Abstract Background Current evidence suggests that the association between non-steroidal anti-inflammatory drug (NSAID) use and colorectal cancer (CRC) risk remains uncertain, and little is known about potential heterogeneity by molecular subtype. Clarifying these associations may provide insights into CRC carcinogenesis. The aim of this systematic review was to evaluate the association between NSAID use and CRC risk by four clinically relevant molecular subtypes. Methods PubMed, Web of Science and Embase were searched for studies assessing NSAID use in relation CRC risk by microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), somatic mutations in the B-Raf proto-oncogene serine/threonine kinase (BRAF) gene or the Kirsten rat sarcoma viral oncogene homolog gene (KRAS) status. Meta-analyses were performed to calculate summary relative risks (sRR). Results Nine studies met the inclusion criteria (n = 4,726 overall study population). NSAID use was associated with a stronger inverse association for BRAF-wildtype CRC (sRR = 0.73; 95% CI: 0.66–0.80) than for BRAF-mutated CRC (sRR = 0.85; 95% CI: 0.71–1.02), although the difference was not statistically significant (P heterogeneity = 0.11). Associations were in a similar direction for CIMP-high (sRR = 0.71; 95% CI: 0.61–0.83) and CIMP-low CRC (sRR = 0.73; 95% CI: 0.66–0.80), with no difference by CIMP status (P heterogeneity = 0.80). Similarly, risk reductions were observed for KRAS-mutated (sRR = 0.78; 95% CI: 0.69–0.88) and KRAS-wildtype (sRR = 0.76; 95% CI: 0.69–0.84), and for both MSI-high (sRR = 0.80; 95% CI: 0.67–0.96) and MSI-low/MSS CRC (sRR = 0.75; 95% CI: 0.69–0.82), with no evidence of heterogeneity by KRAS or MSI status (P heterogeneity = 0.76 and P heterogeneity = 0.51 respectively). Conclusion NSAID use was associated with lower risk of CRC across molecular subtypes, with suggestive differences by BRAF mutation status. These findings highlight the need for further studies to clarify potential subtype-specific associations and their implications for targeted chemoprevention strategies. Citation Format: Mary Jose. Urruchúa-Rodríguez, Toktam Pour, Hermann Brenner, Michael Hoffmeister. Non-steroidal anti-inflammatory drugs use and risk of colorectal cancer molecular subtypes: A systematic review and meta-analysis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: The Rise in Early-Onset Cancers—Knowledge Gaps and Research Opportunities; 2025 Dec 10-13; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(23_Suppl):Abstract nr B036.

Colorectal cancer (CRC) is commonly associated with epigenetic modifications, including altered DNA methylation. Recent studies suggest that tumour-resident bacteria may influence CRC development, yet the impact of bacteria on epigenetic regulation is not understood. This study investigates the effect of lipopolysaccharide (LPS) from Fusobacterium periodonticum and Bacteroides fragilis, bacteria that are abundant in CRC tumours with high CpG island methylator phenotype (CIMP), on DNA methylation in HT29 colorectal cancer cells. HT29 cells were treated with LPS from F. periodonticum, B. fragilis, or a combination of both. DNA methylation was assessed using reduced representation bisulfite sequencing (RRBS), followed by bioinformatic analysis to identify differentially methylated CpG sites. RT-qPCR was used to analyse the expression of selected genes with altered CpG promoter methylation. F. periodonticum LPS treatment induced both hypermethylation and hypomethylation in HT29 cells, with significant hypermethylation observed near specific promoter regions, including PEPD and VAV3, with associated decrease in gene expression of these genes. B. fragilis LPS treatment predominantly induced hypomethylation. Co-treatment with both LPS molecules resulted in distinct methylation patterns, with B. fragilis LPS attenuating F. periodonticum LPS-induced hypermethylation. Bacterial LPS can induce dynamic alterations in DNA methylation profiles in HT29 colorectal cancer cells, leading to changes in gene expression. These findings suggest a novel link between tumour-resident bacteria and DNA methylation in colorectal cancer, highlighting, for the first time, a potential mechanism by which bacteria may influence colorectal carcinogenesis.

Colorectal cancer (CRC) is the third leading cancer-type worldwide and is exhibiting an increasing incidence in developing countries including India. Aberrant activation of Wnt-signalling and Mismatch-Repair inactivation causing microsatellite instability are the two major CRC drivers. CpG island methylator phenotype, the third driver, exhibits extensive overlap with MSI. The mechanisms driving CRC in India are poorly understood. We attempted to characterize sporadic CRC caused by MSI and its complex interplay with other CRC drivers in patients from Telangana, India. To determine the MSI status of our cohort, we analysed the instability statuses of five canonical microsatellites, expression statuses of major MMR genes, and the mutation statuses of the MLH1 and MSH2 genes. Detection of H3K36me3 was performed to assess for defects in MMR-complex recruitment. The methylation statuses of a panel of CIMP markers, in addition to the nuclear-stabilization statuses of β-catenin and p53 were also assessed to identify the involvement of other pathways. Among 230 CRC patients, a majority of whom were males, ≥ 50years old and harbouring late stage tumours in the rectum, low frequency of loss of major MMR genes was observed in MSI + tumors. MSH2 loss was detected in 16% of MSI + samples, a frequency significantly higher than previously reported. Causal genetic aberrations were undetected in 57% of MSI + tumors suggesting novel/non-canonical drivers. A majority of MSI + samples exhibited proficient expression of the four major MMR genes. The results highlight the importance of studying geographically-distinct populations to expand our understanding of CRC origins.

Quantitative Histology of Nonmetastatic Regional Lymph Nodes as a Novel Prognostic Indicator in Microsatellite Instability-High Colorectal Cancer.

Abstract IDH-mutant glioma (IDH-G) is initially slow-growing but invariably progresses to fatal disease. While IDH-G is generally characterized by global hypermethylation at CpG islands - known as the glioma CpG islands methylator phenotype (G-CIMP), recent bulk profiling studies have shown that progressed IDH-G loses the G-CIMP status (shift to G-CIMP-low). However, the impact of G-CIMP loss on glioma transcriptional cell states and their inheritance has not been elucidated. To address the question, we leveraged recent advances in single-cell multi-omics technologies to profile a longitudinal cohort of 35 IDH-G tumor samples (15 pairs), co-capturing full-length transcriptional (by Smart-Seq2) and DNA methylation (by extended-representation bisulfite sequencing (XRBS)), within the same single-nuclei. Compared to reduced-representation bisulfite sequencing (RRBS) used for single-cell DNA methylation profiling in our prior multi-omic single-cell study (Nat Genet., 2021), XRBS provided higher coverage of CpG islands (mean of 378,888 vs 198,345.1, P = 2.2 x 10-16) in this study. Transcriptional and epigenetic comparisons of cell states during IDH-G progression revealed increased stem-like states, decreased differentiation and identified potential cell states regulators. Single-cell DNA methylation analysis demonstrated methylation loss in progressed tumors, however the degree of global DNA methylation was similar between different malignant cells states within individual tumors. This suggests that lower DNA methylation is not due to a change in cell state composition, but rather that it may alter cell state architecture. To address whether decreased methylation in progressed tumors affects cellular dynamics, we applied a quantitative framework to directly measure cell state heritability and plasticity based on transcriptional annotation of high-resolution phylogenetic trees in clinical samples. The analysis suggested that decreased methylation reshapes cellular hierarchy to increased heritability of stem-like states and decreased differentiation. This study provides insight into the impact of DNA methylation on glioma progression, integrating cell states transition dynamics with epigenetic evolution.

Patient: Male, 78-year-oldFinal Diagnosis: Colon adenocarcinomaSymptoms: NoneClinical Procedure: —Specialty: Gastroenterology and HepatologyObjective: Mistake in diagnosisBackgroundSessile serrated lesions (SSLs) are precursors in approximately 20–30% of colorectal cancer (CRC) cases, often characterized by B-Raf proto-oncogene (BRAF) mutations and CpG island methylator phenotype (CIMP), and follow the serrated neoplasia pathway. Histopathological differentiation between SSLs with dysplasia and conventional adenomas is diagnostically challenging because their visual and pathologic features often overlap. SSLs with dysplasia carry a high malignant potential and can progress rapidly to invasive carcinoma, underscoring the need for accurate classification and timely intervention.Case ReportA 78-year-old man with a history of acute myeloid leukemia (treated with bone marrow transplant), Barrett’s esophagus, and ulcerative colitis in remission underwent colonoscopy, which identified an 11-mm polyp. It was resected piecemeal and initially reported as showing features of both a sessile serrated adenoma (SSA) and a tubular adenoma (TA). Given the mixed histology, a follow-up colonoscopy was performed 6 months later, revealing an invasive, moderately-differentiated adenocarcinoma with mismatch repair deficiency (loss of MLH1 and PMS2) and a BRAF V600E mutation. Surgical resection confirmed stage III CRC with lymph node involvement. Retrospective pathological review reclassified the original lesion as an SSL with dysplasia (SSL-D).ConclusionsThis case highlights the diagnostic challenges of SSLs, particularly when lesions are resected piecemeal. For accurate diagnosis and optimal patient management, it is essential that there is effective communication between pathologists and endoscopists about the morphological characteristics of the polyp, and the reading pathologists should communicate with endoscopists for any clarification. A multidisciplinary, collaborative approach is crucial for high-risk lesions.

BackgroundPhysical activity is associated with lower colorectal cancer (CRC) risk, but its association with molecular subtypes defined by genetic and epigenetic alterations of the disease is unclear. Such information may enhance the understanding of the mechanisms related to the benefits of physical activity.MethodsPooled observational (cases: n = 5386; controls: n = 6798; studies n = 5) and genome-wide association data (cases: n = 8178; controls: n = 10 472; studies n = 5) were used. We used multivariable logistic regression models and Mendelian randomization to assess the association between physical activity and the risk of CRC subtypes defined by individual tumor markers (and marker combinations), namely microsatellite instability status, CpG island methylator phenotype status, and BRAF and KRAS mutations. We used case-only analysis to test for differences between molecular subtypes. We applied Bonferroni correction to account for multiple tests.ResultsIn the pooled observational analysis, higher levels of physical activity were associated with lower CRC risk (Obs-per 1SD, odds ratio [OR] = 0.94, 95% confidence interval [CI] = 0.90 to 0.97), with an association that was stronger in males (Obs-per 1SD, OR = 0.91, 95% CI = 0.87 to 0.96) than in females (Obs-per 1SD, OR = 0.97, 95% CI = 0.91 to 1.03; Pinteraction = .04). Higher physical activity was associated with a lower risk of CRC across all molecular subtypes, especially in males. There was no difference in the associations by subtypes by pooled observational or Mendelian randomization analyses. The findings did not differ by study design, anatomical site, and early or late age onset of CRC.ConclusionsOur findings suggest that physical activity is not differentially associated with the 4 major molecular subtypes involved in colorectal carcinogenesis, indicating that its benefits extend broadly across colorectal cancer pathogenesis.

Exosomes play critical roles in cancer diagnosis and treatment as they carry molecular information that reflects the epigenetic state of their parent cells. For the first time, nanoscale epigenetic profiling of individual exosomes derived from colorectal cancer cell lines is demonstrated via photo-induced force microscopy (PiFM). Exosomes from three cell lines with distinct CpG island methylator phenotype (CIMP) status are analyzed at the single-vesicle level. The nano-IR method provides simultaneous high-resolution topographical and spectroscopic data, revealing detailed vibrational signatures that distinguish CIMP-high (HCT116 and HT29) exosomes from CIMP-negative (SW480) ones. Notably, exosomes from CIMP-high cells exhibit red-shifted amide I and nucleic acid region compared to those from CIMP-negative cells, a shift attributed to increased 5-methylcytosine (5mC) modifications, as verified by quantum chemical calculations. Furthermore, these measurements reveal heterogeneity among individual exosomes, suggesting the presence of distinct subpopulations with unique epigenetic profiles, demonstrating the importance of single-vesicle resolution to detect molecular variations that remain obscured in ensemble studies. These findings present the potential of PiFM-based single-vesicle analysis to identify epigenetic markers in exosomes, laying the groundwork for its application in refined cancer diagnostics and targeted therapeutic strategies.

Human colorectal cancer (CRC) encompasses tumors affecting a segment of the large intestine (colon) and rectum. It is the third most commonly diagnosed malignancy and the second leading cause of cancer deaths worldwide. It is a multifactorial disease, whose carcinogenesis process involves genetic and epigenetic alterations in oncogenes and tumor suppressor genes, including genes related to DNA repair. The pathogenic mechanisms are described based on the pathways of chromosomal instability, microsatellite instability, and CpG island methylator phenotype. When detected early, CRC is potentially curable, and its treatment is based on the pathological characteristics of the tumor and factors related to the patient, as well as on drug efficacy and toxicity studies. Therefore, the aim of this study was to review the pathogenesis and molecular subtypes of CRC and to describe the main targets of disease-directed therapy used in patients refractory to current treatments.

Multiomics analysis reveals the genetic and epigenetic features of high-risk NK cell-type chronic active EBV infection.

BACKGROUNDGastric adenocarcinoma with primitive phenotypes has recently attracted increasing attention due to its aggressive nature and challenging diagnosis. Gastric adenocarcinoma with enteroblastic differentiation (GAED) and hepatoid adenocarcinoma (HAC) were previously regarded as gastric adenocarcinoma with primitive enterocyte phenotype (GAPEP). GAPEP is known for its poor prognosis, and the accurate diagnosis of GAPEP directly affects therapeutic decision-making. Despite their poor prognosis and morphological heterogeneity, the molecular drivers of GAPEP, particularly methylation-driven mechanisms, remain poorly explored.AIMTo investigate the clinicopathological and molecular characteristics of GAPEP and establish an integrative diagnostic strategy to guide therapeutic decision-making.METHODSBased on the expression profile and morphology, patients were divided into three groups: GAPEP (including GAED and HAC), conventional gastric cancer (CGC), and CGC expressing primitive phenotypic markers. We analyzed clinicopathological features and overall survival. Data from The Cancer Genome Atlas were also analyzed, and functional enrichment analysis was conducted.RESULTSGAPEP showed diverse morphology, and immunohistochemical staining alone was not adequate for accurate diagnosis. Histologically, GAPEP was characterized by large, polygonal tumor cells with supranuclear or subnuclear vacuoles, a “piano keyboard-like” appearance, and clear or eosinophilic cytoplasms. Compared to CGC and CGC expressing primitive phenotypic markers, GAPEP displayed more aggressive clinical features. Molecular analysis showed significant differences in molecular subtypes, TP53 mutation, ERBB2 amplification, ARID1A mutation, MSI status, and CpG island methylator phenotype category. Genomic analysis revealed that TP53 mutations, APC mutations, and ERBB2 amplifications were more frequent in GAPEP. Genes involved in methylation processes were highly upregulated in GAPEP. HAC and GAED shared similar clinicopathological and genetic characteristics. Functional enrichment analysis highlighted the critical role of methylation in the development of GAPEP.CONCLUSIONThe diversity and aggressiveness of GAPEP are driven by deregulated methylation, necessitating the integration of morphological and immunohistochemical diagnosis. Targeting methylation can provide new therapeutic opportunities for treating this aggressive cancer.

Sporadic colorectal cancer (CRC), the third leading cause of cancer-related death globally, arises through a continuum from normal tissue to adenomas, progressing from low-grade (LGD) to high-grade dysplasia (HGD); yet, the early epigenetic drivers of this transition remain unclear. To investigate these events, we profiled LGD and HGD adenomas using EM-seq, and identified a consensus differential methylation signature (DMS) of 626 regions through two independent bioinformatics pipelines. This signature effectively distinguished LGD from HGD in both tissue and plasma-derived cell-free DNA (cfDNA), highlighting specific methylation patterns. Functional annotation indicated enrichment for regulatory elements associated with transcription factor activity and cell signaling. Applying the DMS to the TCGA CRC dataset revealed three tumor subtypes with increasing hypermethylation and one normal cluster. The most hypermethylated subtype exhibited poor survival, high mutation burden, and disrupted transcriptional networks. While overlapping with classical CpG Island Methylator Phenotype (CIMP) categories, the DMS captured a broader spectrum of methylation alterations. These findings suggest that the DMS captures functionally relevant, antecedent epigenetic alterations in CRC progression, enabling the robust stratification of dysplasia severity and tumor subtypes. This signature holds promise for enhancing preclinical detection and molecular classification, and warrants further evaluation in larger prospective cohorts.

BackgroundDNA methylation plays a crucial role in cancer development and progression and has been linked to genetically and clinically distinct tumor classes, including IDH-mutated and IDH-wildtype adult-type diffuse gliomas. Here, we identify a CpG-island methylator phenotype (CIMP) that characterizes the receptor tyrosine kinase 2 (RTK2) subtype of IDH-wildtype glioblastoma.ResultsThis RTK2-CIMP affects genomic locations and cell functions distinct from those of IDH mutation-associated IDH-CIMP and suppresses the expression of its target genes. The RTK2-CIMP-region chromatin is characterized by a combination of repressive and activating marks, including polycomb-associated H3K27me3 and enhancer-associated H3K4me1, consistent with DNA methylation-mediated silencing of genes with bivalent-state promoters in neural progenitor cells. Functionally, RTK2-CIMP affects neuronal lineage genes and is significantly associated with astrocyte-like glioblastoma, suggesting that RTK2-CIMP is an epigenetic signature of the astrocyte-like cell state. Furthermore, we demonstrate that RTK2-CIMP can be induced by genetic manipulation in glioblastoma cells.ConclusionsOur results suggest that RTK2-CIMP is a key contributor to cell-state plasticity in glioblastoma.

The CpG island methylator phenotype (CIMP) occurs in many colorectal cancers (CRCs). CIMP is closely associated with global hypermethylation and tends to occur in proximal tumours with microsatellite instability (MSI), but its origins have been obscure. A few CRCs carry oncogenic (gain‐of‐function) mutations in isocitrate dehydrogenase IDH1. Whilst IDH1 is an established CRC driver gene, the low frequency of IDH1‐mutant CRCs (about 0.5%) has meant that the effects and molecular covariates of those mutations have not been established. We first showed computationally that IDH2 is also a CRC driver. Using multiple public and in‐house CRC datasets, we then identified IDH mutations at the hotspots (IDH1 codons 132 and IDH2 codons 140 and 172) frequently mutated in other tumour types. Somatic IDH mutations were associated with BRAF mutations and expression of mucinous/goblet cell markers, but not with KRAS mutations or MSI. All IDH‐mutant CRCs were CIMP‐positive, mostly at a high level. Cell and mouse models showed that IDH mutation was plausibly causal for DNA hypermethylation. Whilst the aetiology of hypermethylation generally remains unexplained, IDH‐mutant tumours did not form a discrete methylation subcluster, suggesting that different underlying mechanisms can converge on similar final methylation phenotypes. Although further analysis is required, IDH mutations may be the first cause of hypermethylation to be identified in a common cancer type, providing evidence that CIMP and DNA methylation represent more than aging‐related epiphenomena. Cautious exploration of mutant IDH inhibitors and DNA demethylating agents is suggested in managing IDH‐mutant CRCs. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

In colon cancer, the BRAFV600E mutation is strongly associated with the CpG island methylator phenotype (CIMP). Here, we characterized the contribution of BRAFV600E to maintenance of aberrant DNA methylation using CRISPR-LbCpf1-corrected BRAF (V600E) organoids. DNA methylation analyses identified 5,187 differentially methylated CpGs within CpG islands-82% hypermethylated in BRAFV600E organoids-including CIMP-associated genes and polycomb repressor complex 2 (PRC2) target genes. RNA sequencing showed concordant repression of these genes. Furthermore, BRAFV600E organoids demonstrated high expression of PRC2 core components (EZH2, SUZ12, and EED), showed PRC2-induced H3K27 trimethylation in promoter regions, and maintained a PRC2-associated embryonic phenotype. This phenotype was lost following mutation correction or DNA methylation inhibition. These findings show that BRAFV600E maintains aberrant DNA and histone methylation patterns in advanced colon cancer, likely preserving the transformed phenotype. Silencing of PRC2 target genes may contribute to this phenomenon. Epigenetic therapies may have value in the treatment of BRAFV600E-mutant colon cancer.

ObjectiveTo test the hypothesis that the association of smoking with long-term colorectal cancer incidence may be stronger for tumours with higher mutational and neoantigen loads.Methods and analysisIn the Nurses’ Health Study (1980–2012) and the Health Professionals Follow-up Study (1986–2012), our novel prospective cohort incident-tumour biobank method (PCIBM) used 3053 incident colorectal carcinoma cases including 752 cases with whole-exome sequencing data. Using the multivariable duplication-method Cox regression model with the inverse probability weighting to adjust for the selection bias due to tissue availability, we assessed a differential association of cigarette smoking with colorectal carcinoma incidence by an exome-wide tumour mutational burden (e-TMB) or neoantigen load.ResultsThe association of pack-years smoked with colorectal cancer incidence differed by e-TMB (Pheterogeneity<0.001). Multivariable-adjusted HRs for e-TMB-high (≥10 mutations/megabase) tumours were 1.28 (95% CI 0.72 to 2.28) and 2.56 (95% CI 1.61 to 4.07) for 1–19 and ≥20 pack-years (vs 0 pack-years; Ptrend<0.001), respectively. In contrast, pack-years smoked were not associated with e-TMB-low tumour incidence (Ptrend=0.67). A similar differential association was observed for the neoantigen load (Pheterogeneity=0.017). The differential association by e-TMB appeared consistent in the strata of CpG island methylator phenotype status, BRAF mutation or lymphocytic infiltrates.ConclusionsSmoking is more strongly associated with the long-term incidence of colorectal carcinoma harbouring higher mutational and neoantigen loads. Our PCIBM-based evidence supports the immunosuppressive effect of smoking and the potential of smoking cessation in improving antitumour immunity for cancer prevention and treatment.

B‐cell acute lymphoblastic leukemia (B‐ALL) is a highly heterogeneous disease with a challenging prognosis, particularly in adult patients. We enrolled 88 adult B‐ALL patients with transcriptomic and mutation profiles for classification system identification, and a comprehensive system for B‐ALL patients (COMBAT) was developed. COMBAT stratified patients into three cohorts: (1) COMBAT1, characterized by high stem/myeloid antigen expression, low immune infiltration, high infiltration of endothelial cells, and hypo‐CIMP (CpG island methylator phenotype); (2) COMBAT2, defined as an inflamed subtype with immune exhaustion, moderate myeloid antigen expression, and hypo‐CIMP; and (3) COMBAT3, marked by proliferative profiles with MYC pathway activation and hypomethylation at enhancer regions in patients characterized by CIMP. The molecular features of the three COMBATs were verified in two external cohorts, the GSE34861 (N = 194) and GSE66005 (N = 109) datasets. In univariate analysis, only COMBAT classification presented significance for OS, and patients of COMBAT3 presented significantly superior survival than COMBAT1/2 in Ph‐negative ALL. Ph‐negative ALL patients undergoing allogeneic hematopoietic stem cell transplantation (allo‐HSCT) in the COMBAT3 group showed better overall survival (OS) than those in the COMBAT1‐2 groups (estimated 3‐year OS: 100% vs. 65.6%, P = 0.034), suggesting a prognostic benefit of this subtype. In summary, the COMBAT system redefines the characteristics of adult B‐ALL subtypes and guides the selection of allo‐HSCT for Ph‐negative patients.

Abstract Colorectal cancer (CRC) progression is closely linked to alterations in key signaling pathways and metabolic reprogramming. The WNT signaling pathway plays a critical role in regulating cell development and proliferation, and its abnormal activation is a hallmark of CRC. Plant Homeodomain 6 (PHF6), a chromatin-associated factor, is known for its roles in clonal hematopoiesis, ribosome biogenesis, DNA repair, and aging, and is frequently mutated in lymphoid cancers and leukemia. In this study, RNA-seq and ATAC-seq were conducted to investigate the function of PHF6, we identified PHF6 as a novel regulator of the WNT signaling pathway. We found that PHF6 depletion activate WNT signaling and proliferation of colon cancer cells. In the AOM/DSS model, PHF6 knockout mice exhibited larger polyp formation compared to wild type mice, with polyps extending into the proximal region. Proximal CRC is more prevalent and aggressive and has distinct genetic features compared to distal CRC. Alterations in proximal CRC include the PIK3CA mutation and CpG island methylator phenotype (CIMP)-high frequency, the development of CIMP is followed by global gene expression alteration and metabolic changes. However, the molecular mechanisms of this type of cancer are not fully understood. We observed a reduced levels of LEO1, the transcriptional elongation partner of PHF6 within the PAF1 complex, in the proximal region of PHF6 KO mice. We identified LEO1 and SETD1A as epigenetic regulators of lipid homeostasis in colon cancer cells and patient samples with matched normal. Dysregulation of lipid metabolism in cancer cells impacts tumor initiation, progression, and metastasis. LEO1 KO cells exhibited reduced expression and activation of SREBP1, a master regulator of lipid metabolism, under glucose starvation. These effects were further enhanced by SETD1A knockdown. LEO1 was found to localize to the nucleus and is required for the nuclear localization of SETD1A under nutrient stress. These cells showed an altered cell cycle, resulting in reduced proliferation but increased metastatic potential. These findings suggest that the PHF6-LEO1 axis plays a crucial role in the regional development and metastasis, underscoring its importance in advancing our understanding of tumor biology and guiding targeted therapy development. Citation Format: Su Chan Park, Shinae Park, Ji-Yeon Lee, Taehan Yoon, So Hyun Kwon, Jung-Shin Lee, Eun Jung Park, Ji Min Lee. PHF6-LEO1 axis depletion drives aggressive colorectal cancer through WNT activation and metabolic reprogramming with therapeutic implications [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 184.

Abstract Purpose: Collecting duct carcinoma (CDC) is a rare and aggressive subtype of renal carcinoma, posing significant challenges in diagnosis and treatment. Current treatment relies on platinum-based chemotherapy, yet clinical outcomes remain poor. While targeted therapies and immune checkpoint inhibitors (ICIs) show promise in clear-cell renal cell carcinoma (ccRCC), they have not been evaluated in CDC, and their efficacy remains speculative. This underscores a pressing need for personalized treatment approaches, driven by an in-depth molecular understanding of the disease. Experimental Design: We conducted a comprehensive molecular characterization of 22 CDC cases using whole-exome sequencing, RNA-seq, and DNA methylation profiling. Renal medullary carcinoma, ccRCC, type 2 papillary RCC, and chromophobe RCC were included as comparators. To verify mutations of interest, we retrieved 25 CDC samples from GENIE (v15.0). We also assessed the association of genomic alterations with response to ICIs using data from the BIONIKK trial (NCT02960906) and the published CheckMate (-009, -010, -025) trials. Results: We identified frequent mutations in NF2 (20%), LZTR1 (20%), and SMARCB1 (10%), all located on chromosome 22q, along with 22q copy number loss (52%). Mutations in NF2, SAV1, and WWC1—key components of the Hippo signaling pathway—were observed, implicating its disruption (33%) and leading to overexpression of the YAP signature. Notably, a mutually exclusive pattern between DNA damage-related (DDR) mutations (33%) and 22q loss was observed. Transcriptomic analysis highlighted a cell cycle and immune cell enrichment signature in CDC, distinct from other RCC comparators. TME deconvolution revealed two CDC phenotypes tightly associated with genomic alterations. The immune-hot CDC was characterized by enrichment of CD8 T cells, B cells, and tertiary lymphoid structure signatures, and was enriched in DDR mutations. In contrast, the immune-cold CDC was marked by 22q loss and overexpression of the YAP signature. Furthermore, post-hoc analyses using BIONIKK and CheckMate data demonstrated that combining 22q loss and DDR mutations improves the precision of ICI response predictions. We also found a correlation between 22q loss and a CpG Island Methylator Phenotype (CIMP) pattern in CDC, which resembles the aggressive, immune-evasive CIMP subgroup observed in type 2 papillary RCC. Conclusions: Our comprehensive characterization broadens the molecular understanding of CDC, revealing actionable vulnerabilities such as DDR mutations for immunotherapy and YAP activation for targeted interventions. These findings redefine CDC’s therapeutic possibilities, offering a roadmap for precision oncology in this rare malignancy. Citation Format: Xiaofan Lu, Wenxuan Cheng, Nassim Tawanaie Pour Sedehi, Li Xu, Xiaoping Su, Roger Mouawad, Nizar M. Tannir, Gabriel G. Malouf. Comprehensive molecular characterization reveals hallmarks and therapeutic insights of collecting duct carcinoma [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 1123.

Purpose: The epigenetic deregulation of CpG islands (CGIs) plays a crucial role in cancer initiation and progression. Genome-wide analysis of aberrant methylation patterns in CGIs offers a promising approach for the early cancer detection. However, the clinical utility of whole genome bisulfite sequencing is limited by the need for high sequencing coverage to achieve sensitive detection. To address this challenge, we have developed aMAPP, a method that enriches hypermethylated and/or hypomethylated CGIs. This approach enables the detection and tracing of aberrant methylation patterns, achieving high coverage in CGIs with ultra-low depth sequencing. Methods: NEBNext Ultra II Library Prep. was used for adapter ligation. DNA was then subjected to APOBEC-mediated deamination and/or TET-Assisted Pyridine Borane Sequencing (TAPS) and pre-amplified. Methylation-specific amplification was then conducted using novel, customized proximity primers composed of an anchor, binding to the adapter, and a 6-10 bp probe, designed to bind tandem CpG dinucleotides downstream of the adapter. These primers amplify selectively DNA fragments containing methylated CpGs that remain intact after APOBEC deamination or unmethylated CpGs that remain intact after TAPS. Following Proximity Primer-PCR, MiSeq was conducted, generating approximately 500,000 reads per sample. Results: Proximity primers were shown to amplify APOBEC-treated DNA fragments containing CpGs that match the primer probes, even if these CpGs are 100 bases downstream the adapter. While unmethylated fragments lack CpGs and are not amplified (∆Ctmeth-unmeth≈18). Applying aMAPP to APOBEC-treated methylated gDNA provided large amplification footprint of genome-wide methylated CGIs with remarkable efficiency, yielding 355-fold and 746-fold more CGIs with coverage of ≥5 and ≥10, respectively, compared to standard EM-Seq. Depending on conditions applied, aMAPP captures &amp;gt;80% of all CGIs with high coverage using ultra-low sequencing depth. Similarly, aMAPP applied to TAPS-treated unmethylated DNA captured selectively the unmethylated CGIs. aMAPP can be applied with very low starting material (10-100pg). Upon application to clinical samples, aMAPP detected thousands of CGIs and identified CpG island methylator phenotype (CIMP) and the top 20 hypermethylated CGIs in colorectal cancer. aMAPP could trace differentially methylated CGIs (DMR) in tumor samples present at 0.01% levels within adjacent normal tissue, or within circulating DNA of normal volunteers. Conclusion: aMAPP enriches methylated and/or unmethylated portions of the genome with a unique efficiency, simplicity and flexibility that supersedes previously reported methylation enrichment approaches, enabling focused methylation sequencing at high coverage. Multiple applications in biomarker tracing and discovery can be envisioned in cancer, and beyond cancer. Citation Format: Farzaneh Darbeheshti, Hayet Radia Zeggar, Hamzeh Salmani, Yibin Liu, Viktor A. Adalsteinsson, G. Mike Makrigiorgos. Adapter-anchored methylation specific amplification on genomic DNA using proximity primers (aMAPP) [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 4104.