EZH2 Activity Research Articles

EZH2 affects malignant progression and DNA damage repair of lung adenocarcinoma cells by regulating RAI2 expression

BackgroundLung adenocarcinoma (LUAD) is featured in high morbidity and mortality. Aberrant activation of the histone methyltransferase EZH2 has close association with cancer progression. This research aimed to deeply dive into the role and possible molecular mechanisms of EZH2 and its downstream genes in malignant progression and DNA damage repair of LUAD cells. MethodsExpression of EZH2 in LUAD cells was analyzed by qRT-PCR, and the effects of EZH2 on proliferation, and apoptosis of LUAD cells were examined by CCK-8, colony formation and flow cytometry assays. The downstream targets of EZH2 were predicted by bioinformatics analysis. Then, the targeting relationship between EZH2 and RAI2 was examined by CHIP and luciferase reporter assays. Rescue assay were used to further validate the effect of EZH2/RAI2 on the malignant progression of LUAD cells. The expression levels of EZH2, RAI2 and p53 were examined by Western blot. ResultsUpregulation of EZH2 was identified in LUAD tissues and cells. RAI2 was a downstream target gene of EZH2, and the two were negatively correlated. Silencing EZH2 suppressed proliferation of LUAD cells, promoted expression of p53, cell cycle arrest and apoptosis. While silencing RAI2 could reverse the above-mentioned effects caused by EZH2 silencing. ConclusionThese results demonstrated that EZH2 promoted malignant progression and DNA damage repair of LUAD cells by targeting and negatively regulating RAI2.

LncRNA MALAT1 Regulates USP22 Expression Through EZH2-Mediated H3K27me3 Modification to Accentuate Sepsis-Induced Myocardial Dysfunction

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), has been confirmed to recruit enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) to regulate cardiomyocyte apoptosis in diabetic cardiomyopathy. However, whether the similar regulatory axis exists in sepsis-induced myocardial dysfunction (SIMD) has not been clearly established. The current study sought to define the mechanism governing MALAT1-mediated EZH2 in SIMD. MALAT1 was significantly upregulated in lipopolysaccharide-induced cardiomyocytes. Depletion of MALAT1 by caudal vein injection of small interfering RNA targeting MALAT1 alleviated myocardial injury in SIMD rats, restored cardiac function, reduced oxidative stress production and fibrosis, and inhibited inflammatory factors and apoptosis in myocardial tissues. Moreover, MALAT1 bound to EZH2 and promoted EZH2 activity in the nucleus of cardiomyocytes. EZH2 repressed ubiquitin-specific peptidase 22 (USP22) expression through H3K27me3 modification. EZH2 elevation aggravated the cardiac injury in SIMD rats, while USP22 upregulation inhibited the effect of EZH2, which reduced the cardiac injury in SIMD rats. Taken together, MALAT1 decreased USP22 expression by interacting with EZH2, thereby worsening SIMD, highlighting an attractive therapeutic strategy for SIMD.

Abstract 5708: The DNA methylation landscape of prostate cancer brain metastases

Abstract Introduction: Improved survival in prostate cancer through more effective therapies has also led to an increase in the diagnosis of metastases to infrequent locations such as the brain. In the largest cohort of prostate cancer brain metastases (PCBM) yet studied, we investigated the DNA methylation landscape of PCBM. Methods: Using Illumina EPIC arrays, we profiled the DNA methylation landscapes of 95 metastatic samples from 42 patients, with multi-regional analysis of the metastases from 31 patients. We compared DNA methylation profiles from metastases to the primary prostate cancer (PCa) samples of 18 of these patients. Multi-regional data was obtained for 15 of these. We performed unsupervised analyses of DNA methylation profiles across sample types, and identified differentially methylated CpG sites and regions (DMRs) between PCBM and PCa samples, and PCBM/PCa samples and normal prostate tissue. We integrated our data with targeted-RNA sequencing and whole exome sequencing (WES). Results: We highlight the epigenetic heterogeneity present in different regions of the PCBM from patients for which multi-regional data were available. While substantial DNA methylation changes were detected in PCa compared to normal prostate tissue, epigenetic dysregulation was strikingly more pronounced in PCBM samples. DNA methylation changes in PCBM involved striking hypermethylation particularly enriched at CpG islands, and associated with activity of the PRC2 complex. We observed a marked distinction of the methylation profiles of SPOP mutant and TMPRSS2-ERG samples. SPOP mutant samples acquired substantially more epigenetic aberrations, particularly enriched at CpG islands which were hypermethylated genome-wide compared to TMPRSS2-ERG PCBM samples, as well as SPOP mutant PCa samples. These changes were strongly associated with the activity of EZH2 and MYC, both of which were overexpressed specifically in SPOP mutant PCBM. TMPRSS2-ERG samples showed fewer DNA methylation changes, but those detected were associated with targets of the SP1 transcription factor. Conclusion: We show that epigenetic heterogeneity is present in different regions of PCBM. While the distinct methylation landscapes of SPOP mutant and TMPRSS2-ERG PCa has been previously reported, we show that metastasis-specific DNA methylation changes emerge in each of these settings. These observations suggest that the acquisition of DNA methylation changes may be an important feature of PCBM, and may help to improve our understanding of the processes driving the occurrence of these rare metastases. Citation Format: John Gallon, Antonio Rodriguez-Calero, Sina Maletti, Charlotte Ng, Mark Rubin, Salvatore Piscuoglio. The DNA methylation landscape of prostate cancer brain metastases [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 5708.

Abstract 2922: Targeting triple negative breast cancer with a VHL recruiting EZH2 protein degrader

Abstract EZH2 (enhancer of zeste homolog 2) is the main catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes methylation of histone H3K27. EZH2 is overexpressed in a broad spectrum of cancers including prostate, myeloma and lymphoma, and high expression correlates with poor prognosis. However, EZH2 inhibitors, which do not affect EZH2 protein levels, are ineffective at inhibiting growth of TNBC and other breast cancer cell lines with EZH2 overexpression even though they effectively inhibit the catalytic activity of EZH2/PRC2. It has also been shown that overexpression of EZH2 is a major driver for breast cancer development and progression, and knockdown of EZH2 inhibits proliferation of TNBC and other breast cancer cells. These results together suggest that EZH2 overexpression, but not the methyltransferase activity of EZH2, is critical for driving breast cancer progression. Bivalent inhibitor technologies such as PROTACs (proteolysis targeting chimeras) and hydrophobic tagging have been successfully applied to selective degradation of multiple protein targets. CRBN-recruiting (E7) and VHL-recruiting EZH2 (YM281) EZH2 targeting PROTACs have been reported, but do not show potent efficacy in targeting TNBC. Here, we characterize MS8815, a VHL-recruiting EZH2 degrader, that we created. MS8815 displayed nanomolar efficacy at EZH2 degradation and potent in vitro effects on TNBC cell lines, no off-target interactions and utilized the canonical PROTAC mechanism of action. Based on these promising preliminary results, we report MS8815 as the best-in-class EZH2 PROTAC degrader that is able to efficiently degrade EZH2 and kill TNBC cells. Citation Format: Brandon Dale, Chris Anderson, Kwang-su Park, H. Ümit Kaniskan, Xufen Yu, Jian Jin. Targeting triple negative breast cancer with a VHL recruiting EZH2 protein degrader [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 2922.

Abstract 5436: HH2853 is a selective small molecular dual inhibitor of EZH1/2 with potent anti-tumor activities

Abstract Polycomb Repressive Complex 2 (PRC2) is a multiprotein complex that catalyzes the methylation of lysine 27 on histone H3 (H3K27), and the histone methyltransferases EZH1/2 are the catalytic subunit of PRC2. Aberrant activation of EZH2 drives H3K27 methylation and plays a critical role in cancer initiation and progression. Meanwhile, loss-of-function mutation in the major components of SWI/SNF complex leads to a loss of its suppression ability against PRC2 and subsequently activates EZH2. Targeting EZH2 has been validated as a promising therapeutic strategy for cancer treatment, especially for those with EZH2 gain-of-function (GOF) mutations or alterations of SWI/SNF complex proteins that act as oncogenic drivers. Although when PRC2 was suppressed by EZH2 inhibition, the activity of EZH1 complementarily increased to replace the role of EZH2 and maintained the function of PRC2. In addition, EZH2 inhibition in hematopoietic stem cells of mice has been shown to facilitate the development of heterogeneous hematologic malignancies in an EZH1-dependent manner. As a result, dual inhibition of EZH1 and EZH2 may be more effective than EZH2 inhibition alone in suppressing PRC2 function, as well as overcoming the issues related to the development of secondary malignancies, which has been observed in the clinical development of EZH2 selective inhibitor. Thus, we discovered and developed HH2853,a dual inhibitor of EZH1/2. The potency and selectivity of HH2853 against EZH1/2 were assessed at biochemical and cellular levels, and its anti-tumor activities were evaluated in multiple tumor models in vitro and in vivo in comparison with FDA-approved EZH2 selective inhibitor tazemetostat. In addition, the pharmacokinetic (PK) profile of HH2853 was characterized. HH2853 inhibited the enzymatic activities of wild-type and mutant EZH2 with IC50 values of 2.21-5.36 nM, which was similar to that of tazemetostat. In addition, HH2853 inhibited EZH1 enzymatic activity with an IC50 of 9.26 nM, which was stronger than tazemetostat (IC50: 58.43 nM). In contrast, HH2853 up to 10 μM exhibited only marginal or minor inhibitory activity against 36 histone modification enzymes. At cellular level, HH2853 potently inhibited H3K27 mono-, di- and tri-methylation in multiple cancer cell lines with wild-type or mutant EZH2. In contrast, HH2853 up to 10 μM had no effect on the other types of methylation modifications on histone H3. Moreover, HH2853 potently inhibited the cell viability of multiple cancer cell lines with EZH2 GOF mutation or alterations in SWI/SNF complex, and exhibited superior anti-tumor efficacy in several tumor xenograft models than tazemetostat at a comparable dose level. Furthermore, HH2853 exhibited superior PK property than tazemetostat. Taken together, HH2853 is a selective EZH1/2 dual inhibitor with potent anti-tumor activities and favorable PK properties. HH2853 is currently in phase I clinical development. Citation Format: Xu-Xing Chen, Qian-Qian Shen, Zhao Zhao, Yan-Fen Fang, Jun-Yu Yang, Ying-Lei Gao, Lei Liu, Yixiang Zhang, Yi Chen, Leping Li. HH2853 is a selective small molecular dual inhibitor of EZH1/2 with potent anti-tumor activities [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 5436.

Abstract 3033: Glucose deprivation promotes lung adenocarcinoma de-differentiation due to unbalanced EZH2 activity

Abstract Lung cancer is the most frequent cancer-related cause of death, and adenocarcinoma (LUAD) is the most frequent type. Despite the recent success of immunotherapies, survival of lung cancer patients has not significantly improved in the last decades. New therapies are necessary. We have previously identified sodium-glucose transporter 2 (SGLT2) as the major responsible for glucose uptake in LUAD, and we have showed that treatment with SGLT2 inhibitors significantly delays LUAD development and prolongs survival in murine models. However, our data shows that SGLT2 inhibitors also induce de-differentiation of LUAD cells, leading to a more aggressive phenotype and increased resistance to cisplatin. Glucose deprivation causes reduced αKG levels, leading to reduced activity of αKG-dependent histone demethylases and consequent histone hypermethylation. Supplementation of αKG or inhibition of the histone methyltransferase EZH2 reverse this phenotype, suggesting that this de-differentiated phenotype depends on insufficiency of αKG-dependent histone demethylases and unbalanced EZH2 activity. Consistently, double treatment with an SGLT2 inhibitor and an EZH2 inhibitor significantly reduces the tumor burden in a genetically engineered murine model of LUAD. We further characterized the effect of low glucose-induced tumor de-differentiation, identifying stabilization of hypoxia inducible factor 1α (HIF1α) as a major pathway responsible for the acquisition of a more aggressive phenotype following glucose deprivation. Finally, we identified an HIF1α-dependent transcriptional signature with prognostic significance in human LUAD. Our studies further our knowledge of the relationship between glucose metabolism and cell differentiation in cancer, characterizing the epigenetic adaptation of cancer cells to nutrient deprivation and identifying novel targets to prevent the development of resistance to metabolic therapies. Citation Format: Pasquale Saggese, Aparamita Pandey, Eileen Fung, Giorgio Giurato, Alessandro Weisz, Steven M. Dubinett, Claudio Scafoglio. Glucose deprivation promotes lung adenocarcinoma de-differentiation due to unbalanced EZH2 activity [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 3033.

MEDB-83. A novel epigenetic nanotherapeutic strategy to induce medulloblastoma differentiation

The histone-lysine N-methyltransferase EZH2 is the catalytic component of the PRC2 complex and is overexpressed in several medulloblastoma subtypes. However, its role in medulloblastoma tumorigenesis has been shown to be context-dependent using genetic approaches. Furthermore, pharmacological approaches have been limited by the very poor blood-brain barrier (BBB) penetration of current EZH2 inhibitors in use. Using laser capture microdissection and RNA-Seq analysis of human nodular/desmoplastic SHH medulloblastoma FFPE tissue, we provide data for the spatial epigenetic heterogeneity of primitive/proliferative regions compared to nodular/mature regions. Bioinformatic analysis identifies ~120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3 or SUZ12. ChIP-Seq analysis shows striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Utilizing a genetically-engineered mouse model of SHH medulloblastoma, we show that conditional EZH2 genetic ablation within medulloblastoma cells results in wide-spread tumor cell differentiation (n=31 mice; *p=2e-07). Conversely, conditional EZH2 (Y641F) activation in this GEM model prevents tumor cell differentiation. Notably, we have found that the CDNK2A (p16) locus is an important EZH2 target that regulates tumor cell differentiation. qRT-PCR analysis of SHH medulloblastoma in wild-type and Ezh2 knockout settings show significant reduction in Gli1 and CCND1 and increase p15 and p16 expression in Ezh2 knockout mice compared to Ezh2 wildtype mice (*p<0.05). Importantly, genetic ablation of p16 conditionally in SHH MB EZH2 double knockout mice rescues the widespread tumor cell differentiation (n=9 mice; *p=3e-06) seen in Ezh2 single knockout SHH medulloblastoma mice. Finally, we developed a novel fucoidan-based nanoparticle strategy to deliver the EZH2 inhibitor (EPZ-6438) across the intact BBB of this GEM model to achieve significant extension of mouse survival (median 70 days compared to 19 days in control mice; *p=0.01, Mantel-Cox) with potential utility for other pediatric brain tumors.

CBX4 contributes to HIV-1 latency by forming phase-separated nuclear bodies and SUMOylating EZH2.

The retrovirus HIV-1 integrates into the host genome and establishes a latent viral reservoir that escapes immune surveillance. Molecular mechanisms of HIV-1 latency have been studied extensively to achieve a cure for the acquired immunodeficiency syndrome (AIDS). Latency-reversing agents (LRAs) have been developed to reactivate and eliminate the latent reservoir by the immune system. To develop more promising LRAs, it is essential to evaluate new therapeutic targets. Here, we find that CBX4, a component of the Polycomb Repressive Complex 1 (PRC1), contributes to HIV-1 latency in seven latency models and primary CD4+ T cells. CBX4 forms nuclear bodies with liquid-liquid phase separation (LLPS) properties on the HIV-1 long terminal repeat (LTR) and recruits EZH2, the catalytic subunit of PRC2. CBX4 SUMOylates EZH2 utilizing its SUMO E3 ligase activity, thereby enhancing the H3K27 methyltransferase activity of EZH2. Our results indicate that CBX4 acts as a bridge between the repressor complexes PRC1 and PRC2 that act synergistically to maintain HIV-1 latency. Dissolution of phase-separated CBX4 bodies could be a potential intervention to reactivate latent HIV-1.

Trial in progress: A phase I trial of dual EZH 1/2 inhibitor valemetostat tosylate (DS-3201b) in pediatric, adolescent, and young adult patients with malignant solid tumors.

TPS10059 Background: Enhancer of zeste homolog enzymes (EZH1 and EZH2) form parts of the polycomb repressive complex 2 and regulate gene expression by catalyzing the tri-methylation of lysine 27 residue of histone H3. SMARCB1/INI1 is one of the core components of the SWI/SNF chromatin remodeling complex, with the loss of SMARCB1/INI1 causing the oncogenic activation of EZH2 and EZH1. The inhibition of EZH2 or both EZH2 and EZH1 can be effective against various hematological malignancies and SMARCB1/INI1-deficient solid tumors. Valemetostat tosylate (DS-3201b; valemetostat) is a potential first-in-class dual inhibitor of EZH1 and EZH2 that targets epigenetic regulations by inhibiting both EZH1 and EZH2 enzymes. A phase 2 single-arm study showed that valemetostat demonstrated promising response rates in Japanese patients with relapsed or refractory adult T-cell leukemia/lymphoma (Yoshimitsu M et al., presented at ASH Annual Meeting, 2021). Tumors characterized by SMARCB1/INI1 deficiency (a SWI/SNF mutation), such as malignant rhabdoid tumors, epithelioid sarcoma, or synovial sarcoma are quite frequently observed during childhood and adolescence, among whom valemetostat is expected to show antitumor effects. Methods: This open-label multi-center phase I trial evaluates the safety and efficacy of valemetostat in pediatric, adolescent, and young adult patients with refractory/relapsed solid tumors. The inclusion criteria are relapsed, refractory, or progressive metastatic disease; &gt;3 and &lt;19 years of age during the dose escalation cohort and &lt; 29 years of age in the expanded cohort; performance status of &gt;50 (assessed by Karnofsky Performance score in patients &gt;16 years old, and Lansky Performance score in patients &lt;15 years old); and adequate organ function. Valemetostat is administered orally once a day without interruption. Three dose levels (150, 200, and 250 mg/1.7 m2) are assessed using a 3+3 design during the dose escalation cohort. After determining the recommended phase 2 dose (RP2D) during dose escalation cohort, up to 30 patients will be further enrolled, and the safety and efficacy data of valemetostat are determined in the expanded cohort. The primary endpoint is the incidence of dose limiting toxicity, whereas the secondary endpoints include safety, pharmacokinetics, overall response rate, progression-free survival. The overall response rate of the tumors with SMARCB1/INI1 deficiency or SWI/SNF mutation is also evaluated as a secondary endpoint. Exploratory endpoint includes overall survival. Enrollment into this trial began in March 2020, and enrollment into the dose escalation cohort was completed. Enrollment into the expanded cohort began in November 2021. Clinical Trial Information: jRCT2031190268.

The MiR-101/EZH2 negative feedback signaling drives oxygen-glucose deprivation/reperfusion-induced injury by activating the MAPK14 signaling pathway in SH-SY5Y cells.

MiR-101 has been reported to be involved in neuroinflammation, neuronal injury and neurotoxicity. However, the specific role and mechanism of miR-101 in ischemia/reperfusion (I/R)-induced neuronal injury remain largely unknown. Our study found that after oxygen-glucose deprivation/reperfusion (OGD/R) exposure, the level of miR-101 in SH-SY5Y cells was significantly decreased, which was accompanied by a decrease in cell viability, and an increase in LDH release and apoptosis. MiR-101 overexpression (miR-101 mimics) significantly promoted viability and inhibited LDH release and apoptosis in OGD/R-exposed SH-SY5Y cells. Luciferase reporter assay indicated that enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) was a direct target of miR-101, and EZH2 siRNA obviously increased the viability, inhibited LDH release and apoptosis in OGD/R-exposed SH-SY5Y cells. Besides, EZH2 siRNA could inhibit the activation of mitogen-activatedproteinkinase(MAPK14) signaling pathway and theMAPK14agonist (anisomycin) could reverse EZH2 siRNA-induced increase in cell viability, and decreases in LDH release and apoptosis. Furthermore, when the methyltransferase activity of EZH2 was inhibited by its specific inhibitorGSK126, the level of miR-101 was increased in OGD/R-exposed SH-SY5Y cells. Additionally, EZH2 siRNA upregulated miR-101 expression in OGD/R-exposed SH-SY5Y cells. Taken together, our findings reveal that miR-101/EZH2 negative feedback signaling drives OGD/R-induced injury by activating the MAPK14 signaling pathway in SH-SY5Y cells.

Discovery of precision targeting EZH2 degraders for triple-negative breast cancer

EZH2 is usually overexpressed in TNBC and other tumors, which has a great influence on the occurrence, development and prognosis of tumors. However, current EZH2 inhibitors, including Tazemetostat and GSK126, affect the methyl catalytic capacity of EZH2 and have little effect on the tumorigenic activity of EZH2 itself, resulting in poor efficacy against most solid tumors. Herein, we designed and optimized proteolytic targeting chimeras (PROTACs) precision targeting EZH2. The most active PROTAC molecule U3i has a high affinity for PRC2 complex (KD = 16.19 nM) and show good inhibitory effects on MDA-MB-231 (IC50 = 0.57 μM) and MDA-MB-468 (IC50 = 0.38 μM) cells. Compared with that of the GSK126, the growth inhibitory activities of U3i against these two TNBC cells increased by approximately 20- and 30-fold. Further studies showed that U3i can degrade PRC2 complex in TNBC cells, induce apoptosis, and cause little damage to normal cells. Therefore, U3i is a potential anticancer molecule for TNBC treatment.

Therapeutic Targeting of EZH2 and BET BRD4 in Pediatric Rhabdoid Tumors.

Aberrant activity of the H3K27 modifiers EZH2 and BRD4 is an important oncogenic driver for atypical teratoid/rhabdoid tumor (AT/RT), and each is potentially a possible therapeutic target for treating AT/RT. We, therefore, determined whether targeting distinct histone modifier activities was an effective approach for treating AT/RT. The effects of EZH2 and BRD4 inhibition on histone modification, cell proliferation, and cell invasion were analyzed by immunoblotting, MTS assay, colony formation assay, and cell invasion assay. RNA- and chromatin immunoprecipitation-sequencing were used to determine transcriptional and epigenetic changes in AT/RT cells treated with EZH2 and BRD4 inhibitors. We treated mice bearing human AT/RT xenografts with EZH2 and BRD4 inhibitors. Intracranial tumor growth was monitored by bioluminescence imaging, and the therapeutic response was evaluated by animal survival. AT/RT cells showed elevated levels of H3K27 trimethylation (H3K27me3) and H3K27 acetylation (H3K27ac), with expression of EZH2 and BRD4, and lack of SMARCB1 proteins. Targeted inhibition of EZH2 and BRD4 activities reduced cell proliferation and invasiveness of AT/RT in association with decreasing H3K27me3 and H3K27ac. Differential genomic occupancy of H3K27me3 and H3K27ac regulated specific gene expression in response to EZH2 and BRD4 inhibitions. A combination of EZH2 and BRD4 inhibition increased the therapeutic benefit in vitro and in vivo, outperforming either monotherapy. Overall, histones H3K27me3 and H3K27ac were elevated in AT/RT cells and distributed in distinct chromatin regions to regulate specific gene expression and to promote AT/RT growth. Targeting EZH2 and BRD4 activity is, therefore, a potential combination therapy for AT/RT.

Induction of APOBEC3-mediated genomic damage in urothelium implicates BK polyomavirus (BKPyV) as a hit-and-run driver for bladder cancer

Limited understanding of bladder cancer aetiopathology hampers progress in reducing incidence. Mutational signatures show the anti-viral apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes are responsible for the preponderance of mutations in bladder tumour genomes, but no causative viral agent has been identified. BK polyomavirus (BKPyV) is a common childhood infection that remains latent in the adult kidney, where reactivation leads to viruria. This study provides missing mechanistic evidence linking reactivated BKPyV-infection to bladder cancer risk. We used a mitotically-quiescent, functionally-differentiated model of normal human urothelium to examine BKPyV-infection. BKPyV-infection led to significantly elevated APOBEC3A and APOBEC3B protein, increased deaminase activity and greater numbers of apurinic/apyrimidinic sites in the host urothelial genome. BKPyV Large T antigen (LT-Ag) stimulated re-entry from G0 into the cell cycle through inhibition of retinoblastoma protein and activation of EZH2, E2F1 and FOXM1, with cells arresting in G2. The single-stranded DNA displacement loops formed in urothelial cells during BKPyV-infection interacted with LT-Ag to provide a substrate for APOBEC3-activity. Addition of interferon gamma (IFNγ) to infected urothelium suppressed expression of the viral genome. These results support reactivated BKPyV infections in adults as a risk factor for bladder cancer in immune-insufficient populations.

Inhibition of thedeubiquitinating enzyme USP47 as a novel targeted therapy for hematologic malignancies expressing mutant EZH2.

Activating mutations in EZH2, the catalytic component of PRC2, promote cell proliferation, tumorigenesis, and metastasis through enzymatic or non-enzymatic activity. The EZH2-Y641 gain-of-function mutation is one of the most significant in diffuse large B-cell lymphoma (DLBCL). Although EZH2 kinase inhibitors, such as EPZ-6438, provide clinical benefit, certain cancer cells are resistant to the enzymatic inhibition of EZH2 because of the inability to functionally target mutant EZH2, or because of cells' dependence on the non-histone methyltransferase activity of EZH2. Consequently, destroying mutant EZH2 protein may be more effective in targeting EZH2 mutant cancers that are dependent on the non-catalytic activity of EZH2. Here, using extensive selectivity profiling, combined with genetic and animal model studies, we identified USP47 as a novel regulator of mutant EZH2. Inhibition of USP47 would be anticipated to block the function of mutated EZH2 through induction of EZH2 degradation by promoting its ubiquitination. Moreover, targeting of USP47 leads to death of mutant EZH2-positive cells in vitro and in vivo. Taken together, we propose targeting USP47 with a small molecule inhibitor as a novel potential therapy for DLBCL and other hematologic malignancies characterized by mutant EZH2 expression.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

An EZH2 blocker sensitizes histone mutated diffuse midline glioma to cholesterol metabolism inhibitors through an off-target effect.

BackgroundDiffuse Midline Glioma, H3K27M-mutant (DMG) is a rare, highly aggressive pediatric tumor affecting the brainstem, and is one of the deadliest cancers. Currently available treatment options such as chemotherapy and radiotherapy do only modestly prolong survival. In this pathology, H3K27 mutations deregulate Polycomb Repressive Complex 2 (PRC2), including enzymatic activity of EZH2, which is therefore under investigation as a therapeutic target.MethodsWe used a chemical EZH2 inhibitor, GSK126, small interfering RNAs, and a CRISPR/Cas9 knockout approaches in a series of DMG tumor cell lines to investigate metabolic treatment responses by proteomic analysis. A combination strategy was elaborated and studied in primary and established DMG cells, spheroid 3D cultures, and in vivo in a chick chorio-allantoic membrane DMG assay and an orthotopic intracranial DMG mouse model.ResultsGSK126 shows significant (P < .05–.001) inhibitory effects in in vitro cell proliferation assays and induces apoptosis. Chemical targeting of EZH2 induced expression of proteins implicated in cholesterol metabolism. Low-dose GSK126 treatment together with statins revealed strong growth inhibition in combinatorial treatments, but not in single treatments, both in DMG cells in vitro, in DMG spheroid cultures, and in chick and mouse in vivo models (P < .05). All statistical tests were two-sided.ConclusionsOur results reveal an unexpected GSK126-inducible sensitivity to cholesterol biosynthesis inhibitors in highly aggressive pediatric glioma that warrants further evaluation as treatment strategy. This combinatorial therapy should have few side effects because of the low doses used to achieve significant anti-tumor activity.

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta–gonad–mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

Curcumol inhibits malignant biological behaviors and TMZ-resistance in glioma cells by inhibiting long noncoding RNA FOXD2-As1-promoted EZH2 activation.

Currently, conventional treatment is not sufficient to improve the survival of glioma patients. Hence, adopting novel personalized treatment programs is imperative. Curcumol, a Chinese herbal medicine extract from the roots of Rhizoma Curcumae, has attracted significant interest due to its beneficial pharmacological activities. The current study revealed that curcumol inhibited the proliferation, metastasis, self-renewal ability, and TMZ resistance in glioma cells in vitro and in vivo. Next, the potential molecular mechanisms of curcumol in inhibiting glioma were investigated. We found that the long non-coding RNA (lncRNA) FOXD2-As1 might contribute to the effects of curcumol on glioma cells. Enforced expression of FOXD2-As1 attenuated the curcumol-induced reduction in glioma cell proliferation, metastasis, self-renewal ability, and TMZ resistance. Moreover, the forced expression of FOXD2-As1 reversed the inhibitory effect of curcumol on the binding ability of EZH2 and H3K27me3 modification in the promoter regions of anti-oncogenes. Our results showed for the first time that curcumol is effective in inhibiting malignant biological behaviors and TMZ-resistance of glioma cells by suppressing FOXD2-As1-mediated EZH2 activation. Our study offers the possibility of exploiting curcumol as a promising therapeutic agent for glioma treatment and may provide an option for the clinical application of this natural herbal medicine.

Single Cell Analyses Reveal a Non-Canonical EZH2 Activity As a Main Driver of Retinoic Acid Resistance in PLZF/Rara Leukemia

Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA.We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature (“Rep” cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance.These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

NEK2 Inhibition Enhances the Efficacy of PD-1/PD-L1 Blockade in Multiple Myeloma

IntroductionThe development of new treatments for high-risk multiple myeloma (HRMM) are needed. The PD-1/PD-L1 axis is one of the chief inhibitory immune checkpoints in antitumor immunity. Despite the success of PD-1 (PDCD1) / PD-L1 (CD274) blockade in some neoplasms, use of it as a monotherapy has failed to improve outcome in RRMM. We have previously demonstrated that the cell-cycle-regulated serine-threonine kinase, NEK2 is elevated in HRMM and that inhibition of NEK2 can overcome drug-resistance and prolong survival of xenografted MM cells. Here, we aimed to investigate the possible role of NEK2 in regulating the immune checkpoint response in MM and development of possible anti-PD1/PDL1 combination therapies.MethodsGene expression profiles and pathway enrichment analyses were conducted on oligonucleotide microarray gene expression profiles from over 1000 primary MM samples to evaluate the correlation of NEK2 and immune checkpoint expression levels. To elucidate the underlying mechanism, we used Nek2 -/- mice crossed with EμMyc mice to generate B cell tumor mouse model with NEK2 deficiency. RNA-sequencing analyses of premalignant B cells was compared between EμMyc/Nek2 WT and EμMyc/Nek2 -/- mice. The hub molecular regulators in the NEK2 correlated pathways were further determined by western blot using NEK2 overexpressing and knockdown cell lines and then verified by co-immunoprecipitation with a NEK2 antibody. Lastly, to establish its clinic significance, the efficacy of INH1 (small compound NEK2 inhibitor), (D)-PPA 1 (peptide-based PD-1/PD-L1 interaction inhibitor) or a PD-L1 (monoclonal antibody) was tested in bone marrow BM mononuclear cells from primary MM patients in-vitro as well as in MM xenografts. Tumor burden and T cell immune responses were monitored by M-spike and mass cytometry.ResultsGene expression profiles demonstrated that CD274 expression was significantly higher in the non-proliferative hyperdiploid (HY) subtype of MM, representing between 25-35% of all MM. NEK2 was negatively correlated with CD274 gene expression across all 7 MM subtypes. Gene set enrichment analysis showed that the IFN-γ signaling pathway, which can induce CD274 expression, was significantly enriched in the HY subtype as well as premalignant B cells from EμMyc/Nek2 -/- mice. Elevated expression of EZH2, a histone methyltransferase gene, is also highly correlated wirth NEK2 levels in primary MM. We found that NEK2 inhibition increases CD274 expression as well as reduced EZH2 expression and H3K27me3 levels in MM cell lines. In contrarst, myeloma cells overexpressing NEK2 showed increased expression and activity of EZH2 and H3K27me3 levels. Thus, NEK2 appears to regulate CD274/PD-L1 expression through EZH2-mediated histone methylation. Next we demonstrated that NEK2 and EZH2 directly interact and that overexpression of NEK2 leads to increased methylation of the CD274/PD-L1 gene. We treated BM mononuclear cells from primary MM with PD-1/PD-L1 inhibitor with and without a NEK2 inhibitor. The combination was most effective at eliminating CD138 + myeloma cells while having no effects on T, B and myeloid cell populations.ConclusionOur study showed that expression of CD274/PD-L1 is suppressed in primary HRMM and that CD274/PD-L1 expression is negatively regulated by NEK2 via EZH2-mediated methylation. Inhibition of NEK2 sensitizes myeloma cells to PD-1/PD-L1 blockade, showing either a synergistic or an additive effect in MM cell cytotoxicity. DisclosuresNo relevant conflicts of interest to declare.