Enhancer Of Zeste Homolog 2 Mutations Research Articles

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Tazemetostat in Combination with R-CHOP in Patients with High-Risk, Frontline Follicular Lymphoma (Epi-RCHOP): A Phase II Study from the Lysa

Background: Tazemetostat (TAZ), an enhancer of zeste homolog 2 (EZH2) inhibitor, is approved by the US Food and Drug Administration for the treatment of patients with relapsed/refractory (R/R) follicular lymphoma (FL) whose disease has mutant (MT) EZH2 and who have received ≥2 prior therapies or with R/R FL who have no satisfactory alternative treatment options. We previously reported that the RP2D of TAZ in combination with R-CHOP was 800mg BID in the phase 1 part of Epi-RCHOP study (NCT02889523). Here, we report results of the phase 2 part evaluating TAZ with R-CHOP + rituximab maintenance in a cohort of previously untreated high-risk FL patients. Methods: patients were eligible if aged 18-80 years, had histologically confirmed FL grade 1-3A, high-risk FLIPI (score of 3-5) and were in need of treatment according to GELF criteria. Induction treatment consisted in 6 cycles of RCHOP every 21 days and TAZ 800mg BID every day, starting on day 2 of cycle 1, plus 2 additional 21 days cycles of rituximab+TAZ (cycle 7 and 8). Responses were assessed at end of induction (EOI), and responders then received maintenance 1 (TAZ 6 mo + rituximab 12 mo) and maintenance 2 (12 mo of rituximab alone).The primary efficacy endpoint was PET-based complete response rate based on local assessment at the end of induction or at permanent treatment discontinuation, as determined by Cheson IWG 2014 Lugano Classification (Deauville scale 1-3); in addition bone marrow biopsy (BMB) was mandatory at EOI if involved at baseline and complete metabolic response (CMR) patients with missing assessment at EOI classified as partial responders. Sample size calculation was made with the expectation of an increase of 13% in CR, with an H0 hypothesis of 67% and an H1 assumption of 80%, leading to a theorical sample size of 62 pts, assuming a drop out of 10%. Initial assumption was that CR rate should be >76.4% to consider the combination eligible for further clinical evaluation in high risk FL patients. Results: As of June 14, 2022, 62 patients were enrolled between Aug2020 and Apr2022 across 20 centers (including a drop-out of 12 patients, 70 were enrolled in France, 4 in Belgium). Disease characteristics included: males 61.3%, median age 64.5 years, FLIPI 3/4/5 in 61.3%/32.3%/6.5%, EZH2 mutated 17.3% (n=9/46 assessed had EZH2 mutation; 16 undertermined), BM involvement 68.3%, circulating FL cells in 21%, and increased LDH in 50%. Fifty eight (93%) patients completed induction and initiated maintenance 1, 44 (71%) initiated maintenance 2. Fourteen patients permanently discontinued treatment: 4 before EOI not related to PD (2 COVID19 disease, 1 late onset neutropenia and 1 stopped therapy but accepted FU), and 9 after EOI (5 PD, 2 COVID19 disease, 1 neutropenia, 1 paralytic ileus and 1 patient's refusal). At EOI, 79% (95%CI 66.8; 88.3%) achieved complete metabolic response (CMR) and 16.2% PMR for an overall metabolic response rate of 95.2%, however, according to the stringent protocol definition, 16 patients who achieved CMR had no BMB assessment at EOI and were downgraded to PR leading to 53.2% CR and 41.9% PR rate at EOI. Complete response rates were significantly higher in EZH2 mutants (8/9 CMR 88.9%) versus 17/37 (45.9%) in EZH2 wild-type (WT) patients (Fisher exact test p=0.027). Figure 1 shows the AEs reported by more than 10% of patients. The most common grade 3-4 TEAEs were neutropenia (53.2%, only 3.2% febrile), thrombocytopenia (17.7%), anemia (16.1%). Serious AEs occurred in 14 patients, the two most frequent being Covid19 disease (6.5%) and gastrointestinal hypomotility (6.5%). This latter AE was the most frequent non-hematologic event (40.3%), followed by peripheral neuropathy (35.5%). After amending vincristine dose (Oct2020), 30 digestive AEs were reported (16 related to TAZ, mostly grade 1-2, only 1 grade 3, no sequelae). No SPM was observed. With a median follow-up of 19 months, 7 relapses (5 EZH2 WT and 2 undetermined) including 5 needing subsequent treatment and 2 deaths occurred (1 Covid19 disease, 1 after PD). Median PFS and median DOR were not reached and appeared to be similar irrespective of EZH2 status, 18-months PFS and OS rates were 89.3% and 98.3% respectively. Conclusions: Although the primary endpoint was not met due to missing BM assessments, TAZ + RCHOP combination demonstrated a promising complete metabolic response rate of 79% in this high-risk frontline FL population, with a manageable safety profile, after vincristine dose was reduced.

Abstract A026: Overactivation of histone deacetylases and EZH2 in Wilms tumorigenesis

Abstract Wilms tumor (WT) is the most common childhood kidney cancer. Altered epigenetics is central to oncogenesis in many pediatric cancers. The critical contribution of epigenetic dysregulation to pediatric tumors provides a compelling rationale for the therapeutic potential of epigenetic drugs. Histone deacetylases (HDACs, especially HDAC1/2) and Enhancer of Zeste Homolog 2 (EZH2), a histone H3K27 methyltransferase, have been demonstrated to play a critical role in self-renewal and differentiation of mouse nephron progenitor cells (NPCs). WT was considered as the arrest of NPC differentiation at a pre-epithelialized cell state. Altered expression and mutations of HDACs and EZH2 have been linked to many human cancers, including WT. Thus, they are among the most promising therapeutic targets for cancer treatment. The treatment of Panobinostat, an FDA-approved pan-HDAC inhibitor, leaded to apoptosis and growth suppression of WT cells. EZH2 is highly expressed in WT and has been demonstrated to be associated with WT progression. We reasoned that WT results from unrestrained proliferation of progenitor cells due to overactive HDAC1, HDAC2, and EZH2. To prove it, we collected 5 human WT specimens. Immunostaining demonstrated dramatically higher level of HDAC1, HDAC2, SIX1, and SIX2 in the tumor tissues compared with adjacent normal tissues, strongly suggesting a critical role for the overactive HDAC1/2 in the SIX1/2 activation to promote cell proliferation in WT. SIX1 and SIX2 are associated with the high-risk blastemal subtype and with the presence of undifferentiated blastema in WT. In addition, knockdown of HDAC1/2 by siRNAs or CRISPR gene editing resulted in downregulation of SIX1/2 in human embryonic kidney 293 cells. Our study in mouse model also revealed that deletion of Hdac1/2 resulted in deletion of progenitor pool and downregulation of key progenitor genes. We also demonstrated that Ezh2 is the dominant H3K27 methyltransferase in Six2+ NPCs and required for NPC proliferation. Based on these findings, we reason that SIX1/2 upregulation by HDAC1/2 and EZH2 overactivation may play an important role in Wilms tumorigenesis. Concomitant inhibition of HDACs and EZH2 has proven to be highly synergistic and very potent for the treatment of many types of human cancers. Promisingly, combined treatment benzamide-based HDAC1/2-selective inhibitor Merck60 and Tazemetostat (an FDA-approved EZH2 inhibitor) synergistically suppressed cell growth in a wide range of dose combinations in G401 kidney cancer cells. In summary, our studies demonstrated the overactivation of HDAC1/2 in WT, positive regulation of SIX1/SIX2 by HDAC1/2 during tumorigenesis, and synergic effect of HDAC1/2 and EZH2 inhibition in suppressing tumor cell proliferation. Thus, targeting HDAC1/2 and EZH2 may provide a promising therapeutic approach to treat WT and other pediatric kidney cancer with low toxicity and low side effects. Furthermore, availability of epigenetic drugs will facilitate the translation of this research into effective therapies. Citation Format: Hongbing Liu. Overactivation of histone deacetylases and EZH2 in Wilms tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A026.

Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures.

Somatic heterozygous mutations in the active site of the enhancer of zeste homolog 2 (EZH2) are prevalent in diffuse large B-cell lymphoma (DLBCL) and acute myeloid leukemia (AML). The methyltransferase activity of EZH2 towards lysine 27 on histone H3 (H3K27) and non-histone proteins is dysregulated by the presence of gain-of-function (GOF) and loss-of-function (LOF) mutations altering chromatin compaction, protein complex recruitment, and transcriptional regulation. In this study, a comprehensive multi-omics approach was carried out to characterize the effects of differential H3K27me3 deposition driven by EZH2 mutations. Three stable isogenic mutants (EZH2Y641F, EZH2A677G, and EZH2H689A/F667I) were examined using EpiProfile, H3K27me3 CUT&Tag, ATAC-Seq, transcriptomics, label-free proteomics, and untargeted metabolomics. A discrete set of genes and downstream targets were identified for the EZH2 GOF and LOF mutants that impacted pathways involved in cellular proliferation, differentiation, and migration. Disruption of protein networks and metabolic signatures able to sustain aberrant cell behavior was observed in response to EZH2 mutations. This systems biology-based analysis sheds light on EZH2-mediated cell transformative processes, from the epigenetic to the phenotypic level. These studies provide novel insights into aberrant EZH2 function along with targets that can be explored for improved diagnostics/treatment in hematologic malignancies with mutated EZH2.

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.

Stage-specific dual function: EZH2 regulates human erythropoiesis by eliciting histone and non-histone methylation.

Enhancer of zeste homolog 2 (EZH2) is the lysine methyltransferase of polycomb repressive complex 2 (PRC2) that catalyzes H3K27 tri-methylation. Aberrant expression and loss-of-function mutations of EZH2 have been demonstrated to be tightly associated with the pathogenesis of various myeloid malignancies characterized by ineffective erythropoiesis, such as myelodysplastic syndrome (MDS). However, the function and mechanism of EZH2 in human erythropoiesis still remains largely unknown. Here, we demonstrated that EZH2 regulates human erythropoiesis in a stage-specific, dual-function manner by catalyzing histone and non-histone methylation. During the early erythropoiesis, EZH2 deficiency caused cell cycle arrest in the G1 phase, which impaired cell growth and differentiation. Chromatin immunoprecipitation sequencing and RNA sequencing discovered that EZH2 knockdown caused a reduction of H3K27me3 and upregulation of cell cycle proteindependent kinase inhibitors. In contrast, EZH2 deficiency led to the generation of abnormal nuclear cells and impaired enucleation during the terminal erythropoiesis. Interestingly, EZH2 deficiency downregulated the methylation of HSP70 by directly interacting with HSP70. RNA-sequencing analysis revealed that the expression of AURKB was significantly downregulated in response to EZH2 deficiency. Furthermore, treatment with an AURKB inhibitor and small hairpin RNAmediated AURKB knockdown also led to nuclear malformation and decreased enucleation efficiency. These findings strongly suggest that EZH2 regulates terminal erythropoiesis through a HSP70 methylation-AURKB axis. Our findings have implications for improved understanding of ineffective erythropoiesis with EZH2 dysfunction.

Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

Enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of PRC2 complex, plays an important role in tumor development and progression through its catalytic and noncatalytic activities. Overexpression or gain-of-function mutations of EZH2 have been significantly associated with tumor cell proliferation of triple-negative breast cancer (TNBC) and diffuse large B-cell lymphoma (DLBCL). As a result, it has gained interest as a potential therapeutic target. The currently available EZH2 inhibitors, such as EPZ6438 and GSK126, are of benefit for clinical using or reached clinical trials. However, certain cancers are resistant to these enzymatic inhibitors due to its noncatalytic or transcriptional activity through modulating nonhistone proteins. Thus, it may be more effective to synergistically degrade EZH2 in addition to enzymatic inhibition. Here, through a rational design and chemical screening, we discovered a new irreversible EZH2 inhibitor, IHMT-337, which covalently bounds to and degrades EZH2 via the E3 ligase CHIP-mediated ubiquitination pathway. Moreover, we revealed that IHMT-337 affects cell cycle progression in TNBC cells through targeting transcriptional regulating of CDK4, a novel PRC2 complex- and enzymatic activity-independent function of EZH2. More significantly, our compound inhibits both DLBCL and TNBC cell proliferation in different preclinical models in vitro and in vivo. Taken together, our findings demonstrate that in addition to enzymatic inhibition, destroying of EZH2 by IHMT-337 could be a promising therapeutic strategy for TNBC and other malignancies that are independent of EZH2 enzymatic activity.

Tazemetostat in Combination with Lenalidomide and Rituximab in Patients with Relapsed/Refractory Follicular Lymphoma: Phase 1b Results of Symphony-1

Background: The preferred second-line treatment for patients with relapsed/refractory (R/R) follicular lymphoma (FL) is either lenalidomide in combination with rituximab (R2) or an anti-CD20 antibody in combination with chemotherapy/immunotherapy. Tazemetostat (TAZ), an enhancer of zeste homolog 2 (EZH2) inhibitor, is approved by the US Food and Drug Administration for the treatment of patients with R/R FL whose disease has mutant (MT) EZH2 and who have received ≥2 prior therapies or who have no satisfactory alternative treatment options. EZH2 modulates the B-cell tumor microenvironment, supporting combination approaches to treatment. SYMPHONY-1 (EZH-302; NCT04224493) is a phase 1b/3 study designed to determine the recommended phase 3 dose (RP3D), efficacy, and safety of TAZ + lenalidomide and rituximab (R2) in patients with R/R FL after ≥1 prior therapy. We report the results after complete enrollment of the phase 1b safety run-in to assess safety and clinical activity of TAZ when administered with R2, including data for subsets of patients by mutation status, rituximab-refractory disease, and progression of disease in 24 months (POD24). Methods: Patients aged ≥18 years with histologically confirmed FL grade 1-3A who were previously treated with ≥1 prior systemic chemotherapy, immunotherapy, or chemoimmunotherapy were enrolled. The phase 1b portion enrolled patients regardless of EZH2 mutation status. Phase 1b evaluated TAZ at 3 dose levels (400, 600, and 800 mg orally twice daily [BID]) in 28-day cycles with standard-dose R2. Protocol-mandated dose modifications included simultaneous dose interruption and/or reduction of TAZ and lenalidomide for grade 3 neutropenia and thrombocytopenia. Primary endpoints of the phase 1b portion were safety and determination of the RP3D. Secondary endpoints included pharmacokinetic (PK) parameters. Preliminary efficacy analysis was performed on the response-evaluable population, including best overall response, progression-free survival (PFS), and duration of response (DOR) per investigator assessment, according to Lugano 2014 response criteria. Results: As of June 14, 2022, 44 patients were enrolled and receiving TAZ + R2 (400 [n=6], 600 [n=19], and 800 mg [n=19]). Median age was 67 years, and 31.8% of patients had received >1 prior therapy. Overall, 35/42 patients (83.3%) had wild-type (WT) EZH2 FL. Patients with high-risk disease include 15 patients (34.1%) with rituximab-refractory disease and 12 patients (27.3%) with POD24 disease. Median duration of treatment exposure of tazemetostat was 9.1 months. No dose-limiting toxicities were observed in phase 1b, and no new safety signals were identified as of the data cutoff. The PK of TAZ at all dose levels was not altered by concomitant administration of daily oral lenalidomide 20 mg, and the PK of lenalidomide was not altered by concomitant administration of any of the TAZ doses. The RP3D of TAZ was determined to be 800 mg BID in combination with R2. The most common grade 3-4 TEAE was neutropenia (n=15; 34.1%). The most frequent type of dose modification due to TEAEs for any study treatment was drug interruption (n=30; 68.2%). In the 800-mg cohort, the median dose intensity was 98%, 93%, and 100% for tazemetostat, lenalidomide, and rituximab, respectively. Of 41 patients evaluable for tumor assessment, 21 (51.2%) had a complete response, 19 (46.3%) had a partial response, and 1 (2.4%) had stable disease. The overall response rate (ORR) in the response-evaluable population was 97.6% (n=40). The ORRs in patients with WT EZH2 and MT EZH2 were 97% (n=32) and 100% (n=7), respectively. ORRs in patients with rituximab-refractory disease was 100% (n=14) and in patients with POD24 disease was 100% (n=11). With a median follow-up of 11.2 months, median PFS and median DOR were not reached and appeared to be similar, regardless of rituximab-refractory, POD24, or EZH2 mutation status. Conclusions: TAZ + R2 combination therapy demonstrates a consistent and manageable safety profile, with no new safety signals. The combination therapy maintained its efficacy, regardless of mutation status. Durable responses were seen in patients with high-risk disease, including those with rituximab-refractory and POD24 disease. The 2-arm randomized phase 3 portion will further explore the efficacy and safety of TAZ RP3D 800 mg + R2 in ≈500 patients with R/R FL who completed ≥1 prior systemic therapy.

Immunohistochemical loss of enhancer of Zeste Homolog 2 (EZH2) protein expression correlates with EZH2 alterations and portends a worse outcome in myelodysplastic syndromes

AbstractEZH2 coding mutation (EZH2MUT), resulting in loss-of-function, is an independent predictor of overall survival in MDS. EZH2 function can be altered by other mechanisms including copy number changes, and mutations in other genes and non-coding regions of EZH2. Assessment of EZH2 protein can identify alterations of EZH2 function missed by mutation assessment alone. Precise evaluation of EZH2 function and gene-protein correlation in clinical MDS cohorts is important in the context of upcoming targeted therapies aimed to restore EZH2 function. In this study, we evaluated the clinicopathologic characteristics of newly diagnosed MDS patients with EZH2MUT and correlated the findings with protein expression using immunohistochemistry. There were 40 (~6%) EZH2MUT MDS [33 men, seven women; median age 74 years (range, 55–90)]. EZH2 mutations spanned the entire coding region. Majority had dominant EZH2 clone [median VAF, 30% (1–92)], frequently co-occurring with co-dominant TET2 (38%) and sub-clonal ASXL1 (55%) and RUNX1 (43%) mutations. EZH2MUT MDS showed frequent loss-of-expression compared to EZH2WT (69% vs. 27%, p = 0.001). Interestingly, NINE (23%) EZH2WT MDS also showed loss-of-expression. EZH2MUT and loss-of-expression significantly associated with male predominance and chr(7) loss. Further, only EZH2 loss-of-expression patients showed significantly lower platelet counts, a trend for higher BM blast% and R-IPSS scores. Over a 14-month median follow-up, both EZH2MUT (p = 0.027) and loss-of-expression (p = 0.0063) correlated with poor survival, independent of R-IPSS, age and gender. When analyzed together, loss-of-expression showed a stronger correlation than mutation (p = 0.061 vs. p = 0.43). In conclusion, immunohistochemical assessment of EZH2 protein, alongside mutation, is important for prognostic workup of MDS.

Abstract 3939: Preclinical development of embryonic ectoderm development (EED) inhibitor APG-5918/EEDi-5273 for cancer therapy

Abstract Three core components constitute the polycomb repressive complex 2 (PRC2), a multiprotein complex that catalyzes methylation of histone H3 at lysine 27 (H3K27): (1) enhancer of zeste homolog 2 (EZH2), (2) EED, and (3) suppressor of zeste 12 protein homolog (SUZ12). Dysregulated function of PRC2 is implicated in the development of various cancer types. With regulatory approval of EZH2 inhibitor tazemetostat, a potentially effective successful cancer therapeutic strategy is now available for patients with epitheloid sarcoma and relapsed or refractory follicular lymphoma. However, EZH2 inhibitor activity might be reduced because of acquired resistance through secondary mutations in EZH2 or its inability to inhibit paralogs of EZH2 (including EZH1). Because binding of EED with trimethylated H3K27 (H3K27me3) is required to activate methyltransferase activity of EZH2, allosterically targeting EED is emerging as a novel approach to inhibit PRC2 (Qi et al, Nat Chem Biol 2017). APG-5918/EEDi-5273 is a well-documented, investigational, novel, bioactive, and potent EED inhibitor (Rej et al, JMC 2021). The aim of this study was to further characterize the anticancer therapeutic activity of APG-5918 in the preclinical setting. APG-5918/EEDi-5273 showed high-affinity binding to EED protein in a biochemical assay (IC50 = 1.2 nM). It also exerted potent antiproliferative activity in several EZH2 mutant (EZH2mut) diffuse large B-cell lymphoma (DLBCL) cell lines, with IC50 values in the nanomolar range and inhibitory effects that were approximately 5 times lower than those with tazemetostat. In a mouse xenograft model derived from EZH2mut KARPAS-422 DLBCL cells, single-agent APG-5918 conferred potent and dose-dependent antitumor activity, resulting in durable complete tumor regression that correlated with inhibition of H3K27me3, induction of PRC2 target genes, and drug exposure in tumors. In summary, our results provide scientific rationale for the clinical development of APG-5918/EEDi-5273 in EZH2mut lymphomas and, potentially, other hematologic malignancies and solid tumors. Citation Format: Saijie Zhu, Douglas D. Fang, Qiang Li, Dongmei Yang, Shoulai Gu, Shaomeng Wang, Dajun Yang, Yifan Zhai. Preclinical development of embryonic ectoderm development (EED) inhibitor APG-5918/EEDi-5273 for cancer therapy [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 3939.

SHR2554, an enhancer of zeste homolog 2 (EZH2) inhibitor, in relapsed or refractory (r/r) mature lymphoid neoplasms: A first-in-human phase 1 study.

7525 Background: Dysregulation of histone methyltransferase EZH2 plays critical roles in lymphomagenesis. Emerging evidence shows that EZH2 also contributes to tumor immune evasion. SHR2554 is an oral, small-molecule inhibitor exhibiting potent selectivity for EZH2. We initiated a first-in-human study to assess SHR2554 across a broad spectrum of lymphoid neoplasms that had relapsed or was refractory to standard systemic therapies, including B-cell lymphomas, T-cell lymphomas, and classical Hodgkin lymphoma (cHL). Methods: This multicenter, phase 1 study was composed of 3 parts: dose escalation phase according to mTPI-2 design, dose expansion phase, and clinical expansion phase in selected tumor subtypes. Dose was escalated at 50, 100, 200, and 400 mg BID, and then de-escalated at 300 and 350 mg based on the observed toxicities. Two dose levels (300 and 350 mg) were expanded. Subsequently, follicular lymphoma (FL), peripheral T-cell lymphoma (PTCL), and cHL were selected for clinical expansion at RP2D. Primary endpoints were to assess the safety and determine the MTD and RP2D. Key secondary endpoint included clinical activity at RP2D in different subtypes. Results: As of Sep 10, 2021, 113 heavily pretreated pts were enrolled, with 53 (46.9%) having received ≥3 lines of prior systemic therapies. In dose escalation phase, DLTs occurred in 2 of 3 pts at 400 mg and 1 of 6 pts at 350 mg; thus, 350 mg BID was the MTD. Combined with the safety, tolerability, PK/PD, and preliminary efficacy findings observed in dose escalation and expansion phases, RP2D was determined to be 350 mg BID. As of cutoff date, 41 FL, 22 PTCL, and 21 cHL pts who received SHR2445 at 350 mg BID completed at least one post-baseline efficacy assessment. ORR in FL was 58.5% (95% CI 42.1-73.7); majority of responses (66.7%) were still ongoing, and the estimated median DoR was 9.3 mos (95% CI 5.6-NR). EZH2mut FL pts showed a slightly higher ORR than EZH2WT FL pts (62.5% vs 55.2%). EZH2 mutations were not detected in pts with PTCL or cHL. ORR in PTCL was 63.6% (95% CI 40.7-82.8); majority of responses (71.4%) were still ongoing, and the estimated median DoR was 7.4 mos (95% CI 2.9-NR). All cHL pts had received prior chemotherapy and anti-PD-1/PD-L1 antibodies. Shrinkage in target lesions was shown in 76.2% of cHL pts; ORR was 19.0% (95% CI 5.4-41.9); majority of responses (75.0%) were still ongoing, and the median DoR had not been reached yet. Grade ≥3 treatment-related AEs occurred in 38 of the 113 pts (33.6%), with the most common being decreased platelet count (17.7%), decreased neutrophil count (8.8%), decreased white blood cell count (8.0%), and anemia (6.2%). Conclusions: SHR2554 showed a manageable safety profile and promising anti-tumor activity in pts with r/r lymphomas, supporting further explorations in FL, PTCL, and cHL. Clinical trial information: NCT03603951.

Tazemetostat: EZH2 Inhibitor.

Epigenetic regulation is a novel approach to cancer treatment. Inhibition of enhancer of zeste homolog 2 (EZH2) is a method to provide targeted epigenetic regulation. Tazemetostat is a first-in-class targeted epigenetic regulator that specifically inhibits EZH2. This new FDA-approved oral treatment received accelerated approval for patients with hematologic and solid malignancies. Tazemetostat was first approved for patients 16 years and older with metastatic or locally advanced epithelioid sarcoma not eligible for complete resection based on the results of an international open-label phase II basket trial. Another open-label multicenter phase II trial led to the approval for patients with relapsed or refractory follicular lymphoma with EZH2 mutation who have received at least two prior systemic therapies or patients who have no satisfactory alternative treatment options. Tazemetostat as an oral EZH2 inhibitor provides a new effective and tolerable treatment option for these patients.

A Matching-Adjusted Indirect Comparison of Single-Arm Trials in Patients with Relapsed or Refractory Follicular Lymphoma Who Received at Least Two Prior Systemic Treatments: Tazemetostat was Associated with a Lower Risk for Safety Outcomes Versus the PI3-Kinase Inhibitors Idelalisib, Duvelisib, Copanlisib, and Umbralisib.

IntroductionTazemetostat is an enhancer of zeste homolog 2 (EZH2) inhibitor recommended for patients with relapsed/refractory (R/R) follicular lymphoma (FL) after demonstrating single-agent, antitumor activity in patients with wild-type or mutant EZH2. The phosphoinositide 3-kinase (PI3K) inhibitors idelalisib, copanlisib, umbralisib and (formerly) duvelisib are indicated for third-line, fourth-line, and later (3L/4L+) treatment of R/R FL. The objective of this analysis was to provide an indirect treatment comparison of tazemetostat with each PI3K inhibitor for 3L/4L+ R/R FL treatment.MethodsA systematic literature review was conducted to identify trials for idelalisib (DELTA), duvelisib (DYNAMO), copanlisib (CHRONOS-1 Part B), and umbralisib (UNITY-NHL) in 3L+ R/R FL. Matching-adjusted indirect comparisons were conducted by weighting tazemetostat individual patient data with available baseline characteristics from each comparator trial: age, Eastern Cooperative Oncology Group performance status, disease stage, histology, prior treatment lines, prior stem cell therapy, progression within 24 months, and refractory status to last therapy. Only the tazemetostat trial included patients with grade 3b or transformed FL, or recorded EZH2 mutation status. Primary safety outcomes included risk of grade ≥ 3 treatment-emergent adverse events (TEAEs); primary efficacy outcomes included objective response rate (ORR).ResultsMatched patients treated with tazemetostat had lower relative risk (RR) for all grouped safety outcomes, including any grade ≥ 3 TEAEs, compared with idelalisib (RR = 0.45), duvelisib (RR = 0.35), copanlisib (RR = 0.37), and umbralisib (RR = 0.65; all, p < 0.01), any serious TEAE, and any TEAE leading to dose reduction, drug discontinuation, or interruption. The ORR was not significantly different for tazemetostat versus other treatments (idelalisib 43% vs 56%, p = 0.16; duvelisib 48% vs 47%, p = 0.91; copanlisib 49% vs 61%, p = 0.11; and umbralisib 57% vs 47%, p = 0.10).ConclusionsIn this statistically adjusted comparison, tazemetostat was associated with lower RR for safety outcomes versus idelalisib, duvelisib, copanlisib, and umbralisib, while achieving similar efficacy outcomes.

EZH2 genetic alterations in follicular and peripheral T-cell lymphoma and correlation with immunohistochemistry

Background: Mutations in Enhancer of zeste homolog 2 (EZH2) are common events in lymphoma pathogenesis.1 Recent studies have suggested that the presence of mutations in EZH2 may have prognostic2 and therapeutic implications.

Abstract P088: Combined inhibition of histone deacetylases and EZH2 for the treatment of Wilms tumors

Abstract Wilms tumor (WT) is the most common childhood kidney cancer. It is believed to arise from immature kidney cells–nephron progenitor cells (NPCs)—which have failed to differentiate properly. Altered epigenetics is central to oncogenesis in many pediatric cancers. The critical contribution of epigenetic dysregulation to pediatric tumors provides a compelling rationale for the therapeutic potential of epigenetic drugs. Histone deacetylases (HDACs, especially HDAC1/2) and Enhancer of Zeste Homolog 2 (EZH2), a histone H3K27 methyltransferase, have been demonstrated to play a critical role in self-renewal and differentiation of mouse nephron progenitor cells (NPCs). In addition, altered expression and mutations of HDACs and EZH2 have been linked to many human cancers, including WT. As such, they are among the most promising therapeutic targets for cancer treatment. For sinstance, the treatment of Panobinostat, an FDA-approved pan-HDAC inhibitor, leaded to apoptosis and growth suppression of WT cells. EZH2 is highly expressed in WT and has been demonstrated to be associated with WT progression. We reasoned that WT results from unrestrained proliferation of progenitor cells due to overactive HDAC1, HDAC2, and EZH2. To prove this, we collected 5 human WT specimens. Immunostaining demonstrated dramatically higher level of HDAC1, HDAC2, SIX1, and SIX2 in the tumor tissues compared with adjacent normal tissues, strongly suggesting a critical role for the overactive HDAC1/2 in the SIX1/2 activation to promote cell proliferation in WT. Gain-of-function of SIX1 or SIX2 has been demonstrated to be closely correlated with high proliferation and aberrant differentiation of WT. Importantly, SIX2 marks the cancer stem cell population in WT. In addition, knockdown of HDAC1/2 by siRNAs or CRISPR gene editing resulted in downregulation of SIX1/2 in human embryonic kidney 293 cells and G401 cells derived from a Wilms tumor. Published studies also showed that treatment of Trichostain (a potent pan HDAC inhibitor) significantly reduced the Six1 expression in vertebrate neural crest and Hodgkin lymphoma cells. All these support the regulation of SIX1/2 expression by HDAC1/2. Concomitant inhibition of HDACs and EZH2 has proven to be highly synergistic and very potent for the treatment of many types of human cancers. Promisingly, combined treatment benzamide-based HDAC1/2-selective inhibitor Merck60 and Tazemetostat (an FDA-approved EZH2 inhibitor) synergistically suppressed cell growth in a wide range of dose combinations in 293T and G401 cells. In summary, our studies demonstrated elevated expression and activity of HDAC1/2 in WT, positive regulation of SIX1/SIX2 by HDAc1//2 during tumorigenesis, and synergic effect of HDAC1/2 and EZH2 inhibition in suppressing tumor cell proliferation. Thus, targeting HDAC1/2 and EZH2 may provide a promising therapeutic approach to treat WT and other pediatric kidney cancer with low toxicity and low side effects. Furthermore, availability of epigenetic drugs will facilitate the translation of this research into effective therapies. Citation Format: Hongbing Liu, Nhi Ngo, Chao-Hui Chen, Samir S. El-Dahr. Combined inhibition of histone deacetylases and EZH2 for the treatment of Wilms tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P088.

Molecular and Genetic Characterization of Tumor Samples from Patients with Relapsed or Refractory Follicular Lymphoma Identifies Factors Influencing Response to Tazemetostat

Molecular and Genetic Characterization of Tumor Samples from Patients with Relapsed or Refractory Follicular Lymphoma Identifies Factors Influencing Response to Tazemetostat

Easy or Not-The Advances of EZH2 in Regulating T Cell Development, Differentiation, and Activation in Antitumor Immunity.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2), which regulates downstream gene expression by trimethylation of lysine 27 in histone H3 (H3K27me3). EZH2 mutations or overexpressions are associated with many types of cancer. As inhibition of EZH2 activity could upregulate the expression of tumor suppressor genes, EZH2 has recently become an interesting therapeutic target for cancer therapy. Moreover, accumulating evidence has shown that EZH2 may contribute to the regulation of immune cells, especially T cells. EZH2 regulates T cell development, differentiation, and function, suggesting that EZH2 also regulates immune homeostasis in addition to tumor suppressor genes. Moreover, EZH2 can regulate T cell fate by targeting non-T cell factors such as metabolism, cytokines, and myeloid-derived suppressor cells. The role of EZH2 in this process has not been fully addressed. This review discusses up-to-date research on EZH2-mediated regulation of immunological function and the progress of immunological therapeutic strategies based on this regulation.

Going beyond Polycomb: EZH2 functions in prostate cancer.

The Polycomb group (PcG) protein Enhancer of Zeste Homolog 2 (EZH2) is one of the three core subunits of the Polycomb Repressive Complex 2 (PRC2). It harbors histone methyltransferase activity (MTase) that specifically catalyze histone 3 lysine 27 (H3K27) methylation on target gene promoters. As such, PRC2 are epigenetic silencers that play important roles in cellular identity and embryonic stem cell maintenance. In the past two decades, mounting evidence supports EZH2 mutations and/or over-expression in a wide array of hematological cancers and solid tumors, including prostate cancer. Further, EZH2 is among the most up-regulated genes in neuroendocrine prostate cancers, which become abundant due to the clinical use of high-affinity androgen receptor pathway inhibitors. While numerous studies have reported epigenetic functions of EZH2 that inhibit tumor suppressor genes and promote tumorigenesis, discordance between EZH2 and H3K27 methylation has been reported. Further, enzymatic EZH2 inhibitors have shown limited efficacy in prostate cancer, warranting a more comprehensive understanding of EZH2 functions. Here we first review how canonical functions of EZH2 as a histone MTase are regulated and describe the various mechanisms of PRC2 recruitment to the chromatin. We further outline non-histone substrates of EZH2 and discuss post-translational modifications to EZH2 itself that may affect substrate preference. Lastly, we summarize non-canonical functions of EZH2, beyond its MTase activity and/or PRC2, as a transcriptional cofactor and discuss prospects of its therapeutic targeting in prostate cancer.

Abstract 1131: ORIC-944, a potent and selective allosteric PRC2 inhibitor, demonstrates robust in vivo activity in prostate cancer models

Abstract The polycomb repressive complex 2 (PRC2) is responsible for the methylation of histone 3 at lysine 27 (H3K27) which leads to long-term transcriptional silencing. Through this epigenetic chromatin silencing mechanism, PRC2 plays a key role in regulating cellular functions such as cell growth and differentiation. PRC2 comprises three core subunits: the catalytic subunit enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12). EED directly interacts with histone H3K27me3 and is essential for the histone methyltransferase activity of PRC2. PRC2 dysregulation occurs in multiple solid tumors and hematological malignancies, resulting in elevated levels of PRC2 activity and H3K27 trimethylation, and has been linked to poor prognosis in patients with metastatic prostate cancer. First-generation PRC2 inhibitors which target EZH2 have demonstrated clinical activity in several cancers, yet the pharmacological and ADME properties of these compounds require high doses that only achieve partial target inhibition at clinically active levels and exhibit drug-drug interaction (DDI) liabilities. As an alternative strategy to fully inhibit the PRC2 complex, we developed ORIC-944, a potent and highly selective allosteric inhibitor of PRC2 via binding the EED subunit. This unique EED targeting strategy can more completely inhibit PRC2, for example, in the presence of innate or acquired resistance mutations in EZH2, and by addressing the potential compensatory escape mechanism of EZH1-driven tumor growth. ORIC-944 has potential best-in-class drug properties compared to first generation PRC2 inhibitors, including superior potency and improved DDI liabilities. In diffuse large B-cell lymphoma (DLBCL) xenografts in vivo, ORIC-944 significantly depleted H3K27 trimethylation and induced tumor regressions in a dose-dependent manner. ORIC-944 demonstrated strong tumor growth inhibition as a single agent with once daily oral dosing in both enzalutamide-responsive and enzalutamide-resistant prostate cancer models. ORIC-944 caused a significant reduction in tumor cell proliferation and anti-apoptotic signaling, as measured by Ki67 and survivin, respectively. Moreover, in PK/PD assessments, ORIC-944 strongly depleted H3K27me3 in treated tumors. These in vivo studies established that effective single agent inhibition of PRC2 via EED results in decreased tumor cell growth in PRC2-dependent prostate cancer models. In summary, ORIC-944 is a potent, highly selective, allosteric, orally bioavailable PRC2 inhibitor via the EED subunit that represents a differentiated strategy to block PRC2 activity in selected cancers. We are developing ORIC-944 as a best-in-class PRC2 inhibitor for the treatment of patients with advanced prostate cancer and expect to file an IND in the second half of 2021. Citation Format: Anneleen Daemen, Jessica D. Sun, Aleksandr Pankov, Frank L. Duong, Natalie Yuen, Shravani Barkund, Shelly Kaushik, Jae H. Chang, David M. Briere, Niranjan Sudhakar, Andrew Calinisan, Aaron Burns, John M. Ketcham, Matthew A. Marx, Peter Olson, James G. Christensen, Melissa R. Junttila, Lori S. Friedman. ORIC-944, a potent and selective allosteric PRC2 inhibitor, demonstrates robust in vivo activity in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1131.

Response to the EZH2 Inhibitor Tazemetostat Is Independent of Cell of Origin Determined Via Hans Imunohistochemistry or Nanostring Lymphoma Subtyping Test in EZH2 Wild-Type DLBCL Patients

Response to the EZH2 Inhibitor Tazemetostat Is Independent of Cell of Origin Determined Via Hans Imunohistochemistry or Nanostring Lymphoma Subtyping Test in EZH2 Wild-Type DLBCL Patients