Various methods have been developed for 5-methylcytosine (5mC) sequencing; however, effective ways to enrich hypomethylated DNA regions have been limited. Here, we describe the DEMETER-assisted 5-Methylcytosine Nicking sequencing (DMN-seq) utilizing 5mC-specific glycosylase DEMETER to nick DNA at 5mC sites, enabling 5mC detection at the single-base resolution. Leveraging this nicking activity to deplete hypermethylated sites, we adapt DMN-seq to preferentially enrich and investigate hypomethylated regions in colorectal cancer samples. When applied to cell-free DNA as low as 0.1 ng, DMN-seq significantly expands the scope of cancer biomarkers by capturing hypomethylated regions, with high sensitivity and reproducibility even in low-input clinical samples.

Activation in T cells through their antigen receptors has been closely tied to the strength of recognition of their cognate antigens, dictated by the variable complementarity-determining region loops. We show that, in the human gamma delta (γδ) T cell receptors (TCRs), the gamma constant domains modulate activation intensity independent of variable region antigen recognition. Using single-cell RNA sequencing data, we demonstrate that Cγ usage in vivo corresponds with distinct phenotypes, with cells using Cγ1 having a more differentiated cytotoxic effector phenotype in the thymus and higher granzyme expression in peripheral tissues. TCRs with Cγ1 are also selectively expanded in colorectal tumors. Cγ2 usage is correlated with naïve phenotypes in development and with inhibition and wound healing in the periphery. We propose that the modulation of activation using Cγ1 or Cγ2 translates to differences in γδ T cell phenotype and clonal expansion throughout its life span, providing human γδ T cells another "dial" to modulate their function.

Acyl-CoA synthetase short chain family member 2 (ACSS2) catalyzes the conversion of acetate to acetyl-CoA. Here we show that ACSS2 expression is markedly elevated in all stages of human colorectal cancer (CRC), and Acss2 silencing or genetic ablation leads to a marked reduction in CRC tumor load in allograft and xenograft models and CRC models induced by epithelial Apc deletion or azoxymethane/dextran sodium sulfate treatment. Tumors with ACSS2 depletion exhibit robust DNA damage, excessive apoptosis, and increased recruitment of macrophages and CD8+ T cells to the tumor microenvironment. Treatment with a small-molecule ACSS2 inhibitor markedly suppresses tumor growth in allograft/xenograft and Apc-mutant CRC models. ACSS2 promotes tumor cell growth by blocking DNA damage and apoptosis under nutritional stress. Collectively, these data indicate that the conversion of acetate to acetyl-CoA by ACSS2 is required for CRC progression and ACSS2 is a potential druggable target for CRC management.

The role of m6A RNA methylation of self non-coding RNA remains poorly understood. Here we show that m6A-methylated self U6 snRNA is recognized by YTHDF2 to reduce its stability and prevent its binding to Toll-like receptor 3 (TLR3), leading to decreased inflammatory responses in human and mouse cells and mouse models. At the molecular level, endosomal U6 snRNA binds to the LRR21 domain in TLR3, independent of m6A methylation, to activate inflammatory gene expression, a mechanism that is distinct from that of the best known synthetic TLR3 agonist poly I:C. Both U6 snRNA and YTHDF2 are localized to endosomes via the transmembrane protein SIDT2, where YTHDF2 functions to prevent the U6-TLR3 interaction. We further show that UVB exposure inhibits YTHDF2 by inducing its dephosphorylation and autophagic protein degradation in human keratinocytes and mouse skin. Skin-specific deletion of Ythdf2 in mice enhanced the UVB-induced skin inflammatory response and promoted tumor initiation. Taken together, our findings demonstrate that YTHDF2 plays a crucial role in controlling inflammation by inhibiting m6A U6-mediated TLR3 activation, suggesting that YTHDF2 and m6A U6 are potential therapeutic targets for preventing and treating inflammation and tumorigenesis.

The evolution of diet has played an essential role in shaping human physiology and pathology, including the development of the immune system and responses to environmental stimuli. However, the specific mechanisms underlying the regulatory significance of dietary components in modulating anti-tumor immunity remain largely unknown. Here, we applied a co-culture screen approach using a blood nutrient compound library to identify zeaxanthin, a dietary carotenoid pigment found in many fruits and vegetables that is important for eye health, as an immunomodulator that enhances cytotoxicity of CD8+ T cells against tumor cells. Oral zeaxanthin, but not lutein, a zeaxanthin structural isomer, enhances anti-tumor immunity in vivo. Integrated multi-omics mechanistic studies reveal that zeaxanthin directly promotes T-cell receptor (TCR) stimulation on CD8+ T cell surface, leading to improved intracellular TCR signaling for effector T cell function. Hence, zeaxanthin treatment augments effectiveness of immune checkpoint inhibitor in vivo and human TCR-engineered CD8+ T cells to induce cell death against co-cultured tumor cells. Our findings uncover a previously unknown immunoregulatory function of zeaxanthin, which has translational potential as a dietary element in bolstering immunotherapy. Freya Q. Zhang, Jiacheng Li, Rukang Zhang, Jiayi Tu, Zhicheng Xie, Takemasa Tsuji, Hardik Shah, Matthew O. Ross, Ruitu Lyu, Junko Matsuzaki, Kelly Xue, Fatima Choudhry, Chunzhao Yin, Hamed R. Youshanlouei, Syed Shah, Michael W. Drazer, Marc Bissonnette, Yuancheng Li, Hui Mao, Jun Huang, Lei Dong, Rui Su, Chuan He, Kunle Odunsi, Jing Chen, Hao Fan. Zeaxanthin augments CD8+ effector T cell function by enhancing TCR stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6325.

ABSTRACT We assessed the predictive value of MAdCAM‐1 expression on response to vedolizumab in patients with inflammatory bowel disease. This was a retrospective, single‐center, cohort study including 109 patients with pretreatment inflammation who completed at least three doses of vedolizumab. We described clinical and endoscopic outcomes of patients based on MAdCAM‐1 expression. There was no significant difference in MAdCAM‐1 expression when stratified by histology. Patients in clinical remission at 14 weeks had significantly lower median baseline MAdCAM‐1 expression (37425.3 vs. 46278.9, p < 0.015). There was no difference in pretreatment MAdCAM‐1 expression among patients who later achieved endoscopic or biologic response. In the posttreatment cohort, lower MAdCAM‐1 expression was associated with an increased likelihood of endoscopic or biologic response (36719.5 vs. 44229.9, p < 0.038). However, posttreatment MAdCAM‐1 expression did not significantly differ when stratified for clinical remission at 14 weeks. Ultimately, MAdCAM‐1 immunohistochemistry has limited utility as a predictive biomarker but may provide insights into vedolizumab‐associated bowel healing.

Introduction Peritoneal metastases (PM) are associated with poor prognosis in patients with colorectal cancer (CRC) or appendiceal adenocarcinoma (AA), yet detection of PM is unreliable using current circulating DNA technology. Leveraging novel 5hmC-seal technology to detect ultra-low amounts of DNA in plasma, we demonstrate the feasibility of 5-hydroxymethylcytosine (5hmC) signatures derived from circulating cell-free DNA (cfDNA) as biomarkers for PM. Methods Using a highly sensitive and robust 5hmC sequencing approach on genomic DNA isolated from peripheral blood samples, we developed predictive models to identify biomarkers for peritoneal metastases. Results We obtained genome-wide 5hmC profiles from 71 CRC/AA patients with PM, 41 without PM, and 73 non-cancer controls. Predictive models trained on genomic region 5hmC levels in patients with cancer could distinguish PM status with high sensitivity and moderate specificity (AUC 0.827, sensitivity 92.4%, specificity 46.1%). Pathway enrichment analysis identified epigenetically dysregulated cancer, cell migration, adhesion, and immune-related pathways in PM. Conclusion Novel 5hmC-Seal technology based 5hmC signatures can detect patients with peritoneal metastases from CRC and AA, albeit with reduced specificity. This study lays a foundation for future clinical assay development for PM. Statement of significance We demonstrate high-sensitivity detection of peritoneal metastasis in colorectal and appendiceal adenocarcinomas using 5hmC-Seal of plasma cfDNA. Earlier detection of this condition could expand curative treatments in ∼20,000 affected U.S. patients. ### Competing Interest Statement C.H. is a scientific founder and member of the scientific advisory board and equity holder of Aferna Bio, Inc., and Ellis Bio Inc.; a scientific co-founder and equity holder of Accent Therapeutics, Inc.; and a member of the scientific advisory board of Element Biosciences and Rona Therapeutics. All other authors declare no competing interests. Irving Harris Foundation Kevin Brown Family Foundation Gastrointestinal Research Foundation

m6A RNA methylation is the most prevalent internal modification in mammalian mRNAs and is involved in many biological processes. METTL16 is a recently identified RNA m6A methyltransferase. However, how METTL16 activity is regulated remains poorly understood. Here, we report a previously unrecognized mechanism in regulating METTL16 activity. SSB not only serves as a co-factor for METTL16 in installing m6A RNA methylation by enhancing METTL16 binding to RNA but it also is a direct target of METTL16-mediated m6A RNA methylation, leading to a positive auto-regulatory loop that promotes m6A methylation, SSB expression, and chemoresistance in colorectal cancer cells. Our findings reveal the regulation of METTL16 activity by SSB, providing a basis for the development of future therapeutic strategies that target the METTL16/SSB axis in METTL16-dependent cancers such as colorectal cancer.

This study develops an efficient and accurate pipeline for classifying tumor-derived organoid candidates as benign or malignant, addressing challenges posed by organoid culture and advancing personalized medicine applications. Tumor-derived organoids replicate tumor architecture and molecular features, providing controlled environments to study tumor behavior and therapeutic responses. A key challenge in using organoid models is classifying cellular aggregates as benign or malignant. Benign cells in a tumor source can sometimes outcompete malignant tumor cells in culture, misrepresenting the tumor's characteristics. Also, histological characterization of organoids is challenging due to the absence of histologic clues from tissue organization. Current approaches, such as genomic sequencing, effectively resolve such uncertainties but are costly and time-intensive. We hypothesized that a deep learning (DL) based computational image analysis classification pipeline could improve the accuracy and efficiency of distinguishing organoids as benign or malignant. We developed a novel pipeline to enhance organoid classification methods using ML. Our pipeline was developed and validated on H&E-stained slides obtained from organoids derived from 17 colorectal cancer patients (26 slides: 11 tumor, 15 normal) with various degrees of differentiation and paired normal controls. Ground truth data were established using a comprehensive genomic profiling panel to confirm that tumor organoids matched primary tumor features and, for tumors with high microsatellite instability, mismatch repair protein status. We first trained a segmentation model using a Human-in-the-Loop approach for precise identification of regions of interest. We extracted non overlapping 256x256 tiles at 20x magnification. We used the UNI foundation model to extract (k=1024) relevant features from each tile. We fitted Linear Discriminant Analysis (LDA) models on the UNI feature vectors using leave-one-patient-out cross validation according to patients. Each slide's final classification score was determined by averaging the LDA scores across all tiles from that slide. The LDA classifier achieved 92.3% accuracy and AUROC of 0.964 (0.907-1.000), matching gastrointestinal pathologist performance. Additionally, we visualized the UNI feature vectors through UMAP plots. Plot evaluation demonstrates distinct clustering of unsupervised features into tumor and normal groups. These findings demonstrate the feasibility of coupling organoids with DL methods to facilitate organoid classification. Our approach offers an efficient new strategy for studying organoid morphology and advancing organoid models. Adaptations of this model are expected to be valuable in understanding organoid responses to various therapies, paving the way for improved personalized cancer care. Adi Orlyanchik, Matteo Sacco, James Dolezal, Joseph Kainov, Piao Zhao, Mohammed Aziz Khan, Marina Garassino, Marc Bissonnette, Le Shen, Alexander T. Pearson, Christopher Weber. Digital pathology foundation models enable accurate tumor organoid classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 39.

Circulating cell-free RNA (cfRNA) in plasma represents a promising avenue for cancer detection. We report low-input multiple methylation sequencing, a method for profiling modification patterns in cfRNA, enabling the detection of diverse transfer RNAs and small noncoding RNAs derived from both the human genome and the microbiome. RNA modification patterns in microbiome-derived cfRNA accurately reflect host microbiota activity and hold potential for the early detection of colorectal cancer.

Abstract Purpose: Detection of colorectal carcinomas at a time when there are more treatment options is associated with better outcomes. This prospective case–control study assessed the 5-hydroxymethylcytosine (5hmC) biomarkers in circulating cell-free DNA (cfDNA) for early detection of colorectal carcinoma and advanced adenomas (AA). Experimental Design: Plasma cfDNA samples from 2,576 study participants from the multicenter METHOD-2 study (NCT03676075) were collected, comprising patients with newly diagnosed colorectal carcinoma (n = 1,074), AA (n = 356), other solid tumors (n = 80), and non–colorectal carcinoma/AA controls (n = 1,066), followed by genome-wide 5hmC profiling using the 5hmC-Seal technique and the next-generation sequencing. A weighted diagnostic model for colorectal carcinoma (stage I–III) and AA was developed using the elastic net regularization in a discovery set and validated in independent samples. Results: Distribution of 5hmC in cfDNA reflected gene regulatory relevance and tissue of origin. Besides being confirmed in internal validation, a 96-gene model achieved an area under the curve (AUC) of 90.7% for distinguishing stage I–III colorectal carcinoma from controls in 321 samples from multiple centers for external validation, regardless of primary location or mutation status. This model also showed cancer-type specificity as well as high capacity for distinguishing AA from controls with an AUC of 78.6%. Functionally, differential 5hmC features associated with colorectal carcinoma and AA demonstrated relevance to colorectal carcinoma biology, including pathways such as calcium and MAPK signaling. Conclusions: Genome-wide mapping of 5hmC in cfDNA shows promise as a highly sensitive and specific noninvasive blood test to be integrated into screening programs for improving early detection of colorectal carcinoma and high-risk AA.

Kirsten Rat Sarcoma (KRAS) is the most commonly mutated oncogene in colorectal carcinoma (CRC). We have previously reported the interactions between microsatellite instability (MSI), DNA promoter methylation, and gene expression. In this study, we looked for associations between KRAS mutation, gene expression, and methylation that may help with precision medicine. Genome-wide gene expression and DNA methylation were done in paired CRC tumor and surrounding healthy tissues. The results suggested that (a) the magnitude of dysregulation of many major gene pathways in CRC was significantly greater in patients with the KRAS mutation, (b) the up- and down-regulation of these dysregulated gene pathways could be correlated with the corresponding hypo- and hyper-methylation, and (c) the up-regulation of CDKN2A was more pronounced in tumors with the KRAS mutation. A recent cell line study showed that there were higher CDKN2A levels in 5-FU-resistant CRC cells and that these could be down-regulated by Villosol. Our findings suggest the possibility of a better response to anti-CDKN2A therapy with Villosol in KRAS-mutant CRC. Also, the more marked up-regulation of genes in the proteasome pathway in CRC tissue, especially with the KRAS mutation and MSI, may suggest a potential role of a proteasome inhibitor (bortezomib, carfilzomib, or ixazomib) in selected CRC patients if necessary.

PURPOSE Using the prostate, lung, colorectal, and ovarian (PLCO) Cancer Screening Trial samples, we identified cell-free DNA (cfDNA) candidate biomarkers bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that detected occult colorectal cancer (CRC) up to 36 months before clinical diagnosis. MATERIALS AND METHODS We performed the 5hmC-seal assay and sequencing on ≤8 ng cfDNA extracted from PLCO study participant plasma samples, including n = 201 cases (diagnosed with CRC within 36 months of blood collection) and n = 401 controls (no cancer diagnosis on follow-up). We conducted association studies and machine learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio. RESULTS We successfully obtained 5hmC profiles from these decades-old samples. A weighted Cox model of 32 5hmC-modified gene bodies showed a predictive detection value for CRC as early as 36 months before overt tumor diagnosis (training set AUC, 77.1% [95% CI, 72.2 to 81.9] and validation set AUC, 72.8% [95% CI, 65.8 to 79.7]). Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and race/ethnicity, and significantly outperformed risk factors such as age and obesity (assessed as BMI). Finally, when splitting cases at median weighted prediction scores, Kaplan-Meier analyses showed significant risk stratification for CRC occurrence in both the training set (hazard ratio, [HR], 3.3 [95% CI, 2.6 to 5.8]) and validation set (HR, 3.1 [95% CI, 1.8 to 5.8]). CONCLUSION Candidate 5hmC biomarkers and a scoring algorithm have the potential to predict CRC occurrence despite the absence of clinical symptoms and effective predictors. Developing a minimally invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient outcomes.

Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.

The 5hmC diagnostic model shows cancer type specificity.

Background:Colorectal cancer (CRC) is a leading cause of cancer-related mortality, and CRC detection through screening improves survival rates. A promising avenue to improve patient screening compliance is the development of minimally-invasive liquid biopsy assays that target CRC biomarkers on circulating cell-free DNA (cfDNA) in peripheral plasma. In this report, we identify cfDNA biomarker candidate genes bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that diagnose occult CRC up to 36 months prior to clinical diagnosis using the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial samples.Methods:Archived PLCO Trial plasma samples containing cfDNA were obtained from the National Cancer Institute (NCI) biorepositories. Study subjects included those who were diagnosed with CRC within 36 months of blood collection (i.e., case, n = 201) and those who were not diagnosed with any cancer during an average of 16.3 years of follow-up (i.e., controls, n = 402). Following the extraction of 3 - 8 ng cfDNA from less than 300 microliters plasma, we employed the sensitive 5hmC-Seal chemical labeling approach, followed by next-generation sequencing (NGS). We then conducted association studies and machine-learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio.Results:Despite the technical challenges associated with the PLCO samples (e.g., limited plasma volumes, low cfDNA amounts, and long archival times), robust genome-wide 5hmC profiles were successfully obtained from these samples. Association analyses using the Cox proportional hazards models suggested several epigenetic pathways relevant to CRC development distinguishing cases from controls. A weighted Cox model, comprised of 32-associated gene bodies, showed predictive detection value for CRC as early as 24-36 months prior to overt tumor presentation, and a trend for increased predictive power was observed for blood samples collected closer to CRC diagnosis. Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and self-reported race/ethnicity, and significantly outperformed risk factors such as age and obesity according to BMI (body mass index). Additionally, further improvement of predictive performance was achieved by combining the 5hmC-based model and risk factors for CRC.Conclusions:An assay of 5hmC epigenetic signals on cfDNA revealed candidate biomarkers with the potential to predict CRC occurrence despite the absence of clinical symptoms or the availability of effective predictors. Developing a minimally-invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient survival through higher compliance screening and earlier intervention. Future investigation to expand this strategy to prospectively collected samples is warranted.

Background and Objective: In sporadic colorectal carcinomas (CRC), microsatellite instability (MSI) pathways play important roles. Previously, we showed differences in DNA methylation patterns in microsatellite stable (MSS) colorectal carcinomas and MSI-CRC. In the current study, we explore the similarities and differences in gene expression profiles in MSS and MSI at the gene level and at the pathway level to better understand CRC pathogenesis and/or the potential for therapeutic opportunities. Material and Methods: Seventy-one CRC patients (MSI = 18, MSS = 53) were studied. Paired tumor and adjacent normal tissues were used for genome-wide gene expression assays. Result: At the gene level, we compared the list of differentially expressed genes (fold change (FC) ≥ 3 and FDR < 0.05) in tumor tissues compared to corresponding normal tissue in CRC patients with MSI tumors (190 genes) and MSS tumors (129 genes). Of these, 107 genes overlapped. The list of genes that were differentially expressed in MSI tumors only showed enrichment predominantly in two broad categories of pathways—(a) Inflammation-related pathways including the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, chemokine signaling, nuclear factor kappa B (NFκB) signaling, and cytokine-cytokine interactions, and (b) metabolism-related pathways, including retinol metabolism, steroid hormone biosynthesis, drug metabolism, pentose and glucoronate interconversions, and ascorbate and aldarate metabolism. The genes in inflammation-related pathways were up-regulated whereas genes in metabolism-related pathways were down-regulated in MSI tumor tissue. Pathway-level analysis also revealed similar results confirming the gene enrichment findings. For example, the 150 genes involved in the IL-17 signaling pathway were on average up-regulated by 1.19 fold (CI 1.16–1.21) in MSI compared to 1.14 fold (CI 1.13–1.16) in MSS patients (interaction p = 0.0009). Conclusions: We document an association between MSI status and differential gene expression that broadens our understanding of CRC pathogenesis. Furthermore, targeting one or more of these dysregulated pathways could provide the basis for improved therapies for MSI and MSS CRC.

Supplementary Material, Methods, and Figure Legend