EZH2 Inhibitor Research Articles

EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis

EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis

Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

H3K27me3 of Rnf19a promotes neuroinflammatory response during Japanese encephalitis virus infection

Histone methylation is an important epigenetic modification that affects various biological processes, including the inflammatory response. In this study, we found that infection with Japanese encephalitis virus (JEV) leads to an increase in H3K27me3 in BV2 microglial cell line, primary mouse microglia and mouse brain. Inhibition of H3K27me3 modification through EZH2 knockdown and treatment with EZH2 inhibitor significantly reduces the production of pro-inflammatory cytokines during JEV infection, which suggests that H3K27me3 modification plays a crucial role in the neuroinflammatory response caused by JEV infection. The chromatin immunoprecipitation-sequencing (ChIP-sequencing) assay revealed an increase in H3K27me3 modification of E3 ubiquitin ligases Rnf19a following JEV infection, which leads to downregulation of Rnf19a expression. Furthermore, the results showed that Rnf19a negatively regulates the neuroinflammatory response induced by JEV. This is achieved through the degradation of RIG-I by mediating its ubiquitination. In conclusion, our findings reveal a novel mechanism by which JEV triggers extensive neuroinflammation from an epigenetic perspective.

Exploiting an epigenetic switch in adrenal cancer.

EZH2 inhibitors may slow the progression of an aggressive form of adrenocortical cancer.

Neuroprotective effects of GSK-343 in an in vivo model of MPTP-induced nigrostriatal degeneration

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic nigrostriatal neurons, which causes disabling motor disorders. Scientific findings support the role of epigenetics mechanism in the development and progression of many neurodegenerative diseases, including PD. In this field, some studies highlighted an upregulation of Enhancer of zeste homolog 2 (EZH2) in the brains of PD patients, indicating the possible pathogenic role of this methyltransferase in PD. The aim of this study was to evaluate the neuroprotective effects of GSK-343, an EZH2 inhibitor, in an in vivo model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic degeneration. Specifically, nigrostriatal degeneration was induced by MPTP intraperitoneal injection. GSK-343 was administered intraperitoneally daily at doses of 1 mg/kg, 5 mg/kg and 10 mg/kg, mice were killed 7 days after MPTP injection. Our results demonstrated that GSK-343 treatment significantly improved behavioral deficits and reduced the alteration of PD hallmarks. Furthermore, GSK-343 administration significantly attenuated the neuroinflammatory state through the modulation of canonical and non-canonical NF-κB/IκBα pathway as well as the cytokines expression and glia activation, also reducing the apoptosis process. In conclusion, the obtained results provide further evidence that epigenetic mechanisms play a pathogenic role in PD demonstrating that the inhibition of EZH2, mediated by GSK-343, could be considered a valuable pharmacological strategy for PD.

Design, synthesis and mechanism studies of dual EZH2/BRD4 inhibitors for cancer therapy

Aberrant expression of EZH2 is frequently observed in cancers, and the EZH2 inhibitors are only effective in hematological malignancies and almost noneffective against solid tumors. It has been reported that the combination of EZH2 and BRD4 inhibitors may be a promising strategy to treat solid tumors being insensitive to EZH2 inhibitors. Thus, a series of EZH2/BRD4 dual inhibitors were designed and synthesized. The optimized compound 28, encoded as KWCX-28, was the most potential compound by the SAR studies. Further mechanism studies showed that KWCX-28 inhibited HCT-116 cells proliferation (IC50 = 1.86 µM), induced HCT-116 cells apoptosis, arrested cell cycle arrest at G0/G1 phase and resisted the histone 3 lysine 27 acetylation (H3K27ac) upregulation. Therefore, KWCX-28 was a potential dual EZH2/BRD4 inhibitors for treating solid tumors.

The Heightened Importance of EZH2 in Cancer Immunotherapy

The transcriptional inhibitor histone methyltransferase enhancer of zeste homolog 2 (EZH2) predominantly targets genes involved in tumor suppression. EZH2 is highly expressed in a variety of human malignancies and promotes carcinogenesis and malignant transformation. Recent research has indicated that altering the tumor microenvironment by focusing on epigenetic variables can improve antitumor immunity. Recent research has also revealed that EZH2 has pleiotropic functions in immune and malignant cells. EZH2 inhibition could be a promising strategy to improve the outcomes of current immunotherapies. Based on the role of EZH2 in the immunomodulation of both immune and tumor cells, we evaluated the effect of EZH2 on tumor immunity in this review. We also highlight improvements in combined EZH2-targeted treatment and immunotherapy.

Histone methyltransferase enzyme enhancer of zeste homolog 2 counteracts ischemic brain injury via H3K27me3-mediated regulation of PI3K/AKT/mTOR signaling pathway.

Epigenetic histone methylation plays a crucial role in cerebral ischemic injury, particularly in the context of ischemic stroke. However, the complete understanding of regulators involved in histone methylation, such as Enhancer of Zeste Homolog 2 (EZH2), along with their functional effects and underlying mechanisms, remains incomplete. Here, we employed a rat model of MCAO (Middle cerebral artery occlusion) and an OGD (Oxygen-Glucose Deprivation) model of primary cortical neurons to study the role of EZH2 and H3K27me3 in cerebral ischemia-reperfusion injury. The infarct volume was measured through TTC staining, while cell apoptosis was detected using TUNEL staining. The mRNA expression levels were quantified through quantitative real-time polymerase chain reaction (qPCR), whereas protein expressions were evaluated via western blotting and immunofluorescence experiments. The expression levels of EZH2 and H3K27me3 were upregulated in OGD; these expression levels were further enhanced by GSK-J4 but reduced by EPZ-6438 and AKT inhibitor (LY294002) under OGD conditions. Similar trends were observed for mTOR, AKT, and PI3K while contrasting results were noted for UTX and JMJD3. The phosphorylation levels of mTOR, AKT, and PI3K were activated by OGD, further stimulated by GSK-J4, but inhibited by EPZ-6438 and AKT inhibitor. Inhibition of EZH2 or AKT effectively counteracted OGD-/MCAO-induced cell apoptosis. Additionally, inhibition of EZH2 or AKT mitigated MCAO-induced infarct size and neurological deficit in vivo. Collectively, our results demonstrate that EZH2 inhibition exerts a protective effect against ischemic brain injury by modulating the H3K27me3/PI3K/AKT/mTOR signaling pathway. The results provide novel insights into potential therapeutic mechanisms for stroke treatment.

Structural classification of EZH2 inhibitors and prospects for the treatment of tumor: a review

Structural classification of EZH2 inhibitors and prospects for the treatment of tumor: a review

Interplay between EZH2/β-catenin in stemness of cisplatin-resistant HNSCC and their role as therapeutic targets

The Wnt/β-catenin signaling pathway is associated with the regulation of cancer stem cells, and it can be driven by epigenetic modifications. Here, we aim to identify epigenetic modifications involved in the control of the Wnt/β-catenin signaling and investigate the role of this pathway in the accumulation of cancer stem cells (CSC) and chemoresistance of Head and Neck Squamous Cell Carcinoma (HNSCC). Quantitative-PCR, western blot, shRNA assay, viability assay, flow cytometry assay, spheres formation, xenograft model, and chromatin immunoprecipitation were employed to evaluate the Wnt/β-catenin pathway and EZH2 in wild-type and chemoresistant oral carcinoma cell lines, and in the populations of CSC and non-stem cells. We demonstrated that β-catenin and EZH2 were accumulated in cisplatin-resistant and CSC population. The upstream genes of the Wnt/β-catenin signaling (APC and GSK3β) were decreased, and the downstream gene MMP7 was increased in the chemoresistant cell lines. The inhibition of β-catenin and EZH2 combined effectively decreased the CSC population in vitro and reduced the tumor volume and CSC population in vivo. EZH2 inhibition increased APC and GSK3β, and the Wnt/β-catenin inhibition reduced MMP7 levels. In contrast, EZH2 overexpression decreased APC and GSK3β and increased MMP7. EZH2 and β-catenin inhibitors sensitized chemoresistant cells to cisplatin. EZH2 and H3K27me3 bounded the promoter of APC, leading to its repression. These results suggest that EZH2 regulates β-catenin by inhibiting the upstream gene APC contributing to the accumulation of cancer stem cells and chemoresistance. Moreover, the pharmacological inhibition of the Wnt/β-catenin combined with EZH2 can be an effective strategy for treating HNSCC.

#4425 EZH2 PROMOTES RENAL INTERSTITIAL FIBROSIS THROUGH DOWNREGULATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE 1 MEDIATED GLUCONEOGENESIS

Abstract Background and Aims Renal fibrosis is the common pathological pathway of various chronic kidney diseases progressing to the end stage of renal failure. The methyltransferase enhancer of zeste homolog 2 (EZH2) has been identified as a therapeutic target to inhibit renal interstitial fibrosis. However, the mechanism underlying the role of EZH2 in renal fibrosis is not completely understood. Method Unilateral ureteral obstruction (UUO), unilateral ischemia-reperfusion injury (UIRI) mouse models were established. PCR, cleavage under targets and tagmentation (CUT&Tag) and Western blotting was performed to evaluate the expression of EZH2 and phosphoenolpyruvate carboxykinase 1 (PCK1). Results By using EZH2 inhibitor 3-DZNeP and Ezh2 conditional knockout mice, we confirmed the pro-fibrotic effect of EZH2 in unilateral ureteral obstruction (UUO). Through RNA sequence and cleavage under targets and tagmentation (CUT&Tag) sequence analysis, we found that the phosphoenolpyruvate carboxykinase 1 (PCK1), a critical enzyme in gluconeogenesis, is negatively regulated by EZH2 in fibrotic kidneys, which was further confirmed by quantitative PCR, CUT&Tag and Western blotting. We further showed that deletion or inhibition of EZH2 inhibited renal fibrosis and enhanced PCK1 expression and activity in unilateral ischemia-reperfusion injury (UIRI) and folic acid induced mouse nephropathy. Moreover, the dysregulated production of renal glucose and lactate in mouse UUO kidneys was restored after EZH2 inhibition by 3-DZNeP. Finally, inhibition of PCK1 by 3-mercaptopropionic acid (3-MPA) abrogated the anti-fibrotic effect of 3-DZNeP in UUO kidneys. Conclusion We conclude that EZH2 promotes renal interstitial fibrosis through inhibition of PCK1 mediated gluconeogenesis.

Dysregulation of SWI/SNF Chromatin Remodelers in NSCLC: Its Influence on Cancer Therapies including Immunotherapy.

Lung cancer is the leading cause of cancer death worldwide. Molecularly targeted therapeutics and immunotherapy revolutionized the clinical care of NSCLC patients. However, not all NSCLC patients harbor molecular targets (e.g., mutated EGFR), and only a subset benefits from immunotherapy. Moreover, we are lacking reliable biomarkers for immunotherapy, although PD-L1 expression has been mainly used for guiding front-line therapeutic options. Alterations of the SWI/SNF chromatin remodeler occur commonly in patients with NSCLC. This subset of NSCLC tumors tends to be undifferentiated and presents high heterogeneity in histology, and it shows a dismal prognosis because of poor response to the current standard therapies. Catalytic subunits SMARCA4/A2 and DNA binding subunits ARID1A/ARID1B/ARID2 as well as PBRM1 were identified to be the most commonly mutated subunits of SWI/SNF complexes in NSCLC. Mechanistically, alteration of these SWI/SNF subunits contributes to the tumorigenesis of NSCLC through compromising the function of critical tumor suppressor genes, enhancing oncogenic activity as well as impaired DNA repair capacity related to genomic instability. Several vulnerabilities of NSCLCS with altered SWI/SNF subunits were detected and evaluated clinically using EZH2 inhibitors, PROTACs of mutual synthetic lethal paralogs of the SWI/SNF subunits as well as PARP inhibitors. The response of NSCLC tumors with an alteration of SWI/SNF to ICIs might be confounded by the coexistence of mutations in genes capable of influencing patients' response to ICIs. High heterogenicity in the tumor with SWI/SNF deficiency might also be responsible for the seemingly conflicting results of ICI treatment of NSCLC patients with alterations of SWI/SNF. In addition, an alteration of each different SWI/SNF subunit might have a unique impact on the response of NSCLC with deficient SWI/SNF subunits. Prospective studies are required to evaluate how the alterations of the SWI/SNF in the subset of NSCLC patients impact the response to ICI treatment. Finally, it is worthwhile to point out that combining inhibitors of other chromatin modulators with ICIs has been proven to be effective for the treatment of NSCLC with deficient SWI/SNF chromatin remodelers.

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.

EZH1/2 dual inhibitors suppress HTLV-1-infected cell proliferation and hyperimmune response in HTLV-1-associated myelopathy.

Human T-cell leukemia virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy (HAM), adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated uveitis, and pulmonary diseases. Although both HAM and ATL show proliferation of infected cells, their pathogeneses are quite different. In particular, the pathogenesis of HAM is characterized by hyperimmune responses to HTLV-1-infected cells. Recently, we demonstrated the overexpression of histone methyltransferase EZH2 in ATL cells and the cytotoxic effects of EZH2 inhibitors and EZH1/2 dual inhibitors on these cells. However, these phenomena have never been studied in HAM. Furthermore, what effect these agents have on the hyperimmune response seen in HAM is completely unknown. In this study, we investigated histone methyltransferase expression levels in infected cell populations (CD4+ and CD4+CCR4+ cells) from patients with HAM using microarray and RT-qPCR analyses. Next, using an assay system that utilizes the spontaneous proliferation characteristic of peripheral blood mononuclear cells derived from patients with HAM (HAM-PBMCs), we investigated the effects of EZH2 selective inhibitors (GSK126 and tazemetostat) and EZH1/2 dual inhibitors (OR-S1 and valemetostat, also known as DS-3201), particularly on cell proliferation rate, cytokine production, and HTLV-1 proviral load. We also examined the effect of EZH1/2 inhibitors on the proliferation of HTLV-1-infected cell lines (HCT-4 and HCT-5) derived from patients with HAM. We found elevated expression of EZH2 in CD4+ and CD4+CCR4+ cells from patients with HAM. EZH2 selective inhibitors and EZH1/2 inhibitors significantly inhibited spontaneous proliferation of HAM-PBMC in a concentration-dependent manner. The effect was greater with EZH1/2 inhibitors. EZH1/2 inhibitors also reduced the frequencies of Ki67+ CD4+ T cells and Ki67+ CD8+ T cells. Furthermore, they reduced HTLV-1 proviral loads and increased IL-10 levels in culture supernatants but did not alter IFN-γ and TNF-α levels. These agents also caused a concentration-dependent inhibition of the proliferation of HTLV-1-infected cell lines derived from patients with HAM and increased annexin-V(+)7-aminoactinomycin D(-) early apoptotic cells. This study showed that EZH1/2 inhibitors suppress HTLV-1-infected cell proliferation through apoptosis and the hyperimmune response in HAM. This indicates that EZH1/2 inhibitors may be effective in treating HAM.

Parallel testing of liquid biopsy (ctDNA) and tissue biopsy samples reveals a higher frequency of EZH2 mutations in follicular lymphoma.

Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.

Combining EZH2 inhibition with senescence induction helps immune cells fight pancreatic cancer.

Combining EZH2 inhibition with senescence induction helps immune cells fight pancreatic cancer.

SAM-Competitive EZH2-Inhibitors Induce Platinum Resistance by EZH2-Independent Induction of ABC-Transporters.

T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

Abstract B017: Lineage-specific PRC2 targets and response to EZH2 inhibition in neuroendocrine prostate cancer

Abstract Lineage plasticity is an emerging mechanism of treatment-resistance in prostate cancer, which can manifest clinically as histologic transformation from prostate adenocarcinoma to small-cell neuroendocrine prostate cancer (NEPC). NEPC is associated with poor prognosis, and new treatment strategies are urgently needed. Epigenetic dysregulation has been implicated as a key driver of prostate cancer lineage plasticity, mediated in part by EZH2. EZH2 is a component of the polycomb repressive complex (PRC2) regulating the transcriptional repressive mark H3K27me3. EZH2 also controls cell-fate determination during normal development via bivalent promoters bearing both repressive H3K27me3 and active H3K4me3 marks. EZH2 is targetable, and several EZH2 inhibitors are in clinical development; these trials are currently in biomarker-unselected prostate cancer populations. A thorough understanding of the mechanism of action of EZH2 is imperative for predicting optimal response to EZH2i and for the development of robust combination treatment strategies. Cell viability studies across a diverse panel of prostate cancer models representing adenocarcinoma and NEPC indicated variable responses to tazemetostat (EZH2i) treatment. The castration-resistant prostate adenocarcinoma (CRPC) cell line LNCaP-abl showed a greater than 50% reduction in cell viability upon EZH2i (5uM) while LNCaP and 22Rv1 showed moderate response with 15-25% reduction in cell viability. However, none of our 5 NEPC patient-derived models showed significant response to EZH2i which was also validated in vivo. Hierarchical clustering of RNA-seq profiles of responder and non-responder models revealed distinct cell-type specific EZH2 targets and clusters of genes that may be associated with response to EZH2i. Approximately 15% of the EZH2 targets in the NEPC organoid WCM154 carried bivalency in their promoters including neuronal lineage determining genes such as ASCL1 and LHX2. Gene set enrichment analysis of EZH2 targets bearing bivalency in NEPC revealed association with neurogenesis and cell proliferation pathways. Further upregulation of neuronal-lineage PRC2 targets in NEPC models upon EZH2i may provide justification for lack of lineage reversal and maintenance of terminal differentiation. While there is evidence that EZH2i leads to lineage reversion in adeno-to-NEPC transitioning preclinical models, our data shows that EZH2i was associated with modest response in NEPC models and did not revert lineage plasticity. Our results provide insights into a potential role for bivalent promoters in EZH2-mediated lineage reprogramming and sustenance of terminal differentiation upon EZH2i in NEPC. Understanding lineage-specific action of EZH2i may inform biomarkers in ongoing clinical trials. Genes and pathways dysregulated because of their bivalency might also be exploited as potential candidates to be co-targeted with EZH2i. Citation Format: Varadha Balaji Venkadakrishnan, Richa Singh, Yasutaka Yamada, Kei Mizuno, Adam G. Presser, Sheng-Yu Ku, Henry Long, David Rickman, Himisha Beltran. Lineage-specific PRC2 targets and response to EZH2 inhibition in neuroendocrine prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B017.

Abstract IA016: Epigenetic crosstalk associated with prostate cancer lineage plasticity

Abstract Background: Prostate cancer arises as an androgen driven disease and therefore androgen receptor (AR) targeting therapies have been a major focus of prostate cancer treatment. Lineage plasticity, a process by which differentiated cells lose their identity and acquire alternative lineage programs, has recently been identified as an emerging mechanism of resistance to targeted therapies in prostate cancer. This plasticity can manifest as histologic transformation from an AR-positive prostate adenocarcinoma to neuroendocrine prostate cancer (NEPC). NEPC is clinically aggressive and prognosis is poor. Although NEPC tumors arise clonally from prostate adenocarcinoma and share genomic alterations, there is significant epigenetic deregulation during the transformation process. However, mechanistically, we still do not know how these epigenetic alterations arise and how best to leverage these alterations as a therapeutic opportunity. Methods: To model DNA methylation program associated with the transition from CRPC to NEPC, we performed reduced-representation bisulfite sequencing (RRBS) in a variety of clinically relevant models including a novel genetically engineered mouse model and patient-derived organoids/xenografts (PDO/X). We performed genetic (CRISPR/Cas9 technology) and pharmacologic inhibition of DNA methylation writers (DNMTs), erasures (TETs) and polycomb repressive complex 2 histone methyltransferase EZH2 to query their role in the establishment of the DNA methylation program. To assess EZH2 enzymatic activity, we performed H3K27me3 ChIP-seq in our murine and human models. Results: The transcriptome from adenocarcinoma or NEPC foci in our murine model closely resembled clinical CRPC or NEPC transcriptome, respectively. We also found increased expression of DNA methylation regulators (DNMT1/3B and TET1) in NEPC foci. A clinically relevant methylome signature based on murine model was found to segregate clinical samples into CRPC and NEPC subtypes which was also validated in androgen-deprived isogenic LNCaP cells as well as PDO/Xs. Interestingly, AR-target genes (e.g., FKBP5 and TMPRSS2) were found to be hypermethylated and downregulated while cell fate decision regulators (e.g., FOXA2 and SLIT2) and EZH2 targets were hypomethylated and upregulated in NEPC foci. While we found that genetic or pharmacologic inhibition of DNA methylation writers and erasures impacted the NEPC-associated DNA methylation program, inhibition of EZH2 resulted in significant alterations to the DNA methylation program. In turn, we also found that that DNMT inhibition led to equally significant alterations to EZH2 activity. Conclusion: Our data suggest that there is an interplay between EZH2 and DNA methylation that is associated with the molecular reprogramming associated with the progression from AR-positive adenocarcinoma towards NEPC as well as for the maintenance of the NEPC molecular program. More mechanistic studies are now needed to fully characterize this interplay and to determine if we can harness this synergy for therapeutic potential. Citation Format: David Rickman, Richa Singh, Varadha Balaji Venkadakrishnan, Yasutaka Yamada, Himisha Beltran. Epigenetic crosstalk associated with prostate cancer lineage plasticity [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr IA016.

Sunitinib attenuates reactive MDSCs enhancing anti-tumor immunity in HNSCC

Enhancer of zeste homolog 2 (EZH2) is implicated in promoting HNSCC malignant progression. However, EZH2 inhibitors, when used alone, increase the number of myeloid-derived suppressor cells (MDSCs), which are responsible for enhancing tumor stemness and promoting tumor immune escape. We aimed to determine whether combining tazemetostat (an EZH2 inhibitor) and sunitinib (a MDSC inhibitor) can improve the response rate to an immune-checkpoint-blocking (ICB) therapy. We evaluated the efficacy of the above treatment strategies by bioinformatics analysis and animal experiments. EZH2 overexpression and abundant MDSCs in patients with HNSCC are associated with tumor progression. Tazemetostat treatment alone had limited inhibitory effect on HNSCC progression in the mouse models, accompanied by a surge in the number of MDSCs in the tumor microenvironment. Conversely, the combined use of tazemetostat and sunitinib reduced the number of MDSCs and regulatory T cell populations, promoting intratumoral infiltration of T cells and inhibiting of T cell exhausting, regulating of wnt/β-catenin signaling pathway and tumor stemness, promoting the intratumoral PD-L1 expression and improved the response rate to anti-PD-1 therapy. The combined use of EZH2 and MDSC inhibitors effectively reverses HNSCC-specific immunotherapeutic resistance and is a promising strategy for overcoming resistance to ICB therapy.