Pharmacological Inhibition Of EZH2 Research Articles

Abstract WMP114: EZH2 Inhibitor Improves Cognitive Behavior in CAA

Introduction: Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid-β (Aβ) in the leptomeningeal and cortical vessels. CAA may lead to dementia and stroke; however effective options currently are very limited to treat or halt CAA progression. EZH2 (enhancer of zeste 2 polycomb repressive complex 2) is a histone methyltransferase, a key regulator of inflammation and microglia phagocytosis. The present study investigates the therapeutic potential of an EZH2 inhibitor (GSK343) in CAA mice. Methods: Brain samples were collected from a cohort of 4 WT and 4 TgSwDI (CAA model) male mice and lysed for immunoblot analysis to quantify EZH2 and H3K27me3 expression levels. A second cohort of 10 WT and 20 TgSwDI male mice (15 months old) were randomly assigned into DMSO (vehicle) and GSK343 groups. After performing baseline behavioral testing (Barnes maze and NORT), WT and TgSwDI-DMSO groups were injected with 0.1% DMSO while TgSwDI-GSK343 groups were injected with GSK343 (5 mg/kg) every other day intraperitoneally. After 6 weeks of treatment, behavioral testing was performed, and brain tissues were collected for IHC. Data are presented as Mean±SEM. Results: Immunoblot assays showed elevated expression of EZH2 and H3K27me3 in TgSwDI in comparison to WT mice. TgSwDI mice injected with GSK343 had improvements in the ability to recognize a novel object (58.36%±1.84) as compared to TgSwDI controls (45.04%±3.41) in the NORT ( p = 0.0006). The latency to escape (63.08±2.86) was significantly faster in GSK343-treated mice compared to TgSwDI control mice (77.34±3.43) in the Barnes maze ( p = 0.003). IHC results were in line with behavioral testing as GSK343 reduced the expression of EZH2 and Aβ in the hippocampus and frontal cortex. Conclusions: Our study revealed that EZH2 expression is upregulated in the brain of CAA-model mice. Pharmacological inhibition of EZH2 improved cognitive outcomes in CAA mice, suggesting a possible therapeutic target.

Sustained activation of NF-κB through constitutively active IKKβ leads to senescence bypass in murine dermal fibroblasts

ABSTRACT Although the transcription factor nuclear factor κB (NF-κB) plays a central role in the regulation of senescence-associated secretory phenotype (SASP) acquisition, our understanding of the involvement of NF-κB in the induction of cellular senescence is limited. Here, we show that activation of the canonical NF-κB pathway suppresses senescence in murine dermal fibroblasts. IκB kinase β (IKKβ)-depleted dermal fibroblasts showed ineffective NF-κB activation and underwent senescence more rapidly than control cells when cultured under 20% oxygen conditions, as indicated by senescence-associated β-galactosidase (SA-β-gal) staining and p16INK4a mRNA levels. Conversely, the expression of constitutively active IKKβ (IKKβ-CA) was sufficient to drive senescence bypass. Notably, the expression of a degradation-resistant form of inhibitor of κB (IκB), which inhibits NF-κB nuclear translocation, abolished senescence bypass, suggesting that the inhibitory effect of IKKβ-CA on senescence is largely mediated by NF-κB. We also found that IKKβ-CA expression suppressed the derepression of INK4/Arf genes and counteracted the senescence-associated loss of Ezh2, a catalytic subunit of the Polycomb repressive complex 2 (PRC2). Moreover, pharmacological inhibition of Ezh2 abolished IKKβ-CA-induced senescence bypass. We propose that NF-κB plays a suppressive role in the induction of stress-induced senescence through sustaining Ezh2 expression.

Pharmacological inhibition of human EZH2 can influence a regenerative β-like cell capacity with in vitro insulin release in pancreatic ductal cells

BackgroundTherapeutic replacement of pancreatic endocrine β-cells is key to improving hyperglycaemia caused by insulin-dependent diabetes . Whilst the pool of ductal progenitors, which give rise to the endocrine cells, are active during development, neogenesis of islets is repressed in the human adult. Recent human donor studies have demonstrated the role of EZH2 inhibition in surgically isolated exocrine cells showing reactivation of insulin expression and the influence on the H3K27me3 barrier to β-cell regeneration. However, those studies fall short on defining the cell type active in transcriptional reactivation events. This study examines the role of the regenerative capacity of human pancreatic ductal cells when stimulated with pharmacological inhibitors of the EZH2 methyltransferase.ResultsHuman pancreatic ductal epithelial cells were stimulated with the EZH2 inhibitors GSK-126, EPZ6438, and triptolide using a 2- and 7-day protocol to determine their influence on the expression of core endocrine development marker NGN3, as well as β-cell markers insulin, MAFA, and PDX1. Chromatin immunoprecipitation studies show a close correspondence of pharmacological EZH2 inhibition with reduced H3K27me3 content of the core genes, NGN3, MAFA and PDX1. Consistent with the reduction of H3K27me3 by pharmacological inhibition of EZH2, we observe measurable immunofluorescence staining of insulin protein and glucose-sensitive insulin response.ConclusionThe results of this study serve as a proof of concept for a probable source of β-cell induction from pancreatic ductal cells that are capable of influencing insulin expression. Whilst pharmacological inhibition of EZH2 can stimulate secretion of detectable insulin from ductal progenitor cells, further studies are required to address mechanism and the identity of ductal progenitor cell targets to improve likely methods designed to reduce the burden of insulin-dependent diabetes.

Pharmacological inhibition of EZH2 by ZLD1039 suppresses tumor growth and pulmonary metastasis in melanoma cells in vitro and in vivo

The incidence and mortality rate of malignant melanoma are increasing worldwide. Metastasis reduces the efficacy of current melanoma therapies and leads to poor prognosis for patients. EZH2 is a methyltransferase that promotes the proliferation, metastasis, and drug resistance of tumor cells by regulating transcriptional activity. EZH2 inhibitors could be effective in melanoma therapies. Herein, we aimed to investigate whether the pharmacological inhibition of EZH2 by ZLD1039, a potent and selective S-adenosyl-l-methionine-EZH2 inhibitor, suppresses tumor growth and pulmonary metastasis in melanoma cells. Results showed that ZLD1039 selectively reduced H3K27 methylation in melanoma cells by inhibiting EZH2 methyltransferase activity. Additionally, ZLD1039 exerted excellent antiproliferative effects on melanoma cells in 2D and 3D culture systems. Administration of ZLD1039 (100 mg/kg) by oral gavage caused antitumor effects in the A375 subcutaneous xenograft mouse model. RNA sequencing and GSEA revealed that the ZLD1039-treated tumors exhibited changes in the gene sets enriched from the “Cell Cycle” and “Oxidative Phosphorylation”, whereas the “ECM receptor interaction” gene set had a negative enrichment score. Mechanistically, ZLD1039 induced G0/G1 phase arrest by upregulating p16 and p27 and inhibiting the functions of the cyclin D1/CDK6 and cyclin E/CDK2 complexes. Moreover, ZLD1039 induced apoptosis in melanoma cells via the mitochondrial reactive oxygen species apoptotic pathway, consistent with the changes in transcriptional signatures. ZLD1039 also exhibited excellent antimetastatic effects on melanoma cells in vitro and in vivo. Our data highlight that ZLD1039 may be effective against melanoma growth and pulmonary metastasis and thus could serve as a therapeutic agent for melanoma.

Abstract C015: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC

Abstract T-cell based immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be a powerful way to enhance T cell infiltration into tumors and reactivate a different type of innate Natural Killer (NK) cell anti-tumor immunity that can mediate tumor control. Here, we found that the pancreas tumor microenvironment (TME) suppresses NK cell surveillance following therapy-induced senescence (TIS) with MEK and CDK4/6 inhibitor treatment through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity enhanced the production of pro-inflammatory SASP factors and led to NK and T cell infiltration and immune-mediated tumor control in transplanted and genetically engineered PDAC mouse models. Mechanistically, EZH2 suppression induced expression of SASP factors CCL2 and CXCL9/10 necessary for lymophocyte chemotaxis into the pancreas TME. EZH2 levels were also associated with reduced NK cell numbers, SASP expression, and overall survival in a PDAC patient cohort. Taken together these results demonstrate that EZH2 mediates repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK and T cell surveillance in PDAC to achieve immune-mediated tumor responses. Citation Format: Loretah Chibaya, Katherine C. Murphy, Yvette Lopez Diaz, Kelly D. DeMarco, Haibo Liu, Sneha Gopalan, Melissa Faulkner, Junhui Li, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Elisa de Stanchina, Karl Simin, Lihua J. Zhu, Thomas G. Fazzio, Scott W. Lowe, Marcus Ruscetti. EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C015.

Pharmacological targeting EZH2 to modulate chronic graft-versus-host disease

Pharmacological targeting EZH2 to modulate chronic graft-versus-host disease

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Abstract 3279: Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations

Abstract Background: Lymphomas are characterized by aberrations in epigenetic proteins that contribute to establishing and maintaining the malignant phenotype. Gain of function mutations in EZH2 histone methyltransferase and inactivating mutations in CREBBP acetyltransferase occur in up to 30% of diffuse large B cell lymphomas (DLBCLs). Half of DLBCLs with mutated EZH2 have also mutated CREBBP. In vitro, mutated EZH2 DLBCLs have a lower sensitivity to HDAC inhibitors, HDACi (Mensah et al, 2021). Here, we explored the dual pharmacological inhibition of EZH2 and HDAC for an improved anti-lymphoma activity. Methods: Biochemical evaluation and characterization of target engagement were done using fluorescence polarization, thermal shift, surface plasmon resonance, isothermal titration calorimetry and microscale thermophoresis. Computer modelling was performed using available EZH2 and HDACs crystal structures. Anti-proliferative activity was assessed after 7 days (d) using MTT and live imaging in DLBCL cells (n = 5). For cell cycle analysis by flow cytometry, cells were treated, fixed then stained with 7-AAD. Results: We designed and synthesized 2 dual EZH2/HDAC inhibitors, MC4343 and MC4355, starting from the structures of EZH2 inhibitor tazemetostat (taz) and HDACi vorinostat. In biochemical assays, MC4343 and MC4355 had equal potencies towards EZH2 (0.032 nM) but different specificities towards class I and class II HDACs: MC4355 showed 7.5-fold greater inhibition of HDAC3 compared to MC4343 (0.38 µM and 2.85 µM, respectively) and more potently inhibited HDACs 6 and 8 (0.016, 0.17 µM and no activity, respectively). Computational modelling showed that the coordinative functional group of MC4343, but not of MC4355, caused steric clashes with several HDACs in increasing order: HDAC1 = HDAC3 > HDAC8 >> HDAC4 = HDAC6. These results closely mirrored those obtained from the biochemical analysis. Both compounds inhibited proliferation in DLBCL cell lines irrespective of EZH2 or CREBBP mutational status but EZH2 and/or CREBBP mutants were more sensitive. MC4355 (IC50 range = 0.17 - 1.68 µM; median = 0.2 µM) was more potent than MC4343 (IC50 range = 0.17-2.72 µM; median = 1.78 µM). This was confirmed by live imaging analyses. SUDHL4, with EZH2 Y666N, showed poorer sensitivity to both inhibitors compared to DLBCLs with EZH2 Y646N/S. MC4343 and MC4355 induced cell death and G1 arrest in a dose-dependent manner. Pfeiffer, KARPAS422, WSUDLCL2, most sensitive to taz alone (IC50 = 4, 16, 77 nM), were most sensitive to MC4355 (IC50 = 200, 174, 171 nM). Notably, dual inhibitor treatment of Toledo and SUDHL4 with low sensitivity to taz (IC50 = 9, 14 µM), partially rescued sensitivity (IC50 = 1.7, 1.6 µM). Conclusions: We designed and synthesized 2 novel dual EZH2/HDAC inhibitors, MC4343 and MC4355, with robust anti-proliferative effects in DLBCL. Our data show the efficacy of this novel class of epigenetic agents in lymphomas. Citation Format: Afua Adjeiwaa Mensah, Sergio Valente, Milos Matkovic, Giulio Sartori, Chiara Falzarano, Chiara Tarantelli, Luciano Cascione, Stefano A. Pileri, Andrea Cavalli, Antonello Mai, Francesco Bertoni. Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3279.

CAIRE: A basket multicenter open-label phase 2 study evaluating the EZH2 inhibitor tazemetostat in combination with durvalumab in patients with advanced solid tumors.

TPS2703 Background: Recent studies have shown shown that genetic depletion of EZH2 in Tregs (using FoxP3creEZH2fl/fl mice) and pharmacological inhibition of EZH2 elicits phenotypic and functional alterations of Tregs, leading to an effector-like T cell profile. Further, EZH2 inhibition enhances the cytotoxicity of human Teffs in vitro and the proportion of tumor-infiltrating cytotoxic T cells in vivo in murine models. It has been shown that immune checkpoint inhibition (ICI) increases EZH2 expression in human T cells across various tumor types and that increased EZH2 expression in T cells inversely correlate with clinical outcome. Upregulation of EZH2 mediated by immune checkpoint inhibition in T cells modulates T cell responses and diminishes the effectiveness of immunotherapy. Consistent with this mechanism, pharmacologic inhibition of EZH2 has been shown to increase effector-like T cell responses and enhances effectiveness of immune checkpoint therapy in tumor-bearing mice [16-17]. Our group also reported first clinical data from a participant with sarcoma showing strong T cell infiltration after treatment with tazemetostat (Italiano et al Lancet Oncol 2018). Altogether, these findings pave the way for clinical trials combining ICI with the specific EZH2 inhibitor, tazemetostat in solid tumors. Methods: CAIRE (NCT04705818) is a multi-cohort, four single-arm phase 2, multicenter, open-label study investigating tazemetostat combined with durvalumab in patients with advanced cancers: pancreatic adenocarcinoma (cohort A), microsatellite stable colorectal cancer (cohort B), solid tumors with presence of tertiary lymphoid structures (assessed centrally) (cohort C), soft-tissue sarcomas (cohort D). Cohort A will enroll ̃32 patients and cohorts B, C and D ̃47 patients respectively. Eligible patients must have no standard treatment options available or a contraindication to standard treatment options, and ECOG PS 0–1. Patients with prior exposure to EZH2 inhibitor and/or PD1/PDL1 monoclonal antibodies are excluded. Tazemetostat will be administered per-os, twice daily (800 mg x 2), continuously. Treatment by Tazemetostat will start on Day 1 (of cycle 1). Durvalumab will be administered by intravenous infusion (1120 mg) on day 1, every 3 weeks. Treatment by Durvalumab will start on Day 1 of cycle 2. The primary endpoints are disease control rate within 24 weeks of treatment as per RECIST 1.1 in cohort A and B; objective response rate within 24 weeks of treatment as per RECIST 1.1 in cohort C, and 6-months non progression rate for cohort D. Secondary endpoints include adverse events (AEs)/serious AEs, duration of response, and progression-free survival. Pharmacodynamic and other biomarkers will be explored. The first patient received study drug on July, 30, 2022 and 1 site across France are currently enrolling patients. Clinical trial information: NCT04705818.

Abstract PD3-04: Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype

Abstract Triple-negative breast cancer (TNBC) is a collection of biologically diverse cancers characterized by distinct transcriptional patterns, biology and immune composition. TNBCs display transcriptional diversity with at least four tumor-intrinsic subtypes that include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Using integrative analyses of transcriptomic, epigenetic, proteomic and phospho-proteomic patterns we have identified subtype-specific vulnerabilities, which advanced our understanding of the cellular origins of TNBC subtypes. In mesenchymal subtype tumors we observed high mutation load and genomic instability, absence of immune cells, low PD-L1 expression, loss of global DNA methylation and transcriptional repression of antigen presentation genes through the polycomb repressor complex 2 (PRC2). Using cell line models, we demonstrate that MHC-I expression is epigenetically silenced by the PRC2 in mesenchymal TNBC cell lines and inhibition of EZH2 restores expression. Pharmacological inhibition of EZH2 enhances chemotherapy efficacy in syngeneic murine tumor models providing a rationale for using PRC2 inhibitors in PD-L1 negative mesenchymal tumors. Citation Format: Brian D Lehmann, Antonio Colaprico, Tiago C Silva, Jianjiao Chen, Hanbing An, Yuguang Ban, Lily Wang, Jamaal L James, Justin Balko, Paula I Gonzalez-Ericsson, Melinda E Sanders, Bing Zhang, Jennifer A Pietenpol, Xi S Chen. Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-04.

EZH2 Mediates Proliferation, Migration, and Invasion Promoted by Estradiol in Human Glioblastoma Cells.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors. 17β-estradiol (E2) increases proliferation, migration, and invasion of human GBM cells; however underlying mechanisms are no fully understood. Zeste 2 Enhancer Homologous enzyme (EZH2) is a methyltransferase part of Polycomb 2 repressor complex (PRC2). In GBM, EZH2 is overexpressed and involved in the cell cycle, migration, and invasion processes. We studied the role of EZH2 in the pro-oncogenic actions of E2 in human GBM cells. EZH2 gene silencing and pharmacological inhibition of EZH2 blocked proliferation, migration, and invasion of GBM cells induced by E2. We identified in silico additional putative estrogen response elements (EREs) at the EZH2 promoter, but E2 did not modify EZH2 expression. In silico analysis also revealed that among human GBM samples, EZH2 expression was homogeneous; in contrast, the heterogeneous expression of estrogen receptors (ERs) allowed the classification of the samples into groups. Even in the GBM cluster with high expression of ERs and those of their target genes, the expression of PCR2 target genes did not change. Overall, our data suggest that in GBM cells, pro-oncogenic actions of E2 are mediated by EZH2, without changes in EZH2 expression and by mechanisms that appear to be unrelated to the transcriptional activity of ERs.

Targeting Chemotherapy to Decondensed H3K27me3-Marked Chromatin of AML Cells Enhances Leukemia Suppression.

Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death-promoting and growth-inhibitory genes.In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities. SIGNIFICANCE: Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy.See related commentary by Adema and Colla, p. 359.

Abstract PO-124: EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses

Abstract T cell-targeting immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that cellular senescence and its associated secretory phenotype (SASP), which leads to production of proinflammatory cytokines, chemokines, and growth factors, can be a powerful way to reactivate a different type of Natural Killer (NK) cell immunity. Recently, we found that RAS pathway targeting MEK and CDK4/6 inhibitors can induce senescence in KRAS mutant lung tumor and PDAC models; however, therapy-induced senescence (TIS) led to NK cell-mediated tumor regressions only in the lungs. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for PDAC immunotherapy. Syngeneic murine KRAS-driven lung tumor and PDAC cells were transplanted into lungs, pancreas, or liver of C57BL/6 mice. Following 2-week treatment with the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P) to induce senescence, immune responses were assessed by flow cytometry, and the SASP profile assessed by RNA- and ATAC-seq analysis. An NK1.1-targeted antibody was also used to deplete NK cells and evaluate treatment efficacy. shRNA-mediated EZH2 knockdown and treatment with EZH2 methyltransferase inhibitors in murine and human PDAC cells and mouse models was used to determine the role of EZH2 in SASP regulation by qPCR and cytokine array. The effects of EZH2 blockade on NK cell immunity were determined in co-culture migration and cytotoxicity assays in vitro and in PDAC transplant models in vivo by flow cytometry. The efficacy of T/P treatment in combination with EZH2-targeting shRNAs or inhibitors was measured by ultrasound tumor measurements and survival in PDAC-bearing animals. Tumors propagated in the pancreas TME display transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15,-18) and chemotaxis (CCL2,-7,-8; CXCL9,-10) following TIS, and do not undergo anti-tumor NK immune surveillance as compared to tumors grown in the lungs and liver. We identified induction of EZH2 and repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell infiltration and anti-tumor cytotoxicity in PDAC models. Remarkably, EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in PDAC-bearing mice that were reversed following NK cell depletion. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with TIS could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor control. Citation Format: Loretah Chibaya, Yvette Lopez-Diaz, Haibo Liu, Katherine C. Murphy, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Lakhena Leang, Elisa de Stanchina, Lihua J. Zhu, Scott W. Lowe, Marcus Ruscetti. EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-124.

STEM-07. PRC2 MAINTAINS A CANCER STEM CELL PHENOTYPE IN GLIOBLASTOMA MULTIFORME VIA CHROMATIN MODIFICATION OF H3K27me3

Abstract Multi-potent stem-like cells (i.e. cancer stem cells, CSCs) are critical determinants of tumor propagation, therapeutic resistance, and recurrence in glioblastoma (GBM). Modifications in chromatin architecture play a fundamental role in the tumor cell phenotype of GBM. The polycomb repressor complex 2 (PRC2) is a key histone modifier that supports multi-potency and oncogenesis via H3K27 trimethylation (H3K27me3). Understanding how these epigenetic modifications cooperatively drive cancer cell stemness should unveil new targets for therapeutic development in GBM. Using a combination of next-generation sequencing, bioinformatics, and molecular approaches we identified EZH2, the catalytic domain of the PRC2 complex, as a critical mediator of reprograming events in GBM cells. We found that EZH2 is highly induced in response to transgenic Oct4/Sox2 with global increases in H3K27me3. Pharmacological inhibition of EZH2 diminishes self-renewal capacity of GBM neurospheres concurrent with a reduction in gene expression levels of markers and drivers of stemness. Furthermore, we identified and validated a set of 6 putative tumor suppressor genes repressed by Oct4 and Sox2 in a PRC2-dependent manner. We identified miR-217 as an EZH2 regulator in GBM cells and miR-217 reconstitution using advanced nanoparticle formulations re-activates the PRC2-repressed tumor suppressors, inhibited tumor growth and enhanced the effects of ionizing radiation in an orthotopic model of GBM. Taken together, these data show that PRC2-mediated chromatin changes in H3K27me3 help regulate the stem-cell phenotype induced by Oct4 and Sox2 in GBM cells and predict that targeting EZH2 could have therapeutic benefit in GBM.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

SUMMARYThe dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients.

Contrasting Stage Dependent Requirements for the Histone Methyltransferase EZH2 during the Evolution of Acute Myeloid Leukemia

Epigenetic regulators function in a context-dependent manner and are commonly mutated in tumors. Activating mutations and overexpression of the histone-methyltransferase, enhancer of zeste, homolog 2 (EZH2), occur in lymphoma and other malignancies, whilst loss-of-function mutations are predicted in myeloid malignancies. We study this apparent contradiction, examining the importance of cellular context and disease stage for Ezh2 loss, genetically deleting Ezh2 during the induction or maintenance of a single malignancy, Acute Myeloid Leukemia (AML).We initially focused on the role of Ezh2 in AML maintenance, using conditional Ezh2fl/fl mice. Following in vitro retroviral immortalization of haematopoietic stem and progenitor cells (HSPCs) from these with AML-fusion oncogenes: MLL-AF9 or AML1-ETO9a (reflecting two separate models of AML clinically associated with good and poor prognosis, markedly differing in mechanism of leukemic transformation and representing AML heterogeneity), these were then transduced with p-babe-Cre-puro vector (or empty vector) to delete Ezh2 . Strikingly, Ezh2 loss completely abolished cellular growth and colony formation, whilst empty vector transduced cells maintained their clonogenic output. To interrogate Ezh2 as a target in AML maintenance in vivo, we generated secondary MLL-AF9 or AML1-ETO9a leukemias through transplantation of bulk leukemia derived from Ezh2fl/fl ; Mx1-Cre primary leukemic mice. Following engraftment, deletion of Ezh2 was achieved by pIpC administration. Loss of Ezh2 in vivo markedly impaired both secondary MLL-AF9 and AML1-ETO9a leukemia progression. These data suggest that Ezh2 is an oncogene across multiple disparate subtypes of established AML and propose it as a possible therapeutic target in this context .Addressing this hypothesis, we examined the role of pharmacological inhibition of EZH2 in AML, utilizing GSK343, an S-Adenosyl Methionine competitive EZH2 inhibitor. Through colony formation and functional assays, we demonstrated in vitro sensitivities of MLL-AF9 transformed murine BM HSPCs, primary murine Ezh2+/+ MLL-AF9 tumours , the AML1-ETO fusion driven human cell line Kasumi-1 and primary leukemic cells isolated from patients with various AML genotypes to GSK343 at similar IC50 (~10µM) across all cells. In contrast, minimal effects of GSK343 were seen on normal primary CD34+ HSCs clonogenic function. Together these data provide evidence of therapeutic potential, validating EZH2 as a target in AML.To characterise the role of Ezh2 during AML induction in vivo, we utilized Ezh2fl/fl; Mx1-Cre mice to generate Ezh2-/- mice and compared them to Ezh2+/+ controls. HSPCs from these were retrovirally transduced with MLL-AF9 or AML1-ETO9a and transplanted into lethally irradiated wild-type recipients, and leukemias allowed to develop. Remarkably in the context of disease induction, and in stark contrast to the maintenance experiments, Ezh2 functioned as a tumor suppressor gene, with its loss significantly accelerating leukemia generation across both subtypes of AML (compared to control).To interrogate the molecular mechanisms of these findings we performed integrated genomic analysis with differential global gene expression analysis via RNA-sequencing (in Ezh2+/+ vs Ezh2-/- non-transformed HSPCs, Ezh2+/+ vs Ezh2-/- AML1-ETO9a and Ezh2+/+ vs Ezh2-/- MLL-AF9 leukemias) and ChIP-sequencing of the histone modifications H3K27me3 and H3K4me3. These revealed that during disease induction, Ezh2 represses a subset of bivalent promoters that resolve towards gene activation upon Ezh2 loss, inducing a number of genes including the transcription factor Plag1 (with known oncogenic function in CBFB-MYH11 murine leukemogenesis). Of note, overexpression of Plag1 phenocopied Ezh2 loss i n vivo to significantly accelerate MLL-AF9 leukemia induction.In addition, we demonstrated virtual mutual exclusivity of genes de-repressed in established MLL-AF9 leukemias following pharmacological Ezh2 inhibition compared to those de-repressed in the induction of Ezh2-/- MLL-AF9 leukemias. This de-repression of entirely separate gene programmes explains the starkly contrasting phenotypic outcomes of targeting Ezh2 in AML induction and maintenance, and further provides reassurance that EZH2 inhibition in established AML is a safe strategy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the Histone Methyltransferase EZH2 Enhances Protumor Monocyte Recruitment in Human Mesothelioma Spheroids.

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes’ recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.

Feedback activation of NF-KB signaling leads to adaptive resistance to EZH2 inhibitors in prostate cancer cells

BackgroundProstate cancer (PCa) is the most common malignant tumor in developed countries, which has seriously threatened men’s lifestyle and quality of life. The up-regulation of EZH2 is associated with advanced PCa and poor prognosis, making it a promising therapeutic target. However, the EZH2 inhibitors-based treatment is basically ineffective against PCa, which limits its clinical application.MethodsMicroarray data (GSE107779) from LNCaP cells treated with either siRNA against EZH2 or a EZH2 inhibitor EPZ6438 was analyzed by Limma R package. Western blot, real-time PCR and luciferase reporter assays were used to determine the EZH2-SOX9-TNFRSF11A axis and the activity of NF-κB signaling in PCa cells. CCK-8 assay was used to determine the viability of PCa cells following various treatments.ResultsGenetic ablation or pharmacological inhibition of EZH2 leads to feedback activation of NF-κB signaling in PCa cells. EZH2-dependent SOX9 expression regulates the activation of NF-κB signaling. TNFRSF11A, also known as receptor activator of NF-κB (RANK), is a downstream target of SOX9 in PCa cells. SOX9 recognizes two putative SOX9 response elements in the promoter region of TNFRSF11A gene to drive TNFRSF11A expression and downstream NF-κB signaling activation. Suppression of the NF-κB signaling by either TNFRSF11A silencing or BAY11-7082 treatment rendered PCa cells to EZH2 inhibitors.ConclusionCollectively, our finding reveals a EZH2-SOX9-TNFRSF11A axis in the regulation of activity of NF-κB signaling in PCa cells and suggests that a combination of EZH2 inhibitors and BAY11-7082 would be an effective approach for the treatment of PCa patients in the future.

Abstract P759: Microglial EZH2 Inhibition Leads to Neuroprotection After Stroke in Aged Mice

Introduction: Ischemic stroke results in activation of microglia, which may polarize towards a pro-inflammatory (M1) phenotype and/or an anti-inflammatory (M2) phenotype. Enhancer of zeste homolog (EZH) 2 is a histone-lysine N-methyltransferase enzyme. Accumulating evidence has suggested EZH2 is key modulator of microglia polarization by epigenetically regulating gene expression. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke in vivo . We further tested the therapeutical potential of an EZH2 inhibitor after stroke. Methods: Aged male mice were subjected to 60-minutes middle cerebral artery stroke. Tamoxifen administration was started 30 days prior to stroke to induce genetic deletion of microglial EZH2 in CX3CR1-creER/EZH2-floxed mice. EZH2 floxed mice were used as controls. EZH2 inhibitor GSK343 was I.P. injected (once a day for two consecutive days), starting three hours after stroke onset. Mice were sacrificed for immunohistochemistry and crystal violet staining (brain infarct assay) after behavior tests at 3 days after stroke. Results: The expression of microglial EZH2 was significantly abrogated in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm 2 , N=5/each group, P<0.01). EZH2 deletion reduced brain infarct volume (29.27±2.23% vs. 6.07±0.88%, N=7/each group, P<0.001) and improved functional outcome assayed by adhesive removal test (59±13.1 sec, N=7 in floxed control vs. 26.28±4.1 sec, N=12 in KO, P<0.01). Mechanistically, microglial EZH2 deletion led to a decrease in expression of M1 marker iNOS (170±14.78 vs. 76.65±11.38 cells/mm 2 , N=4/each group, P<0.05), an increase in M2 marker Arg1 (96.64±11.48 vs. 203.3±22.02 cells/mm 2 , N=4/each group, P<0.05) co-stained in microglia (Iba1). Finally, GSK343 treatment robustly reduced infarct volume (37.1±2.97% vs. 21.9±2.92%) and increased latency to fall in hang-wire test (23.5±3.5 vs. 45.9±7.1 sec) (N=8/group, p<0.05) 3 days after stroke. Conclusions: Both genetic deletion of EZH2 in microglia and pharmacological inhibition of EZH2 improved stroke outcome in aged. The effect may be due to limiting microglial M1 polarization and enhancing M2 polarization.