM survival and OCLp differentiation and resorption. Differentiating OCLp did not protect MM cells from GSK126 anti-MM effects, nor did the MM cells prevent GSK126 from blocking OCL differentiation. However, mature OCL added to 3D-MM co-cultures increased the IC50 MM inhibition dose of both bortezomib and GSK126 by 40% and 50%, respectively. Further their synergy on MM cells was reduced by 70%.3D-MM co-cultures with total bone marrows harvested from mice of different ages (1-12 months old) showed selective protection from GSK126, but not bortezomib, on MM viability. Furthermore, the resistance to GSK126 was age-dependent. Cultures of bone marrows from older mice exhibited enhanced protection of MM cells from GSK126 as compared to younger marrows. Using confocal microscopy, we found that in addition to soluble factors, physical interaction between MM and bone marrow cells reduced the effectiveness of the epigenetic drug targeting against MM. Depletion of the BMSC population from the total bone marrows using CD45+ selection before establishing the 3D MM co-cultures resulted in diminished protection of MM cell survival from GSK126. Consistent with this, addition of both primary murine and MM-patient derived BMSCs to MM cultures significantly protected MM cells from EZH2 inhibition. In addition to cell-cell contacts, the pro-survival factor IL6 released by mature OCL and BMSCs, has been implicated in mediating chemo-resistance of MM cells. In agreement with this, addition of soluble IL6 to MM-3D cultures significantly protected MM cells from GSK126 inhibition.Here we show that various cell compartments of the bone microenvironment exhibit differential and drug-specific protection for MM cells from EZH2 inhibition. In addition to direct bone marrow-MM cell interactions, soluble IL6 also exhibits resistance to GSK126. Our novel 3D-MM system enables us to rapidly screen drug combinations, and simultaneously evaluate the influence of bone-microenvironmental interactions on MM drug resistance and bone marrow cell responses to the drugs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 808: Opportunity for therapeutic expansion in mantle cell lymphoma: Tazemetostat combination synergy status in preclinical MCL models

Abstract Tazemetostat (EPZ-6438) is a potent, selective, orally bioavailable small molecule inhibitor of EZH2, the enzymatic subunit of the polycomb repressive complex 2, which is currently being evaluated in multiple phase II clinical trials for the treatment of non-Hodgkin lymphoma, mesothelioma and molecularly defined solid tumors. Objective clinical responses have been observed in phase II studies of tazemetostat, including patients with B-cell lymphomas. EZH2 has been shown to play a key role in the maturation of B-cells and, consistent with this phenomenon, multiple B-cell malignancies are dependent on EZH2 for survival. In preclinical studies in mantle cell lymphoma (MCL), a disease arising from mantle zone cells surrounding germinal centers, treatment with EZH2 inhibitors, in in vitro and in vivo models demonstrated antiproliferative activity, suggesting EZH2 may be a promising therapeutic target for MCL. MCL is a distinct B-cell non-Hodgkin lymphoma characterized by the chromosomal translocation t(11;14) that leads to overexpression of cyclin D1. The disease most often presents at an advanced stage, and while initial responses occur, nearly all cases relapse or become resistant to frontline therapy and progress with poor prognosis. Novel targeted therapies are currently entering the clinic in combination with standard of care treatments, but heterogeneous response rates and durations of response show that there remains an unmet medical need in this population. The most widely used MCL treatment regimens include combinations of two or more Standard of Care (SOC) agents. In order to support potential future clinical investigations with tazemetostat we established in vitro assays to assess its combinatorial activity with 24 traditional and emerging therapies across seven MCL cell lines. All compounds were evaluated for their single agent activity in order to determine the appropriate starting doses to investigate synergy. We then looked at both a seven-day pretreatment with tazemetostat alone followed by a four-day cotreatment with combination partner as well as a seven-day cotreatment including both drugs simultaneously. Synergy status was determined via Loewe, BLISS and additional mathematical models. Robust synergy was observed with glucocorticoid receptor agonists, immunomodulatory drugs, venetoclax and a variety of B-cell receptor pathway modulators in both dosing regimens. In addition, we show that EPZ011989 (an EZH2 tool compound) induces tumor growth inhibition in a MCL xenograft model, and has combination benefit with ibrutinib. Preclinical data suggest that the sensitivity to tazemetostat as a single agent in MCL cell lines can be greatly enhanced in combination with new and emerging therapies. These combination approaches may be worthy of exploration in the clinic for the treatment of MCL. Citation Format: Tami L. Hood, Kat Cosmopoulos, Allison Drew, Kelli Armstrong, Jennifer Johnson, Smith Jesse, Alejandra Raimondi. Opportunity for therapeutic expansion in mantle cell lymphoma: Tazemetostat combination synergy status in preclinical MCL models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 808.

Abstract 1384: The epigenetic modifier EZH2 represses antitumor immunity in melanoma

Abstract Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remains a key obstacle. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution. EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Aberrant EZH2 in cancer results in the silencing of hundreds of tumor-suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2. Using ChIP-seq and microarrays to reveal downstream EZH2 target genes, we identified many tumor-suppressor genes including some that play a role in the antitumor immune response. Interestingly, xenografts in immune-deficient NOD SCID mice showed no significant inhibition in tumor growth when treated with an EZH2 inhibitor. However, growth inhibition was achieved when the same experiment was performed in nude mice, which retain some mediators of innate immunity. The effect of EZH2 overexpression in melanoma on immune-mediated rejection was studied in an inducible in vivo model. Subcutaneous B16 tumors expressing model antigen were initially rejected but relapsed in the majority of immunodeficient mice transferred with CD4 T cells. Long-term (120-day) survival was reduced from 40% to 22% if the tumors also overexpressed EZH2. Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the antitumor immune response. The progression of several preclinical, small-molecule inhibitors of EZH2 remains promising. Citation Format: Jessamy Tiffen, Elena Shklovskaya, Stuart Gallagher, Dilini Gunatilake, Peter Hersey. The epigenetic modifier EZH2 represses antitumor immunity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1384.

42O - A phase 1 study of CPI-1205, a small molecule inhibitor of EZH2, preliminary safety in patients with B-cell lymphomas

42O - A phase 1 study of CPI-1205, a small molecule inhibitor of EZH2, preliminary safety in patients with B-cell lymphomas

A phase 1b/2 study of CPI-1205, a small molecule inhibitor of EZH2, combined with enzalutamide (E) or abiraterone/prednisone (A/P) in patients with metastatic castration resistant prostate cancer (mCRPC).

TPS398 Background: EZH2 is frequently mutated and overexpressed in human cancer. High levels of expression are correlated with disease stage, aggressiveness, and poor outcome in patients with prostate cancer (Varambally, 2002). EZH2 inhibition restores androgen receptor expression and sensitivity to antiandrogen therapy in preclinical models of advanced prostate cancer, suggesting epigenetic reprogramming as an approach to overcome resistance to antiandrogen therapy (Ku, 2017). CPI-1205 is a potent, selective and cofactor-competitive EZH2 inhibitor, that inhibits both wild-type and mutant EZH2 catalytic activity in a reversible manner. Preclinical studies have shown profound anti-proliferative effects of CPI-1205 in treating both lymphoma and prostate cancer cell models. Antitumor activity of EZH2 methyltransferase inhibitors has been observed in patients with non-Hodgkin’s lymphoma. These observations support the clinical evaluation of CPI-1205 with standard doses of A/P or E in mCRPC. Methods: We present a Phase 1b/2 study to explore the safety and efficacy of two regimens of CPI-1205 in combination with either E or A/P. Key eligibility criteria include progressive mCRPC in patients previously treated with a second generation androgen inhibitor, ECOG 0-1 and measurable or non- measurable disease. Key exclusion criteria include > 1 second generation androgen inhibitor. During the phase 1b, sequentially enrolled cohorts will receive CPI-1205 at continuous – 28-day cycles combined with either standard dose of E (160mg PO once daily) or A/P (1000mg PO once daily/ 5 mg BID). The primary objective is to determine the maximum tolerated dose and recommended phase 2 dose (RP2D) of each regimen. Secondary objectives include safety, pharmacokinetic, pharmacodynamics profiles and activity (PSA, circulating tumor cells, imaging) in mCRPC. Once RP2D is established, we will start a Phase 2 trial of a 2nd generation androgen inhibitor combined with CPI-1205.