Abstract Background: Colorectal cancer (CRC) arises from both genetic and epigenetic alterations. Epidemiological and animal studies suggest early-life exposures, including gut microbiota changes during weaning, can influence long-term CRC risk. However, the mechanisms linking early microbial interactions to durable disease protection remain unclear. We hypothesize that early microbial exposure epigenetically programs long-lived intestinal stem cells (ISCs), shaping immune regulation and disease susceptibility. Methods: Lgr5-GFP reporter mice were used to isolate colonic ISCs and intestinal epithelial cells (IECs) under specific-pathogen-free (SPF) and germ-free (GF) conditions. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing were performed to assess microbiota-associated transcriptional changes mediated by DNA methylation from weaning into adulthood. Gut microbe transplant (GMT) experiments defined the critical developmental window during which ISC epigenetics are most sensitive to microbial influences. A maternal low-dose penicillin (LDP) model combined with shotgun metagenomics identified specific bacterial taxa contributing to epigenetic effects. Dextran sulfate sodium (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced CRC models were used to evaluate long-term consequences. Results: We identified 683 differentially methylated regions (DMRs) that were persistently associated with the microbiome in both adult ISCs and IECs. Among these, 51% were located in enhancers, and the majority DMRs (79%) exhibited loss of methylation under SPF conditions. The hypomethylated genes were enriched for immune and host defense functions, including MHC class II genes (Cd74, H2-Aa, H2-Eb1, and Ciita). Loss of methylation occurred post-weaning and correlated with increased gene expression. GMT experiments demonstrated the post-weaning as the optimal window to restore methylation patterns, compared to adolescence or adulthood. Mechanistically, a transient IFN-γ burst during weaning drove epigenetic reprogramming via the IFN-γ-STAT3-TET3 axis. In addition, microbiota-derived metabolites, including short-chain fatty acids (SCFAs), α-ketoglutarate (α-KG), and methionine-γ-lyase-dependent products, reinforced the establishment of proper methylation patterns. Early-life LDP exposure reduced Gram-positive bacterial abundance, altered IFN-γ expression, disrupted MHC-II epigenetics, and increased susceptibility to colitis and CRC. Conclusion: Early-life microbial and immune signals establish durable epigenetic programs that protect against CRC. Timed microbial interventions may provide long-lasting protection against adult-onset diseases. Citation Format: Li Yang, Shirui Zhou, Xiaomin Chen, Fabiola Gutierrez, Stephanie Fowler, Lanjing Zhang, Julia M. Salamat, Karen Riggins, Jiejun Shi, Lanlan Shen. Early-life gut microbiota programs intestinal stem cell epigenetics to protect against colon cancer later in life [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1955.

During weaning, the transition to solid food diversifies the gut microbiome, triggering a programmed immune response critical for long-lasting mucosal immunity. Previous work showed that the gut microbiome mediates epigenetic development in intestinal stem cells (ISCs) during suckling, but what happens during weaning is unclear. Here, genome-wide profiling revealed that weaning-driven microbiome changes shape the DNA methylome and transcriptome of murine ISCs in an IFNγ-dependent manner. Specifically, we observe demethylation of enhancer elements essential for MHC class II genes, which results in a transcriptional memory that persists through differentiation into adulthood. IFNγ blockade, or low-dose penicillin to target Gram-positive bacteria, in early life impaired microbiome-mediated epigenetic control and mucosal immunity, and exacerbated colitis. Murine organoids primed with IFNγ showed rapid, amplified transcriptional responses upon secondary stimulations. These findings reveal that early-life events alter the gut microbiome and these changes reprogramme ISC epigenetic memory to shape mucosal immunity.

Abstract Background: Lung cancer is a leading cause of cancer death worldwide; however, current targeted therapies have limited efficacy for most patients. p16 epimutation, characterized by epigenetic silencing of p16 by promoter DNA hypermethylation, is common in lung cancer. Elucidating how p16 epimutation drives lung tumorigenesis may uncover new therapeutic opportunities. Methods: We developed a mouse model combining the conditional Cre-inducible KrasG12D-mutation with epigenetically engineered p16 epimutation and conducted time-course study to analyze the tumor phenotype and survival. Organoids derived from normal and tumorous lung tissue were used to assess the role of p16 epimutation in tumor initiation and maintenance. Moreover, spatial transcriptomic analysis was used to characterize tumor heterogeneity and define distinct cell populations. To test the therapeutic potential of reversing p16 epimutation, we evaluated the efficacy of hypomethylating agents 5-Aza-2’-Deoxycytidine (DAC) and GSK3685032 on p16 reactivation in organoid and in vivo. Furthermore, we developed mice enabling conditional, inducible and site-specific DNA demethylation using a CRISPR-dCas9-Tet1 based epigenetic editing system. We assessed the effect of targeted p16 promoter demethylation on tumor progression in vivo. Results: Mice with combined Kras-mutation and p16 epimutation developed malignant tumors more rapidly and had significantly shorter survival than mice with Kras-mutation only (Median survival: 161 days vs. 223 days, n=50, p=0.0003). Histopathological analyses confirmed the tumor progression to adenocarcinoma with distinct papillary and intraluminal growth patterns in combined mice, but not in Kras-mutation only mice. Spatial transcriptomic analysis confirmed that tumors with this growth patterns were enriched in transitional airway progenitors co-expressing Sox2, Nkx2-1, and ciliated markers. Moreover, normal and tumor organoids derived from the combined mice proliferated robustly whereas organoids from Kras-mutation only mice stopped growing shortly after establishment. While global hypomethylation agents significantly reactivated p16 and inhibited proliferation in tumor organoids, these agents failed to block tumor development in vivo (Fold change of tumor burden to control: DAC 1.41 ± 0.11, GSK3685032 1.33 ± 0.18, n=5, p=0.07). In contrast, CRISPR-mediated targeted p16 promoter demethylation significantly reduced both tumor number and size in vivo compared with control mice (Tumor number: 16.17 vs. 6.33 per mouse, n=6, p=0.04). Conclusion: Our work identifies p16 as a bona fide epigenetic driver and therapeutic target for Lung cancer. Importantly, this study provides proof-of-concept that precise, locus-specific DNA demethylation can restore tumor suppressor function, highlighting a promising strategy for targeted epigenetic therapy in lung cancer patients. Citation Format: Xiaomin Chen, Li Yang, Eduardo Lopez, Lili Ma, Chao Cheng, Lanjing Zhang, Lanlan Shen. Age-related p16 epimutation is a targetable driver of Kras-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1964.

Abstract Introduction: DNA methylation is a key epigenetic mechanism that regulates development and disease, including cancer. However, current tools for precise, locus-specific epigenetic gene editing remain limited. Because phenotypic outcomes often result from interactions among multiple genes, there is a critical need for systems that can modulate methylation across several genomic loci simultaneously. Equally important, tight spatial and temporal control of such epigenetic modulation is essential to accurately dissect causal relationships between DNA methylation and gene function. To address these challenges, we developed mouse models that enable controlled, cell type-specific, and time-dependent epigenetic modulation in vivo. Methods: We applied the CRISPR-dCas9-SunTag system for targeted DNA methylation editing, using TET1 for demethylation and DNMT3A/3L for methylation. To generate mouse models, we employed homologous recombination to introduce knock-in constructs at genomic safe harbors. Specifically, the dCas9-SunTag-TET1-GFP system was inserted into the Rosa26 locus and the dCas9-SunTag-DNMT3A/3L-mCherry system was integrated into the Hipp11 (H11) locus. Expression of the dCas9-based epigenetic editors was regulated through recombinase systems: Cre or Flp recombination activated expression, while Dre recombination removed the entire construct, thereby terminating expression. Specific guide RNA (gRNAs) directed targeted demethylation or methylation at single or multiple genomic sites. Results: For both mouse lines, correct knock-in was confirmed by Southern blot and DNA sequencing. Germline transmission was successful, and offspring developed normally with the expected Mendelian ratios. Mouse embryonic fibroblasts (MEFs) derived from these lines were used to validate inducible expression mediated by Cre or Flp recombination. Inducibility was confirmed by Cas9 Western blot, RT-PCR analysis of TET1 or DNMT3A/3L transcripts, and fluorescent reporter expressions. Using gRNA targeting multiple promoters, including tumor suppressor genes p16 and Hic1 and oncogenes Tfap2a and VEGF, we observed robust and site-specific DNA methylation editing. Furthermore, targeted methylation of the p16 promoter in MEFs resulted in transcriptional silencing and bypass of the senescence checkpoint. Conclusion: We successfully established mouse models that enable precise, locus-specific manipulation of DNA methylation through inducible CRISPR-dCas9-SunTag-based epigenetic editing. These models provide a versatile platform for dissecting the causal relationships between DNA methylation and gene function in vivo. Given their flexibility and inducible control, these systems will be broadly applicable to researchers investigating functional epigenomics, developmental regulation, and the epigenetic mechanisms underlying human diseases. Citation Format: Julia M. Salamat, Li Yang, Xiaomin Chen, Eduardo Lopez, Lanlan Shen. Novel mouse models enabling locus-specific manipulation of DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1952.

The extent of genetic variation and its influence on gene expression across multiple tissue and cellular contexts is still being characterized, with germline Structural Variants (SVs) being historically understudied. DNA methylation also represents a component of normal germline variation across individuals. Here, we combine germline SVs (by short-read sequencing) with tumor DNA methylation across 1292 pediatric brain tumor patients. For thousands of methylation probes for CpG Islands (CGIs) or enhancers, rare and common SV breakpoints upstream or downstream associate with differential methylation in tumors spanning various histologic types, a significant subset involving genes with SV-associated differential expression. Cancer predisposition genes involving SV-associated differential methylation and expression include MSH2, RSPA, and PALB2. SV breakpoints falling within CGIs or histone marks H3K36me3 or H3K9me3 associate with differential CGI methylation. Genes with SVs and CGI methylation associated with patient survival include POLD4. Our results capture a class of normal phenotypic variation having disease implications.

BACKGROUND:The incidence of early-onset colorectal cancer (EOCRC) has been rising at an alarming rate in the USA, and EOCRC disproportionately affects racial/ethnic minorities. Here, we construct comprehensive profiles of EOCRC DNA methylomes at base-pair resolution for a cohort of Hispanic and African American patients. RESULTS:We show the epigenetic landscape of these EOCRC patients differs from that of late-onset colorectal cancer patients, and methylation canyons in EOCRC tumor tissue preferentially overlapped genes in cancer-related pathways. Furthermore, we identify epigenetic alterations in metabolic genes that are specific to our racial/ethnic minority EOCRC cohort but not Caucasian patients from TCGA. Top genes differentially methylated between these cohorts included the obesity-protective MFAP2 gene as well as cancer risk susceptibility genes APOL3 and RNASEL. CONCLUSIONS:In this study, we provide to the scientific community high-resolution DNA methylomes for a cohort of EOCRC patients from underrepresented populations. Our exploratory findings in this cohort highlight epigenetic mechanisms underlying the pathogenesis of EOCRC and nominate novel biomarkers for EOCRC in underrepresented populations.

Supplementary Figure S2 shows an enhanced 1C metabolic pathway that contributes to tumor growth in response to dietary methyl donor supplementation.

Supplementary Table S4 shows 18 differentially expressed metabolites in tumor samples induced by dietary supplementation.

Structural variation heavily influences the molecular landscape of cancer, in part by impacting DNA methylation-mediated transcriptional regulation. Here, using multi-omic datasets involving >2400 pediatric brain and central nervous system tumors of diverse histologies from the Children's Brain Tumor Network, we report hundreds of genes and associated CpG islands (CGIs) for which the nearby presence of somatic structural variant (SV) breakpoints is recurrently associated with altered expression or DNA methylation, respectively, including tumor suppressor genes ATRX and CDKN2A. Altered DNA methylation near enhancers associates with nearby somatic SV breakpoints, including MYC and MYCN. A subset of genes with SV-CGI methylation associations also have expression associations with patient survival, including BCOR, TERT, RCOR2, and PDLIM4. DNA methylation changes in recurrent or progressive tumors compared to the initial tumor within the same patient can predict survival in pediatric and adult cancers. Our comprehensive and pan-histology genomic analyses reveal mechanisms of noncoding alterations impacting cancer genes.

Supplementary Figure S4 shows scRNA-seq analysis which reveals the immune landscape of colon tumors from supplemented mice.

The extent to which non-genetic environmental factors, such as diet, contribute to carcinogenesis has been long debated. One potential mechanism for the effects of environmental factors is through epigenetic modifications that affect gene expression without changing the underlying DNA sequence. However, the functional cooperation between dietary factors and cancer-causing epigenetic regulation is largely unknown. Here, we use a mouse model of age-dependent p16 epimutation, in which the p16 gene activity is directly controlled by promoter DNA methylation. We show p16 epimutation is modulated by folate and cofactors in dietary supplementation, which leads to increased colon cancer risk. Importantly, our findings provide functional evidence concerning the safety of folate fortification in the general population.

Supplementary Figure S3 shows dietary methyl donor supplementation markedly increases tumor cell proliferation and immune cell infiltration.

Supplementary Table S3 shows 25 differentially expressed metabolites in serum induced by dietary supplementation.

Supplementary Table S1 shows comparison of diets between current and previous studies.

Supplementary Figures 1-2 from ΔDNMT3B Variants Regulate DNA Methylation in a Promoter-Specific Manner

Colorectal cancer (CRC) is the third leading cause of death from cancer in both men and women in the United States. Late onset colorectal cancer (LOCRC) or CRC diagnosed in older adults >50 years old has continued to decline while early onset colorectal cancer (EOCRC) or colorectal cancer in patients under the age of 50 continues to rise at an alarming rate since the mid-1990s. In fact, EOCRC is now the second most common cancer and the third leading cause of cancer mortality in people <50 years of age in the USA. The incidence of EOCRC has been on the rise over the past four decades and is expected to increase by >140% by 2030. Notably, Blacks and Hispanics are disproportionately affected with the EOCRC diagnosis. Despite this, minority patients remain underrepresented in clinical trials and translational research. It has been proposed that environmental exposures including westernized diet, metabolic factors, and the microbiome might be responsible for the rise in incidence of EOCRC. Epigenetic modifications are well-known mechanisms by which the environment can modulate gene expression without changing the DNA sequence. One such modification is DNA methylation, which controls DNA accessibility, chromatin formation, and gene transcriptional activity. Unfortunately, however, little is known about the role of DNA methylation in EOCRC and much less is understood of EOCRC in minority populations. Therefore, we identified a unique cohort of 11 EOCRC patients within the Baylor College of Medicine/Harris Health System which is comprised of majority minority EOCRC population (7- Hispanic, 2 Non-Hispanic (NH) Black, and 1 NH-White EOCRC patients). Using whole-genome bisulfite sequencing, we comprehensively characterized the DNA methylome at single-base resolution in our 11 EOCRC patient samples. Our analyses revealed distinct EOCRC methylation patterns including both global hypomethylation and promoter-specific hypermethylation, which were not previously captured in The Cancer Genome Atlas (TCGA) Analyses. Among the differentially methylated regions (DMRs), we discovered that DNA and RNA transcription pathways were enriched in EOCRCs at advanced stage. Furthermore, we identify differential DMRs patterns among ethnicities. Taken together, our work supports a pathogenic role for DNA methylation in EOCRC. We are the first group to take this comprehensive unbiased approach to analyze the methylome in an underrepresented EOCRC cohort of patients. Citation Format: Karen Riggins, Jason Sheng Li, Benjamin Musher, Li Yang, Patricia Castro, Wedad Alfarkh, Neda Zarrin-Kamah, Michael Scheurer, Chad Creighton, Wei Li, Lanlan Shen. Methylome-wide profiling of early-onset colorectal cancer in underrepresented populations [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C057.

Legend for Supplementary Figures S1-S5.

Supplementary Table 1 from RIL, a LIM Gene on 5q31, Is Silenced by Methylation in Cancer and Sensitizes Cancer Cells to Apoptosis

Supplementary Figures 1-3, Tables 1-5 from Epigenetic Profiles Distinguish Malignant Pleural Mesothelioma from Lung Adenocarcinoma