Epigenetic Repression Research Articles

EPCO-35. THE ZINC FINGER IN THE CEREBELLUM (ZIC1/4) LOCUS IS A CONTEXT DEPENDENT DRIVER GENE IN MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a malignant paediatric brain tumor comprised of 4 molecularly and clinically distinct subgroups: WNT, SHH, Group 3 and Group 4. Previous studies have reported recurrent, mutually exclusive epigenetic modifier mutations converging in these subgroups, as well as aberrant H3K27me3 depositions within these tumors. While these observations hint at aberrant epigenetic mechanisms driving MB pathobiology, detailed understanding of MB chromatin landscape remains elusive to date. As such, chromatin landscape of MB was defined by conducting H3K27Ac ChIP-Seq in 102 primary MB tumors and H3K27me3 ChIP-Seq in 63 overlapping tumors. Chromatin landscape of Medulloblastoma was subgroup specific and H3K27me3 modification recurrently repressed ZIC1 in Group 3 and 4 MB, a driver gene that is also recurrently mutated within Group 4 and SHH MB. Validation cohort comprised of 251 MB samples with matching RNA-Seq and WGS data demonstrated that the ZIC1 - ZIC4 locus (ZIC1/4) undergoes recurrent epigenetic repression of single alleles in 25% of Group 3, 50% of Group 4 but rarely in other subgroups. Strikingly, ZIC1 mutations in the Group 4 tumors always occurred in the context of repression of the wildtype allele, revealing a recurrent convergence of genetic and epigenetic mechanisms targeting both copies of ZIC1. Overexpression of ZIC1 and ZIC4 together, or ZIC1 alone to a lesser extent, lead to reduced proliferation of Group 3 MB cells in vitro and in vivo. Intriguingly, while Group 4 ZIC1 mutations exhibited loss of function phenotypes, SHH ZIC1 mutations exhibited gain of function phenotypes. Overexpression of ZIC1 in Granule Progenitor Cells (GPC), which give rise to SHH MB, resulted in increased proliferation of cells in contrast to Group 3 MB cells. Taken together, we demonstrate that multiple epigenetic and genetic mechanisms converge to govern context dependent driver roles for ZIC1 and ZIC4 underlying MB pathobiology.

Thioredoxin-interacting protein-activated intracellular potassium deprivation mediates the anti-tumour effect of a novel histone acetylation inhibitor HL23, a fangchinoline derivative, in human hepatocellular carcinoma

IntroductionHyperactivated histone deacetylases (HDACs) act as epigenetic repressors on gene transcription and are frequently observed in human hepatocellular carcinoma (HCC). Although multiple pharmacological HDAC inhibitors (HDACis) have been developed, none is available in human HCC. ObjectivesTo investigate the pharmacological effects of a fangchinoline derivative HL23, as a novel HDACi and its molecular mechanisms through TXNIP-mediated potassium deprivation in HCC. MethodsBoth in vitro assays and orthotopic HCC mouse models were used to investigate the effects of HL23 in this study. The inhibitory activity of HL23 on HDACs was evaluated by in silico studies and cellular assays. Chromatin immunoprecipitation (ChIP) was conducted to confirm the regulation of HL23 on acetylation mark at TXNIP promoter. Genome-wide transcriptome analysis together with bioinformatic analysis were conducted to identify the regulatory mechanisms of HL23. The clinical significance of TXNIP and HDACs was evaluated by analysing publicly available database. ResultsHL23 exerted compatible HDACs inhibition potency as Vorinostat (SAHA) while had superior anti-HCC effects than SAHA and sorafenib. Both in vitro and in vivo studies showed HL23 significantly suppressed HCC progression and metastasis. HL23 significantly upregulated TXNIP expression via regulating acetylation mark (H3K9ac) at TXNIP promoter. TXNIP was responsible for anti-HCC activity of HL23 through mediating potassium channel activity. HDAC1 was predicted to be the target of HL23 and HDAC1lowTXNIPhigh could jointly predict promising survival outcome of patients with HCC. Combination treatment with HL23 and sorafenib could significantly enhance sorafenib efficacy. ConclusionOur study identified HL23 as a novel HDACi through enhancing acetylation at TXNIP promoter to trigger TXNIP-dependent potassium deprivation and enhance sorafenib efficacy in HCC treatment.

55. Epigenetic therapy-induced transposable element antigens in Glioblastoma

Among the CNS neoplasms, glioblastoma (GBM) is the most common and most lethal. Profiling the GBM histologic, genetic and epigenetic landscapes enables clinical stratification of distinct patient populations with prognostic and therapeutic implications. Despite accounting for nearly half the genome content, transposable elements (TEs) have been underexplored in cancer genomic studies owing to their constitutive epigenetic repression in somatic cells. However, clinical application of DNA methyltransferase (DNMTi) and histone deacetylase inhibitors (HDACi) have demonstrated potent activation of TE-derived alternative transcriptional start sites. Here, we show that decitabine (DNMTi) and panobinostat (HDACi) work both alone and synergistically to increase TE-expression by reversing their epigenetic repression in glioblastoma stem cells (GSCs). When translated, transcripts initiated at cryptic TE-derived alternative transcription start sites are processed and presented on cell surface HLA-I, potentially enabling immunotherapy targeting of these tumor-specific antigens. Specifically, we performed epigenomic and transcriptomic profiling of three patient-derived GSC lines, quiescent and replicating fibroblast and normal human astrocyte controls treated with 1uM decitabine DNMTi and 100nM panobinostat HDACi. In silico immunogenomics tools were then applied to assess the antigenicity of translated TE-derived transcripts. Finally, HLA-I peptidome immunoprecipitation, followed by liquid chromatography tandem mass-spectrometry (LC-MS/MS) were used to validate TE-derived peptides at the proteomic level. Beyond corroborating previously observed cryptic TE-promoter activation by DNMTi and HDACi, we demonstrate immunopeptidomic changes in epigenetic therapy-treated GSCs and posit the potential synergistic value of epigenetic therapy in immuno-oncology treatment regimens.

NRSF/REST lies at the intersection between epigenetic regulation, miRNA-mediated gene control and neurodevelopmental pathways associated with Intellectual disability (ID) and Schizophrenia

Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association remain to be determined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a genetic disorder linked with neurodevelopmental disorders and associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA and leads to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Significantly, the EHMT1-regulated miRNA gene set not only controls NRSF/REST but is enriched for association for Intellectual Disability (ID) and schizophrenia. This reveals a broad molecular interaction between H3K9 demethylation, NSRF/REST regulation and risk for ID and Schizophrenia.

Exploration of nuclear body-enhanced sumoylation reveals that PML represses 2-cell features of embryonic stem cells

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml−/− mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.

Virally mediated mechanisms of HLA class I loss in Merkel cell carcinoma and implications for viral epitope presentation

Background and aims: Loss of human leukocyte antigen class I (HLA-I) is a striking feature of Merkel cell carcinoma (MCC) and a well-characterized mechanism of immune escape. However, HLA-I can be upregulated in MCC with IFN-γ, histone deacetylase inhibitors, or demethylating agents, suggestive of epigenetic repression of class I antigen presentation genes. We hypothesized that restoration of HLA-I should increase MCC immunogenicity and facilitate viral epitope presentation. Moreover, given the paucity of mutations in Merkel cell polyomavirus-positive (MCPyV+) MCC in particular, we suspected that viral antigen-mediated signaling may suppress HLA-I surface expression in MCPyV+ MCC.

Epigenetic repression of THBD transcription by BRG1 contributes to deep vein thrombosis

BackgroundDeep vein thrombosis (DVT) with its major complication, pulmonary embolism, is a global health problem. Endothelial dysfunction is involved in the pathogenesis of DVT. We have previously demonstrated that endothelial specific deletion of Brahma-related gene 1 (BRG1) ameliorates atherosclerosis and aneurysm in animal models. Whether endothelial BRG1 contributes to DVT development remains undetermined. MethodsDVT was induced in mice by ligation of inferior vena cava. Deletion of BRG1 in endothelial cells was achieved by crossing the Cdh5-ERT-Cre mice with the Brg1loxp/loxp mice. ResultsHere we report that compared to the wild type mice, BRG1 conditional knockout (CKO) mice displayed substantially decreased DVT susceptibility characterized by decreased weight and size of thrombus and reduced immune infiltration. In endothelial cells, thrombomodulin (THBD) expression was significantly decreased by TNF-α stimulation, while BRG1 knockdown or inhibition recovered THBD expression. Further analysis revealed that BRG1 deficiency decreased the CpG methylation levels of the THBD promoter induced by TNF-α. Mechanistically, BRG1 directly upregulated DNMT1 expression after TNF-α treatment in endothelial cells. More importantly, administration of a small-molecule BRG1 inhibitor PFI-3 displayed potent preventive and therapeutic potentials in the DVT model. ConclusionsOur findings implicate BRG1 as an important regulator of DVT pathogenesis likely through epigenetic regulation of THBD expression in endothelial cells and provide translational proof-of-concept for targeting BRG1 in DVT intervention.

DNA hypermethylation of Kiss1r promoter and reduction of hepatic Kiss1r in female rats with type 2 diabetes

ABSTRACT Kisspeptin, produced from the brain and peripheral tissues, may constitute an important link in metabolic regulation in response to external cues, such as diet. The kisspeptin system is well described in the brain. However, its function and regulation in the peripheral tissues, especially in relation to metabolic disease and sex differences, remain to be elucidated. As Kiss1 and Kiss1r, encoding for kisspeptin and kisspeptin receptors, respectively, are altered by overnutrition/fasting and regulated by DNA methylation during puberty and cancer, epigenetic mechanisms in metabolic disorders are highly probable. In the present study, we experimentally induced type 2 diabetes mellitus (DM2) in female Wistar rats using high-fat diet/streptozocin. We analysed expression and DNA methylation of Kiss1 and Kiss1r in the peripheral tissues, using quantitative-reverse-transcription PCR (qRT-PCR) and pyrosequencing. We discovered differential expression of Kiss1 and Kiss1r in peripheral organs in DM2 females, as compared with healthy controls, and the profile differed from patterns reported earlier in males. DM2 in females was linked to the increased Kiss1 mRNA in the liver and increased Kiss1r mRNA in the liver and adipose tissue. However, Kiss1r promoter was hypermethylated in the liver, suggesting gene silencing. Indeed, the increase in DNA methylation of Kiss1r promoter was accompanied by a reduction in Kiss1r protein, implying epigenetic or translational gene repression. Our results deliver novel evidence for tissue-specific differences in Kiss1 and Kiss1r expression in peripheral organs in DM2 females and suggest DNA methylation as a player in regulation of the hepatic kisspeptin system in DM2.

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

BackgroundEpigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target.MethodsFunctional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays.ResultsNeuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo.ConclusionsWe report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma.

PRC1-independent binding and activity of RYBP on the KSHV genome during de novo infection.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes lifelong infection in humans by establishing latency after primary infection. Latent infection is a prerequisite for both persistent infection and the development of KSHV-associated cancers. While viral lytic genes are transiently expressed after primary infection, their expression is significantly restricted and concomitant with the binding of host epigenetic repressors Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) to lytic genes. PRC1 and PRC2 mediate the repressive histone marks H2AK119ub and H3K27me3, respectively, and maintain heterochromatin structure on KSHV lytic genes to inhibit their expression. In contrast to PRC2, little is known about the recruitment and role of PRC1 factors on the KSHV genome following de novo infection. Thus, the goal of this study was to examine the function of PRC1 factors in the establishment of KSHV latency. To address this question, we performed an shRNA screen targeting 7 different components of the canonical and non-canonical PRC1 complexes during primary KSHV infection. We found that RYBP, a main subunit of the non-canonical PRC1 complexes, is a potent repressor of KSHV lytic genes that can bind to the viral genome and inhibit lytic genes as early as 4 hours post infection. Surprisingly, our ChIP analyses showed that RYBP binds to lytic viral gene promoters in a PRC1-independent manner, does not affect PRC1 activity on the KSHV genome, and can reduce the level of histone marks associated with transcription elongation. Our data also suggest that RYBP can repress the viral lytic cycle after primary infection by inhibiting the transcription elongation of the lytic cycle inducer KSHV gene RTA. Based on our results we propose that RYBP uses a PRC1-independent mechanism to block KSHV RTA expression thereby promoting the establishment of KSHV latency following de novo infection.

Promising use of immune cell-derived exosomes in the treatment of SARS-CoV-2 infections.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection is persistently threatening the lives of thousands of individuals globally. It triggers pulmonary oedema, driving to dyspnoea and lung failure. Viral infectivity of coronavirus disease 2019 (COVID‐19) is a genuine challenge due to the mutagenic genome and mysterious immune‐pathophysiology. Early reports highlighted that extracellular vesicles (exosomes, Exos) work to enhance COVID‐19 progression by mediating viral transmission, replication and mutations. Furthermore, recent studies revealed that Exos derived from immune cells play an essential role in the promotion of immune cell exhaustion by transferring regulatory lncRNAs and miRNAs from exhausted cells to the active cells. Fortunately, there are great chances to modulate the immune functions of Exos towards a sustained repression of COVID‐19. Engineered Exos hold promising immunotherapeutic opportunities for remodelling cytotoxic T cells’ function. Immune cell‐derived Exos may trigger a stable epigenetic repression of viral infectivity, restore functional cytokine‐producing T cells and rebalance immune response in severe infections by inducing functional T regulatory cells (Tregs). This review introduces a view on the current outcomes of immunopathology, and immunotherapeutic applications of immune cell‐derived Exos in COVID‐19, besides new perspectives to develop novel patterns of engineered Exos triggering novel anti‐SARS‐CoV‐2 immune responses.

Expression and therapeutic targeting of BMI1 in canine gliomas

The BMI1 proto-oncogene, polycomb ring finger protein (BMI1) is a key component of the epigenetic polycomb repressor complex 1, and has been associated with aggressive behaviour and chemotherapeutic resistance in various malignances including human gliomas. Similar to humans, spontaneous canine gliomas carry a poor prognosis with limited therapeutic options. BMI1 expression and the effects of BMI1 inhibition have not been evaluated in canine gliomas. Here, we demonstrate that BMI1 is highly expressed in canine gliomas. Although increased BMI1 protein expression correlated with higher glioma grade in western blot assays, this correlation was not observed in a larger sample set using immunohistochemical analysis. The BMI1 inhibitor, PTC-209, suppressed BMI1 expression in established canine glioma cell lines and resulted in antiproliferative activity when used alone and in combination with chemotherapeutic agents. PTC-209 targeting of BMI1 activated the retinoblastoma (RB) pathway through downregulation of total and phosphorylated RB, independent of INK4A/ARF signalling, likely through BMI1-inhibition mediated upregulation of p21. These data support the rationale for targeting of BMI1 signalling and the use of canine glioma as a translational therapeutic model for human disease.

EZH2 regulates a SETDB1/ΔNp63α axis via RUNX3 to drive a cancer stem cell phenotype in squamous cell carcinoma.

Enhancer of zeste homolog 2 (EZH2) and SET domain bifurcated 1 (SETDB1, also known as ESET) are oncogenic methyltransferases implicated in a number of human cancers. These enzymes typically function as epigenetic repressors of target genes by methylating histone H3 K27 and H3-K9 residues, respectively. Here, we show that EZH2 and SETDB1 are essential to proliferation in 3 SCC cell lines, HSC-5, FaDu, and Cal33. Additionally, we find both of these proteins highly expressed in an aggressive stem-like SCC sub-population. Depletion of either EZH2 or SETDB1 disrupts these stem-like cells and their associated phenotypes of spheroid formation, invasion, and tumor growth. We show that SETDB1 regulates this SCC stem cell phenotype through cooperation with ΔNp63α, an oncogenic isoform of the p53-related transcription factor p63. Furthermore, EZH2 is upstream of both SETDB1 and ΔNp63α, activating these targets via repression of the tumor suppressor RUNX3. We show that targeting this pathway with inhibitors of EZH2 results in activation of RUNX3 and repression of both SETDB1 and ΔNp63α, antagonizing the SCC cancer stem cell phenotype. This work highlights a novel pathway that drives an aggressive cancer stem cell phenotype and demonstrates a means of pharmacological intervention.

Maternal SMCHD1 controls both imprinted Xist expression and imprinted X chromosome inactivation

Embryonic development is dependent on the maternal supply of proteins through the oocyte, including factors setting up the adequate epigenetic patterning of the zygotic genome. We previously reported that one such factor is the epigenetic repressor SMCHD1, whose maternal supply controls autosomal imprinted expression in mouse preimplantation embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1’s role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.

Suppression of Allergic Asthma by Loss of Function of Miz1-mediated Th1 Skewing.

Asthma is the most prevalent chronic respiratory disease worldwide. There is currently no cure, and it remains an important cause of morbidity and mortality. Here we report that lung-specific loss of function of the transcription factor Miz1 (c-Myc-interacting zinc finger protein-1) upregulates the pro-T-helper cell type 1 cytokine IL-12. Upregulation of IL-12 in turn stimulates a Th1 response, thereby counteracting T-helper cell type 2 response and preventing the allergic response in mouse models of house dust mite- and OVA (ovalbumin)-induced asthma. Using transgenic mice expressing Cre under a cell-specific promoter, we demonstrate that Miz1 acts in lung epithelial cells and dendritic cells in asthma. Chromatin immunoprecipitation coupled with high-throughput DNA sequencing or quantitative PCR reveals the binding of Miz1 on the Il12 promoter indicating direct repression of IL-12 by Miz1. In addition, HDAC1 (histone deacetylase 1) is recruited to the Il12 promoter in a Miz1-depdenent manner, suggesting epigenetic repression of Il12 by Miz1. Furthermore, Miz1 is upregulated in the lungs of asthmatic mice. Our data together suggest that Miz1 is upregulated during asthma, which in turn promotes asthma pathogenesis by preventing Th1 skewing through the transcriptional repression of IL-12.

T Cell-Intrinsic Vitamin A Metabolism and Its Signaling Are Targets for Memory T Cell-Based Cancer Immunotherapy.

Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T-cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

Changing paradigms in oncology: Toward noncytotoxic treatments for advanced gliomas.

Glial‐lineage malignancies (gliomas) recurrently mutate and/or delete the master regulators of apoptosis p53 and/or p16/CDKN2A, undermining apoptosis‐intending (cytotoxic) treatments. By contrast to disrupted p53/p16, glioma cells are live‐wired with the master transcription factor circuits that specify and drive glial lineage fates: these transcription factors activate early‐glial and replication programs as expected, but fail in their other usual function of forcing onward glial lineage‐maturation—late‐glial genes have constitutively “closed” chromatin requiring chromatin‐remodeling for activation—glioma‐genesis disrupts several epigenetic components needed to perform this work, and simultaneously amplifies repressing epigenetic machinery instead. Pharmacologic inhibition of repressing epigenetic enzymes thus allows activation of late‐glial genes and terminates glioma self‐replication (self‐replication = replication without lineage‐maturation), independent of p53/p16/apoptosis. Lineage‐specifying master transcription factors therefore contrast with p53/p16 in being enriched in self‐replicating glioma cells, reveal a cause‐effect relationship between aberrant epigenetic repression of late‐lineage programs and malignant self‐replication, and point to specific epigenetic targets for noncytotoxic glioma‐therapy.

Abstract 1599: Genes subject to changes in heterogeneity can function as regulators of metastasis

Abstract Tumor cell heterogeneity has been strongly implicated in metastatic progression of solid tumors such as triple-negative breast cancer (TNBC), leading to resistance and recurrence. Raf Kinase Inhibitory Protein (RKIP or PEBP1) effectively suppresses metastasis in TNBC but is lost in many patients. We hypothesize that RKIP works in part by reducing the variability of gene expression in TNBC cells. To test whether RKIP reduces overall transcriptional heterogeneity, we compared the variation in gene expression between individual cells in metastatic (low RKIP) versus non-metastatic (high RKIP) TNBC tumors. Single-cell RNA-sequencing revealed transcriptional heterogeneity was decreased with RKIP overexpression. Many cancer regulatory genes were more homogeneous and up-regulated in the RKIP-expressing cells. Among these enriched and homogenously expressed genes is KMT5C, a histone methyltransferase that facilitates epigenetic transcriptional repression. Cell, mouse, and clinical studies suggest KMT5C expression blocks invasion, extravasation, and colonization of tumor cells, consistent with a role as a novel suppressor of metastasis. These studies reveal the importance of genes involved in heterogeneity as potential regulators of metastatic progression in cancer. Citation Format: Dongbo Yang, Christopher Dann, Andrea Valdespino, Lydia Robinson-Mailman, Mengjie Chen, Sebastian Pott, Marsha Rosner. Genes subject to changes in heterogeneity can function as regulators of metastasis [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 1599.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [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 SY24-03.

Abstract 3885: USP7 is an interaction partner of EZHIP and potential druggable target in PFA ependymomas

Abstract Ependymomas (EPN) are tumors of the central nervous system (CNS) that can arise in the supratentorial brain (ST-EPN), hindbrain or posterior fossa (PF-EPN), or anywhere in the spinal cord (SP-EPN), both in children and adults. Molecular profiling has identified distinct subgroups and subtypes in each of the anatomic compartments. In the posterior fossa, three subgroups have been identified: PFA, PFB and PF-SE. Of them, PFA ependymomas are characterized by a young median age at diagnosis, an overall balanced genome and a bad clinical outcome (56% 10-year overall survival). Standard therapy consists of tumor resection and radiotherapy, but working chemo- or targeted therapies will be essential to improve patient outcomes. Recently, we and others identified enhancer of zeste inhibiting protein (EZHIP) as potential main driver of PFA tumorigenicity. By inhibiting EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), EZHIP prevents the distribution of the epigenetic repressor mark H3K27me3. However, since EZHIP does not possess any known enzymatic functions itself, it does not serve as the druggable target urgently needed for PFA tumors. Thus, interaction partners are one main focus of ongoing PFA research. In this project, we focused on the ubiquitin-specific protease 7 (USP7), a well-known regulator of cancer pathways with a variety of inhibitors available, which was observed to interact with EZHIP in non-PFA cell lines before. We were able to confirm this interaction of EZHIP and USP7 in PFA cells in vitro by co-immunoprecipitation and mass spectrometry, and could show that the EZHIP-USP7 interaction is independent of EZH2, a separate interactor of both. Functionally, we are testing the hypothesis that USP7 de-ubiquitinates EZHIP, thereby preventing its proteasomal degradation and thus stabilizing the protein. As EZHIP expression is essential to PFA cell survival, we aim to target EZHIP indirectly by interfering with its stability regulation via USP7. RNAi-based knockdown (KD) experiments have been used to test a susceptibility of PFA cells to a loss of USP7, focusing on effects on cell proliferation and apoptosis, but also investigating gene expression changes provoked by a change in USP7 levels. Moreover, multiple USP7 inhibitors already highly affect the survival of different PFA cell lines in vitro. Ongoing experiments with PFA patient-derived xenograft (PDX) models will hopefully confirm the strong effect of USP7 inhibitors in PFA, and help to improve targeted therapy for PFA patients. Citation Format: Anne Jenseit, Aylin Camgöz, Monika Mauermann, Stefan M. Pfister, Marcel Kool. USP7 is an interaction partner of EZHIP and potential druggable target in PFA ependymomas [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 3885.