Epigenetic Silencing Research Articles

OTUB2 silencing promotes ovarian cancer via mitochondrial metabolic reprogramming and can be synthetically targeted by CA9 inhibition

Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.

Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

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

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

Polycomb protein binding and looping in the ON transcriptional state.

Polycomb group (PcG) proteins mediate epigenetic silencing of important developmental genes by modifying histones and compacting chromatin through two major protein complexes, PRC1 and PRC2. These complexes are recruited to DNA by CpG islands (CGIs) in mammals and Polycomb response elements (PREs) in Drosophila. When PcG target genes are turned OFF, PcG proteins bind to PREs or CGIs, and PREs serve as anchors that loop together and stabilize gene silencing. Here, we address which PcG proteins bind to PREs and whether PREs mediate looping when their targets are in the ON transcriptional state. While the binding of most PcG proteins decreases at PREs in the ON state, one PRC1 component, Ph, remains bound. Further, PREs can loop to each other and with presumptive enhancers in the ON state and, like CGIs, may act as tethering elements between promoters and enhancers. Overall, our data suggest that PREs are important looping elements for developmental loci in both the ON and OFF states.

Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes

BackgroundActivation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies.MethodsThis study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads.ResultsWe show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival.ConclusionsAltogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

SUMO protease FUG1, histone reader AL3 and chromodomain protein LHP1 are integral to repeat expansion-induced gene silencing in Arabidopsis thaliana.

Epigenetic gene silencing induced by expanded repeats can cause diverse phenotypes ranging from severe growth defects in plants to genetic diseases such as Friedreich's ataxia in humans. The molecular mechanisms underlying repeat expansion-induced epigenetic silencing remain largely unknown. Using a plant model with a temperature-sensitive phenotype, we have previously shown that expanded repeats can induce small RNAs, which in turn can lead to epigenetic silencing through the RNA-dependent DNA methylation pathway. Here, using a genetic suppressor screen and yeast two-hybrid assays, we identified novel components required for epigenetic silencing caused by expanded repeats. We show that FOURTH ULP GENE CLASS 1 (FUG1)-an uncharacterized SUMO protease with no known role in gene silencing-is required for epigenetic silencing caused by expanded repeats. In addition, we demonstrate that FUG1 physically interacts with ALFIN-LIKE 3 (AL3)-a histone reader that is known to bind to active histone mark H3K4me2/3. Loss of function of AL3 abolishes epigenetic silencing caused by expanded repeats. AL3 physically interacts with the chromodomain protein LIKE HETEROCHROMATIN 1 (LHP1)-known to be associated with the spread of the repressive histone mark H3K27me3 to cause repeat expansion-induced epigenetic silencing. Loss of any of these components suppresses repeat expansion-associated phenotypes coupled with an increase in IIL1 expression with the reversal of gene silencing and associated change in epigenetic marks. Our findings suggest that the FUG1-AL3-LHP1 module is essential to confer repeat expansion-associated epigenetic silencing and highlight the importance of post-translational modifiers and histone readers in epigenetic silencing.

Abstract P05: Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2

Abstract Overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance, and poor survival outcomes. Previously, we have established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes in NKTL. As EZH2 enzymatic inhibitors have proven ineffective against NKTL, we embarked on the development of novel EZH2 Proteolysis Targeting Chimera (PROTAC) compounds. EZH2 PROTACs consist of an EZH2- and E3-ligase binding domain which promotes targeted ubiquitination of the EZH2 protein thereby marking it for proteasomal degradation. Our initial efforts focused on the iterative synthesis of EZH2 PROTAC candidates optimizing factors such as permeability, solubility and stable ternary complex formation. Through extensive screening, we identified C2911 and C1311, which successfully trigger the dose-dependent degradation of EZH2 and PRC2 components concomitant with a significant loss of survival and induction of apoptosis in both NKTL and DLBCL. These effects were rescued by pre-treatment with proteasome inhibitor Bortezomib or when E3 ligase activity was disrupted through siRNA knockdown. In NKTL, we show that EZH2 PROTAC treatment decreased JAK3 and phospho-STAT5 protein expression and identified the suppression of the JAK-STAT-MYC axis as a potential mechanism of action. EZH2 PROTAC treatment effectively reactivates transcription of canonical EZH2 target genes such as MYT1 and ANPEP and downregulates expression of oncogenes c-Myc and E2F1. Pharmacokinetic studies in vivo demonstrate that these PROTACs exhibit good plasma stability, half-life (>4h) and low clearance thereby establishing potential for future clinical development. The efficacy of EZH2 PROTACs in xenograft mice are currently being evaluated. Collectively, our data supports the discovery of novel EZH2 PROTACs and the critical role it plays in successfully targeting the oncogenic role of EZH2 in NKTL or other non-canonical EZH2-driven cancers through protein depletion where enzymatic-only inhibition was ineffective. We are further investigating the mechanisms stabilizing EZH2 protein levels to enhance PROTAC-mediated cytotoxicity. Citation Format: Nurulhuda Mustafa, Zubaida Talal Alnaseri, Darrel Tan Egk Jian, David Quach Thanh Truong, Wan Hsin Lim, Wee Joo Chng. Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2 [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P05.

Blessing or curse: how the epigenetic resolution of host-transposable element conflicts shapes their evolutionary dynamics.

Transposable elements (TEs) are selfish genetic elements whose antagonistic interactions with hosts represent a common genetic conflict in eukaryotes. To resolve this conflict, hosts have widely adopted epigenetic silencing that deposits repressive marks at TEs. However, this mechanism is imperfect and fails to fully halt TE replication. Furthermore, TE epigenetic silencing can inadvertently spread repressive marks to adjacent functional sequences, a phenomenon considered a 'curse' of this conflict resolution. Here, we used forward simulations to explore how TE epigenetic silencing and its harmful side effects shape the evolutionary dynamics of TEs and their hosts. Our findings reveal that epigenetic silencing allows TEs and their hosts to stably coexist under a wide range of conditions, because the underlying molecular mechanisms give rise to copy-number dependency of the strength of TE silencing. Interestingly, contrary to intuitive expectations that TE epigenetic silencing should evolve to be as strong as possible, we found a selective benefit for modifier alleles that weaken TE silencing under biologically feasible conditions. These results reveal that the dual nature of TE epigenetic silencing, with both positive and negative effects, complicates its evolutionary trajectory and makes it challenging to determine whether TE epigenetic silencing is a 'blessing' or a 'curse'.

Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer.

While pancreatic ductal adenocarcinomas (PDACs) are addicted to KRAS-activating mutations, inhibitors of downstream KRAS effectors, such as the MEK1/2 kinase inhibitor trametinib, are devoid of therapeutic effects. However, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway may induce vulnerabilities of therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by trametinib unveiled the induction of endogenous retroviruses (ERVs) escaping epigenetic silencing, leading to the production of double-stranded RNAs and the increased expression of interferon (IFN) genes. We tracked ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated nonsilenced retroelements and synergized with IRF1 (interferon regulatory factor 1) in the activation of IFNs and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the rational design of combination therapies in immuno-oncology.

“Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer”

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin.We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human KRAS-mutant NSCLC cell lines A549 and H460 and mouse KRAS-mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the transcription factor (TF) LRF, a known epigenetic silencer, was examined as a modulator of non-coding RNAs expression. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060–109,747,960 mouse chromosomal region to micro-RNA upregulated species. Furthermore, the novel epigenetic miR-148b-3p/lncPVT1/ZBTB7A axis was identified, which differentiates human cell line of lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes.The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease progression, NSCLC subtypes discrimination and advancement to patient-tailored treatments.

A systematic review of high impact CpG sites and regions for MGMT methylation in glioblastoma [A systematic review of MGMT methylation in GBM]

BackgroundMGMT (O 6 -methylguanine-DNA methyltransferase) promoter methylation is a commonly assessed prognostic marker in glioblastoma (GBM). Epigenetic silencing of the MGMT gene by promoter methylation is associated with greater overall and progression free survival with alkylating agent regimens. To date, there is marked heterogeneity in how MGMT promoter methylation is tested and which CpG sites are interrogated.MethodsTo further elucidate which MGMT promoter CpG sites are of greatest interest, we performed comprehensive searches in PubMed, Web of Science, and Embase and reviewed 2,925 article abstracts. We followed the GRADE scoring system to assess risk of bias and the quality of the studies we included.ResultsWe included articles on adult glioblastoma that examined significant sites or regions within MGMT promoter for the outcomes: overall survival, progression free survival, and/or MGMT expression. We excluded systemic reviews and articles on lower grade glioma. fifteen articles met inclusion criteria with variable overlap in laboratory and statistical methods employed, as well as CpG sites interrogated. Pyrosequencing or BeadChip arrays were the most popular methods utilized, and CpG sites between CpG’s 70–90 were most frequently investigated. Overall, there was moderate concordance between the CpG sites that the studies reported to be highly predictive of prognosis. Combinations or means of sites between CpG’s 73–89 were associated with improved OS and PFS. Six studies identified CpG sites associated with prognosis that were closer to the transcription start site: CpG’s 8, 19, 22, 25, 27, 32,38, and CpG sites 21–37, as well as low methylation level of the enhancer regions.ConclusionThe following systematic review details a comprehensive investigation of the current literature and highlights several potential key CpG sites that demonstrate significant association with OS, PFS, and MGMT expression. However, the relationship between extent of MGMT promoter methylation and survival may be non-linear and could be influenced by potential CpG hotspots, the extent of methylation at each CpG site, and MGMT enhancer methylation status. There were several limitations within the studies such as smaller sample sizes, variance between methylation testing methods, and differences in the various statistical methods to test for association to outcome. Further studies of high impact CpG sites in MGMT methylation is warranted.

Abstract 2864: Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain tumor in children. It is categorized into four molecular subgroups, each with unique genetic and epigenetic alterations, molecular signatures, and differences in clinical features and treatment responses. Targeted therapy for MB has failed in the clinic, prompting new studies of mechanisms involved in MB tumorigenesis and progression. Experimental Procedure: The transcriptomic information of the MB patients was obtained from the publicly available RNAseq datasets and was analyzed for differential expression and clustering analysis. Patient-derived orthotopic xenografts of medulloblastoma tumors with high- and low-REST expression were histologically and biochemically studied for autophagy markers. Furthermore, the SHH-MB cell lines were studied to get a mechanistic understanding of the REST-driven autophagy in this MB subgroup. Summary: Our data showed that autophagy, a process responsible for cellular recycling, and genes associated with this pathway exhibit distinctive expression patterns in human MB subgroups. Specifically, we demonstrate that autophagy is induced in one of the MB subgroups (Sonic Hedgehog-SHH) in response to elevated expression of the RE1-silencing transcription factor (REST), a transcriptional repressor and a canonical regulator of neuronal differentiation genes. REST is also a driver of SHH-MB metastasis. Pharmacological inhibition of autophagy decreases MB cell growth, suggesting it is a pro-survival pathway in REST-driven SHH MBs. In mechanistic studies, we found REST-dependent autophagy induction to involve upregulation of the hypoxia-inducible factor 1-alpha (HIF1α) due to REST-mediated silencing of the von Hippel-Lindau (VHL) gene. The VHL gene product promotes HIF1α ubiquitination and proteasomal degradation, and its loss in SHH-MBs with elevated REST expression drives HIF1α stabilization and nuclear localization to activate target autophagy-related genes. Conclusion: Our work is the first to link VHL to MB pathology. VHL loss of function in cancers has been mainly attributed to mutational events. Here, we report a novel mechanism of VHL loss, namely its epigenetic silencing by REST. Our findings provide the foundation for future pre-clinical investigations to examine the feasibility of autophagy blockade as a therapeutic strategy for REST-driven SHH MBs. Citation Format: Ashutosh Singh, Donghang Cheng, Jyothishmathi Swaminathan, Yan Zheng, Nancy Gordon, Vidya Gopalakrishnan. Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2864.

Abstract 1446: Gastric cancer with microsatellite instability: Ex vivo generation of syngeneic mouse models

Abstract The DNA mismatch repair (MMR) system is a highly conserved pathway crucial for maintaining the genomic integrity. MMR impairment in sporadic tumors is due to MLH1 epigenetic silencing or mutations in other MMR genes (such as MSH2, MSH6, PMS2) and leads to microsatellite instability (MSI). Two independent molecular classifications reported the existence of a MSI subgroup in gastric cancer (GC), including 22-23% of all cases. GC MSI inter and intra-tumoral heterogeneity represents a significant challenge for precise patient stratification and treatment. Even if MSI status in GC is usually associated with a good prognosis, some reports described a subpopulation of MSI GC patients displaying a worse outcome. Moreover, while clinical trials testing immune checkpoint inhibitors in MSI tumors showed effectiveness across all treatment lines, the outcomes in GC patients were not as remarkable as in other cancer types. From the analysis of MSI GC patients enrolled in phase III clinical trials testing anti-PD1 monotherapy, it emerged that nearly half of the tumors are intrinsically resistant and complete and long-lasting responses are achieved in very few cases. Tumor mouse immunocompetent models allow to mimic the complex interactions between a fully functional immune system and a developing tumor. MSI GC syngeneic mouse models could thus represent a valuable tool to better understand the molecular complexity and predict the clinical behavior of this subgroup. To obtain MSI GC mouse models, we generated non-transformed organoids from normal gastric mucosa of BALB/c mice as a starting point for the induction of a neoplastic transformation ex vivo. To induce MMR deficiency, we inactivated either Mlh1 or Msh2 genes using CRISPR/Cas9 genome editing. Importantly, we did not introduce any other alteration in known oncogenes and tumor suppressor genes, thus allowing the natural emergence of driver mutations in the context of the genetic instability. Over time, MMR deficient models, but not the WT controls, exhibited MSI and increased their mutational burden. Furthermore, when cultured in stringent culture conditions (two-dimensional culture, growth factor deprivation), only MMR deficient cells generated tumor masses upon subcutaneous injection in immunodeficient NOD SCID mice. Importantly, cells retrieved from the tumor masses (n = 8) exhibited mutations in a relevant fraction of genes associated with the MSI subgroup by TCGA (21 out of 35) – including Kras, Arid1a, Erbb3 and Tpr53 – thus offering a good representation of the human MSI GC mutational landscape. Given the immunogenicity of the Cas9 protein, we finalized our models optimizing the genome editing protocol to avoid a persistent Cas9 expression. Cas9-free MMR deficient models were able to generate tumors in immunocompetent BALB/c mice, thus representing a valuable resource to investigate tumor development, progression and responses to immunotherapy in vivo. Citation Format: Daniela Conticelli, Gabriele Picco, Cristina Migliore, Claudia Orrù, Fabrizio Maina, Emanuela Boccuni, Simona Corso, Silvia Giordano. Gastric cancer with microsatellite instability: Ex vivo generation of syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1446.

Abstract 97: A E2F6 ceRNA network suppresses dendritic cell function, via PBX1/IL-10 signaling, in ovarian cancer

Abstract It is reported that long-term use of estrogen could increase the risk of ovarian cancer. However, the role of estrogen in immunoevasion is not fully explored. We have previously demonstrated that estrogen-mediated upregulation of E2F6, and, c-Kit, by epigenetic silencing of miR-193a, and a competing endogenous (ceRNA) mechanism. In this study, we found that PBX1, a transcriptional activator of the immunosuppressive cytokine, IL-10, is also a target of miR-193a. Importantly, overexpression of the E2F6 3’UTR upregulates both E2F6 and, PBX1, as well as IL10 in ovarian cancer cell lines, suggesting that ceRNA mechanism exists between E2F6 and PBX1. These phenomena are further supported by our stochastic simulation of the estrogen-mediated E2F6 ceRNA network on the distribution of E2F6 and PBX1 mRNA in cancer cells, which is consistent with the TCGA ovarian cancer RNA-Seq dataset. Importantly, monocyte-derived dendritic cell activation of T-cell function was inhibited by pretreatment of conditioned media derived from ovarian cancer cells overexpressing E2F6 3’UTR; such inhibition was rescueable by an anti-IL-10 antibody. Clinically, IL10 level was higher in ovarian cancer patients with higher E2F6 and PBX1, and in ovarian cancer cell lines overexpressed with E2F6 3’UTR. Taken together, these results showed that E2F6 could suppress anti-tumor immune response of dendritic cell, E2F6 ceRNA network. Epigenetic intervention in restoring the expression of miR-193a may be able to enhance anti-tumor immune response against ovarian cancer. Citation Format: Michael W.Y. Chan, Yin-Chen Chen, Ching-Wen Lin, Frank Cheng, Ching-Cher Sanders Yan, Chao-Ping Hsu, Yu-Min Chuang, Jie-Ting Low, Xiaojing Ma, Yao-Ting Huang, Chia-Bin Chang, Chin Li, Hung-Cheng Lai, Shu-Fen Wu, Shih-Hsun Hung, Je-Chiang Tsai. A E2F6 ceRNA network suppresses dendritic cell function, via PBX1/IL-10 signaling, in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 97.

Abstract 1425: Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status

Abstract Background: Epigenetic silencing of MHC-I, and poor T cell infiltration and function in the tumor microenvironment (TME) are prevalent in solid malignancies and associate with lack of response to immune checkpoint blockade (ICB). Novel combination therapies may overcome these barriers, thereby providing clinical benefit across solid tumors. STAR0602 is a first-in-class bifunctional therapeutic molecule, that simultaneously engages a nonclonal mode of T cell receptor (TCR) activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. Here, we examined the tumor-suppressive ability and mode of action of a murine surrogate of STAR0602 (mSTAR1302) in combination with the class I HDAC inhibitor Entinostat, an epigenetic modulator that enhances tumor immune cell recognition, in multiple tumor models, including ICB-refractory. Methods: mSTAR1302 and/or Entinostat were administered to mice bearing ICB-responsive breast EMT6 wild-type (wt) and ICB-refractory EMT6 β2-microglobulin (B2m)-deleted and CT26 (Kras G12Dmut colon) tumors. Anti-tumor efficacy, survival, and protective memory were monitored. Immune cell depletion studies were conducted. Comprehensive proteomic and immune profiling of tumor, tumor-draining lymph node (tdLN), and spleen cell populations was conducted. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: Combination therapy demonstrated significant enhancement in tumor suppression and prolonged survival in CT26 (MHC-I+), EMT6 wt (MHC-I+) and EMT6 β2m-deleted (MHC-Inull) tumors relative to single agent therapy, as well as enhanced tumor-specific protective memory responses. Tumor suppression was independent of CD4+ T cell immunity and associated with sustained activation and expansion of Vβ13 CD8+ T cells. Conclusions: Ongoing mechanistic studies indicate combination therapy to elicit complimentary mechanisms of tumor suppression beyond CD8+ T cells. Ongoing proteomic and single-cell transcriptomic investigation of MHC-I competent and null tumors will provide a deeper insight into the mode of action of this combination therapy. Collectively, these data support the use of STAR0602 in combination with Entinostat for the treatment of solid malignancies, including for patients harboring ICB-refractory tumors devoid of MHC class I. Citation Format: Katherine E. Lothstein, Asma S. Khelifa, Masaya Miyamoto, Christine M. Minnar, Lisa Poppe, Nicholas Roller, Andrew Bayliffe, Ke Liu, Jacques Moisan, Madan Katragadda, Zhen Su, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1425.

Abstract 4893: Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas

Abstract Introduction: Colorectal cancer (CRC) is second most common cause of cancer-related mortality in the USA and most patients with advanced tumors lack effective therapeutic options. Epigenetic silencing resulting in reduced expression of O6-methylguanine-DNA methyltransferase (MGMT) has been described in diverse tumor types including CRC and it is linked with reduced DNA repair capability and increased sensitivity to alkylating agents such as temozolomide. However, the potential role and clinical significance of MGMT overexpression in cancer cells has not been studied. Methods: We used spatially resolved multiplex quantitative immunofluorescence (mQIF) analysis to concurrently measure the levels of MGMT protein, γH2AX and CD8+ tumor infiltrating lymphocytes (TILs) in a cohort of 111 CRCs and paired non-tumor colorectal mucosa from the same patient represented in tissue microarray format. To assess the genomic and immunologic context of MGMT expression, we studied the methylome, mutation profiling and mRNA expression of 526 CRCs from TCGA. The immunologic landscape of tumors was studied using GSEA and multiple transcriptomic-based tumor immune infiltration scores such as TIMER, CIBERSORT, etc. Finally, the genomic alterations and immunogenicity of SW620 human CRC cells (with baseline MGMT hypermethylation) with or without MGMT transfection were studied using in vitro co-culture preparations coupled to ultra-deep DNAseq and flow cytometry analysis. Results: On mQIF analysis, cancer-cell MGMT protein upregulation was identified in 73.9% of CRCs and was associated with reduced levels of γH2Ax in malignant cells and lower CD8+ TILs. 26.1% of cases showed MGMT protein downregulation. The genomic analysis of the TCGA cohort revealed that the mean nonsynonymous mutational load was significantly lower in MGMT-high than low tumors with marked differences in the type of variants and frequency of oncogenic mutations. The transcriptomic analysis revealed that MGMT overexpressing tumors showed lower immune infiltration than the MGMT-low group and reduced representation of immune associated signatures including interferon gamma response and TNF-alpha signaling. In contrast, DNA repair, TP53 pathways, were enriched. The exogenous expression of MGMT in SW620 cells reduced the number of nonsynonymous mutations by 15.6% (141 vs 119 mean mutations) and was also associated with low T-cell activation (CD69+/CD8+ T-cells) and cancer-cell elimination on allogenic co-cultures with human PBMCs. Conclusion: MGMT protein upregulation is common in human CRCs and is associated with reduced DNA damage response, distinct genomic features, and adaptive immune evasion. To our knowledge, this study is the first to report the biological and clinical characteristics of CRCs with MGMT overexpression that could support diagnostic and therapeutic developments. Citation Format: Barani Kumar Rajendran, Janie Zhang, Shruti Desai, Joanna Gibson, Jassim DiPalermo, Patricia LoRusso, Yong Kong, Hongyu Zhao, Michael Cecchini, Kurt Alex Schalper. Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4893.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

BackgroundNaturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers.MethodsWe conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation.ResultsSimilar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs.ConclusionsThese data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.Graphical abstract

Cellular and clinical impact of protein phosphatase enzyme epigenetic silencing in multiple cancer tissues

BackgroundProtein Phosphatase Enzymes (PPE) and protein kinases simultaneously control phosphorylation mechanisms that tightly regulate intracellular signalling pathways and stimulate cellular responses. In human malignancies, PPE and protein kinases are frequently mutated resulting in uncontrolled kinase activity and PPE suppression, leading to cell proliferation, migration and resistance to anti-cancer therapies. Cancer associated DNA hypermethylation at PPE promoters gives rise to transcriptional silencing (epimutations) and is a hallmark of cancer. Despite recent advances in sequencing technologies, data availability and computational capabilities, only a fraction of PPE have been reported as transcriptionally inactive as a consequence of epimutations.MethodsIn this study, we examined promoter-associated DNA methylation profiles in Protein Phosphatase Enzymes and their Interacting Proteins (PPEIP) in a cohort of 705 cancer patients in five tissues (Large intestine, Oesophagus, Lung, Pancreas and Stomach) in three cell models (primary tumours, cancer cell lines and 3D embedded cancer cell cultures). As a subset of PPEIP are known tumour suppressor genes, we analysed the impact of PPEIP promoter hypermethylation marks on gene expression, cellular networks and in a clinical setting.ResultsHere, we report epimutations in PPEIP are a frequent occurrence in the cancer genome and manifest independent of transcriptional activity. We observed that different tumours have varying susceptibility to epimutations and identify specific cellular signalling networks that are primarily affected by epimutations. Additionally, RNA-seq analysis showed the negative impact of epimutations on most (not all) Protein Tyrosine Phosphatase transcription. Finally, we detected novel clinical biomarkers that inform on patient mortality and anti-cancer treatment sensitivity.ConclusionsWe propose that DNA hypermethylation marks at PPEIP frequently contribute to the pathogenesis of malignancies and within the precision medicine space, hold promise as biomarkers to inform on clinical features such as patient survival and therapeutic response.

Neuronal miR-9 promotes HSV-1 epigenetic silencing and latency by repressing Oct-1 and Onecut family genes

Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.