Search

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

Differential H3K4me3 enrichment profiling after KDM5i, DAC, or both

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

KDM5i alone and in combination with DAC upregulates cancer hallmark & immunomodulatory pathways

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.

KDM5i and DAC do not synergize in MCF10A, nor alter cell cycle in MCF-7

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.

KDM5i increases H3K4 trimethylation alone and in combination with DAC.

DAC & KDM5i induced expression changes

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

Legends for Supplementary Figures and Tables

PDF file - 108KB

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.

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.

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.

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.

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.

Aberrant promoter DNA-hypermethylation and repressive chromatin constitutes a frequent mechanism of gene inactivation in cancer. There is great interest in dissecting the mechanisms underlying this abnormal silencing. Studies have shown changes in the nuclear organization of chromatin in tumor cells as well as the association of aberrant methylation with long-range silencing of neighboring genes. Furthermore, certain tumors show a high incidence of promoter methylation termed as the CpG island methylator phenotype. Here, we have analyzed the role of nuclear chromatin architecture for genes in hypermethylated inactive versus nonmethylated active states and its relation with long-range silencing and CpG island methylator phenotype. Using combined immunostaining for active/repressive chromatin marks and fluorescence in situ hybridization in colorectal cancer cell lines, we show that aberrant silencing of these genes occurs without requirement for their being positioned at heterochromatic domains. Importantly, hypermethylation, even when associated with long-range epigenetic silencing of neighboring genes, occurs independent of their euchromatic or heterochromatic location. Together, these results indicate that, in cancer, extensive changes around promoter chromatin of individual genes or gene clusters could potentially occur locally without preference for nuclear position and/or causing repositioning. These findings have important implications for understanding relationships between nuclear organization and gene expression patterns in cancer. Cancer Res; 70(20); 8015–24. ©2010 AACR.

Epigenetic silencing of genes in association with aberrant promoter DNA hypermethylation has emerged as a significant mechanism in the development of human cancers. Such genes are also often targets of the polycomb group repressive complexes in embryonic cells. The polycomb repressive complex 2 (PRC2) has been best studied in this regard. We now examine a link between PRC1 and cancer-specific gene silencing. Here, we show a novel and direct association between a constituent of the PRC1 complex, CBX7, with gene repression and promoter DNA hypermethylation of genes frequently silenced in cancer. CBX7 is able to complex with DNA methyltransferase (DNMT) enzymes, leading us to explore a role for CBX7 in maintenance and initiation of gene silencing. Knockdown of CBX7 was unable to relieve suppression of deeply silenced genes in cancer cells; however, in embryonal carcinoma (EC) cells, CBX7 can initiate stable repression of genes that are frequently silenced in adult cancers. Furthermore, we are able to observe assembly of DNMTs at CBX7 target gene promoters. Sustained expression of CBX7 in EC cells confers a growth advantage and resistance to retinoic acid–induced differentiation. In this setting, especially, there is increased promoter DNA hypermethylation for many genes by analysis of specific genes, as well as through epigenomic studies. Our results allow us to propose a potential mechanism through assembly of novel repressive complexes, by which the polycomb component of PRC1 can promote the initiation of epigenetic changes involving abnormal DNA hypermethylation of genes frequently silenced in adult cancers. [Cancer Res 2009;69(15):6322–30]

SRY-box containing gene 17 (Sox17) is a member of the high mobility group (HMG) transcription factor superfamily, which plays critical roles in the regulation of development and stem/precursor cell function, at least partly through repression of Wnt pathway activity. Modulators controlling aberrant Wnt signaling activation are frequently disrupted in human cancers through complementary effects of epigenetic and genetic changes. Our recent global analysis of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that SOX17 gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. Here, we report that CpG island methylation-dependent silencing of SOX17 occurs in 100% of CRC cell lines, 86% of colorectal adenomas, 100% of stage I and II CRC, 89% of stage III CRC, 89% of primary esophageal cancer, and 50% of non–small cell lung cancer. Overexpression of SOX17 in HCT116 CRC cells inhibits colony growth and β-catenin/T-cell factor–dependent transcription. Structure-based deletion analysis further shows the presence of a Wnt signaling repression domain in the SOX17 HMG box. Together, our studies suggest that SOX17 is a negative modulator of canonical Wnt signaling, and that SOX17 silencing due to promoter hypermethylation is an early event during tumorigenesis and may contribute to aberrant activation of Wnt signaling in CRC. [Cancer Res 2008;68(8):2764–72]

Herein, we report a model for how BRAF mutations, in contrast to KRAS mutations, specifically associate with and induce, in mouse colon organoids without a microenvironment, the phenotype of human sporadic right-sided colon adenocarcinomas (COAD). The human BRAF mutant COAD are particularly distinguished by having an increased incidence of promoter CpG island methylation, termed CpG island methylator phenotype (CIMP)-High. This latter phenotype contrasts with the majority of the COAD which are classified in the CIMP-low to intermediate groups and have mostly KRAS mutations. To understand how these mutations influence tumor evolution and the methylation landscape, we modeled the early carcinogenesis of colorectal cancer by inducing BRAFV600E and KRASG12D mutations individually in 3D organoids prepared from mouse proximal colon. The induction of BRAFV600E mutation, but not KRASG12D mutation, showed various features of progressive transformation. In vitro induction of BRAFV600E drove the organoids to derive cystic changes in morphology and promoted adoption of a stem cell niche independency characterized by growth in the absence of added stem cell niche factors including Wnt3a, R-Spondin, and Noggin. This independency is not due to increased secretion of niche factors by the Paneth-like cells in BRAFV600E mutant organoids. In addition, BRAFV600E organoids showed dysplastic changes such as high nuclear to cytoplasmic ratio and abnormal budding. The most exciting finding is that induction of BRAFV600E mutation, but not KRASG12D mutation, induced complete transformation of the organoids forming xenograft tumors in NOD/SCID mice. The tumors exhibit histological characteristics of human mucinous adenocarcinoma, which is the tumor type highly associated with BRAFV600E mutation in human COAD. Gene expression analyses revealed up-regulation of intestinal stem cell signature genes and down-regulation of genes related to intestinal differentiation in BRAFV600E organoids. In addition, BRAFV600E organoids showed increased expression levels of Wnt pathway target genes indicating an enhanced and sustained Wnt-signaling. Analyses of CpG-island methylation in a panel of genes showed increased CpG-island methylation only in the BRAFV600E mutant organoids. In conclusion, in mouse colon organoids, BRAFV600E drives a human right-sided COAD phenotype with adoption of a stem cell niche independency, activation of the Wnt pathway, and induction of CpG island methylation. Citation Format: Byunghak Kang, Julie In, Nicholas Zachos, David Huso, Shinji Maegawa, Jean-Pierre Issa, Hariharan Easwaran, Stephen B. Baylin. Oncogenic BRAFV600E drives stem cell niche factors-independent growth and tumorigenic transformation in colon organoids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4273.

Herein, we define a model for how cigarette smoke and chronic inflammation induce human lung cancer via evolution of a co-ordinated pattern of progressive cancer-associated epigenetic abnormalities which prime cells for addiction to a single key genetic alteration, KRAS mutation. Non-clonogenic, non-tumorigenic, epigenetically stable human bronchial epithelial cells (HBEC) were exposed to cigarette smoke condensate (CSC) for 15 months. Earlier studies have established a requirement for the simultaneous disruption of three oncogenes to fully transform these cells. Genome-wide DNA methylation, expression, chromatin changes, as well as binding to chromatin of key epigenetic regulators and cell phenotypic features were examined over time in exposed versus non-exposed cells. CSC exposure acutely causes, within 10 days, a change we have previously associated with DNA damage, tightening of DNA methyltransferase 1 (DNMT1) and EZH2 to chromatin. While the EZH2 binding decreases with prolonged exposure, DNMT1 remains tightly bound to chromatin. Chronic exposure causes progressive, but stochastically variable, global DNA methylation changes which begin by six months and progress over the time course of the study to include hypermethylation of gene promoters which are frequent in human lung cancer. ChIP-seq analyses reveal, preceding the above methylation changes, that promoters of such methylated genes have an initial recruitment of EZH2, which begins at 10 days and then decreases with time. In contrast, recruitment of EZH2 increases with time and remains dominant for these same genes in the non-exposed controls. Following 10 months of exposure, CSC treated cells begin to clone in soft agar, often a feature of transformation, yet do not form tumors in immunodeficient mice. At this time point, gene expression studies show the top signaling pathway change is strong activation of MAP-kinase and KRAS pathways. Yet genome-wide, exome sequencing reveals no known lung cancer driver gene mutations. Remarkably, overexpression of mutant KRAS alone now markedly enlarges the soft agar colonies, and the cells are now fully transformed and form tumors in mice. Our study reveals, in a chronic cigarette exposure model relevant to the time course for evolution of KRAS mutant human lung adenocarcinoma, a key initial role for smoking induced epigenetic changes which facilitate addiction to the oncogene. Citation Format: Michelle P. Vaz, Stephen Y. Hwang, Ashwini Patil, Jillian Phallen, Lauren Murphy, Cynthia A. Zahnow, Victor E. Velculescu, Hariharan Easwaran, Stephen B. Baylin. Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2773.

Chronic inflammation has been shown to play a significant role in mediating early epigenetic changes that contribute to the development of cancer. Prolonged exposure to inflammatory stimuli such as cigarette smoke causes genetic and epigenetic alterations in the lung, thereby contributing to the development of lung cancer. However, the full timing, and precise mechanisms leading to the evolution of these epigenetic alterations have not been well defined. The goal of this study is to model the progressive chromatin and DNA methylation alterations which associate with key gene expression changes during the progression to lung cancer. Cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in medium with or without cigarette smoke condensate (CSC) for 15 months and genome-wide DNA methylation, expression, chromatin changes plus binding to chromatin of key epigenetic regulators and cell phenotypic features are examined over time. At each time point, non-exposed cultured cells are also examined to match their “aging” on plastic changes to the CSC-specific changes. By 10 months, exposure to CSC induces distinct phenotypic changes in which cells become less translucent and have features of EMT. Preceding these changes, exposure acutely causes, within 10 days, DNA methyltransferase 1 (DNMT1) and EZH2 to become tightly bound to chromatin. Over the first month of exposure, EZH2 binding decreases while DNMT1 remains tightly bound to chromatin after 15 months. Progressive, but stochastically variable global DNA methylation changes begin by 6 months and are observed over the entire time course of the study. As early as 6 months, two types of increases in DNA methylation emerge at unmethylated CpG-island promoters, those specific to CSC treated cells and those which evolve in the “aging” cells. The latter changes often increase further with time in the CSC exposed cells. The early CSC-specific methylated genes are biased to homeobox genes, transcription factors and developmental genes. Increased methylation of key tumor suppressor genes is observed beginning at 10 months of exposure at a time when the CSC exposed cells begin to clone in soft agar. This change is suggestive of transformation but the cells do not yet form tumors when explanted into immunodeficient mice. The genes that get methylated at this time point are biased to those regulating the Hedgehog, Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. Gene expression studies show that activation of MAP-kinase and KRAS pathway dominate by the time point the above transformation changes have ensued. The CSC specific DNA methylation changes seem to track best in TCGA with patterns for lung adenocarcinoma, although there is definite overlap with those for squamous cell lung cancer. ChIP-seq analyses reveal important chromatin changes that precede the above methylation changes. Promoters of genes methylated by CSC during the course of the treatment show an initial recruitment of EZH2 at their promoters following CSC exposure, which begins at 10 days and then decreases with time. However, in the untreated “aging” cells, recruitment of EZH2 increases with time and remains dominant for the genes that become DNA hypermethylated with CSC exposure, which have limited EZH2 binding. We thus define a model for lung cancer evolution due to CSC exposure wherein key molecular changes appear, and switch regulatory features with time, to evolve a more co-ordinated pattern of cancer-associated epigenetic features which may help drive inflammation induced pulmonary carcinogenesis. Citation Format: Michelle Vaz, Stephen Y. Hwang, Ashwini Patil, Hariharan Easwaran, Stephen B. Baylin. Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to early steps of transformation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B18.

Leukemia often displays substantial intratumor heterogeneity, comprising subpopulations of cells with different self-renewing and leukemia initiation capacities. Epigenetic mechanisms have been implicated in stem cell fate regulation during normal hematopoiesis. Although aberrant epigenetic changes have been well documented in hematological malignancies, the role epigenetic mechanisms play in governing stem-like properties, such as leukemia initiating capacity, and possible cell fate transitions between stem-like and non-stem-like leukemia subpopulations is unclear. Using next-generation sequencing and microarray technologies, we conducted an integrated analysis of DNA methylation, histone modifications and transcriptional profiles to investigate the epigenomic differences between experimentally characterized CD34-positive (CD34pos) leukemia-initiating and CD34-negative (CD34neg) non-leukemia-initiating subpopulations in a human acute myelogenous leukemia (AML) cell line. We demonstrated that multiple normal and leukemic stem cell gene signatures are significantly enriched in CD34pos leukemia initiating cells as opposed to CD34neg AML cells. We also observed concordant chromatin state transitions at the promoters of stem cell signature genes. The stem-like state of CD34pos leukemia initiating cells is also characterized by a number of genes with bivalent histone modifications at their promoters. Many genes undergoing bivalent domain resolution and transcriptional activation in CD34neg AML cells are involved in differentiation-related pathways. Most importantly, epigenomic analysis showed that treatment with epigenetic modifying agents, such as 5-aza-2'-deoxycytidine, appeared to facilitate cell fate transitions, induce marked changes in chromatin configurations, and reverse the enrichments of stem cell signatures in CD34pos leukemia-initiating cells. This provides an important molecular basis for the diminished in vitro clonogenecity and in vivo leukemogenecity of AML cells after 5-aza-2'-deoxycytidine treatment in our previous studies. Our data supports the important role of epigenetic mechanisms in underpinning cell fate and functional properties of leukemia subpopulations. The epigenetic regulation of leukemic stem-like properties might differ from that of normal hematopoietic stem cell state since the same 5-aza-2'-deoxycytidine treatment did not seem to negatively affect clonogenecity of normal hematopoietic cells. Understanding the dynamics of epigenetic state transitions between leukemia subpopulations may offer novel targets for leukemia treatment, and facilitate development of epigenetic therapy targeting leukemia initiating cells. Citation Format: Hsing-Chen Tsai, Hariharan Easwaran, Stephen B. Baylin. Epigenetic regulation of stem cell fate in leukemic subpopulations. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B27.

DNA methylation, one of the key epigenetic modifications in the mammalian genome, plays fundamental roles in development and disease. Although DNA methylation and DNA methyltransferases (DNMTs) have been studied intensively, our knowledge about DNA demethylation is just starting to emerge. The Ten-eleven translocation (TET) family of proteins are newly identified methylcytosine dioxygenases/demethylases that can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and promote DNA demethylation either passively through DNA replication, or actively via thymine DNA glycosylase (TDG)-mediated base excision repair. TET-mediated DNA demethylation has also been shown to play important roles in diverse biological processes and tumorigenesis. Human TET2 is frequently mutated in myeloid malignancies, while loss of 5hmC and TETs expressions have been broadly observed in many other cancer types. Recent studies suggested that TETs may be regulated by microRNAs and calpain proteases, but, overall, the regulation of TET activities is not well outlined. Here we show, in human ovarian cancer cells, that TET2 activity may be significantly modulated by post-translational modifications. TET2 is acetylated by p300/CBP at K110/111, and this acetylation can be reversed by HDAC1/2 and SIRT1/2 deacetylases. Acetylation increases TET2 stability by protecting it from ubiquitination and proteasome-mediated degradation. Interestingly, DNMT1 physically associates with and stabilizes TET2 protein, and a TET2 acetylation deficient mutant (2KR) disrupts this association, indicating that acetylation of TET2 may stimulate DNMT1 binding which prevents TET2 from degradation. In addition, we show that acetylation increases TET2 enzymatic activity. Treatment of cells with HDAC inhibitors TSA/NAM, or knocking down HDAC1/2, significantly increases global 5hmC levels. Consistent with these latter observations, the 2KR mutant almost completely abolishes TET2 catalytic activity, and this mutant protein is less efficient in forming dimer with wild type TET2. Thus acetylation events may increase the activity of TET2 through enhanced dimerization of the protein. Taken together, our study reveals a novel acetylation-dependent regulatory mechanism controlling the function of TET2. Citation Format: Yang Zhang, Yi Cai, Wenbing Xie, Hariharan Easwaran, Stephen B. Baylin. Acetylation regulates TET2 stability and enzymatic activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2864. doi:10.1158/1538-7445.AM2015-2864

DNA methylation is one of several epigenetic mechanisms used by cells to control gene expression. The de novo methyltransferases, DNMT3a and DNMT3b, establish new methylation patterns during early development. DNMT1, the maintenance methyltransferase, ensures propagation of hemi-methylated DNA. Human cancers are characterized by aberrant DNA methylation patterns, including global hypomethylation and hypermethylation of promoters at tumor suppressor genes. This latter change leads to transcriptional silencing. Experimentally, DNMT1 is capable of transcriptional repression without its methyltransferase activity, partially through interactions with histone-modifying enzymes. We hypothesized DNMT3B may also be capable of modulating gene expression independently of its methyltransferase activity, by recruiting repressive epigenetic proteins to the promoters of endogenous DNMT3B targets. Using an isogenic colorectal cancer cell culture model, we investigated the functional consequences of the removal and re-introduction of DNMT3B on target gene expression, methylation, and chromatin status. A stable genetic knock out cell line, transient shRNA knock down, and pharmacologic inhibition resulted in upregulation of a subset of candidate DNMT3B targets identified by gene expression arrays and validated by quantitative RT-PCR. Interestingly, reintroduction of both wild-type and catalytically dead mutant DNMT3B in the knockout cells transcriptionally repressed the same loci. Infinium 450K arrays were performed to assess DNA methylation status. As expected, the loss of DNMT3B did not result in significant changes in global methylation at TSS, CpG islands, shores, shelves, and UTRs. However, in selected of the above targets, locus-specific demethylation was identified. Overexpression of both wild-type and catalytically dead mutant DNMT3B failed to restore local methylation, in concert with transcriptional repression of these loci, further suggesting an alternative regulatory mechanism to explain transcriptional changes. Interactions of DNMT3B with other repressive proteins could account for the above transcriptional repression. ChIP studies revealed LSD1, a histone demethylase that represses transcription, is lost in the 3B knockout cells but is recruited back to the promoters of repressed DNMT3B target genes with DNMT3B insertion. These data strongly suggest DNMT3B possesses a non-canonical function as a scaffold protein for transcriptional repressors. Citation Format: Khadijah A. Mitchell, Hariharan Easwaran, Stephen B. Baylin. DNMT3B (a de novo DNA methyltransferase) epigenetically regulates gene expression, independent of its DNA methyltransferase activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4779. doi:10.1158/1538-7445.AM2014-4779

Epigenetic alterations driving carcinogenesis and cancer progression can be specifically targeted by the demethylating agent azacitidine (Aza) and the histone deacetylase inhibitor entinostat. While this treatment combination has been effective in a limited number of patients (pts) with treatment-refractory non-small cell lung cancer (NSCLC), we observed clinical benefit in 5 of 5 patients who received immunotherapy with PD-1/PD-L1 pathway blockade immediately following epigenetic therapy. Three of 5 pts developed partial tumor regressions (RECIST criteria, duration 10+ to 20+ mo.) and 2 pts had stable disease ≥6 mo. This compares to the objective response + SD rates of NSCLC to monotherapy with anti-PD-1 (18% + 5%) or anti-PD-L1 (10% + 12%). To understand how epigenetic therapy may synergize with blockade of the immunosuppressive PD-1 pathway, we used genome wide methylation and expression profiling on 8 NSCLC cell lines treated with low dose Aza. We discovered complex immunomodulatory effects of Aza with up-regulation of diverse immune related pathways including Jun/Jnk, NFKB, viral defense, type I interferon signaling, the inflammasome, antigen processing and presentation and immune evasion including up-regulation of PD-L1 expression. Multiple cancer-testes antigens were also up-regulated, thereby conferring de novo antigenicity. Supporting the idea that Aza acts specifically through inhibition and degradation of DNA methyltransferase proteins, colon cancer cells genetically haplo-insufficient for DNMT1 and devoid of DNMT3b mirror the immunomodulatory effects of Aza. Upstream events potentially controlling these pathways were defined, and prominent among them was up-regulation of the transcription factor, interferon regulatory factor 7 (IRF7), a DNA hypermethylated gene. These data were used to query hundreds of primary NSCLC samples from the Cancer Genome Atlas project (TCGA). A low basal expression signature of interferon pathway related genes was significantly associated with low IRF7 expression and promoter methylation in squamous tumors. Another hypermethylated transcription factor, PITX1, which inhibits a subset of type I interferon signaling genes, tracked with non-squamous cancers. Together, these findings support a model in which epigenetic modulation activates innate and adaptive immune responses within the tumor microenvironment together with induction of counter-regulatory immune checkpoint ligands which can be therapeutically blocked with antibodies. Based on these findings, a clinical trial testing the efficacy of DNMT and HDAC inhibition combined with PD-1 pathway blockade is under development. This work will form the basis for an immune-classification of NSCLC, as well as biomarker discovery for a novel therapeutic paradigm combining epigenetic and immunotherapy with potentially synergistic activity against the world's most deadly malignancy. Supported by Stand Up to Cancer. Citation Format: John Wrangle, Wei Wang, Alexander Koch, Hariharan Easwaran, Helai Mohammad, Princy Parsana, Frank Vendetti, Kristen Rodgers, Xiaoyu Pan, Kirsten Harbom, Cynthia Zahnow, Janis Taube, Julie Brahmer, Peter Jones, Suzanne Topalian, Charles Rudin, Malcolm Brock, Drew Pardoll, Stephen Baylin. Epigenetic therapy and sensitization of lung cancer to immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2013-4619

A key question in cancer epigenetics is how cancers of multiple types simultaneously acquire global losses and gains of DNA methylation. The increases often occur in promoter CpG islands and can be associated with transcriptional inactivation of key tumor suppressor genes. Here we demonstrate a molecular response to oxidative damage, a key factor in cancer risk states, which could help explain the gains and losses of DNA methylation that occur during tumor progression. Within 30 minutes of exposing cells to the reactive oxygen species, hydrogen peroxide (H2O2), the major maintenance enzyme for DNA methylation, DNMT1, becomes notably more tightly bound to chromatin. Concomitantly, DNMT1 targets SIRT1 to chromatin and both proteins become part of a large multi-protein complex(es) which also contains the de novo DNA methyltransferase, DNMT3B. This oxidative damage-induced complex also contains specific polycomb group members, which, in the context of SIRT1, define the Polycomb Repressive Complex 4 (PRC4), previously found only in embryonic and adult stem cells and cancer cells. By examining chromatin on a genome-wide scale, we demonstrate that H2O2 treatment preferentially damages GC-rich regions of DNA. This damage causes a shift in localization of the above complex(es) from non-GC-rich to GC-rich regions of the genome including promoter CpG islands of which some are DNA hypermethylated in cancer cells. Additionally, this treatment causes a decrease in nascent RNA transcript levels of these genes that have oxidative damage-induced enrichment of the silencing complex in their CpG island-containing promoters. Thus, oxidative damage induces formation and localization of a silencing complex that may in part explain cancer specific aberrant losses and gains in DNA methylation associated with gene transcriptional silencing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-185. doi:10.1158/1538-7445.AM2011-LB-185

It has been firmly established that cancer gene expression partly mimics the gene expression signature in embryonic stem cells (ESC). Since most cancers arise in adult stem or progenitor cells, rather than ESC, we now compare cancer chromatin to both embryonic and adult cell renewal systems to understand the relationships between DNA hypermethylation, gene expression, and chromatin states. DNA hypermethylation at CpG island promoters of hundreds of genes, including classic tumor suppressors, is a major modulator of gene expression in human cancers. Past studies suggest ∼ 50% of these are frequently marked by polycomb complex (PcG) transcriptional repressors, but not DNA methylation, in embryonic stem cells (ESC). In ESC, PcG occupancy is predominantly in the context of “bivalent chromatin”, wherein the active transcription mark H3K4me3 and the repressive PcG mark H3K27me3 are simultaneously present. Genes so marked are in a low, but poised, transcription state important for stemness and self-renewal, characteristics shared with tumor cells. Using whole genome ChIP-seq and DNA methylation arrays, we find between 70 to 80% of genes with DNA hypermethylation in cancer have bivalent chromatin not only in ESC, but also in adult stem cells. For these genes, there appears to be an epigenetic switch in cancer wherein bivalent chromatin is replaced by DNA hypermethylation resulting in tighter repression of gene expression. Many of these DNA methylated cancer genes are constituents of the recently proposed “PRC module” of the “ESC cancer signature.” Our data suggest a “DNA methylation module” that recapitulates the “stem cell signature” and that DNA hypermethylation may be a key mechanism that confers cell-renewal and stemness to tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-174. doi:10.1158/1538-7445.AM2011-LB-174