Pharmacological Inhibition Of EZH2 Research Articles

LincRNA-immunity landscape analysis identifies EPIC1 as a regulator of tumor immune evasion and immunotherapy resistance.

Through an integrative analysis of the lincRNA expression and tumor immune response in 9,626 tumor samples across 32 cancer types, we developed a lincRNA-based immune response (LIMER) score that can predict the immune cells infiltration and patient prognosis in multiple cancer types. Our analysis also identified tumor-specific lincRNAs, including EPIC1, that potentially regulate tumor immune response in multiple cancer types. Immunocompetent mouse models and in vitro co-culture assays demonstrated that EPIC1 induces tumor immune evasion and resistance to immunotherapy by suppressing tumor cell antigen presentation. Mechanistically, lincRNA EPIC1 interacts with the histone methyltransferase EZH2, leading to the epigenetic silencing of IFNGR1, TAP1/2, ERAP1/2, and MHC-I genes. Genetic and pharmacological inhibition of EZH2 abolish EPIC1's immune-related oncogenic effect and its suppression of interferon-γ signaling. The EPIC1-EZH2 axis emerges as a potential mechanism for tumor immune evasion that can serve as therapeutic targets for immunotherapy.

Mutant p53 drives clonal hematopoiesis through modulating epigenetic pathway

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. Here we discover that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Furthermore, genetic and pharmacological inhibition of EZH2 decreases the repopulating potential of p53 mutant HSPCs. Thus, we uncover an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract TP121: Inhibition of Ezh2 Simultaneously Blocks Pro-Inflammatory Polarization and Enhances Vegfa Expression in Male and Female Microglia

Background: Microglia are the innate immune cells of the central nervous system and are poised to be the first responders to ischemic stroke by polarizing to a damaging pro-inflammatory or a reparative anti-inflammatory phenotype. Oxygen glucose deprivation (OGD) alters microglia epigenetic regulation to promote an anti-inflammatory phenotype and up-regulation of Vegfa which may enhance angiogenesis after ischemic stroke. Interestingly, OGD down-regulates Enhancer of Zeste Homologue 2 (Ezh2), a histone lysine N -methyltransferase associated with repression of anti-inflammatory genes to promote pro-inflammatory polarization. Hypothesis: We hypothesized that OGD drives microglial anti-inflammatory polarization through down-regulation of Ezh2 . Methods: We cultured male and female primary microglia from neonatal P0.5-P3 C57Bl/6 mice in phenol red-free DMEM (Gibco) and charcoal stripped FBS (Sigma). Cultures were treated with GSK343 (Cayman Chemical) or DMSO control and exposed cells to OGD (95% N 2 , 5% CO 2 ) for 8hrs. Microglia were collected for RNA (Qiagen) and analyzed using real-time PCR (Bio-Rad). Results: We found that OGD up-regulated anti-inflammatory genes, notably Gdf15 , Il1rn , Irf4 , and Vegfa while down-regulating pro-inflammatory genes including Tnfa , Il1b , Il6 , and Ccl2 (n=14, p<0.05). Ezh2 expression was down-regulated with 8hr OGD (p<0.001). Pharmacological inhibition of Ezh2 with GSK343 in OGD resulted in exaggerated down-regulation of Il1b and Il6 (p<0.01) as well as enhanced up-regulation of Vegfa (p<0.05). Notably, there were no sex differences identified in our work (n=6-7, p>0.05). Conclusion: Our data shows that OGD-induced down-regulation of Ezh2 is necessary for down-regulation of Il1b and Il6 and up-regulation of Vegfa . This may be a novel therapeutic target for ischemic stroke available to both men and women to simultaneously diminish pro-inflammatory gene expression and enhance angiogenesis and functional recovery.

Modulation of EZH2 expression in T cells improves efficacy of anti-CTLA-4 therapy.

Enhancer of zeste homolog 2-mediated (EZH2-mediated) epigenetic regulation of T cell differentiation and Treg function has been described previously; however, the role of EZH2 in T cell-mediated antitumor immunity, especially in the context of immune checkpoint therapy, is not understood. Here, we showed that genetic depletion of EZH2 in Tregs (FoxP3creEZH2fl/fl mice) leads to robust antitumor immunity. In addition, pharmacological inhibition of EZH2 in human T cells using CPI-1205 elicited phenotypic and functional alterations of the Tregs and enhanced cytotoxic activity of Teffs. We observed that ipilimumab (anti-CTLA-4) increased EZH2 expression in peripheral T cells from treated patients. We hypothesized that inhibition of EZH2 expression in T cells would increase the effectiveness of anti-CTLA-4 therapy, which we tested in murine models. Collectively, our data demonstrated that modulating EZH2 expression in T cells can improve antitumor responses elicited by anti-CTLA-4 therapy, which provides a strong rationale for a combination trial of CPI-1205 plus ipilimumab.

Loss of tumor suppressor IGFBP4 drives epigenetic reprogramming in hepatic carcinogenesis.

Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.

Pharmacological inhibition of EZH2 disrupts the female germline epigenome

BackgroundRecently discovered drugs that target epigenetic modifying complexes are providing new treatment options for a range of cancers that affect patients of reproductive age. Although these drugs provide new therapies, it is likely that they will also affect epigenetic programming in sperm and oocytes. A promising target is Enhancer of Zeste 2 (EZH2), which establishes the essential epigenetic modification, H3K27me3, during development.ResultsIn this study, we demonstrate that inhibition of EZH1/2 with the clinically relevant drug, tazemetostat, severely depletes H3K27me3 in growing oocytes of adult female mice. Moreover, EZH2 inhibition depleted H3K27me3 in primary oocytes and in fetal oocytes undergoing epigenetic reprogramming. Surprisingly, once depleted, H3K27me3 failed to recover in growing oocytes or in fetal oocytes.ConclusionTogether, these data demonstrate that drugs targeting EZH2 significantly affect the germline epigenome and, based on genetic models with oocyte-specific loss of EZH2 function, are likely to affect outcomes in offspring.

CARM1-expressing ovarian cancer depends on the histone methyltransferase EZH2 activity

CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 is often overexpressed in human cancers. However, clinically applicable cancer therapeutic strategies based on CARM1 expression remain to be explored. Here, we report that EZH2 inhibition is effective in CARM1-expressing epithelial ovarian cancer. Inhibition of EZH2 activity using a clinically applicable small molecule inhibitor significantly suppresses the growth of CARM1-expressing, but not CARM1-deficient, ovarian tumors in two xenograft models and improves the survival of mice bearing CARM1-expressing ovarian tumors. The observed selectivity correlates with reactivation of EZH2 target tumor suppressor genes in a CARM1-dependent manner. Mechanistically, CARM1 promotes EZH2-mediated silencing of EZH2/BAF155 target tumor suppressor genes by methylating BAF155, which leads to the displacement of BAF155 by EZH2. Together, these results indicate that pharmacological inhibition of EZH2 represents a novel therapeutic strategy for CARM1-expressing cancers.

Effects of histone methyltransferase inhibition in endometriosis.

Although the histone methyltransferase EZH2 and its product H3K27me3 are well studied in cancer, little is known about their role and potential as therapeutic targets in endometriosis. We have previously reported that endometriotic lesions are characterized by global enrichment of H3K27me3. Therefore, we aimed to (1) characterize the expression levels of EZH2 in endometriotic tissues; (2) assess H3K27me3 enrichment in candidate genes promoter regions; and (3) determine if pharmacological inhibition of EZH2 impacts migration, proliferation, and invasion of endometriotic cells. Immunohistochemistry of an endometriosis-focused tissue microarray was used to assess the EZH2 protein levels in tissues. Chromatin immunoprecipitation-qPCR was conducted to assess enrichment of H3K27me3 in candidate gene promoter regions in tissues. Immunofluorescence was performed to assess the effect of an EZH2-specific pharmacological inhibitor on H3K27me3 global enrichment in cell lines. To measure effects of the inhibitor in migration, proliferation, and invasion in vitro we used Scratch, BrdU, and Matrigel assays, respectively. Endometriotic lesions had significantly higher EZH2α nuclear immunostaining levels compared to eutopic endometrium from patients (glands, stroma) and controls (glands). H3K27me3 was enriched within promoter regions of candidate genes in some but not all of the endometriotic lesions. Inhibition of EZH2 reduced H3K27me3 levels in the endometriotic cells specifically, and also reduced migration, proliferation but not invasion of endometriotic epithelial cells (12Z). These findings support future preclinical studies to determine in vivo efficacy of EZH2 inhibitors as promising nonhormonal treatments for endometriosis, still an incurable gynecological disease.

Sestrin1, a tumor suppressor that can be rescued

ABSTRACTSESTRIN1 is a tumor suppressor in follicular lymphoma that controls mTORC1 activity and it is inactivated by chromosomal deletions or epigenetically silenced by mutant EZH2Y641X. Pharmacological inhibition of EZH2 promotes SESTRIN1 re-expression and it restores its tumor suppressive activity, suggesting the possibility to epigenetically control mTORC1 activity.

The histone lysine methyltransferase Ezh2 is required for maintenance of the intestine integrity and for caudal fin regeneration in zebrafish

The histone lysine methyltransferase EZH2, as part of the Polycomb Repressive Complex 2 (PRC2), mediates H3K27me3 methylation which is involved in gene expression program repression. Through its action, EZH2 controls cell-fate decisions during the development and the differentiation processes. Here, we report the generation and the characterization of an ezh2-deficient zebrafish line. In contrast to its essential role in mouse early development, loss of ezh2 function does not affect zebrafish gastrulation. Ezh2 zebrafish mutants present a normal body plan but die at around 12 dpf with defects in the intestine wall, due to enhanced cell death. Thus, ezh2-deficient zebrafish can initiate differentiation toward the different developmental lineages but fail to maintain the intestinal homeostasis. Expression studies revealed that ezh2 mRNAs are maternally deposited. Then, ezh2 is ubiquitously expressed in the anterior part of the embryos at 24 hpf, but its expression becomes restricted to specific regions at later developmental stages. Pharmacological inhibition of Ezh2 showed that maternal Ezh2 products contribute to early development but are dispensable to body plan formation. In addition, ezh2-deficient mutants fail to properly regenerate their spinal cord after caudal fin transection suggesting that Ezh2 and H3K27me3 methylation might also be involved in the process of regeneration in zebrafish.

The histone methyltransferase EZH2 as a druggable target in SHH medulloblastoma cancer stem cells.

The histone methyltransferase EZH2 plays a role in maintenance of the stem component of cancer, and its overexpression and/or mutation typically drives tumor aggressiveness, drug resistance and patients’ poor prognosis. In this study, we use mouse and human medulloblastoma stem-like cells belonging to the Sonic Hedgehog subgroup (SHH MB-SLCs) and demonstrate that genetic suppression of EZH2 reduces the level of its histone mark H3K27me3 and lowers proliferation and self-renewal. We designed an EZH2 inhibitor (EZH2i) as a simplified analog of EPZ005687 and GSK2816126, MC3629, and we tested its biological activity in SHH MB-SLCs. Pharmacological inhibition of EZH2 impairs SHH MB cells proliferation and self-renewal, and induces apoptosis in vitro. Finally, we generated xenograft MB-SLCs orthotopic tumors in nude mice to test MC3629 in vivo. In treated mice, we observed impairment of tumor growth, together with induction of apoptosis and reduction of proliferation and stemness.Overall, these findings describe EZH2 as a druggable target in MB and provide insight into the biological activity of MC3629 as an EZH2i.

Abstract 1944: Tazemetostat displays synergistic antiproliferative activity with backbone therapies in preclinical models of AT/RT and MRT

Abstract Malignant Rhabdoid Tumors (MRT) and Atypical Teratoid Rhabdoid Tumors (AT/RT) are typically pediatric cancers which are rare and aggressive with extremely high unmet medical need. At a molecular level MRT and AT/RT tumors are almost universally characterized by loss of the INI1 protein (also known as SNF5 or SMARCB1), a component of the Switch Sucrose Non-fermentable (SWI/SNF) multimeric chromatin modifying complex. In many cell types, the SWI/SNF complex and the PRC2 complex have an antagonistic relationship in the regulation of tumor suppressor genes, cell cycle checkpoints, hedgehog and myc pathway genes, among others [Wilson et al, Cancer Cell. 2010 Oct 19;18(4):316-28.]. This explicated the possibility of a novel treatment modality for these tumors, based on inhibition of EZH2 - the catalytic subunit of PRC2. Tazemetostat (EPZ-6438) is a potent and orally bioavailable small molecule inhibitor of EZH2 currently in phase 2 clinical trials in adult patients with non-Hodgkin lymphoma, INI1-negative tumors and mesothelioma. Dependency of INI1-negative tumors on EZH2 catalytic activity was demonstrated in preclinical models of MRT, where robust tumor regressions were induced with tazemetostat treatment [Knutson et al, Proc Natl Acad Sci USA. 2013 May 7;110(19):7922-7]. Consistent with this, tazemetostat demonstrated activity in relapsed or refractory patients with INI1-negative tumors enrolled in the adult phase 1 clinical trial. In addition, a phase 1 dose escalation study in pediatric patients with INI1-negative solid tumors is currently ongoing. Backbone therapies for MRT and AT/RT are comprised of cytotoxic chemotherapy which may or may not be administered in the context of stem cell transplant, in addition to surgical resection and radiation therapy. In support of combination clinical scenarios including EZH2 inhibition in this setting, we sought to explore the antiproliferative effects of combining tazemetostat with current small molecule treatment therapies in cell line models of AT/RT and MRT. Synergistic activity was observed when tazometostat was combined with individual components of chemotherapeutic regimens and targeted therapies such as vincristine, doxorubicin, alisertib and HDAC inhibitors.Further, we investigated the effects of ionizing radiation together with tazemetostat treatment. Application of X-ray irradiation concomitantly or after tazemetostat treatment, induced robust antiproliferative activity and reduction in clonogenic potential of both AT/RT cell lines tested.Taken together these results suggest that pharmacological inhibition of EZH2 enhances the activity of backbone therapy and may have an advantage over monotherapy in INI1-negative cancers supporting the therapeutic potential of combination regimens that include tazemetostat in these tumors. Citation Format: Christine R. Klaus, Jeffrey A. Keats, Jesse J. Smith, Richard Chesworth, Robert A. Copeland, Scott Ribich, Alejandra Raimondi. Tazemetostat displays synergistic antiproliferative activity with backbone therapies in preclinical models of AT/RT and MRT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2017-1944

The histone methyltransferase EZH2 is a therapeutic target in small cell carcinoma of the ovary, hypercalcaemic type.

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodelling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodelling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumour samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry, with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells. In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis, and neuron-like differentiation. EZH2 inhibitors suppressed tumour growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular mechanisms regulated by the histone methyltransferase EZH2 in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a highly aggressive cancer. Although surgical resection remains the treatment of choice in localized ACC, clinical management of metastatic disease is ineffective. It is thus essential to identify the actors of malignant ACC progression to allow development of more effective therapeutic strategies. Deregulation of epigenetic factors is frequently involved in tumor progression. We have recently shown that the histone methyltransferase EZH2 is the most deregulated epigenetic histone modifier in ACC. EZH2 is over expressed downstream of TP53/RB1/E2F1 and is associated with tumor proliferation and poor prognosis. Pharmacological inhibition of EZH2 inhibits H295R cells growth and aggressive behaviour and induces apoptosis. This suggests that EZH2 is involved in malignant progression and could represent an interesting therapeutic target. Expression of the pro-apoptotic factor NOV/CCN3 is decreased in ACC, which is correlated with poor prognosis. Our molecular analyses show that EZH2 inhibition by siRNA or pharmacological treatment with DZNep increases expression of NOV/CCN3, suggesting that EZH2 over expression may favour malignant progression in ACC by inhibition of apoptosis stimulators. NOV has previously been identified as a negative target of the nuclear receptor SF1 in ACC cells, although the molecular mechanisms underlying this inhibition were unidentified. Interestingly, in prostate cancer, NOV expression is inhibited by the androgen receptor, through recruitment of EZH2 and deposition of the H3K27me3 mark. We are now conducting a combination of transfection and ChIP experiments to evaluate a similar cooperation between SF1 and EZH2 to repress NOV expression and block apoptosis in ACC.

Abstract LB-204: Neoadjuvant Ezh2 inhibition amplifies an inflammatory program and sensitizes Kras-driven NSCLC to anti-NF-κB inhibition

Abstract Kras mutant non-small cell lung cancer (NSCLC) is a leading cause of death but has limited therapeutic options. We investigated the impact of Polycomb repressive complex 2 (PRC2) inhibitors as single agent or as neoadjuvant treatment for subsequent anti-inflammatory therapy. Using orthotopic grafts, we show that Kras cells highly expressing the PRC2 enzymatic component Ezh2 display sensitivity to its inhibition. Ezh2 inhibition leads to transcriptional amplification of an inflammatory program involving NF-kB. Pharmacological inhibition of Ezh2 rerouted Kras tumor progression towards exploiting PRC2/NF-kB target genes as inferred by in vivo RNAi screen but also enhanced the response of Kras NSCLC to a combination of Nimesulide and Bortezomib. These data suggest that low PRC2 levels in Kras-driven NSCLC promote an epigenetic switch, providing a rationale for exploring a novel therapeutic option in which Ezh2 simultaneously acts as biomarker for therapeutic decision and as a neoadjuvant target for therapy. Citation Format: Michela Serresi, Bjorn Siteur, Matteo Cesaroni, Natalie Proost, Danielle Hulsman, Maarten van Lohuizen, Gaetano Gargiulo. Neoadjuvant Ezh2 inhibition amplifies an inflammatory program and sensitizes Kras-driven NSCLC to anti-NF-κB inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-204.

Deregulation of polycomb repressor complex 1 modifier AUTS2 in T-cell leukemia.

Recently, we identified deregulated expression of the B-cell specific transcription factor MEF2C in T-cell acute lymphoid leukemia (T-ALL). Here, we performed sequence analysis of a regulatory upstream section of MEF2C in T-ALL cell lines which, however, proved devoid of mutations. Unexpectedly, we found strong conservation between the regulatory upstream region of MEF2C (located at chromosomal band 5q14) and an intergenic stretch at 7q11 located between STAG3L4 and AUTS2, covering nearly 20 kb. While the non-coding gene STAG3L4 was inconspicuously expressed, AUTS2 was aberrantly upregulated in 6% of T-ALL patients (public dataset GSE42038) and in 3/24 T-ALL cell lines, two of which represented very immature differentiation stages. AUTS2 expression was higher in normal B-cells than in T-cells, indicating lineage-specific activity in lymphopoiesis. While excluding chromosomal aberrations, examinations of AUTS2 transcriptional regulation in T-ALL cells revealed activation by IL7-IL7R-STAT5-signalling and MEF2C. AUTS2 protein has been shown to interact with polycomb repressor complex 1 subtype 5 (PRC1.5), transforming this particular complex into an activator. Accordingly, expression profiling and functional analyses demonstrated that AUTS2 activated while PCGF5 repressed transcription of NKL homeobox gene MSX1 in T-ALL cells. Forced expression and pharmacological inhibition of EZH2 in addition to H3K27me3 analysis indicated that PRC2 repressed MSX1 as well. Taken together, we found that AUTS2 and MEF2C, despite lying on different chromosomes, share strikingly similar regulatory upstream regions and aberrant expression in T-ALL subsets. Our data implicate chromatin complexes PRC1/AUTS2 and PRC2 in a gene network in T-ALL regulating early lymphoid differentiation.

Functional characterization of EZH2 in pancreatic carcinogenesis

ID: 1413. Dual specificity kinase TTK in pancreatic neuroendocrine cancer Marina Hutzler , Harald Schmidt , Brajesh Kaistha , Rita Lawlor , Aldo Scarpa , Thomas Gress , Malte Buchholz 1 Gastroenterology and Endocrinology, Philipps University Marburg, Marburg, Germany Dipartimento ad Attivit a Integrata (DAI) di Patologia e Diagnostica, University of Verona, Verona, Italy Introduction: Pancreatic neuroendocrine tumours are a rare but still very challenging disease, as despite ongoing research, surgical resection still is the only reliable cure. Therefore, about 80 % of patients have a terminal illness and their tumours are still difficult to manage with the currently available chemoand biotherapies obtaining tumour growth stabilization rather than inducing regression. Gene expression analyses of primary tissues from PNET patients identified a number of potential target genes for the development of new therapeutic strategies. Amongst others, we found strong upregulation of the dual specificity kinase TTK, which has been shown before to play an important role during mitosis by inhibiting transition to anaphase until all chromosomes have been attached to the spindle apparatus correctly. Aims: The aim of this study is to elucidate the role of TTK and its value for PNET therapy. Materials & methods: Quantitative realtime PCR, RNAi, cell proliferation and viability assays, Westernblots Results: TTK is strongly overexpressed in pancreatic neuroendocrine tumour tissue. Functional in vitro analyses by siRNA-mediated knockdown of TTK in human as well as in rat PNET cell lines resulted in decreased proliferation and viability of cells, leading to increased apoptosis rates which could be further confirmed by Westernblot analyses of key molecules of the classical apoptosis pathway (Caspase-3 and PARP cleavage). Conclusion: Overexpression of TTK dual specificity kinase in pancreatic neuroendocrine tumours is important for proliferative phenotype of tumour cells and inhibition of TTK leads to apoptotic cell death. Therefore TTK could be a promising target for the treatment of neuroendocrine tumours. Abstract ID: 1416, Oral-3.ID: 1416, Oral-3. Context-dependent epigenetic regulation of NFATc1 transcription in pancreatic plasticity Elisabeth Hessmann , Nai-ming Chen , Clara Lubeseder-Martellato , Jochen Gaedcke , Jens Siveke , Albrecht Neesse , Volker Ellenrieder 1 Department of Gastroenterology and Gastrointestinal Oncology, University Medical Center Goettingen, Germany 2 II. Med. Klinikum rechts der Isar, TU Munich, Germany Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Germany West German Cancer Center Essen, Germany Introduction: Cellular plasticity of the pancreas highly contributes to its enormous regenerative potential upon acinar insults, but also causes therapeutic resistance in pancreatic cancer (PDAC) treatment. On the molecular level, regulation of cellular plasticity involves epigenetic mechanisms that transcriptionally control key-signatures of tissue adaptation. Transcriptional activation of the oncogenic Nuclear Factor of Activated T-cell (NFAT) c1 transcription factor significantly contributes to PDAC cell plasticity. Nevertheless, how epigenetic alterations converge on transcriptional regulation of NFATc1 remains elusive. Aims: To investigate how epigenetic regulation impacts on NFATc1 transcription in pancreatic plasticity. Materials&methods: NFATc1 function and regulationwere studied in the regenerative pancreas upon caerulein-induced acinar cell damage and in various in vitroand in vivo PDAC-models. EZH2-dependency of NFATc1 expression was determined upon genetic or pharmacological EZH2Abstracts / Pancreato S10 inhibition. NFATc1 promoter activity was investigated by ChIP-Assays in pancreatic tissue and cells. Co-expression of EZH2 and NFATc1 was validated in micro-dissected PDAC cells from human samples. Results: Pancreatic tissue regeneration requires silencing of the NFATc1 promoter by the Polycomb-Repressor-Complex member EZH2 to circumvent organ atrophy and exocrine insufficiency. In contrast, EZH2 activation in PDAC shows inverse effects on NFATc1 promoter regulation and promotes PDAC cell plasticity by enhancing NFATc1 transcription. Oncogenic mutation of Kraswas identified as the crucial genetic hit responsible for the functional switch in EZH2-dependent regulation of NFATc1 transcription. Conclusion: These data complement previous descriptions of Polycomb-independent EZH2 functions and emphasize how epigenetic mechanisms integrate into genomic alterations in PDAC. Consequently, pharmacological inhibition of EZH2 represents a beneficial strategy to overcome oncogenic NFATc1 activity in PDAC.

Abstract B16: Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated ovarian cancers.

Abstract ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in ~50% of ovarian clear cell carcinomas and ~30% of ovarian endometrioid carcinomas. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated, but not wild-type, ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents an urgently needed, novel treatment strategy for ARID1A mutated ovarian cancers. Citation Format: Benjamin Bitler, Ie-Ming Shih, Rugang Zhang. Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B16.

Genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in multiple myeloma reveals the importance of Polycomb gene targeting and highlights EZH2 as a potential therapeutic target.

Multiple myeloma (MM) is a malignancy of the antibody-producing plasma cells. MM is a highly heterogeneous disease, which has hampered the identification of a common underlying mechanism for disease establishment as well as the development of targeted therapy. Here we present the first genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in MM patient samples, defining a common set of active H3K4me3-enriched genes and silent genes marked by H3K27me3 (H3K27me3 alone or bivalent) unique to primary MM cells, when compared to normal bone marrow plasma cells. Using this epigenome profile, we found increased silencing of H3K27me3 targets in MM patients at advanced stages of the disease, and the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also demonstrated that pharmacological inhibition of EZH2 had anti-myeloma effects in both MM cell lines and CD138+ MM patient cells. In addition, EZH2 inhibition decreased the global H3K27 methylation and induced apoptosis. Taken together, these data suggest an important role for the Polycomb repressive complex 2 (PRC2) in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM.