Cancer Epigenome Research Articles

Abstract B030: Combined inhibition of BET proteins and PARP promotes impaired DNA damage repair response and cell cycle dysregulation in preclinical models of bladder cancer

Abstract Background: Bladder cancer (BC) remains a common and deadly malignancy, with a projected 81,180 new diagnoses and 17,100 deaths in the United States in 2022. According to the MSK/TCGA Bladder Cancer dataset, mutations in DNA damage response (DDR) genes that are part of the homologous recombination (HR) pathway occurred in up to 55% of patients, representing a potential therapeutic target in BC. In previous work, we used UNC’s EpiG Diamond compound library to show that, as a class, inhibition of the methyl-lysine reader bromodomain and extra-terminal domain (BET) proteins potently abrogate BC cell line viability. Here, we evaluated mechanisms related to HR inhibition that could explain pan-BET inhibitor OTX-015’s potency in BC. Methods: 5637 and J82 BC cells were treated with 1 µM OTX-015 or 0.1% DMSO control for 48-hours, total RNA was extracted, and then bulk RNA-sequencing (RNA-seq) was performed on an Illumina NovaSeq 6000 (Novogene, Sacramento, CA). Expression data was analyzed using Geneious Prime v2022.2.1 (Biomatters, San Diego, CA). Cells were then treated with 1 μM OTX-015, 5 μM of the PARP inhibitor olaparib, or combination for gene and protein expression analysis. Gene expression changes in BRCA1, BRCA2, PALB2, and RAD51, as well as MYC as a positive control, were confirmed by RT-PCR in biological triplicates after 48-hour incubation, normalized to an SDHA housekeeper and compared to a 0.1% DMSO control. Western blotting evaluated OTX-015 and olaparib effects on BRCA1, BRCA2, c-MYC, PALB2, and RAD51 protein expression after 72-hour incubation, using a GAPDH loading control and compared to a 0.1% DMSO control. Last, cells were treated with the same treatment schema for 24-72 hours, fixed with methanol, stained with propidium iodide (25 μg/mL), and flow cytometry evaluated effects on cell cycle using a ThermoFisher Attune NxT and FlowJo v10.8.1 (BD Life Sciences, Ashland, OR). Results: Bulk RNA-seq analyses revealed significantly altered expression of HR pathway genes in 5637 and J82 cells treated with OTX-015±olaparib. In 5637 cells, OTX-015 alone significantly reduced BRCA1, BRCA2, PALB2 and RAD51 expression, (n=3; P<0.001 for all), but only PALB2 and RAD51 expression was significantly reduced after combination treatment (n=3; P<0.001 for both). In J82 cells, OTX-015 alone and combination treatment significantly reduced BRCA1, PALB2 and RAD51 expression, (n=3; P<0.001 for all). Similarly, both OTX-015 and combination treatment resulted in reduced BRCA1, c-MYC, PALB2 and RAD51 protein expression in both cell lines. In both cell lines, combination treatment resulted in a substantially increased G2/M fraction at 48 hours compared to 0.1% DMSO (5637 cells: 62.9% vs 12.6% G2/M; J82 cells: 28.2% vs 17.8% G2/M). Conclusions: These data indicate that OTX-015-mediated inhibition of RAD51 and PALB2 could be responsible for impaired HR. Cell cycle data revealed a G2/M stall, rather than an S phase stall, suggesting mechanisms independent from impaired HR could be responsible for cell cycle dysregulation. Citation Format: Ryan M. Kemper, Manfred Meng, Daniel J. Crona. Combined inhibition of BET proteins and PARP promotes impaired DNA damage repair response and cell cycle dysregulation in preclinical models of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B030.

Abstract IA021: Targeting epigenetic regulators of oncogenic transcription factors

Abstract The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodeling and is altered in over 20% of cancers. We recently developed and evaluated a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supraphysiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumor growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers. Citation Format: Arul M. Chinnaiyan. Targeting epigenetic regulators of oncogenic transcription factors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA021.

Abstract IA003: Catalytic and noncatalytic mechanisms of chromatin modifiers

Abstract Many mechanisms regulate chromatin structure to ensure that genes are active or silent. Some are considered epigenetic, ensuring the heritable transmission of a specific chromatin state. One of the mechanisms of chromatin regulation is based on posttranslational modifications of histones. Chemical groups are deposited, read, and removed by protein complexes that regulate chromatin structure. Histone modifications correlate with and sometimes directly affect critical processes such as transcription, replication, recombination, and repair. They are the subject of many clinical studies, as aberrant histone modifications and deregulation of enzymes responsible for their deposition occur in many cancers. These modifications rarely function in isolation, and there is often crosstalk between them that results in a coordinated transcriptional output. The central question in the field is to understand the regulation of histone modifications and associated molecular complexes, their role in gene expression, and their pathology in cancer. Another critical question is to discover and characterize the intricate relationships between different histone modifications and histone modifying enzymes at the mechanistic level. I will discuss our progress in understanding the roles of histone methylation and how it interfaces with other chromatin modifications that play critical roles in gene regulation. Citation Format: Karim-Jean Armache. Catalytic and noncatalytic mechanisms of chromatin modifiers. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA003.

Abstract B017: Dysregulation of ultra long-range DNA methylation canyon interactions in human acute myeloid leukemia

Abstract DNA methylation Canyons (DMCs) are genomic regions devoid of DNA methylation over 3.5kb in hematopoietic stem cells (HSCs). DMCs form ultra long-range interactions spanning decades of megabases specifically in HSCs. These ultra-long DMC interactions between DMCs relies on the strong Polycomb binding inside DMC regions. DMCs are hypermethylated and partially activated in cancer. However, its 3D genomic alterations have not been examined extensively. Here we profiled the high-resolution 3D genomic interactions in 7 primary human acute myeloid leukemia (AMLs). Focusing on DMCs' epigenome state, we found that DMCs in all AMLs are hypermethylated and lose the strong Polycomb enrichment within DMCs in comparison with HSC. Based on the DMC interactions, we discovered two subtypes of AMLs with distinct 3D genomic dysregulation of DMCs’ long-range interactions. In the first AML subtype, DMCs’ interactions are still strong as HSPC but gained leukemia-specific long-range interactions. This subtype is enriched for WT1 mutations and is characterized by the expression of CD34 on blast cells. This particular subtype displayed a global basal level increase in the H3K27me3 mark in the genome. This particular subtype of AML is also sensitive to the EZH2 inhibitor treatment. The EZH2 inhibitor treatment leads to the differentiation of blasts and reduces the colony-forming ability of this subtype of AML. Further dissection shows the EZH2 inhibitors could eradicate the leukemia-specific ultra-long range interactions. In the second subtype of AML, 3D genomic interactions between DMCs significantly lost in AMLs with high HOX cluster gene expression (MLL-r and NPM1c+ AMLs). We further found repressive DMC genes, which are developmental transcriptional factors(TFs) in organ formation are activated in these AMLs. Interestingly, these developmental TFs are involved in forming leukemia-specific long-range interactions independent of CTCF-mediated TADs and chromosomal loops. Among those TFs, IRX3 and IRX5 are frequently activated in MLL-r, NPM1c+ AML and MPAL. These two genes have also been shown before to immortalize bone marrow progenitor cells and block differentiation of these progenitor cells. The loci of these two genes form strong long-range connections to an upstream FTO enhancer mimicking the 3D genomic structure observed in developing muscle and brains. Citation Format: Xue Qing D. Wang, Fan Feng, Yali Dou, Jie Liu, Xiaotian Zhang. Dysregulation of ultra long-range DNA methylation canyon interactions in human acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B017.

Abstract B007: Endogenous retrotransposable elements induction in SMARCA4-deficient ovarian cancer is associated with immunomodulatory changes in the tumor microenvironment

Abstract Introduction: Ovarian cancer (OC) is the most lethal gynecologic cancer, and there is an urgent need to develop new therapies. Patient responses to immune checkpoint blockade have overall been disappointing, which is likely due to the immunosuppressive tumor microenvironment (TME) of most OCs that may be in part under the influence of epigenetic regulation. There is evidence that OCs that harbor loss-of-function mutations in the SWI/SNF chromatin remodeling complex catalytic subunit, SMARCA4, may be more immunogenic and amenable to immunotherapy, however the mechanisms by which this occurs is unclear. Proposed mechanisms include de-methylation of normally silenced noncoding elements. The aim of our study is to investigate the epigenetic changes of retrotransposable elements (RTEs) induced by SMARCA4 mutations that modulate the TME. Experimental Procedures: Stable isogenic SMARCA4-knockout (KO) murine (ID8) and human (OAW28) OC models were generated using CRISPR-Cas9 lentiviral transduction and clonal selection. Transcriptomic analyses of these cells were performed using RNA sequencing using ribosomal depletion protocol and validated using qRT-PCR. Moreover, differentially expressed coding genes in RNA sequencing were identified according to fold change between KO and control cells of RPKM (Reads Per Kilobase of transcript, per Million mapped reads) values. Gene Ontology enrichment analysis was then performed to identify the most altered pathways. Results: CRISPR-Cas9 KO of SMARCA4 in OC cells led to an increased fraction of reads coming from repetitive elements (repeatome). Within the repeatome, short interspersed nuclear elements (SINEs) have increased fraction of mapped reads in KO compared to wildtype OAW28 cells. Quantification analyses of known repeats demonstrate significant upregulation of endogenous retroviruses (ERVs) in KO compared to control ID8 cells. Certain RTEs known to be associated with viral mimicry and increased immunogenicity were upregulated in KO cells. Top 10 upregulated genes were significantly enriched in pathways related to interferon and viral responses. Further quantification analyses demonstrated that the dsRNA sensing pathway was activated in SMARCA4-deficient OCs leading to downstream increase in interferon-response genes. Knockdown of dsRNA sensor mitochondrial antiviral signaling protein (MAVS) downregulates the SMARCA4 loss-induced interferon-response activation. Conclusion: De-repression of normally silenced RTEs and subsequent increased immunogenicity of SMARCA4-deficient OCs suggest a potential new immunomodulatory biomarker for targeted therapy investigation. A mechanistic understanding of the role of SMARCA4 in OCs enhances our knowledge of the epigenetic processes that govern immune-stimulating responses in the TME. Studies are underway to explore combination epigenetic therapy and immunotherapy in these cancers. Citation Format: Melica N. Brodeur, Siyu Sun, Yingjie Zhu, Benjamin Greenbaum, Britta Weigelt, Dmitriy Zamarin. Endogenous retrotransposable elements induction in SMARCA4-deficient ovarian cancer is associated with immunomodulatory changes in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B007.

Abstract PR008: An epigenetic memory of inflammation controls context-dependent lineage plasticity and KRAS-driven tumorigenesis in the pancreas

Abstract Tissue homeostasis depends on responses to environmental insults to restore cellular phenotype, microenvironment composition, and tissue architecture. Inflammation is essential to the disruption of homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define a capacity for an environmental insult to control future cell-fate decisions in a context-dependent manner. The ability of epigenetic memory to potentiate tumor initiation both broadens the relationship between inflammation and cancer and raises the possibility that inducing epigenetic ‘amnesia’ of an inflammatory insult could be leveraged as a novel cancer prevention strategy. Citation Format: David J. Falvo, Adrien Grimont, Paul Zumbo, Julie L. Yang, Alexa Osterhoudt, Grace Pan, Andre F. Rendeiro, John Erby Wilkinson, Friederike Dundar, Olivier Elemento, Rhonda K. Yantiss, Doron Betel, Richard Koche, Rohit Chandwani. An epigenetic memory of inflammation controls context-dependent lineage plasticity and KRAS-driven tumorigenesis in the pancreas. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR008.

Abstract A027: Identification and role of mega-trans-factors in epithelial ovarian cancer

Abstract Epithelial ovarian cancer (EOC) accounts for over 90% of ovarian cancer cases and results in 14,000 U.S. deaths annually. It is the leading cause of death attributed to gynecological cancers. Aside from recent scientific advances, the 5-year survival rate for late-stage invasive EOC has been near 30% for several decades. Our computational transcriptional regulatory analysis in low-grade serous ovarian cancers (LGSOCs) vs. high-grade serous ovarian cancers (HGSOCs) identified a master regulatory complex, Megatrans complex (MTC), the objective of this study is to identify role of MTC in ovarian cancer, never reported before. MTC is estrogen-responsive master regulatory complex containing a multitude of DNA-binding transcription factors (MTC_TFs), including ER-alpha, RAR-alpha, GATA3, AP2-gamma, STAT1, AP1, RUNX1, and FOXA1. Given that we found MTC gene targets are downregulated in HGSOC, and that some target genes are prognostic for poor patient survival, understanding the function of MTC and modulating its activity may improve prognostic outcomes for HGSOC patients. We hypothesize that different combinations of MTC_TFs produce distinct regulatory signatures in different grades of ovarian cancers. In the grade, PEO4, cell line, we have confirmed MTC formation using a gel filtration assay and identified the presence of MTC_TFs by western blot. We discovered tumor suppressor, PITX1, as a novel MTC_TF in ovarian cancer, not previously reported in a breast cancer MTC. Methylation analysis revealed PITX1 and ER-alpha binding to hypomethylated, but repressed promoters and ChIP-seq analysis revealed PITX1 co-occupying 98.8% of ER-alpha binding sites, suggesting a repressive role of PITX1 on ER-alpha regulated genes. We are performing additional ChIP-seq experiments of MTC_TFs in HGSOC and LGSOC cell lines to test whether the MTC_TF binding distinguishes their regulatory signatures. Our findings of a nuclear hormone-regulated MTC complex controlling the transcriptional landscape of ovarian cancer are expected to facilitate new breakthroughs in disease treatment. Citation Format: Sushil Kumar Jaiswal, Kevin Fedkenheuer, Laura Elnitski. Identification and role of mega-trans-factors in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A027.

Durable Clinical Impacts and Mechanisms of Action and Resistance in EZH1/2-Targeting Epigenetic Therapy

Background: Epigenomes allow the rectification of disordered gene expressions, thereby providing new targets for pharmacological interventions in cancer therapy. The clinical utility of targeting histone H3 lysine tri-methylation (H3K27me3) as an epigenetic hallmark has been demonstrated. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumor cells remain unclear. Results and Discussion: We conducted intensive molecular monitoring of three patients enrolled in the valemetostat first-in-human phase 1 clinical trial (NCT02732275) for adult T-cell leukemia/lymphoma (ATL). Valemetostat was administered orally once daily (200 mg/day) until a sign of disease progression could be observed. The number of abnormal lymphocytes (Ably) in these three patients drastically decreased after one week of treatment. All patients could stay on valemetostat single agent for more than 2 years with acceptable safety and durable response. PBMC samples (n = 55) were collected from these three patients immediately before (Pre) and during treatment, as well as during the progressive disease (PD) stage. Flow cytometry-based histone methylation assay demonstrated that tumor H3K27me3 level was generally high in the Pre-samples, but the valemetostat treatment reduced it to a normal level in all patients. In addition, ChIP sequencing (ChIP-seq) could directly assess how valemetostat affects the tumor epigenome. The pre-treatment tumor cells showed an overall increasing trend in H3K27me3. For all H3K27me3 peaks (51,683 peaks), 85.9% of the peak signals decreased at the average fold-change of 0.742 after the valemetostat treatment. Focusing on the gene loci, an overall decrease could be observed around the TSS and across the gene body. The characteristic H3K27me3 clusters initially observed in the tumor cells were demethylated. We also conducted integrative multilayered single-cell analyses (scATAC-seq and scRNA-seq) and showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, such as tumor suppressor genes (TSGs), in all patients. These results demonstrated that valemetostat sufficiently restored the epigenome of the tumor cells close to the healthy state, leading to clinical improvements. Although all tested cases showed a durable response for more than 2 years, these responses were eventually interrupted due to the recurrence of ATL. We identified characteristic somatic mutations of EZH2 during clonal repopulation at PD. The identified amino acid substitutions were all located at the interface between EZH2 and valemetostat. Direct evaluation of the H3K27me3 level in the PD sample harboring the acquired EZH2 mutation showed that tumor cells in a hypermethylated state were repopulating. The scATAC-seq analysis also showed strong chromatin compaction of the same gene set occurred in the rapidly propagating resistant clone, indicating that the clonally selected mutations should be responsible for resistance. In patients free of the EZH2 resistant mutations, we detected TET2 mutation or elevated DNMT3A expression in the propagated resistant clones. Genome-wide DNA methylation analysis revealed increased DNA methylation near the TSS in the resistant clones. The genes characterized by the H3K27me3-related chromatin condensation were restored by valemetostat treatment but were again strongly re-condensed by mCpG gain at PD. Consequently, the gene regulation of several important TSGs restored by valemetostat was repressed again. Furthermore, in the single-cell platform, we successfully identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Conclusion: This study illustrates, for the first time, the molecular and cellular dynamics in patients in response to a molecular-targeting inhibitor designed for histone methyltransferases. Eliminating H3K27me3 leads to the reprogramming of the cancer epigenome, thereby exerting a sustained benefit on tumors. Targeting chromatin homeostasis might provide opportunities for further sustained epigenetic therapies.

The co-evolution of the genome and epigenome in colorectal cancer

Colorectal malignancies are a leading cause of cancer-related death1 and have undergone extensive genomic study2,3. However, DNA mutations alone do not fully explain malignant transformation4–7. Here we investigate the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,370 samples from 30 primary cancers and 8 concomitant adenomas and generated 1,207 chromatin accessibility profiles, 527 whole genomes and 297 whole transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent somatic chromatin accessibility alterations, including in regulatory regions of cancer driver genes that were otherwise devoid of genetic mutations. Genome-wide alterations in accessibility for transcription factor binding involved CTCF, downregulation of interferon and increased accessibility for SOX and HOX transcription factor families, suggesting the involvement of developmental genes during tumourigenesis. Somatic chromatin accessibility alterations were heritable and distinguished adenomas from cancers. Mutational signature analysis showed that the epigenome in turn influences the accumulation of DNA mutations. This study provides a map of genetic and epigenetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.

DNMT and EZH2 inhibitors synergize to activate therapeutic targets in hepatocellular carcinoma

The development of more effective targeted therapies for hepatocellular carcinoma (HCC) patients due to its aggressiveness is urgently needed. DNA methyltransferase inhibitors (DNMTis) represented the first clinical breakthrough to target aberrant cancer epigenomes. However, their clinical efficacies are still limited, in part due to an “epigenetic switch” in which a large group of genes that are demethylated by DNMTi treatment remain silenced by polycomb repressive complex 2 (PRC2) occupancy. EZH2 is the member of PRC2 that catalyzes the placement of H3K27me3 marks. EZH2 overexpression is correlated with poor HCC patient survival. We tested the combination of a DNMTi (5-aza-2′-deoxycytidine, DAC) and the EZH2 inhibitor (EZH2i) GSK126 in human HCC cell lines on drug sensitivity, DNA methylation, nucleosome accessibility, and gene expression profiles. Compared with single agent treatments, all HCC cell lines studied showed increased sensitivity after receiving both drugs concomitant with prolonged anti-proliferative changes and sustained reactivation of nascently-silenced genes. The increased number of up-regulated genes after combination treatment correlated with prolonged anti-proliferation effects and increased nucleosome accessibility. Combination treatments also activate demethylated promoters that are repressed by PRC2 occupancy. Furthermore, 13–31% of genes down-regulated by DNA methylation in primary HCC tumors were reactivated through this combination treatment scheme in vitro. Finally, the combination treatment also exacerbates anti-tumor immune responses, while most of these genes were downregulated in over 50% of primary HCC tumors. We have linked the anti-tumor effects of DAC and GSK126 combination treatments to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets and provided a rationale for treatment efficacy for HCC patients.

Chromatin accessibility of primary human cancers ties regional mutational processes and signatures with tissues of origin.

Somatic mutations in cancer genomes are associated with DNA replication timing (RT) and chromatin accessibility (CA), however these observations are based on normal tissues and cell lines while primary cancer epigenomes remain uncharacterised. Here we use machine learning to model megabase-scale mutation burden in 2,500 whole cancer genomes and 17 cancer types via a compendium of 900 CA and RT profiles covering primary cancers, normal tissues, and cell lines. CA profiles of primary cancers, rather than those of normal tissues, are most predictive of regional mutagenesis in most cancer types. Feature prioritisation shows that the epigenomes of matching cancer types and organ systems are often the strongest predictors of regional mutation burden, highlighting disease-specific associations of mutational processes. The genomic distributions of mutational signatures are also shaped by the epigenomes of matched cancer and tissue types, with SBS5/40, carcinogenic and unknown signatures most accurately predicted by our models. In contrast, fewer associations of RT and regional mutagenesis are found. Lastly, the models highlight genomic regions with overrepresented mutations that dramatically exceed epigenome-derived expectations and show a pan-cancer convergence to genes and pathways involved in development and oncogenesis, indicating the potential of this approach for coding and non-coding driver discovery. The association of regional mutational processes with the epigenomes of primary cancers suggests that the landscape of passenger mutations is predominantly shaped by the epigenomes of cancer cells after oncogenic transformation.

Corrupted devolution: How normal cells are reborn as cancer precursors.

Cancers are genetically divergent but intriguingly display similar patterns of epigenetic deregulation, including global DNA hypomethylation and hypermethylation of promoter CpG islands. Early developmental programmes mirror this cancer epigenome suggesting that reactivation of embryonic programmes is essential to the initiation of cancer. We propose a scenario where two waves of dedifferentiation underlie key cell transitions: the first from normal to cancer, and the second driving malignancy. The possibility that early developmental programmes underpin both normal development and the switch to cancer has huge therapeutic implications. The reignition of embryonic programmes and pluripotency networks in seemingly healthy tissues could provide unique cellular targets, to eliminate pre-cancerous or cancer promoting cells before they have the opportunity to form tumours. We conclude that focusing on epigenetic gatekeepers, and peri-implantation cellular identities, could transform the diagnosis and prevention of cancer, especially if these programmes crosscut many cancer types, solid and haematological.

CRISPR/Cas9-mediated epigenetic editing tool: An optimized strategy for targeting de novo DNA methylation with stable status via homology directed repair pathway

With the development of epigenetic engineering tools for manipulating DNA methylation at the desired locus, the stable maintenance of targeted methylation status in edited cells after division poses a major obstacle. Transient methylation levels, along with the reversible inhibition of the desired genes, by some epigenetic editing techniques, limit their further application in functional and therapeutic studies of critical regulators in the cancer epigenome. In this study, we utilized CRISPR/Cas9-mediated the homology-directed repair (HDR) pathway following double-strand breaks (DSBs) information to target de novo methylation of the vital epigenetic biomarker, O6-methylguanine-DNA methyltransferase (MGMT). Based on synthetic single and double stranded methylated repair templates, DNA methylation patterns can be incorporated into the endogenous MGMT gene. In addition, we observed upregulation of DNA methyltransferases (DNMTs) in edited HeLa cells. In particular, we employed an optimized method of using the DNA ligase IV inhibitor SCR7 to markedly enhance the insertion efficiency induced by the long methylated repair template, contributing to the induction of stable methylation alterations through epigenetic recombination after cell division. Additionally, our study confirmed that CRISPR/Cas9-mediated target methylation in a site-specific manner enabled the maintenance of gene silencing in vitro and in vivo. Collectively, we show that a combination of CRISPR/Cas9 components, SCR7 treatment, and the long methylated HDR template can enhance CRISPR/Cas9-directed epigenomic editing efficiency and further induce stable effects on methylation modifications and transcriptional suppression.

New approaches to targeting epigenetic regulation in prostate cancer.

Many clinical trials are currently underway to target the epigenome of castration-resistant prostate cancer. In this review, we summarize the major epigenetic alterations that occur during prostate cancer progression, describe their biological consequences, and highlight potential of therapies that target epigenetic regulators for use in patients. Epigenetic alterations frequently occur in tumour suppressor genes, DNA repair genes, and genes that regulate cell proliferation and differentiation. Unlike genetic alterations, epigenetic changes are reversible, making them promising targets for cancer therapy. Epigenetic regulators can be divided into three broad groups: writers, readers, and erasers , each with specific drug targets that are being assessed in phase I and II clinical trials for prostate cancer. CBP/p300, and BRD4 are coregulators of the androgen receptor and inhibit androgen signalling, making bromodomain extra-terminal inhibitors and CBP/p300 inhibitors attractive targets in prostate cancer. Enhancer of zeste homolog 2, a histone methyltransferase, is also a potential target in castrate-resistant prostate cancer. An emerging direction is to combine epigenetic inhibitors with other compounds to enhance their efficacy. Preclinical studies indicate that the epigenome is a potential target in prostate cancer, and clinical trials are testing multiple agents that target the epigenome in different ways. However, the process of translating these therapies into the clinic is ongoing and none have yet been approved for castrate-resistant prostate cancer.

Identification of metastasis-related long non-coding RNAs in lung cancer through a novel tumor mesenchymal score

Long non-coding RNAs (lncRNAs) have been proven to play critical roles in epithelial-mesenchymal transition (EMT) and metastasis of lung cancer. However, the biological functions and related mechanisms of lncRNAs are unclear. In addition, the EMT-based prognosis prediction in lung cancer still lacks investigation. Here, we established the methodology of identifying critical metastasis-related lncRNAs using comprehensive datasets of cancer transcriptome, genome and epigenome, and also provided tools for prognosis prediction in lung cancer. Initially, important mesenchymal marker genes were identified to compose the tumor mesenchymal score, which predicted patient prognosis in lung cancer, especially lung adenocarcinoma (LUAD). The score was also correlated with several crucial biological and physiological processes, such as tumor immune and hypoxia. Based on the score, lung cancer patients was classified into epithelial and mesenchymal subtypes, and lncRNAs which exhibited expressional dysregulation, promotor methylation alteration and copy number variation between the two subtypes in LUAD were identified and underwent further prognostic analyses. Finally, we identified 14 lncRNAs as EMT-related and significant biomarkers in prognosis prediction of LUAD. As validation, lncRNA RBPMS-AS1 was proven to be co-expressed with epithelial biomarkers, suppressive for A549 cell migration, invasion and EMT, and also significantly associated with better outcomes of LUAD patients, suggesting the potential of RBPMS-AS1 to serve as a lncRNA epithelial biomarker in metastasis of LUAD. Based on the identified lncRNAs, an EMT-linked lncRNA prognostic signature was further established. Taken together, our study provides robust predictive tools, potential lncRNA targets and feasible screening strategies for future study of lung cancer metastasis.

The Activation of Protamine 1 Using Epigenome Editing Decreases the Proliferation of Tumorigenic Cells.

DNA methyltransferases (DNMT) and histone deacetylases (HDAC) inhibitors are used as cancer epigenome drugs. However, these epigenetic drugs lack targeting specificity and could risk inducing genome instability and the expression of oncogenes. Therefore, there is a need to develop new therapeutic strategies where specific cancer genes can be targeted for silencing or activation. The CRISPR/dCas9 system represents a promising, powerful therapeutic tool because of its simplicity and specificity. Protamine 1 (PRM1) is exclusively expressed in sperm and has a vital role in the tight packaging of DNA, thus inducing transcriptional silencing in sperm cells. We hypothesized that the activation of the PRM1 gene in tumorigenic cells would lead to DNA condensation and reduce the proliferation of these cells. To test our hypothesis, we transfected human embryonic kidney cells 293T with a dCas9-P300 plasmid that adds acetyl groups to the promoter region of PRM1 via specific gRNAs plasmids. RNA-Seq analysis of transfected cells revealed high specificity of targeted gene activation. PRM1 expression resulted in a significant decrease in cell proliferation as measured by the BrdU ELISA assay. To confirm that the activation of PRM1 was due to acetyl groups deposited to H3K27, a ChIP-qPCR was performed. The acetylation of the PRM1 promoter region targeted by dCas9-p300 in transfected cells was higher than that of the control cells. Interestingly, the targeted promoter region for acetylation showed reduced DNA methylation. These findings demonstrate the efficacy of epigenome editing in activating PRM1 in non-expressing tumorigenic cells, which could be used as a promising therapeutic strategy in cancer treatment.

Abstract 2180: Deciphering the Warburg effect: Redox is the key to tumor differentiation

Abstract It was proposed by Warburg that the cause and consequence of the Warburg effect were the injury of respiration and cell dedifferentiation, respectively. However, the underlying mechanism was unclear due to limited understanding of metabolic control of epigenetics. Inhibition of mitochondrial electron transport chain (ETC) causes reduction of NAD+/NADH ratio. Recently it was discovered that this redox stress not only drives lactate production from pyruvate to enhance the Warburg effect, but also promotes the conversion of α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), turning the substrate of DNA demethylase TET into an inhibitor of TET, suggesting a potential link between ETC inhibition and dysregulated DNA methylation in cancer. In this study we use a mitochondrial uncoupler niclosamide ethanolamine (NEN) to block the Warburg effect and remodel cancer epigenome. NEN treatment activates ETC and accelerates glutaminolysis to produce αKG, which promotes promoter CpG island demethylation. This epigenomic reprograming not only upregulates tumor suppressor genes such as p53, inhibits N-Myc expression, but also activates neuron differentiation markers to promote differentiation in neuroblastoma cells. Hypoxia is one common microenvironmental stress existing in all solid tumor. NEN treatment reduces 2HG generation from αKG under hypoxia, highlighting its application potential in preventing DNA hypermethylation in vivo. Associated with 2HG reduction, NEN treatment also represses HIF signaling under hypoxia. In addition, NEN treatment inhibits reductive carboxylation flux. Moreover, dietary supplementation with NEN reduces orthotopic neuroblastoma growth and N-Myc expression in vivo. Together, these results validated Warburg’s hypothesis that mitochondrial respiration is the key to maintain normal differentiation. Restoring ETC activity with mitochondrial uncoupler is an effective metabolic/epigenetic therapy against neuroblastoma (Jiang et al., BioRxiv 2021). Citation Format: Jiang Haowen, Rachel L. Greathouse, Bo He, Yang Li, Albert M. LI, Balint Forgo, Selene Banuelos, Joshua Gruber, Hiroyuki Shimada, Bill Chiu, Jiangbin Ye. Deciphering the Warburg effect: Redox is the key to tumor differentiation [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 2180.

Enhancer methylation dynamics drive core transcriptional regulatory circuitry in pan-cancer.

Accumulating evidence has demonstrated that enhancer methylation has strong and dynamic regulatory effects on gene expression. Some transcription factors (TFs) can auto- and cross-regulate in a feed-forward manner, and cooperate with their enhancers to form core transcriptional regulatory circuitries (CRCs). However, the elaborated regulatory mechanism between enhancer methylation and CRC remains the tip of the iceberg. Here, we revealed that DNA methylation could drive the tissue-specific enhancer basal transcription and target gene expression in human cancers. By integrating methylome, transcriptome, and 3D genomic data, we identified enhancer methylation triplets (enhancer methylation-enhancer transcription-target gene expression) and dissected potential regulatory patterns within them. Moreover, we observed that cancer-specific core TFs regulated by enhancers were able to shape their enhancer methylation forming the enhancer methylation-driven CRCs (emCRCs). Further parsing of clinical implications showed rewired emCRCs could serve as druggable targets and prognostic risk markers. In summary, the integrative analysis of enhancer methylation regulome would facilitate portraying the cancer epigenomics landscape and developing the epigenetic anti-cancer approaches.

Abstract A015: Inferring phylogenetic trees based on copy number aberrations from methylation array

Abstract Background: The development of methylation array platforms has allowed large scale studies on cancer epigenomes. Apart from intended analyses on methylation patterns, it is also possible to infer copy number profiles by leveraging the signal from methylation array data. To disentangle complex mixtures of phylogenetically related clones, it is best to jointly analyze different samples (different regions, timepoints, etc.) from the same tumor. However, current methods for studying copy number aberrations (CNAs) from methylation arrays consider tumor samples independently. In this project, we introduce Multi-Sample EPIC Phylogenetic Reconstruction (MultiSEPhyR), aiming to investigate the copy number profiles and clonal compositions from multi-sample methylation array data. Material and methods: As part of the GCGR (Glioma Cellular Genetic Resource; gcgr.org.uk) project, we analyze 53 samples from 26 cases with multiple methylation array data of primary glioblastomas, tumor-derived cell lines and xenograft re-derived cell lines. Copy Number Aberrations (CNAs) called from the methylation array data are validated using WGS data from a subset of the cohort. By jointly clustering the segmented LogR ratios from related samples, we can define tumor clones based on clustered CNA events. We use a two-step approach, where we firstly identify clonal events to obtain sample purity and tumor ploidy and use these values to subsequently infer the copy number state and the clonality of the remaining events. Results and Discussions: On a simulated dataset, MultiSEPhyR could accurately estimate sample purity and identify both clonal and sub-clonal CNA events. We then apply our method on GCGR dataset and show similar extent of intra-tumor heterogeneity in primary glioblastomas and derived cell lines. We also investigate clonal dynamics in each case. In the cell lines, while the heterogeneity is well captured, some sub-clonal CNA events may exhibit significantly different clonality compared to the primary glioblastomas. For example, we observe a clonal expansion of the clones harboring a deletion in chromosome 6 in xenograft re-derived cell lines. Conclusion We present a systematic way to call CNAs and resolve clonal compositions from methylation array data, leading to a cost-efficient way to study tumor evolution. Citation Format: Chuling Ding, Javier Herrero. Inferring phylogenetic trees based on copy number aberrations from methylation array [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A015.

Abstract P3-05-06: Genome-wide DNA methylation analysis identifies novel biomarkers associated with risk of relapse beyond oncotype DX recurrence-score risk assessment within HR+/HER2- early-stage breast cancer patients

Abstract Background: Early-stage, node-negative, hormone receptor positive (HR+) HER2 negative (HER2-) breast cancer (BC) patients comprise a subset with good prognosis. Based on gene expression panels, these patients may skip chemotherapy and complete adjuvant endocrine therapy as an individual genomic basis assessment. However, up to 15% of patients categorized as non-high risk by Oncotype DX (Recurrence Score, RS≤25) may experience disease relapse. The field of cancer epigenomics is emerging as a dynamic tool to complement prognostic expression analyses and to provide further insight into the biological underpinnings of early HR+/HER2- BC. Methods: A genome-wide DNA methylation profiling in tumor tissue samples, from node-negative HR+/HER2- BC patients with available Oncotype DX RS data, was analyzed using the Illumina MethylationEPIC 850K BeadChip. We evaluated all differential methylation patterns independently of their position to establish a bioinformatic analytical pipeline. The online tools KM Plotter and ROC Plotter were used to assess the impact of survival outcomes (5-year relapse-free survival, RFS) and response to therapy of the candidate genes. Individual patient data from TCGA was used to analyze the correlation between methylation and mRNA levels in HR+/HER2- BC patients. Statistical significance was defined by p<0.05; no multiple testing correction was applied. Results: 31 patients were evaluated (mean age 51.4, SD 13.3). Four samples were discarded after quality control analysis. Sixteen tumor patients presented a RS ≤25, of which 6 patients had experienced relapse with a median follow-up of 7.1 years. Of the 773,193 loci studied, significant differential methylation was observed between the relapse and non-relapse groups at 19665 cgs (2,5%). Genes were ranked according to differential methylation patterns, and those with Delta ≥|0.25|were selected. Ninety-nine genes met these criteria. Of them, 85 (86%) were hypomethylated and 14 (14%) were hypermethylated in the relapse group. We studied methylation-expression correlations and the impact on RFS in independent datasets. Among the hypomethylated genes, VAC14 (HR 0.71, CI 0.63-0.82, p 9.7e-7), NDUFS6 (HR 0.68, CI 0.59-0.76, p 1.5e-8), and C7orf50 (HR 0.74, 0.6-0.91, p 0.0059) were found significantly associated with a worse RFS and exhibited a negative correlation between methylation levels and gene expression. Among the hypermethylated genes, MFSD7 (HR 1.31, CI 1.15-1.49, p 6.1e-5), PLA2G3 (HR 1.15, CI 1.09-1.40, p 0.0016), and FGFR2 (HR 1.27, CI 1.11-1.45, p 3.6e-4) were significantly associated to a better RFS and showed a strong negative correlation. Additionally, NDUFS6 mRNA levels were found to be increased in patients with worse response to adjuvant chemotherapy (p 0.012, 1.5x higher; ROC 0.733), while C7orf50 expression levels was associated with a better outcome following adjuvant ET (p 0.012, 1.3x higher, ROC 0.795). Conclusion: In this exploratory study, 6 differentially methylated genes were identified to discriminate patients with non-high RS who may be at a higher risk of relapse. An effort is ongoing towards the development of an analytical pipeline including a more extensive annotation of both experimental and validation cohorts. Citation Format: Jesus Fuentes-Antrás, Vanesa García-Barberán, Nicolas Costa-Fraga, Fernando Moreno, Aida Bao-Caamano, Aitor Rodríguez-Casanova, Alfonso López de Sá, Alicia De Luna, Igor Lopez-Cade, Carmen Ramirez-Ruda, Alejando Pascual, Pedro Perez-Segura, Balázs Győrffy, Alberto Ocaña, Angel Diaz-Lagares, Jose Angel Garcia-Saenz. Genome-wide DNA methylation analysis identifies novel biomarkers associated with risk of relapse beyond oncotype DX recurrence-score risk assessment within HR+/HER2- early-stage breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-05-06.