EZH2 Inhibitor Research Articles

The role of systemic therapy in advanced skull base chordomas: overview of the current state and the MD Anderson protocol.

The role of systemic therapy in primary or advanced and metastatic chordoma has been traditionally limited because of the inherent resistance to cytotoxic therapies and lack of specific or effective therapeutic targets. Despite resection and adjuvant radiation therapy, local recurrence rates in clival chordoma remain high and the risk of systemic metastases is not trivial, leading to significant morbidity and mortality. Recently, molecular targeted therapies (MTTs) and immune checkpoint inhibitors (ICIs) have emerged as promising therapeutic avenues in chordoma. In recent years, preclinical studies have identified potential targets based on intrinsic genetic dependencies, epigenetic modulators, or newly identified tumor-associated cell populations driving treatment resistance and recurrence. Nonetheless, the role of systemic therapies in the neoadjuvant or adjuvant setting for primary, locally progressive, and distant metastatic chordomas is still being investigated. Herein, an overview of current and emerging systemic treatment strategies in advanced clival chordoma is provided. Furthermore, several molecular biomarkers have been recently uncovered as potential predictors of the response to specific molecular therapeutics. The authors describe the recently discovered role of 1p36 and 9p21 deletions as biomarkers capable of guiding drug selection. Then they discuss completed and ongoing clinical trials of MTTs, including several tyrosine kinase inhibitors used as monotherapy or in combination, such as imatinib, sorafenib, dasatinib, and lapatinib, among others, as well as mammalian target of rapamycin inhibitors such as everolimus and rapamycin. They present their experience and other recent studies demonstrating vast benefits in advanced chordoma from ICIs. Additionally, they provide a brief overview of novel systemic strategies such as adoptive cell transfer (CAR-T and NK cells), oncolytic viruses, epigenetic targeting (KDM6, HDAC, and EZH2 inhibitors), and several promising preclinical studies with high translational potential. Finally, the authors present their institutional multidisciplinary protocol for the incorporation of systemic therapy for both newly diagnosed and recurrent chordomas based on molecular studies including upfront enrollment in MTT trials in patients with epidermal growth factor receptor upregulation or INI-1 deficiency or enrollment in ICI clinical trials for patients with high tumor mutational burden or high PD-L1 expression on tumor cells or in the tumor microenvironment.

2,2- dimethylbenzopyran derivatives containing pyridone structural fragments as selective dual-targeting inhibitors of HIF-1α and EZH2 for the treatment of lung cancer

We formerly reported that EZH2 inhibitors sensitized HIF-1 inhibitor-resistant cells and inhibited HIF-1α to promote SUZ12 transcription, leading to enhanced EZH2 enzyme activity and elevated H3K27me3 levels, and conversely, inhibition of EZH2 promoted HIF-1α transcription. HIF-1α and EZH2 interacted to form a negative feedback loop that reinforced each other's activity. In this paper, a series of 2,2- dimethylbenzopyran derivatives containing pyridone structural fragments were designed and synthesized with DYB-03, a HIF-1α inhibitor previously reported by our group, and Tazemetostat, an EZH2 inhibitor approved by FDA, as lead compounds. Among these compounds, D-01 had significant inhibitory activities on HIF-1α and EZH2. In vitro experiments showed that D-01 significantly inhibited the migration of A549 cells, clone, invasion and angiogenesis. Moreover, D-01 had good pharmacokinetic profiles. All the results about compound D-01 could lay a foundation for the research and development of HIF-1α and EZH2 dual-targeting compounds.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

Identification and functional analysis of the hub Ferroptosis-Related gene EZH2 in diabetic kidney disease

BackgroundDiabetic kidney disease (DKD) is a common microvascular complication and one of the main causes of death in diabetes. Ferroptosis, an iron-dependent mode of cell death characterized by lipid ROS accumulation, was found to be associated with a number of diseases and has great potential for kidney diseases. It has great value to identify potential ferroptosis-related genes and their biological mechanisms in DKD. MethodsWe obtained the GSE30122 dataset from Gene Expression Omnibus (GEO) database and ferroptosis-related genes from the Ferrdb database. After differential expression analysis, and three machine learning algorithms, the hub ferroptosis-related gene EZH2 was identified. In order to investigate the function of EZH2, Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA) and single cell analysis were conducted. The expression of EZH2 was validated in DKD patients, HK-2 cell models and DKD mouse models. EZH2 knockdown HK-2 cells and HK-2 cells treated with GSK126 were performed to verify whether EZH2 affected ferroptosis in DKD. CHIP assay was used to detect whether EZH2 regulated ferroptosis by suppressing SLC7A11. Molecular docking was performed to explore EZH2 and four traditional Chinese medicine (Sennoside A, Berberine, Umbelliferone, Platycodin D) related to ferroptosis in DKD treatment. ResultsAccording to the GSE30122 dataset in GEO and ferroptosis-related genes from the Ferrb database, we obtained the hub ferroptosis-related gene EZH2 in DKD via diversified machine learning methods. The increasing of EZH2 expression was shown in single cell analysis, DKD patients, DKD mouse models and high glucose induced DKD cell models. Further study showed that EZH2 knockdown and inhibition can alleviate HG-induced ferroptosis in vitro. CHIP assay showed EZH2-mediated epigenetic silencing regulated the expression of SLC7A11. Molecular docking results showed that EZH2 had strong binding stability with Sennoside A, Berberine, Umbelliferone, and Platycodin D. ConclusionOverall, our data shouwed that histone H3K27 methyltransferase EZH2 could regulate the renal tubular epithelial cell ferroptosis by suppressing SLC7A11 in DKD, which may serve as a credible reliable indicator for diagnosing DKD and a potential target for treatment.

Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells.

The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank-Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial-mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

Abstract LB403: Suppression of antigen presentation in pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that is diagnosed based on the tumor cell's resemblance to embryonic or poorly differentiated skeletal muscle progenitors. Multiple immunotherapy clinical trials have found no evidence of immune reactivity against RMS, which has been attributed to the relatively low tumor mutation burden in these cancers. However, we made an unexpected observation that many patient RMS tumors had a near absence of MHC-I expression, a key requisite for anti-tumor CD8+ T lymphocytes and many immunotherapies. In this study, we explore the etiology of low antigen presentation in RMS and systematically evaluate therapeutic interventions that sensitize RMS to immunotherapy. Methods: Surface MHC-I expression was evaluated on pediatric tumor cell lines (n=65) from five major pediatric tumor types. RNA-seq, whole exome and genome sequencing data were analyzed from RMS tumors (total of n=247) and iPSC models of skeletal muscle development (PMID: 32011235). Epigentic drugs targeting DNA methylation (decitabine), EZH2 (tazemetostat), Class I/IV HDACs (mocetinostat), and LSD1 (GSK-LSD1) were used at sub-cytotoxic doses on RMS cell lines (n=10) and analyzed for surface MHC-I expression with flow cytometry and gene expression. T cell cytotoxicity assays were performed using engineered human T cells expressing an optimized TCR which recognizes an HLA*A2:01 restricted peptide from PRAME. Results: RMS cell line models exhibited a range of MHC-I expression with most having low or undetectable MHC-I expression, similar to that of a known MHC-I negative tumor type MYCN-amplified neuroblastoma. Exome sequencing of RMS tumors showed no evidence of loss-of-function alterations in essential antigen processing and presentation (APP) pathway genes. Gene expression profiling by RNAseq and western blotting, however, revealed an absence of core APP genes in many RMS tumors and cell lines. We uncovered that during a specific window of embryonic myogenesis, APP is suppressed and that RMS tumors mimicking this state of development show the deepest loss of APP. Multiple classes of epigenetic drugs were evaluated to rescue APP in RMS. Treatment of RMS using a DNA demethylating agent or HDAC inhibitor exhibited the strongest MHC-I restoration across RMS models with no changes observed using LSD1 or EZH2 inhibitors. As a proof-of-concept, we demonstrate that targeting the RMS antigen PRAME using TCR therapy is enhanced by the inclusion of epigenetic therapy. Conclusions and Significance: We revealed that epigenetic suppression of APP is a new hallmark of RMS and normal embryonic skeletal muscle development. Pharmacological reversal of APP pathway downregulation could be achieved using drugs targeting DNA methylation or class I/IV HDACs, and enhanced T cell cytotoxicity against RMS when used in combination. Overall, these findings may explain the historical lack of RMS responsiveness to immunotherapy and unlocks new immunotherapy strategies against RMS that rely on targeting MHC-I antigens. Citation Format: David Milewski, Hsien-Chao Chou, Vineela Gangalapudi, Meijie Tian, Yong Kim, Young Song, Jun Wei, Javed Khan. Suppression of antigen presentation in pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB403.

Abstract LB339: Transient EZH2 suppression by tazemetostat during in vitro expansion maintains T cell stemness and improves adoptive T cell therapy

Abstract The histone methyltransferase enhancer of zeste homolog 2 (EZH2) plays important roles in T cell differentiation, proliferation and function. Previous studies demonstrated that genetic deletion of EZH2 in CD8+ or total T cells impairs their antiviral and antitumor activity, cytokine production and expansion upon rechallenge. Contrary to the detrimental role of deleting T cell-intrinsic EZH2, here we demonstrate that transient inhibition of EZH2 in T cells prior to the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetostat, delays their dysfunctional progression and preserves T cell stemness and polyfunctionality while having no negative impact on cell proliferation. Tazemetostat induces T-cell epigenetic reprogramming and increases the expression of the self-renewal T cell transcription factor TCF1 by reducing its promoter H3K27 methylation preferentially in rapidly dividing T cells. In a murine melanoma model, T cells depleted of EZH2 induce poor tumor control, whereas T cells pretreated with tazemetostat exhibited superior anti-tumor immunity, especially when used in combination with anti-PD-1 blockade after adoptive transfer. Collectively, these data highlight the potential of transient epigenetic reprogramming by EZH2 inhibition to enhance adoptive T cell immunotherapy. Citation Format: Yingqin Hou, Peng Wu. Transient EZH2 suppression by tazemetostat during in vitro expansion maintains T cell stemness and improves adoptive T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB339.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium

BackgroundHistone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium.MethodsWe studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2−) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice.ResultsIncubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2− generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice.ConclusionsEZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.Graphical

EZH2 as a major histone methyltransferase in PDGF-BB-activated orbital fibroblast in the pathogenesis of Graves’ ophthalmopathy

Graves’ ophthalmopathy (GO) is an extra-thyroidal complication of Graves’ disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.

Design, synthesis and activity evaluation of quinolinone derivatives as EZH2 inhibitors

The enhancer of zeste homologue 2 (EZH2) is the core catalytic subunit of polycomb repressive complex 2, which catalyzes lysine 27 methylation of histone H3.Herein, a series of quinolinone derivatives were designed and synthesized based on the structure of Tazemetostat as the lead compound. Compound 9l (EZH2WT IC50 = 0.94 nM) showed stronger antiproliferative activity in HeLa cells than the lead compound. Moreover, compound 9e (EZH2WT IC50 = 1.01 nM) significantly inhibited the proliferation and induced apoptosis in A549 cells.

270 Inducing Neural Maturation and Increasing Survival in SHH Medulloblastoma Using EZH2 Inhibition in a Mouse Model

INTRODUCTION: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Genetically, MB can be divided into four subgroups of which the SHH subtype histologically shows nodular architecture. Within this nodular architecture, there are islands of mature cells, with more abundant neuropil and a low proliferation rate, scattered among sheets of primitive cells. METHODS: We performed laser capture microdissection followed by whole transcriptome analysis, spatial transcriptomics using Digital Spatial Profiling, whole genome DNA methylation and ChIP-Seq analysis of mature and primitive areas from 8 medulloblastomas. We developed a genetically-engineered mouse model of SHH MB showing spontaneous maturation and lack of maturation with a conditional EZH2 genetic ablation or EZH2 overactivation respectively. Finally, we developed a fucoidan-based nanoparticle drug delivery across the blood brain barrier (BBB) for targeted molecular inhibition. RESULTS: Using whole transcriptome and DNA methylation analysis, we identified ∼120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3. ChIP-Seq analysis showed striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Medulloblastoma specific EZH2 genetic ablation resulted in diffuse tumor cell differentiation and prolonged survival in mice (n = 10 per group, log-rank p = 0.01). Conversely, conditional EZH2 (Y641F) activation prevented medulloblastoma differentiation. A fucoidan-based nanoparticle successfully delivered the EZH2 inhibitor (EPZ-6438) across the murine BBB to achieve significant extension of mouse survival (median 70 days compared to 21 days in control mice; *p = 0.01, Mantel-Cox). CONCLUSIONS: Spontaneous maturation of medulloblastoma cells can be induced by inhibition of EZH2. Fucoidan-based nanoparticle delivery systems allows tumor specific targeted delivery across the BBB extending survival in mice.

Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling.

DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.

GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Pharmacology and pharmacokinetics of tazemetostat.

Tazemetostat, a novel oral selective inhibitor of enhancer of zeste homolog 2 (EZH2), was approved by the Food and Drug Administration (FDA) in 2020 for use in patients with advanced epithelioid sarcoma or relapsed/refractory (R/R) EZH2-mutated follicular lymphoma. These indications were approved by the FDA trough accelerated approval based on objective response rate and duration of response that resulted from phase 2 clinical trials. Tazemetostat competes with S-adenosylmethionine (SAM) cofactor to inhibit EZH2, reducing the levels of trimethylated lysine 27 of histone 3 (H3K27me3), considered as pharmacodynamic marker. Tazemetostat is orally bioavailable, characterized by rapid absorption and dose-proportional exposure, which is not influenced by coadministration with food or gastric acid reducing agents. It highly distributes in tissues, but with limited access to central nervous system. Tazemetostat is metabolized by CYP3A in the liver to 3 major inactive metabolites (M1, M3, and M5), has a short half-life and is mainly excreted in feces. Drug-drug interactions were shown with moderate CYP3A inhibitors as fluconazole, leading the FDA to recommend a 50% dose reduction, while studies investigating coadministration of tazemetostat with strong inhibitors/inducers are ongoing. No dosage modifications are recommended based on renal or hepatic dysfunctions. Overall, tazemetostat is the first-in-class EZH2 inhibitor approved by the FDA for cancer treatment. Current clinical studies are evaluating combination therapies in patients with several malignancies.

Abstract 4752: Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4752.

Combination of EZH2 and ATM inhibition in BAP1-deficient mesothelioma.

More than half of mesothelioma tumours show alterations in the tumour suppressor gene BAP1. BAP1-deficient mesothelioma is shown to be sensitive to EZH2 inhibition in preclinical settings but only showed modest efficacy in clinical trial. Adding a second inhibitor could potentially elevate EZH2i treatment efficacy while preventing acquired resistance at the same time. A focused drug synergy screen consisting of 20 drugs was performed by combining EZH2 inhibition with a panel of anti-cancer compounds in mesothelioma cell lines. The compounds used are under preclinical investigation or already used in the clinic. The synergistic potential of the combinations was assessed by using the Bliss model. To validate our findings, in vivo xenograft experiments were performed. Combining EZH2i with ATMi was found to have synergistic potential against BAP1-deficient mesothelioma in our drug screen, which was validated in clonogenicity assays. Tumour growth inhibition potential was significantly increased in BAP1-deficient xenografts. In addition, we observe lower ATM levels upon depletion of BAP1 and hypothesise that this might be mediated by E2F1. We demonstrated the efficacy of the combination of ATM and EZH2 inhibition against BAP1-deficient mesothelioma in preclinical models, indicating the potential of this combination as a novel treatment modality using BAP1 as a biomarker.

Abstract 4404: EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2

Abstract The polycomb protein EZH2 is a commonly deregulated or mutated transcriptional regulator in various cancers. In Chronic Myeloid Leukemia (CML) it has been reported to be upregulated and associated with transcriptional reprograming, rendering CML leukemic stem cells (LSCs) sensitive to EZH2 inhibitor treatment. Mechanistically this seems to be linked to the silencing of apoptotic target genes by EZH2-mediated histone H3 lysine 27 tri-methylation (H3K27me3). We hypothesized that the regulation of apoptotic genes is not the only pathway responsible for its oncogenic function, and that EZH2 might also act as a modulator of the mRNA splicing landscape. To test this, we treated the CML cell line K562 with the EZH2 inhibitors GSK126 and EPZ-6438 and performed RNA-seq followed by splicing analysis. Using two different bioinformatics tools, we detected differential splicing of several hundreds of splicing events. However, differentially spliced genes were not associated with increased EZH2 or H3K27me3 signals, suggesting an indirect mechanism of splicing regulation. Indeed, we found 11 splicing factors transcriptionally upregulated upon EZH2 inhibitor treatment. CELF2 was identified as top candidate to mediate part of the EZH2 inhibitor induced phenotype. Upon overexpression, we observed 1) reduced cell growth, viability, and colony formation of K562 cells, 2) a change in the splicing landscape, significantly overlapping with EZH2 inhibitor induced changes, 3) the downregulation of cMYC signaling, also found upon EZH2 inhibitor treatment. To test if CELF2 is indeed acting as EZH2-regulated tumor suppressor in CML, we extended our study to CML patient samples: we performed RNA-seq followed by splicing and gene expression analysis in seven CML patient-derived CD34+ samples and eight healthy donor-derived CD34+ samples, as well as three CML patient samples transduced with CELF2-containing expression constructs. While we found Celf2 mRNA downregulated in CML derived samples, overexpression led to decreased colony formation and cMYC signaling, validating our findings from K562 cells. We also performed an analysis of CML associated splicing changes and found hundreds of deregulated splicing events, of which a significant portion overlapped with CELF2 overexpression mediated changes, suggesting direct regulation by CELF2. Based on this data we propose a novel role of EZH2, where it exerts part of its oncogenic function in CML through the transcriptional repression of splicing factors, in particular CELF2, leading to splicing deregulation, increased cMYC signaling, and enhanced cell growth and viability. Finally, data from other publicly available EZH2 inhibitor treated cancer models suggests that splicing modulation by EZH2 might not be restricted to CML. Citation Format: Reinhard Brunmeir, Ying Li, Baohong Lin, Leong Qiao Zheng, Henry Yang, Wee Joo Chng. EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4404.

Abstract 7228: In-vitro efficacy of Dordaviprone/GSK126 combination therapy on castration resistant and neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is an aggressive histologic subtype associated with poor prognosis that commonly arises in later stages as a mechanism of treatment resistance. Activation of oncogenic drivers, in combination with epigenetic changes (such as EZH2 overexpression and DNA methylation) further promotes tumor proliferation and expression of downstream neuroendocrine lineage pathways (in part controlled by transcription factors, including SOX2, ASCL1, and BRN2). Importantly, EZH2 inhibitors (EZH2i) can restore AR expression in CRPC (Ku et al., 2017). Imipridones, including Dordaviprone (ONC201), show promise in treating neuroendocrine tumors by interacting with DRD2 and CLpP (Anderson et al., 2022). These results warrant investigation into the potential for synergism in imipridone/EZH2i combination therapies. In the PCa cell lines LNCap, PC3, 22Rv1, and DU145 the combination of ONC201 and EZH2i GSK126 was assessed. Cell viability data after treatment with ONC201 and GSK126 reveals several dose ranges with potential synergistic activity after 72h. Preliminary western blots after 72 hours of GSK126 treatment indicate modulation of proteins relevant to the mechanism of action of imipridones, specifically DR5 and CLpP, in LNCap, PC3, and 22Rv1. Notably, the decrease in DR5 expression warrants investigation into the effects of EZH2i on TRAIL sensitivity and whether imipridones may rescue DR5 expression to maintain TRAIL sensitivity. Subsequent experiments will investigate dosing and time point-based effects on viability and protein expression, in addition to assessing the synergy of other imipridones and EZH2i’s. Further, while the cell lines PC3, 22Rv1, and DU145 are castration-resistant, the neuroendocrine prostate cell line NCI-H660 will be used as a positive control as it expresses higher neuroendocrine features, including DRD2. Additionally, the modulation in DR5 expression warrants inquiry into the effects of combination therapy on tumor-immune cell interactions. Further, we will manipulate the expression of neuroendocrine transcription factors to assess changes in therapy sensitivity. Citation Format: Connor Purcell, Praveen Srinivasan, Maximilian Pinho-Schwermann, William MacDonald, Elizabeth C. Ding, Vida Tajiknia, Wafik El-Deiry. In-vitro efficacy of Dordaviprone/GSK126 combination therapy on castration resistant and neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7228.

Abstract 6981: p38-mediated EZH2 phosphorylation at T367 regulates HER2 pathway in triple negative breast cancer

Abstract Background: Triple negative breast carcinomas (TNBC) are highly aggressive tumors with limited effective treatments. Clinical studies showed that targeting low HER2 levels in TNBC with anti-HER2 agents has survival benefit. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase component of the Polycomb Repressive Complex2 that regulates differentiation genes in normal and neoplastic cells. EZH2 phosphorylation at T416 was shown to maintain the TNBC phenotype. We have reported that p38-induced EZH2-T367 phosphorylation enhances metastasis and that high pEZH2-T367 expression is significantly associated with human TNBC tumors compared to other breast cancer subtypes. In this study we tested the hypothesis that pEZH2-T367 may regulate the HER2 pathway in TNBC and that inhibition of pEZH2-T367 may upregulate HER2 signaling, with clinical implications. Methods: MDA-MB-231 (mesenchymal-like TNBC) and CCN6KO cells derived from MMTV-Cre;Ccn6fl/fl (CCN6KO) TNBC mouse model generated in our lab were transduced with vector or EZH2 shRNA knockdown (KD). MDA-MB-231 EZH2 KD cells were rescued with lentivirus containing EZH2-wild-type (WT) or T367A phosphorylation-deficient mutant. Gene and protein expression was analyzed by RNA sequencing, q-RT-PCR, immunoblot and immunohistochemistry. Vector and EZH2-KD rescue MDA-MB-231 cells were treated with anti-HER2 (trastuzumab or pertuzumab 100 ug/ml) followed by Hoestch proliferation assays. TNBC cells were treated with vehicle, EZH2 inhibitor (EPZ-6438, 20uM), p38 inhibitor (p38i, SB202190, 20uM), EPZ+p38i, anti-HER2 (100 ug/ml), and EPZ+p38i+anti-HER2, followed by Hoestch cell proliferation assays. Results: EZH2-T367A deregulates the expression of a 94-gene network of HER2 pathway genes including EREG, SERPINB5, MERTK, IL6R, SOCS3, and FASCN1 compared to EZH2-WT. EZH2-T367A increased protein levels of HER2, EREG, and PP2AB, a major HER2 regulator and was sufficient to sensitize TNBC cells to the anti-proliferative effects of anti-HER2. Supporting the role of pEZH2-T367, we found that the combination of p38i and EPZ significantly sensitizes TNBC cells to anti-HER2 therapies. Conclusions: We demonstrate that T367 phosphorylation mediates the effect of EZH2 on HER2 signaling in TNBC. Our data support a role for pEZH2-T367 in maintaining the HER2-negative phenotype in and suggest that blocking this phosphorylation event may render TNBC tumors amenable to anti-HER2 treatment strategies. Citation Format: Maria E. Gonzalez, Ahmad Eido, Celina G. Kleer. p38-mediated EZH2 phosphorylation at T367 regulates HER2 pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6981.

Abstract 2680: Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis

Abstract Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic subtype of breast cancer. Lack of known targets, patient-to-patient variations of target antigens and resistance to immune checkpoint inhibitors make TNBC one of the most challenging cancers to treat. TNBC is enriched in tumor-infiltrating neutrophils (TINs). TINs are key contributors to pro-tumorigenic processes linked to “immunologically cold” tumors, in part by preventing recruitment of T-lymphocytes (TILs). Prior studies identified the Enhancer of zeste homolog 2 (EZH2) as a prominent methyltransferase expressed in primary and metastatic TNBC cells. EZH2 is an upstream regulator of the stimulator of interferon genes (STING), a critical innate sensor mediating anti-tumor responses that is inhibited in cold tumors. To date, EZH2/STING signaling in TINs is relatively unknown. Here, we hypothesized that EZH2 inhibition and subsequent STING activation could exert anti-tumor activity by modulating TINs. We derived EZH2-knockout (KO) and EZH2-overexpressed (OX) cell lines from the well-established 4T1 TNBC mouse cell line. We demonstrate a 10-fold decrease in lung metastasis and significant reduction of 4T1 EZH2 KO tumor growth compared to the parent 4T1 cell line. Concomitantly, the TIN:TIL ratio was significantly reduced in 4T1 EZH2 KO vs. control tumors. Overexpression of EZH2 in 4T1 TNBC cells did not affect the primary tumor growth or lung metastasis. A therapeutic study using the repeated injection of the STING agonist MSA-2 after tumor engraftment in vivo also demonstrated a decrease in lung metastasis and tumor growth in MSA-2 vs. vehicle injection with both 4T1 parent and EZH2 KO cell lines. Together, our findings suggest a key role for STING pathway modulation and TIN/TIL poise in primary TNBC progression and lung metastasis. Citation Format: Lenore Monterroza, Maria Parrilla, Sarah Samaranayake, Dormarie Rivera, Brian Dobosh, Periasamy Selvaraj, Rabindra M. Tirouvanziam. Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2680.