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It is unknown why cancers of the same tissue-type but in anatomically distinct locations exhibit different molecular dependencies for tumorigenesis. Proximal and distal colon cancers (CC) exemplify such characteristics, with BRAFV600E predominantly occurring in proximal CC along with increased DNA methylation phenotype. Using mouse colon organoids, we show that proximal and distal colon stem cells have distinct transcriptional programs regulating stemness and differentiation. The homeobox transcription factor, Cdx2, frequently silenced by DNA methylation in proximal colon cancers, was identified as a key mediator of the differential transcriptional programs. Cdx2-mediated proximal colon-specific transcriptional program is concurrently tumor suppressive, and its loss is critical for BRAFV600E-driven transformation of proximal colon stem cells. Human proximal CC with CDX2-downregulation showed similar transcriptional program as the mouse proximal organoids with Cdx2 loss. Our results suggest a model in which developmental transcription factors maintain tissue-location specific transcriptional programs that create tissue-type origin specific dependencies for tumor initiation.

Contains Tables S1 and S2. Supplementary Table 1: List of PCR primers. Supplementary Table 2: Sensitivity of Breast Cancer Cell Lines to CPI-455 Cell Viability was measured following exposure to CPI-455 for 8 days.

PDF file - 2177KB, Scatter Plots of Gene Re-expression with Decitabine or Trichostatin-A in Eight Non-Small Cell Lung Cancer Cell Lines.

PDF file - 287KB, DNA Methylation of CDO1, HOXA9, and TAC1 is Highly Sensitive for Stage I NSCLC in the Cancer Genome Atlas.

Effects of KDM5i and DAC on enhancers and de novo super-enhancers

PDF file - 399KB, Kaplan-Meier Survival Curves for Non-Small Cell Lung Cancer for p16 methylation in combination with APC, RASSF1, or CDH13.

Purpose: Non–small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the world. Novel diagnostic biomarkers may augment both existing NSCLC screening methods as well as molecular diagnostic tests of surgical specimens to more accurately stratify and stage candidates for adjuvant chemotherapy. Hypermethylation of CpG islands is a common and important alteration in the transition from normal tissue to cancer.Experimental Design: Following previously validated methods for the discovery of cancer-specific hypermethylation changes, we treated eight NSCLC cell lines with the hypomethylating agent deoxyazacitidine or trichostatin A. We validated the findings using a large publicly available database and two independent cohorts of primary samples.Results: We identified >300 candidate genes. Using The Cancer Genome Atlas (TCGA) and extensive filtering to refine our candidate genes for the greatest ability to distinguish tumor from normal, we define a three-gene panel, CDO1, HOXA9, and TAC1, which we subsequently validate in two independent cohorts of primary NSCLC samples. This three-gene panel is 100% specific, showing no methylation in 75 TCGA normal and seven primary normal samples and is 83% to 99% sensitive for NSCLC depending on the cohort.Conclusion: This degree of sensitivity and specificity may be of high value to diagnose the earliest stages of NSCLC. Addition of this three-gene panel to other previously validated methylation biomarkers holds great promise in both early diagnosis and molecular staging of NSCLC. Clin Cancer Res; 20(7); 1856–64. ©2014 AACR.

Related Article from Biomarkers for EGFR-Antagonist Response: In the Genes and on the Genes!

PDF file - 100KB, DNA Methylation of CDO1, HOXA9, and TAC1 is Highly Sensitive for Stage I NSCLC in the Cancer Genome Atlas.

Patients with non–small cell lung carcinoma containing epidermal growth factor receptor (EGFR)–activating mutations benefit from EGFR-antagonist therapy. EGFR-antagonist sensitivity is also correlated with epithelial–mesenchymal transition, which Walter and colleagues show to be marked by DNA-methylation changes. If validated, these markers could help identify patients with wild-type EGFR who will benefit from EGFR therapy. Clin Cancer Res; 18(8); 2121–3. ©2012 AACR.

XLSX file - 25KB, List of Infinium 450K Methylation Probes from Figure 1.

Legends for Supplementary Figures and Tables

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Supplementary Table 1 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 3 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 1 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Table 2 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 4 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 5 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

One of the most important goals of developing timely detection and effective intervention strategies for lung cancer is to understand the biology and molecular mechanisms involved in early-stage evolution of this disease. Chronic inflammation is a major driver of lung cancer and contributes to its pathogenesis by inducing genetic and epigenetic abnormalities, as well as shifts in key immune cell populations. However, the evolution of these changes and the mechanisms underlying the early stages in development of pre-neoplasias and progression to invasive tumors have not been well delineated. Monolayer cell culture systems, while providing valuable insights into lung cancer development, lack the complexity of an intact organ system. This study aims to overcome these limitations by using a 3D lung organoid model to delineate the role of epigenetic alterations in driving initiation of non-small cell lung cancer. The stem cell enriched nature of the organoids makes it an ideal system to evaluate the implications of epigenetic alterations in these key cell types and their role in cancer initiation. To better identify the epigenetic alterations that play key roles in lung cancer initiation, cigarette smoke condensate (CSC) was applied to normal lung organoids to mimic chronic inflammatory exposure. Epigenomic and transcriptomic alterations were evaluated by genome-wide DNA methylation analysis, ATAC-seq, and RNA-seq. Flow cytometry and confocal microscopy were used to evaluate changes in composition of cell populations comprising the organoids. Long-term CSC exposure caused distinct morphological changes in organoid structure and cellular composition, accompanied by an increased proliferative potential. Analysis of changes in cell composition revealed increases in Krt14 expression in CSC treated organoids, leading to key shifts in basal stem cell populations from a TP63+KRT5+ to a TP63+KRT5+KRT14+ population. This was accompanied by reduction in differentiated cell types, as analyzed by qRT-PCR. Analysis of genome wide DNA methylation showed increases in promoter DNA methylation in key genes associated with lung tumorigenesis. Most importantly, co-culture studies involving culturing CSC treated and control organoids with key immune cells revealed that chronic CSC treatment caused modulation of the microenvironment from a pro- to an anti-inflammatory state. Our results suggest that chronic inflammation causes key shifts in populations of lung stem cells with an associated decrease in differentiation potential. These changes are accompanied by DNA methylation and gene expression changes suggestive of an increased tumorigenic potential. Finally, these changes in the CSC treated organoids are associated with the ability to modulate the function of key immune cells associated with lung tumorigenesis from a pro- to an anti-inflammatory phenotype. Results from our study, will aid in developing novel biomarker-based methodologies to distinguish and treat lung cancer in its early stages. Citation Format: Na Wang, Ray-Whay Chiu Yen, Hamza Khan, Malcolm V. Brock, Hariharan Easwaran, Stephen B. Baylin, Michelle Vaz. Characterizing the role of inflammation-induced epigenetic alterations in modulating the immune microenvironment during lung cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB046.

Recent studies have mapped key genetic changes in colorectal cancer (CRC) that impact important pathways contributing to the multistep models for CRC initiation and development. In parallel with genetic changes, normal and cancer tissues harbor epigenetic alterations impacting regulation of critical genes that have been shown to play profound roles in the tumor initiation. Cumulatively, these molecular changes are only loosely associated with heterogenous transcriptional programs, reflecting the heterogeneity in the various CRC molecular subtypes and the paths to CRC development. Studies from mapping molecular alterations in early CRC lesions and use of experimental models suggest that the intricate dependencies of various genetic and epigenetic hits shape the early development of CRC via different pathways and its manifestation into various CRC subtypes. We highlight the dependency of epigenetic and genetic changes in driving CRC development and discuss factors affecting epigenetic alterations over time and, by extension, risk for cancer.

Cancer recurrence after surgery remains an unresolved clinical problem1–3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4–6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy. In mouse models of pulmonary metastasis, adjuvant epigenetic therapy targeting myeloid-derived suppressor cells disrupts the premetastatic microenvironment after resection of primary tumours and inhibits the dissemination of residual tumour cells.

Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We now link cancer-specific transcription factor (TF) expression dysregulation to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their pre-malignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF-expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMP), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). CIMP/CEMP coordination appears to be driven by augmented downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The dysregulated expression of TFs related to CIMP/CEMP subtypes occurs early during CRC development, detectable in pre-malignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes. Citation Format: Yuba R. Bhandari, Vinod Krishna, Rachael Powers, Sehej Parmar, Sara-Jayne Thursby, Ekta Gupta, Ozlem Kulak, Prashanth Gokare, Joke Reumers, Liesbeth Van Wesenbeeck, Kurtis E. Bachman, Stephen B. Baylin, Hariharan Easwaran. Transcription factor expression repertoire basis for epigenetic and transcriptional subtypes of colorectal cancers. [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 A012.

Gastric cancer (GC) remains the third leading cause of cancer-related death worldwide. While inflammation is a well-established driver of gastric tumorigenesis, only a small subset of GC patients responds to immunotherapy. One proposed mechanism of immune escape is silencing tumor-antigen expression, and thereby avoiding immune recognition. DNA methyltransferases (DNMTs) are the enzymes responsible for DNA methylation and hence, for the epigenetic silencing of gene expression of tumor-antigens and other immune-related molecules. Interestingly, they are often overexpressed in solid tumors. Epigenetic drugs inhibiting these DNMTs have shown anti-tumor effects in combination with immune checkpoint inhibitors. Here we are studying the role of DNMTs, specifically DNMT3A, in GC mouse models and the possibility of combining DNMT inhibitors with immunotherapy for the treatment of GC. This project utilises various mouse models of GC. We established a Dnmt3a-overexpressing inflammation-driven GC mouse model (gp130FF, A33Dnmt3a). In a second mouse model, mutant Kras, Pi3kca and Tp53 expression results in highly advanced invasive gastric carcinoma formation (KPT model). We successfully established GC organoids of one of these triple mutant tumors, which can be injected subcutaneously into wild type mice and result in allograft tumor formation. DNMT3A overexpression was detected in human GC specimen and associated with bad overall survival of patients. Gastric adenomas of our gp130FF, A33Dnmt3a mouse model have a 10-fold elevated Dnmt3a expression. Importantly, tumor-specific Dnmt3a overexpression significantly increased gastric tumor burden. We have conducted DNA methylation and gene expression analyses to identify differently methylated and expressed genes between gp130FF and gp130FF, A33Dnmt3a gastric adenoma cells. Treatment with the DNMT inhibitor decitabine reduced tumor growth in the inflammation-driven gp130FF gastric adenoma mouse model. In the KPT model, we have identified DNMT3A as being highly expressed in the invasive front of tumors, their liver metastases as well as in the allograft tumors established by the KPT organoids. In this model of advanced GC, treatment with the DNMT inhibitor decitabine significantly decreased tumor growth and we are currently investigating the efficacy of decitabine in combination with anti-PD-1 and/or anti-CTLA-4 immunotherapy. Taken together, we provide evidence for a driver function of Dnmt3a in gastric tumorigenesis and gastric tumor growth. In addition, our findings encourage further studies to investigate the potential of DNMT inhibitors for the treatment of GC. Citation Format: Anne Huber, Christine Dijkstra, Yuba Bhandari, Hariharan Easwaran, Stephen Baylin, Matthias Ernst, Moritz Eissmann. DNA methyltransferase 3A promotes inflammation-associated gastric cancer growth and presents a therapy target for gastric 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 A008.

Summary Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Advanced age is strongly correlated with both increased cancer incidence and general immune decline. The immune tumor microenvironment (ITME) has been established as an important prognostic of both therapeutic efficacy and overall patient survival. Thus, age-related immune decline is an important consideration for the treatment of a large subset of cancer patients. Current studies of aging-related immune alterations are predominantly performed on non-cancerous tissue, requiring additional study into the effects of age on tumor immune infiltration. We leverage large scale transcriptional data sets from The Cancer Genome Atlas and the Genotype-Tissue Expression project to distinguish normal age-related immune alterations from age-related changes in tumor immune infiltration. We demonstrate that while there is overlap between the normal immune aging phenotype and that of the ITME, there are several changes in immune cell abundance that are specific to the ITME, particularly in T cell, NK cell, and Macrophage populations. These results suggest that aged immune cells are more susceptible to tumor suppression of cytotoxic immune cell infiltration and activity than normal tissues, which creates an unfavorable ITME in older patients in excess of normal immune decline with age and may inform the application of existing and emerging immunotherapies for this large population of patients. We additionally identify that age-related increases in tumor mutational burden are associated with decreased DNA methylation and increased expression of the immune checkpoint genesPDL1, CD80,andLAG3which may have implications for therapeutic application of immune checkpoint blockade in older patients.

While the methods available for single-cell ATAC-seq analysis are well optimized for clustering cell types, the question of how to integrate multiple scATAC-seq data sets and/or sequencing modalities is still open. We present an analysis framework that enables such integration across scATAC-seq data sets by applying the CoGAPS Matrix Factorization algorithm and the projectR transfer learning program to identify common regulatory patterns across scATAC-seq data sets. We additionally integrate our analysis with scRNA-seq data to identify orthogonal evidence for transcriptional regulators predicted by scATAC-seq analysis. Using publicly available scATAC-seq data, we find patterns that accurately characterize cell types both within and across data sets. Furthermore, we demonstrate that these patterns are both consistent with current biological understanding and reflective of novel regulatory biology.

SUMMARY To improve our understanding of ovarian cancer, we performed genome-wide analyses of 45 ovarian cancer cell lines. Given the challenges of genomic analyses of tumors without matched normal samples, we developed approaches for detection of somatic sequence and structural changes and integrated these with epigenetic and expression alterations. Alterations not previously implicated in ovarian cancer included amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and TGF-beta receptor pathway members, and rearrangements of YAP1-MAML2 and IKZF2-ERBB4. Dose-response analyses to targeted therapies revealed unique molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, MYC amplifications to PARP inhibitor BMN673, and SMAD3/4 alterations to MEK inhibitor MEK162. Genome-wide rearrangements provided an improved measure of sensitivity to PARP inhibition. This study provides a comprehensive and broadly accessible resource of molecular information for the development of therapeutic avenues in ovarian cancer., In Brief The overall survival of patients with late-stage ovarian cancer is dismal. To identify therapeutic opportunities, Papp et al. integrate genomic, epigenomic, and expression analyses to provide a resource of molecular abnormalities in ovarian cancer cell lines and use these to identify tumors sensitive to PARP, MEK, and PI3K inhibitors., Graphical Abstract

SUMMARY Both tumors and aging alter the immune landscape of tissues. These interactions may play an important role in tumor progression among elderly patients and may suggest considerations for patient care. We leverage large-scale genomic and clinical databases to perform comprehensive comparative analysis of molecular and cellular markers of immune checkpoint blockade (ICB) response with patient age. These analyses demonstrate that aging is associated with increased tumor mutational burden, increased expression and decreased promoter methylation of immune checkpoint genes, and increased interferon gamma signaling in older patients in many cancer types studied, all of which are expected to promote ICB efficacy. Concurrently, we observe age-related alterations that might be expected to reduce ICB efficacy, such as decreases in T cell receptor diversity. Altogether, these changes suggest the capacity for robust ICB response in many older patients, which may warrant large-scale prospective study on ICB therapies among patients of advanced age., In brief Erbe et al. use multi-omics databases to evaluate biomarkers that are used to predict the response of patients with cancer to ICB in the context of aging. Erbe et al. find that biomarkers associated with ICB response are enriched in tumors from older patients relative to their younger counterparts., Graphical Abstract

Colorectal cancer (CRC) is classified into distinct subtypes based on genetic, epigenetic and/or transcriptional subtypes. The underlying mechanisms that drive these subtypes are unknown. By integrating whole genome DNA methylation, gene expression, chromatin patterns, and enhancer elements in CRCs, we show that expression of large numbers of transcriptional factors (TFs) is deregulated in CRCs and these expression patterns drive epigenetic and transcriptional subtypes of CRCs. The first key result emerging is a new understanding of important DNA methylation patterns central to classifying CRCs. Of these, different degrees of cancer associated promoter methylation have been recognized that suppress expression, and/or inducibility of important tumor suppressor genes. Stratified by high to low degrees of hypermethylation, this pattern is termed CpG-island promoter DNA methylation phenotypes or CIMP. We now tightly link CIMP to a cancer-specific hypermethylation at enhancer regulatory elements we term, CpG- enhancer methylation phenotype (CEMP). CIMP and CEMP are driven by decreased expression of multiple TFs with binding sites in these regulatory elements, and the involved TFs are frequently identical between the two regions. In contrast, a different set of TF factors are downregulated in non-CIMP tumors, again in conjunction with methylation patterns linking promoter and enhancer target sites for the involved TFs. Further, enhancer regulatory elements that are hypomethylated in cancer relative to normal tissue are associated with increased expression of another distinct set of TFs with target sites in the corresponding enhancers. Importantly, the TF expression patterns, and the above CIMP, CEMP, and hypomethylation patterns, are established early in the pre-malignant adenomas. Importantly, pan-cancer analyses showed that the above CIMP and CEMP subtypes occur across important cancer types in addition to CRC, again in parallel with marked downregulation of TFs that have target sites in the methylated regulatory elements. All of the above newly defined patterns also provide a basis for the transcriptome-based consensus molecular subtypes (CMS) of CRC. Overall, our studies may provide key understanding of how cancers, and their initiation and progression, are diseases which at the end of the day depend on abnormal patterns of TF expression, and how these relate to methylation alterations at target enhancer and promoter regions. Citation Format: Yuba Bhandari, Rachael Powers, Sehej Parmar, Vinod Krishna, Kurtis E. Bachman, Steve Baylin, Hariharan Easwaran. Transcription factor repertoire basis for epigenetic and consensus molecular subtypes of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2107.

// Nilofer S. Azad 1 , Anthony el-Khoueiry 2 , Jun Yin 3 , Ann L. Oberg 3 , Patrick Flynn 4 , Douglas Adkins 5 , Anup Sharma 1 , Daniel J. Weisenberger 2 , Thomas Brown 1 , Prakriti Medvari 1 , Peter A. Jones 6 , Hariharan Easwaran 1 , Ihab Kamel 1 , Nathan Bahary 7 , George Kim 8 , Joel Picus 9 , Henry C. Pitot 3 , Charles Erlichman 3 , Ross Donehower 1 , Hui Shen 6 , Peter W. Laird 6 , Richard Piekarz 9 , Stephen Baylin 1 and Nita Ahuja 1 1 Johns Hopkins University, Baltimore, MD, USA 2 University of Southern California, Los Angeles, CA, USA 3 Mayo Clinic, Rochestor, MN, USA 4 Metro-Minnesota CCOP, Minneapolis, MN, USA 5 Washington University, St. Louis, MO, USA 6 Van Andel Research Institute, Grand Rapids, MI, USA 7 University of Pittsburgh, Pittsburgh, PA, USA 8 Mayo Clinic, Jacksonville, FL, USA 9 Washington University, St. Louis, MO, USA 10 Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD, USA Correspondence to: Nilofer S. Azad, email: // Keywords : epigenetics, colorectal cancer, DNA methyltransferases inhibitors, histone deacetylase inhibitors Received : August 11, 2016 Accepted : October 25, 2016 Published : February 05, 2017 Abstract Purpose: Therapy with demethylating agent 5-azacitidine and histone deacetylase inhibitor entinostat shows synergistic re-expression of tumor-suppressor genes and growth inhibition in colorectal (CRC) cell lines and in vivo studies. Experimental Design: We conducted a phase II, multi-institutional study of the combination in metastatic CRC patients. Subcutaneous azacitidine was administered at 40 mg/m2 days 1-5 and 8-10 and entinostat was given 7 mg orally on days 3 and 10. An interim analysis indicated toxicity crossed the pre-specified safety boundary but was secondary to disease. A 2 nd cohort with added eligibility restrictions was accrued: prior therapies were limited to no more than 2 or 3 (KRAS-mutated and KRAS-wildtype cancers, respectively) and

TET proteins, by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are hypothesized, but not directly shown, to protect promoter CpG islands (CGIs) against abnormal DNA methylation (DNAm) in cancer. We define such a protective role linked to DNA damage from oxidative stress (OS) known to induce this abnormality. TET2 removes aberrant DNAm during OS through interacting with DNA methyltransferases (DNMTs) in a "Yin-Yang" complex targeted to chromatin and enhanced by p300 mediated TET2 acetylation. Abnormal gains of DNAm and 5hmC occur simultaneously in OS, and knocking down TET2 dynamically alters this balance by enhancing 5mC and reducing 5hmC. TET2 reduction results in hypermethylation of promoter CGIs and enhancers in loci largely overlapping with those induced by OS. Thus, TET2 indeed may protect against abnormal, cancer DNAm in a manner linked to DNA damage.

Summary UHRF1 facilitates the establishment and maintenance of DNA methylation patterns in mammalian cells. The establishment domains are defined, including E3 ligase function, but the maintenance domains are poorly characterized. Here, we demonstrate that UHRF1 histone- and hemimethylated DNA binding functions, but not E3 ligase activity, maintain cancer-specific DNA methylation in human colorectal cancer (CRC) cells. Disrupting either chromatin reader activity reverses DNA hypermethylation, reactivates epigenetically silenced tumor suppressor genes (TSGs), and reduces CRC oncogenic properties. Moreover, an inverse correlation between high UHRF1 and low TSG expression tracks with CRC progression and reduced patient survival. Defining critical UHRF1 domain functions and its relationship with CRC prognosis suggests directions for, and value of, targeting this protein to develop therapeutic DNA demethylating agents.

We addressed the precursor role of aging-like spontaneous promoter DNA hypermethylation in initiating tumorigenesis. Using mouse colon-derived organoids, we show that promoter hypermethylation spontaneously arises in cells mimicking the human aging-like phenotype. The silenced genes activate the Wnt pathway, causing a stem-like state and differentiation defects. These changes render aged organoids profoundly more sensitive than young ones to transformation by Braf(V600E), producing the typical human proximal BRAF(V600E)- driven colon adenocarcinomas characterized by extensive, abnormal gene-promoter CpG-island methylation, or the methylator phenotype (CIMP). Conversely, CRISPR-mediated simultaneous inactivation of a panel of the silenced genes markedly sensitizes to Braf(V600E)-induced transformation. Our studies tightly link aging-like epigenetic abnormalities to intestinal cell fate changes and predisposition to oncogene-driven colon tumorigenesis.

Cancer recurrence after surgery remains an unresolved clinical problem

DNA methylation is a key epigenetic mark in the heritable regulation of gene expression, with important roles in normal development and disease. Genomewide alterations in DNA methylation patterns are universal feature across cancers. Studies in the last few years have shown that similar alterations occur during various normal physiological processes, such as aging. Understanding mechanisms involved in DNA methylation alterations is critical for understanding cancer etiology. In this chapter, we discuss recent work on the nature of the genomic region-specific DNA methylation alterations, its functional implications, and the mechanisms underlying these alterations.

2543 Background: Immune checkpoint blockade (ICB) immunotherapy in some cases elicits striking patient responses, but its efficacy appears to be dependent on several incompletely understood factors. Most studies of ICB therapies in elderly patients have concluded that they received no reduced benefit or even increased benefit compared to the younger patients analyzed, despite the systemic age-related immune changes that might be expected to produce a less effective immune response, such as loss of the capacity to generate new naive T cells. To understand and apply these results, it is necessary to investigate the relationship of age and the immune tumor microenvironment. Methods: We apply bioinformatics methods to genomic, transcriptomic, and clinical data from 9,523 patients across 31 cancer types from TCGA, 15,557 patients with breast, colon, or head and neck cancers from Caris Life Sciences, and 37,961 patients across 8 cancer types collected by GENIE. From these data we apply multivariate linear models across and within individual tumor types to estimate age-related associations to tumor mutational burden (TMB), T cell receptor diversity (miTCR), differential gene expression (edgeR), pathway enrichment (mSigDB and fgsea), and immune cell type infiltration (Quantiseq and MIXTURE). Results: Our analysis of large-scale molecular and clinical databases associates patient age with changes in several major biomarkers of ICB response. Notably, a robust correlation between increased tumor mutational burden and age was found across three different large cohorts (TCGA, Caris Life Sciences, and GENIE) in most ICB-approved cancer types. In the TCGA data, TMB increased with age pan-cancer (p < 1x10-16) and in 7 of 9 ICB-approved cancer types. These associations were validated in the larger cohort of patient samples in GENIE, which demonstrated correlations between increased TMB levels and patient age in all eight ICB-approved cancer types assayed (Table), as well as in the Caris colorectal (q < 0.001) and breast (q < 0.001) cancer cohorts. Significant associations of age to other biomarkers of ICB response (checkpoint gene expression, immune infiltration, and immune related pathway signaling) will be presented. Conclusions: These results provide context for the efficacy of ICB in elderly patients, highlight potential biomarkers for the treatment of elderly patients with immunotherapies, and strongly suggest the value of large-scale prospective study of elderly cancer patients treated with ICB.[Table: see text]

Background: Tumor initiation and development occur not necessarily by genetic changes alone, but in the context of the epigenome and cell microenvironment. Growing evidence indicate that cancer risk factors, such as aging and inflammation, may primarily modulate the epigenome, causing predisposition to cancer development by driver mutations. Thus, there is a need to study and model epigenetic changes preceding and associating with cancer development. We have developed ex vivo models for investigating the evolution and roles of DNA methylation changes during normal cell divisions and how these epigenetic changes play critical roles in early stages of tumor development. Results: We showed that spontaneous epigenetic changes, specifically promoter-DNA hypermethylation, have a very important role in tumor initiation by oncogenic driver mutations. We addressed the precursor role of aging-like spontaneous promoter-DNA hypermethylation in initiating tumorigenesis. Using mouse colon-derived organoids, we show promoter hypermethylation spontaneously arises in cells mimicking the human “aging-like” phenotype. The silenced genes activate the Wnt pathway, causing a stemlike state and differentiation defects. These changes render aged organoids profoundly more sensitive than young ones to transformation by BrafV600E producing the typical human proximal BRAFV600E-driven colon adenocarcinomas characterized by extensive, abnormal gene promoter CGI methylation or the methylator phenotype (CIMP). Conversely, CRISPR-mediated simultaneous inactivation of a panel of the silenced genes markedly sensitized to BrafV600E-induced transformation. We showed that “spontaneous” promoter hypermethylation, arising in an “aging-like” scenario, sufficiently affects genes with roles in negative regulation of Wnt pathway. Inactivation of these genes leads to increased stem cell state and defects in differentiation. Conclusion: Our studies tightly link aging-like epigenetic abnormalities to intestinal cell fate changes and predisposition to oncogene-driven colon tumorigenesis. The organoid-based model opens up an opportunity to study mechanisms underlying epigenetic heterogeneity arising during normal cellular processes and their role in cancer predisposition. Citation Format: Yong Tao, Byunghak Kang, Daniel Petkovich, Steve Baylin, Hariharan Easwaran. Organoid models for deciphering roles of the evolving landscape of epigenetic heterogeneity during aging in cancer development [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A36.

Background Divergent findings regarding the prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) patients exist in current literature. We aim to review data from published studies in order to examine the association between CIMP and CRC prognosis. Materials and methods A comprehensive search for studies reporting disease-free survival (DFS), overall survival (OS), or cancer-specific mortality of CRC patients stratified by CIMP is carried out. Study findings are summarized descriptively and quantitatively, using adjusted hazard ratios (HRs) as summary statistics. Results Thirty-three studies reporting survival in 10 635 patients are included for review. Nineteen studies provide data suitable for meta-analysis. The definition of CIMP regarding gene panel, marker threshold, and laboratory method varies across studies. Pooled analysis shows that CIMP is significantly associated with shorter DFS (pooled HR estimate 1.45; 95% confidence interval (CI) 1.07–1.97, Q = 3.95, I2 = 0%) and OS (pooled HR estimate 1.43; 95% CI 1.18–1.73, Q = 4.03, I2 = 0%) among CRC patients irrespective of microsatellite instability (MSI) status. Subgroup analysis of microsatellite stable (MSS) CRC patients also shows significant association between shorter OS (pooled HR estimate 1.37; 95% CI 1.12–1.68, Q = 4.45, I2 = 33%) and CIMP. Seven studies have explored CIMP's value as a predictive factor on stage II and III CRC patient's DFS after receiving adjuvant 5-fluorouracil (5-FU) therapy: of these, four studies showed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) patients, one concluded to the contrary, and two found no significant correlation. Insufficient data was present for statistical synthesis of CIMP's predictive value among CRC patients receiving adjuvant 5-FU therapy. Conclusion CIMP is independently associated with significantly worse prognosis in CRC patients. However, CIMP's value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies.

The H3K4 demethylase KDM5B is amplified and overexpressed in luminal breast cancer, suggesting it might constitute a potential cancer therapy target. Here, we characterize, in breast cancer cells, the molecular effects of a recently developed small-molecule inhibitor of the KDM5 family of proteins (KDM5i), either alone or in combination with the DNA-demethylating agent 5-aza-2′-deoxycytidine (DAC). KDM5i treatment alone increased expression of a small number of genes, whereas combined treatment with DAC enhanced the effects of the latter for increasing expression of hundreds of DAC-responsive genes. ChIP-seq studies revealed that KDM5i resulted in the broadening of existing H3K4me3 peaks. Furthermore, cells treated with the drug combination exhibited increased promoter and gene body H3K4me3 occupancy at DAC-responsive genes compared with DAC alone. Importantly, treatment with either DAC or DAC+KDM5i induced a dramatic increase in H3K27ac at enhancers with an associated significant increase in target gene expression, suggesting a previously unappreciated effect of DAC on transcriptional regulation. KDM5i synergized with DAC to reduce the viability of luminal breast cancer cells in in vitro assays. Our study provides the first look into the molecular effects of a novel KDM5i compound and suggests that combinatorial inhibition along with DAC represents a new area to explore in translational epigenetics. Significance: This study offers a first look into the molecular effects of a novel KDM5 inhibitory compound, suggesting how its use in combination with DNA methylation inhibitors presents new opportunities to explore in translational cancer epigenetics. Cancer Res; 78(5); 1127–39. ©2017 AACR.

Cancer is a complex disease, driven by aberrant activity in numerous signaling pathways in even individual malignant cells. Epigenetic changes are critical mediators of these functional changes that drive and maintain the malignant phenotype. Changes in DNA methylation, histone acetylation and methylation, non-coding RNAs, post-translational modifications are all epigenetic drivers in cancer, independent of changes in the DNA sequence. These epigenetic alterations, once thought to be crucial only for the malignant phenotype maintenance, are now recognized as critical also for disrupting essential pathways that protect the cells from uncontrolled growth, longer survival and establishment in distant sites from the original tissue. In this review, we focus on DNA methylation and chromatin structure in cancer. While associated with cancer, the precise functional role of these alterations is an area of active research using emerging high-throughput approaches and bioinformatics analysis tools. Therefore, this review describes these high-throughput measurement technologies, public domain databases for high-throughput epigenetic data in tumors and model systems, and bioinformatics algorithms for their analysis. Advances in bioinformatics data integration techniques that combine these epigenetic data with genomics data are essential to infer the function of specific epigenetic alterations in cancer, and are therefore also a focus of this review. Future studies using these emerging technologies will elucidate how alterations in the cancer epigenome cooperate with genetic aberrations to cause tumorigenesis initiation and progression. This deeper understanding is essential to future studies that will precisely infer patients’ prognosis and select patients who will be responsive to emerging epigenetic therapies.

// Frank P. Vendetti 1 , Michael Topper 1 , Peng Huang 1 , Irina Dobromilskaya 1 , Hariharan Easwaran 1 , John Wrangle 1 , Stephen B. Baylin 1 , J. T. Poirier 2 , Charles M. Rudin 2 1 The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD 2 Memorial Sloan Kettering Cancer Center, NY 10065, New York Correspondence to: Charles M. Rudin, e-mail: rudinc@mskcc.org Keywords: epigenetic, azacitidine, entinostat, priming, chemosensitivity, non-small cell lung cancer (NSCLC) Received: July 25, 2014 Accepted: November 04, 2014 Published: November 25, 2014 ABSTRACT Recent clinical data in lung cancer suggests that epigenetically targeted therapy may selectively enhance chemotherapeutic sensitivity. There have been few if any studies rigorously evaluating this hypothesized priming effect. Here we describe a series of investigations testing whether epigenetic priming with azacitidine and entinostat increases sensitivity of NSCLC to cytotoxic agents. We noted no differences in chemosensitivity following treatment with epigenetic therapy in in vitro assays of viability and colony growth. Using cell line and patientderived xenograft (PDX) models, we also observed no change in responsiveness to cisplatin in vivo . In select models, we noted differential responses to irinotecan treatment in vivo . In vitro epigenetic therapy prior to tumor implantation abrogated response of H460 xenografts to irinotecan. Conversely, in vitro epigenetic therapy appeared to sensitize A549 xenografts (tumor growth inhibition 51%, vs. 22% in mock-pretreated control). In vivo epigenetic therapy enhanced the response of adenocarcinoma PDX to irinotecan. Taken together, these data do not support broadly applicable epigenetic priming in NSCLC. Priming effects may be context-specific, dependent on both tumor and host factors. Further preclinical study is necessary to determine whether, and in which contexts, priming with epigenetic therapy has potential to enhance chemotherapeutic efficacy in NSCLC patients.

Reversing the DNA methylation abnormalities by targeting the maintenance DNA methylation machinery represents a sought-after therapy paradigm in both liquid and solid tumors. UHRF1, a multi-domain protein with both chromatin reader and writer functions, is essential for targeting DNA methyltransferase 1 (DNMT1) to replicating DNA to maintain DNA methylation in both normal and cancer cells. Dysregulation of the DNMT1-UHRF1 axis is being increasingly linked to malignant transformation and progression across cancer types. Our recent study shows that UHRF1 depletion, alone or in combination with low doses of a DNMT inhibitor, effectively induces DNA demethylation and tumor suppressor genes (TSGs) reactivation. These findings collectively suggest UHRF1 represents a promising target for developing next-generation DNA demethylation agents for cancer therapy. Understanding the collective contribution of UHRF1’s domains for maintenance methylation is essential for the major translational goal of blocking maintenance of abnormal DNA methylation in established cancers and throughout cancer initiation and progression. While the UHRF1 domains responsible for de novo or re-methylation have been identified, the domain requirements specifically for maintaining normal and cancer-specific DNA methylation are poorly characterized. Herein, by developing a carefully timed genetic complementation assay, we systematically interrogate the roles for each major domain of UHRF1 for maintaining genome-wide DNA methylation in human colorectal cancer (CRC) cells. Distinct from the domain required for establishing methylation, we find that UHRF1 histone-binding and hemimethylated DNA reader domains, but not E3 ligase activity, are essential for maintaining cancer-specific DNA methylation in both HCT116 and RKO cells. Disrupting either one of the essential domains phenocopies UHRF1 depletion for global DNA demethylation and reactivation of epigenetically silenced TSGs. Supporting the essential roles of abnormal DNA methylation in sustaining the key oncogenic properties of CRC cells, we further show genetic perturbation of either histone- or hemimethylated DNA-binding activities of UHRF1 dramatically impairs CRC proliferation, invasion, and metastasis in vitro and in vivo. Moreover, for eight UHRF1 chromatin-reader domain regulated TSGs, we reveal a strong negative correlation between high UHRF1 expression, promoter hypermethylation, and their low expression with disease progression and overall survival in CRC patients in both TCGA and two independent CRC cohorts (n=363 and 390, respectively). Taken together, our data identify important differences from domains dominant for de novo DNA methylation, and provide important implications for the oncogenic functions of UHRF1 in CRC, and for credentialing UHRF1 as a desirable target for cancer drug development and means to personalize this. Citation Format: Xiangqian Kong, Jie Chen, Wenbing Xie, Stephen M. Brown, Yi Cai, Kaichun Wu, Daiming Fan, Yongzhan Nie, Yong Tao, Ray-Whay Chiu Yen, Hariharan Easwaran, Michael J. Topper, Scott B. Rothbart, Limin Xia, Stephen B. Baylin. Defining UHRF1 domains that support maintenance of human colon cancer DNA methylation and tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 947.

Background Bivalent chromatin refers to overlapping regions containing activating histone H3 Lys4 trimethylation (H3K4me3) and inactivating H3K27me3 marks. Existence of such bivalent marks on the same nucleosome has only recently been suggested. Previous genome-wide efforts to characterize bivalent chromatin have focused primarily on individual marks to define overlapping zones of bivalency rather than mapping positions of truly bivalent mononucleosomes. Results Here, we developed an efficacious sequential ChIP technique for examining global positioning of individual bivalent nucleosomes. Using next generation sequencing approaches we show that although individual H3K4me3 and H3K27me3 marks overlap in broad zones, bivalent nucleosomes are focally enriched in the vicinity of the transcription start site (TSS). These seem to occupy the H2A.Z nucleosome positions previously described as salt-labile nucleosomes, and are correlated with low gene expression. Although the enrichment profiles of bivalent nucleosomes show a clear dependency on CpG island content, they demonstrate a stark anti-correlation with methylation status. Conclusions We show that regional overlap of H3K4me3 and H3K27me3 chromatin tend to be upstream to the TSS, while bivalent nucleosomes with both marks are mainly promoter proximal near the TSS of CpG island-containing genes with poised/low expression. We discuss the implications of the focal enrichment of bivalent nucleosomes around the TSS on the poised chromatin state of promoters in stem cells. Electronic supplementary material The online version of this article (doi:10.1186/s12920-016-0221-6) contains supplementary material, which is available to authorized users.

We define how chronic cigarette smoke-induced time-dependent epigenetic alterations can sensitize human bronchial epithelial cells for transformation by a single oncogene. The smoke-induced chromatin changes include initial repressive polycomb marking of genes, later manifesting abnormal DNA methylation by 10 months. At this time, cells exhibit epithelial-to-mesenchymal changes, anchorage-independent growth, and upregulated RAS/MAPK signaling with silencing of hypermethylated genes, which normally inhibit these pathways and are associated with smoking-related non-small cell lung cancer. These cells, in the absence of any driver gene mutations, now transform by introducing a single KRAS mutation and form adenosquamous lung carcinomas in mice. Thus, epigenetic abnormalities may prime for changing oncogene senescence to addiction for a single key oncogene involved in lung cancer initiation.