NAR Cancer

IDH1 R132 mutations are among the most frequent hotspot mutations in cancer, but their mutational origins have remained unclear. Here, we provide evidence that IDH1 R132C, the predominant IDH1 mutation in cholangiocarcinoma, acute myeloid leukemia, and melanoma, likely arises through APOBEC3-mediated mutagenesis. IDH1 R132C is a TpC>TpT substitution on the lagging-strand DNA template within a hairpin-forming sequence context, consistent with APOBEC3 susceptibility. In vitro assays showed that APOBEC3A can deaminate the relevant cytosine, and APOBEC3A and APOBEC3B were relatively highly expressed in tumor types with recurrent IDH1 R132C mutations. IDH1 R132G, a TpC>TpG substitution at the same site, may similarly result from APOBEC3 activity. By contrast, IDH1 R132H, the predominant IDH1 mutation in lower grade glioma and glioblastoma, is a CpG>TpG substitution at a methylated cytosine on the leading-strand DNA template, a pattern more consistent with DNA polymerase epsilon replication error. Concordantly, tumor types enriched for IDH1 R132H showed relatively low POLE expression. Together, these in vitro and bioinformatic analyses provide insight into the distinct mutational mechanisms that likely underlie recurrent IDH1 hotspot mutations in cancer.

SMAD4 is a central transcriptional effector of the TGF-{beta} signaling pathway and a frequently inactivated tumor suppressor gene in various cancers. Missense mutations in its MH2 domain are among the most prevalent somatic alterations in colorectal cancer (CRC). These mutations are associated with disease progression and poor prognosis, yet their precise mechanistic consequences have remained incompletely characterized. Here, we show that CRC-derived SMAD4 MH2 hotspot mutations (D351H, S357P, R361C, and R361H) selectively impair co-activator recruitment without disrupting chromatin occupancy. RNA-seq profiling demonstrated broad suppression of TGF-{beta} target gene expression across all mutants. Notably, the mutations confer distinct degrees of TGF-{beta} pathway unresponsiveness: R361H is completely refractory to TGF-{beta} stimulation, whereas R361C and S357P retain partial transcriptional responsiveness suggesting allele-specific differences in the severity of co-activator interface disruption. Genome-wide chromatin binding analysis by greenCUT&RUN confirmed that all mutants maintain wild-type-like genomic occupancy, as expected given that the MH1 DNA-binding domain is intact in each case. Proximity-dependent biotinylation mass spectrometry in COLO205 cells revealed that all four mutants exhibit markedly reduced interactions with the CREBBP/EP300 histone acetyltransferase complex and BRD4 relative to wild-type SMAD4 identifying disrupted co-activator engagement. Collectively, our findings establish that SMAD4 MH2 mutations impair TGF-{beta}-induced transcription by selectively reducing CREBBP/EP300 recruitment, which provides a molecular mechanism for the loss-of-function SMAD4 phenotype in CRC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/735541v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@14f542eorg.highwire.dtl.DTLVardef@11fd220org.highwire.dtl.DTLVardef@1c3aa1org.highwire.dtl.DTLVardef@14d5a8e_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundCodon usage bias, the non-random usage of synonymous codons in coding sequences, represents a fundamental feature of genomic organization that has been largely understudied in cancer biology. Pancreatic ductal adenocarcinoma (PDAC), the predominant subtype of pancreatic cancer, is characterized by aggressive disease progression and limited therapeutic options, necessitating novel approaches to understand its molecular pathogenesis. Leveraging publicly available single-cell RNA sequencing data, we performed comprehensive codon usage analyses across different cellular populations in PDAC. ResultsEmploying a variety of computational codon usage indices uncovered the connections between cancer-specific cellular state features and codon usage signatures. Our findings reveal that malignant pancreatic cells express genes with significantly higher GC content, demonstrate preferential usage of optimal codons through increased frequency of preferred synonymous codons, and exhibit a marked preference for more cost-effective amino acids. Analysis of transcript-level bulk RNA-seq data from PDAC tumors revealed that these codon optimization patterns extend to alternative isoform usage, with highly expressed isoforms displaying increased codon optimality and enhanced mRNA stability. ConclusionThese codon usage-dependent adaptations operating at both gene expression and transcript isoform levels may enable malignant cells to enhance gene expression rates, potentially leading to increased translational efficiency and protein production. These insights into the codon usage landscape of PDAC may provide potential biomarkers for disease monitoring and treatment response prediction.

Glioblastoma (GBM) is a highly lethal brain cancer with limited therapeutic durability, where the majority of patients develop recurrent or persistent disease after standard chemoradiotherapy. Meanwhile, tRNA-derived fragments (tRFs) have become increasingly relevant to cancer biology; however, their clinical relevance in GBM remains undefined. Here, we report that a specific family of tRFs, 5-tRNA halves (tiR5s) dominates the small RNA landscape of GBM patient tumors and associates with worse overall survival, post-therapeutic disease persistence, and pro-invasive proteogenomic pathways across two independent GBM patient cohorts. This association between elevated tiR5 levels and therapeutic resistance re-emerges in radiation-resistant GBM xenograft models. Our findings reveal that tiR5s are an underappreciated molecular feature of highly aggressive GBM tumors, supporting further investigation into their biological roles and prognostic utility in GBM. HighlightsO_LItiR5s are the predominant tRF family in primary GBM patient tumors C_LIO_LIElevated tiR5 expression distinguishes primary GBM tumors that develop persistent disease after first-line therapy C_LIO_LIRadiation-resistant GBM PDX models show elevated tiR5 expression C_LIO_LIElevated tiR5 expression associates with poor overall patient survival and pro-invasive molecular programs in GBM patient tumors C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/738483v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@184ddc1org.highwire.dtl.DTLVardef@1faadc2org.highwire.dtl.DTLVardef@a5ae02org.highwire.dtl.DTLVardef@1431506_HPS_FORMAT_FIGEXP M_FIG C_FIG

Gastric cancer is driven by aberrant kinase signaling that supports uncontrolled proliferation and metabolic adaptation. Calcium/calmodulin dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, its role in regulating metabolic programs that sustain tumor growth remains incompletely understood. In this study, we employed an integrated multi-omics approach with a primary focus on untargeted metabolomics to investigate the consequences of CAMKK2 inhibition in gastric cancer cells. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells resulted in significant suppression of proliferation, clonogenic growth, migration, and invasion, accompanied by pronounced nuclear abnormalities and multinucleation indicative of mitotic defects. Global metabolomic profiling revealed extensive and time-dependent metabolic reprogramming following CAMKK2 inhibition, characterized by a marked depletion of nucleotide intermediates, including purine and pyrimidine metabolites required for DNA synthesis. Pathway enrichment analysis highlighted suppression of nucleotide metabolism, lipid metabolism, and central carbon metabolic pathways, indicating a broad impairment of biosynthetic capacity. Integration with proteomic and phosphoproteomic datasets demonstrated that metabolic alterations were accompanied by downregulation of DNA replication machinery and attenuation of kinase signaling pathways governing cell cycle progression. Protein metabolite interaction and docking analyses further supported functional coupling between nucleotide metabolites and key replication-associated enzymes, revealing disruption of metabolite enzyme interactions upon CAMKK2 inhibition. Collectively, these findings identify CAMKK2 as a critical regulator of metabolic programs that support DNA replication and cell cycle progression. Its inhibition induces replication stress through coordinated depletion of nucleotide pools and disruption of replication-associated signaling, leading to impaired proliferation and mitotic failure. These results highlight CAMKK2 as a potential therapeutic target for exploiting metabolic vulnerabilities in gastric cancer.

Understanding which genes are involved in mutagenesis is essential for developing cancer prevention and treatment strategies; establishing protectors of the genome has revolutionized cancer biology. Here, we describe metallothionein (MT) proteins as previously uncharacterized protectors against mutagenesis. MT is a heavy metal binding protein essential for zinc homeostasis and protection against heavy metal cytotoxicity. Because zinc binds approximately 10-15% of the proteome and is critical for processes such as DNA repair and mitochondrial health, MT loss is expected to disrupt these processes. We hypothesized that MT loss induces genomic instability by impairing DNA repair and mitochondrial function. In this study, the consequences of MT deficiency in high-grade serous ovarian cancer (HGSC) were investigated by knockdown of the most highly expressed MT, MT2A. Loss of MT2A resulted in the impaired DNA repair pathway base excision repair (BER), leading to increased mutagenesis. MT2A deficiency produced mitochondrial dysfunction, characterized by a decrease in mitochondrial membrane potential, glycolysis, oxidative phosphorylation, amino acids, and an imbalance of nucleobases. Together, these defects reflect cellular states associated with increased cancer aggressiveness. These findings identify MT as a fundamental hub maintaining genomic and metabolic integrity.

The standard-of-care for patients with higher-risk non-muscle invasive bladder cancer (NMIBC) after tumour resection is intravesical administration of Bacillus Calmette-Guerin (BCG). While this form of adjuvant immunotherapy has improved recurrence-free and progression-free survival, a large proportion of patients experience recurrences within a year of diagnosis. The reasons for this high rate of early recurrence following BCG therapy remain unclear; however, inadequate activation of systemic immunity may be a contributing factor. To address this, we analysed the transcriptomic and chromatin accessibility profiles of peripheral blood mononuclear cells obtained from patients with NMIBC at single-cell resolution before BCG immunotherapy and after five induction doses of BCG. Monocytes from patients who experienced disease recurrence within a year of initiation of BCG therapy (BCG non-responders) exhibited a pro-inflammatory phenotype consistent with age-related immunosenescence prior to BCG immunotherapy. Moreover, inflammation-associated pathways that were active before initiation of BCG therapy in the BCG non-responders were down-regulated after five instillations of BCG. In contrast, these pathways were quiescent before BCG therapy in patients who remained disease-free for at least a year but were markedly up-regulated after five doses of BCG. Genomic regions with accessible chromatin were enriched in activator protein 1 (AP-1) binding sequences in monocytes from BCG-non-responders prior to BCG therapy. AP-1 is a central regulator of the inflammatory phenotype associated with immunosenescence. Our findings indicate that a pre-existing state of innate immunosenescence underlies early disease recurrence following BCG. Patients unlikely to benefit from BCG may be offered alternative therapies early in their disease journey. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/723215v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1f7c844org.highwire.dtl.DTLVardef@7cea65org.highwire.dtl.DTLVardef@1008d23org.highwire.dtl.DTLVardef@131f973_HPS_FORMAT_FIGEXP M_FIG C_FIG

Noncoding regulatory variants contribute to colorectal cancer (CRC) susceptibility, yet their functional interpretation remains difficult.This is mainly attributed to regulatory effects being context-dependent and most noncoding regions lack reliable genomic annotations. We have developed a computational framework that aids in prioritizing promoter-associated variants using Evo2, a large-scale autoregressive DNA foundation model. In the framework, variants were mapped to promoter regions ({+/-}1,024 bp) across [~]1,250 CRC-associated genes and scored using Evo2-derived delta scores, the difference in sequence probability between reference and alternate alleles. Promoter variants showed greater predicted regulatory impact than non-promoter variants (median delta = 0.015 vs. 0.002; overall mean = 0.018, SD = 0.011). Applying a distributional threshold (delta > 0.020; top [~]25%) identified 287 high-impact variants across 198 CRC-associated genes. These genes were enriched in CRC-relevant pathways such as Wnt signaling, p53 signaling, and cell cycle regulation and 36.4% (72/198) overlapped known cancer genes (2.3-fold enrichment, p = 8.7x10-6). Independent validation showed high-impact variants were enriched at CRC GWAS loci and overlapped transcription factor binding sites ([~]32%) and motif-disrupting positions ([~]21%), supporting their functional relevance. Together, these results show that sequence-based foundation models can scalably prioritize noncoding regulatory candidates in CRC without supervised training or predefined annotations.

The human genome consists of nearly 50% repetitive DNA, referred to for decades as "junk DNA". These repetitive sequences, usually under the strict control of epigenetic silencing, have been observed to be aberrantly expressed in cancer. Some of these expressed sequences, e.g., transposable elements (TEs), can induce innate immune responses when de-repressed following treatment with epigenetic therapies. As a result, epigenetic therapy has been suggested to augment cancer therapies. TEs are traditionally ignored in most RNA-seq studies and their expression is often excluded from publicly available data sources. Thus, the vast amount of publicly available RNA-seq data is an untapped resource for exploring the role of TE expression in cancer and cancer treatment. Here, we present a uniform re-analysis of over 7,000 RNA-seq samples, encompassing more than 2,000 differential expression experiments across 220 cancer cell lines and 700 drug treatments. We observed that TE expression is more prone to batch effects than gene expression alone, necessitating the use of meta-analysis techniques to probe the dataset for global trends. We confirm that DNMTi and HDACis are powerful inducers of TEs. We also show that non-epigenetic compounds such as CDK and topoisomerase inhibitors can also induce robust up-regulation of transposable elements and confirm that this TE induction is consistent with viral mimicry response. We make all of the reprocessed data, web application, and database publicly available at: https://dataexplorer.coriell.org/TEDEdb/

Squamous cell carcinomas (SCCs) in the lung, head and neck, cervix, and esophagus are characterized by widespread chromosome-arm aneuploidies, most frequently recurrent 3q-gain. However, how these alterations influence cancer development and therapeutic vulnerabilities remains unclear. To identify aneuploidy-driven therapeutic targets, we performed genome-wide CRISPR interference (CRISPRi) and drug-repurposing screens in isogenic immortalized lung epithelial cells harboring chromosome 3-disomy or 3q-gain. Both screens converged on a mevalonate pathway dependency specific to 3q-gain cells, which exhibited heightened sensitivity to sterol regulatory element-binding protein (SREBP) disruption. Rescue experiments demonstrated that these vulnerabilities were on target and that pathway inhibition preferentially causes apoptosis in 3q-gain cells. Transcriptomic and lipidomic profiling revealed 3q-gain-associated alterations in SREBP activation, cholesterol and fatty-acid biosynthesis, and lipid composition. Perturbing SREBP signaling impaired viability in SCC cell lines and suppressed tumor growth in xenografts with 3q-gain. These findings identify an aneuploidy-driven, targetable vulnerability in SCC. SignificanceHere, we demonstrate that SCC-recurrent 3q-gain is a selective vulnerability to SREBP-pathway inhibition. We identify an aneuploidy-driven therapeutic liability in squamous tumors for lipid-targeted precision therapies, providing a framework for targeted treatment in SCC.

BackgroundRecent evidence suggests that cancer can exhibit splicing alterations that give rise to tumour-specific isoforms. One example is NUMB, which produces four isoforms (p72, p71, p66, and p65) through alternative splicing of exons 3 and 9. Traditionally considered a tumour suppressor, it also has been considered an oncogene. We propose that this duality is due to isoform-specific expression. ResultsUsing public databases, we identified a tumour-associated switch in NUMB isoform expression: p72/p71 are upregulated in tumours, whereas p66/p65 are more expressed in non-tumour tissues. These isoforms correlate differently with cellular processes. NUMBL, a NUMB homolog, behaves similarly to p65. We identified two transcriptional clusters: one characterized by high expression of p72/p71, and another by p66/p65/NUMBL. Each group was associated differently with the Notch, WNT/{beta}-catenin, Hedgehog, and Hippo signalling pathways, suggesting isoform-specific regulatory roles. Analysis of breast cancer cell lines (CCLE) led to a NUMB score based on isoform expression, which classified cell lines into biologically distinct groups. The p72/p71-enriched group showed distinct signatures, pathway activity, and drug sensitivity. Applying this score to TCGA-BRCA samples revealed a significant link between high NUMB-score and poor survival, confirmed by Kaplan-Meier analysis. ConclusionsNUMB emerges as a potential oncogenic contributor and biomarker in splicing-based personalised medicine, highlighting isoform-specific expression as a clinically relevant determinant of tumour behaviour, pathway activity, and therapeutic response.

We previously reported that the murine lncRNA Epr is essential for maintaining colon mucosal integrity and permeability. Mice lacking Epr in the colon are more susceptible to colitis and tumor development. Additionally, we demonstrated that human EPR expression is reduced in ulcerative colitis and in a small cohort of colon adenocarcinoma patients. Here, we present evidence that human and mouse EPR share several key physiological features: preferential binding to the KH1 domain of their interacting protein, KSRP; specific expression in canonical and immature goblet cells of the large intestine; and a functional role in intestinal goblet cell development. The correlation between EPR levels and survival in large cohorts of metastatic colon adenocarcinoma patients, together with the capacity of human EPR to inhibit cell proliferation and induce apoptosis in two distinct human colon adenocarcinoma cell lines, suggests that EPR may serve as both a valuable prognostic marker for goblet cell-derived adenocarcinomas and a potential therapeutic target.

MicroRNAs (miRNAs) act as crucial post-transcriptional regulators of large gene networks, and their aberrant expression drives key oncogenic processes such as epithelial-mesenchymal transition (EMT), angiogenesis, immune evasion, and metastasis. Oncogenic miRNAs that lose tissue specificity during malignant transformation represent promising therapeutic targets, as their restricted expression in healthy organs could minimize off-target effects. To identify these candidates, this study performed a comprehensive pan-cancer analysis integrating tissue-specificity profiles of healthy tissues from the GTEx project with tumor data from the TCGA, TARGET, CGCI, and CPTAC cohorts. By combining profiling with differential expression analysis between tumor and matched normal samples, cross-cohort integration revealed that malignant transformation is characterized by a widespread loss of tissue-specific miRNA expression. Among these altered patterns, a cluster of nine oncomiRs was identified: miR-105-5p, miR-1269a, miR-196a-5p, miR-9-5p, miR-96-5p, miR-210-3p, miR-301b-3p, miR-592, and miR-135b-5p. These specific miRNAs were significantly and recurrently upregulated across various solid tumors. Functional enrichment analysis of their experimentally validated targets demonstrated a clear convergence on shared oncogenic pathways, particularly those governing hypoxia response, PI3K/AKT signaling, EMT, angiogenesis, and immune modulation. Author SummaryMicroRNAs are tiny molecules in our cells that act like master switches, capable of adjusting entire networks of genes at once. Because they have such powerful control, cancer cells often hijack them to grow and spread throughout the body. Normally, many microRNAs are highly specialized, operating only in specific organs to maintain healthy tissue function. In this study, we looked across thousands of patient samples from various cancer types to see what happens to this specialization during tumor development. We discovered that when healthy cells transform into cancer, they lose this strict tissue-specific control. Specifically, we identified a group of nine microRNAs that break free from their normal boundaries and become active in tumors where they shouldnt be. Crucially, these nine molecules are responsible for turning on traits that are essential for tumor growth. Since these microRNAs are normally restricted to very specific healthy tissues, targeting them in tumors where they have gone rogue could become a precise and valuable strategy to halt cancer progression while potentially minimizing side effects in the rest of the body.

Structural variants (SVs) in cancer are associated with defects in DNA repair and replication stress, but the mechanisms generating common SV types remain unresolved. We propose that large (>100 kb) tandem duplications originate through a novel sister-fork breakage-fusion mechanism. To capture replication-related context beyond breakpoints, we developed an algorithm to characterize replication timing, origin density, and fork direction across SV-spanned regions, features that refine and differentiate previously defined SV signatures. Large tandem duplications frequently overlap replication origins from which forks proceed bidirectionally; combined with independent evidence from APOBEC strand asymmetry, this pattern is compatible uniquely with the proposed mechanism. Although tandem duplications in CCNE1-amplified and CDK12-mutant cancers also concentrate around origins and highly transcribed genes, they display distinct contexts: CDK12-mutant SVs arise near later-firing origins, whereas those in CCNE1--amplified tumors often coincide with genes in specific strand configurations, suggesting different causes of fork stalling. Incorporating replication features into signature analysis enabled the discovery of new SV signatures, which we used to build SVIG, a multi-class classifier of SV phenotypes. SV signatures attributed to replication stress may help guide therapies targeting this vulnerability.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, yet GIST-specific omics evidence remains scattered across small cohorts and is not represented as a dedicated disease project in major cancer genomics resources, limiting reproducible target exploration. Here, we present dbGIST (https://www.dbgist.com), a dedicated GIST-focused multi-omics resource built to make dispersed GIST evidence searchable, analyzable, and reusable. dbGIST harmonizes data from 37 centers and 1,991 samples, including pathologically verified in-house cohorts, across genomics, bulk transcriptomics, proteomics, phosphoproteomics, and single-cell transcriptomics, and couples these data with curated clinical annotations covering survival, mutation status, risk stratification, metastasis or recurrence, mitotic index, tumor site and size, and imatinib response. The platform supports cohort-level molecular-clinical association, survival, enrichment, immune-infiltration, drug-sensitivity, and single-cell analyses through interactive visualizations, downloadable source data, and public APIs for programmatic access to reusable analysis outputs and visualization-ready data. An optional LLM-assisted interface helps users navigate analyses and interpret outputs. Using MCM7 as a case study, dbGIST linked a resource-derived candidate to survival, risk features, metastatic or recurrent disease, imatinib-response phenotypes, proliferative cell states, and in vitro GIST-cell behavior. dbGIST therefore provides a traceable and interoperable resource for target exploration and precision oncology research in GIST.

BackgroundRetrotransposable elements (RE) comprise approximately 45% of the human genome and are typically repressed by DNA methylation to preserve genomic integrity. In cancer, global DNA hypomethylation can lead to RE derepression, resulting in genomic instability and activation of innate immune pathways through viral mimicry. While individual RE classes have been examined in clear cell renal cell carcinoma (ccRCC), the integrated epigenetic landscape of multiple RE families and their clinical relevance remain incompletely characterized. MethodsWe performed a genome-wide prediction of DNA methylation across three major RE classes (Alu, LINE-1, and LTR elements) using a validated computational framework applied to Illumina methylation array data from two independent ccRCC tumor cohorts. Integrated unsupervised clustering of RE methylation profiles was used to define the epigenetic subtypes. Associations with clinicopathologic variables, tumor immune microenvironment composition (DNA Methylation-derived), hypoxia signaling, innate immune activation, and overall survival were evaluated. Prognostic relevance was assessed using multivariable Cox regression models adjusting for age, sex, AJCC stage or AUA risk group, and immune and angiogenic tumor microenvironment features. Key findings were then externally validated in CPTAC-ccRCC and independently replicated in an institutional Dartmouth Cancer Center (DCC) cohort with matched methylation and RNA-sequencing data. ResultsIntegrated clustering identified three reproducible RE methylation subtypes, Repressed, Transient, and Active. In the discovery cohort, the Active subtype showed significantly worse overall survival than the Repressed subtype, with a graded survival pattern across RE methylation states that persisted after multivariable adjustment. RE hypomethylation was associated with reduced EPAS1 (HIF2A) expression, increased immune infiltration, elevated PD-1 expression, and heightened cGAS-STING and interferon signaling, consistent with an immune-inflamed yet immunosuppressed tumor state. In the external CPTAC validation cohort, RE methylation subtypes recapitulated key molecular features and showed supportive survival trends. In the independent DCC replication cohort, an Active RE state was again associated with poorer survival, lower EPAS1 expression, increased PD-1 expression, greater CD8 T-cell and Treg infiltration, and elevated T-cell exhaustion signatures, supporting the reproducibility of the prognostic and immune-exhausted phenotype across cohorts. ConclusionsWe identified RE methylation subtypes with distinct molecular, immunologic, and prognostic features in ccRCC. External validation in CPTAC and independent replication in DCC support the robustness of this RE methylation framework across large-scale and institutional cohorts. These findings highlight the prognostic potential of RE methylation profiles and support their integration into molecular classification strategies to improve risk stratification in ccRCC.

BackgroundTranscriptional regulation is shaped by both genomic variants and the environment. Yet, how the regulatory effects of genomic variants are reconfigured by dynamic epigenomic changes during tumorigenesis remains incompletely understood. MethodsWe investigated methylation context-dependent links between genotype and gene expression in colorectal cancer (CRC) using paired tumor and normal-adjacent tissue (NAT) from 80 patients, thereby controlling for germline genomic background. By integrating promoter-targeted bisulfite sequencing with RNA-seq, we systematically compared expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (mQTLs). To capture regulatory complexity beyond simple mediation, we implemented a memo-eQTL framework that explicitly models genotype x DNA methylation (GxM) interactions. ResultsWe observed extensive tissue specificity in both eQTL and mQTL landscapes; tumor-specific eGenes were significantly enriched for hallmark oncogenic pathways, including WNT and MAPK signaling. Standard mediation models explained only a minority of genotype-expression relationships, whereas our explicit interaction framework revealed widespread reconfiguration of methylation-dependent genetic effects in tumors. Memo-eQTL mapping (FDR < 0.05) identified 18 NAT and 73 tumor eGenes with significant GxM interactions, and results were consistent at a more permissive threshold (FDR < 0.2). We further developed a patient-level memo-eQTL score and found that interaction-based regulatory disruption in NAT, but not in tumor, significantly correlated with clinical stage (P = 0.035). ConclusionsGenetic regulation in cancer is reorganized through context-dependent GxM interactions. Importantly, GxM signatures in NAT are specifically linked to disease progression, offering new insights into field cancerization and the clinical consequences of regulatory reprogramming in CRC.

Platinum resistance remains a major barrier in Ovarian cancer (OC) treatment[1]. While hyperactivation of DNA damage response (DDR) is a hallmark of chemoresistance[2], the underlying epigenetic mechanisms driving this adaptation remain poorly understood. Here, we identify a novel post-transcriptional regulatory axis involving miR-221-5p that governs two critical DDR effectors: RAD18, which mediates DNA damage tolerance through trans-lesion synthesis (TLS)[3][4], and RAD51, the central recombinase for homologous recombination (HR)[5][6]. Although the miR-221/222 cluster is traditionally categorized as oncogenic[7][8], we demonstrate that the miR-221-5p arm functions as a potent tumor suppressor in OC. Bioinformatic and luciferase reporter assays confirmed that miR-221-5p directly targets the 3'UTRs of both RAD18 and RAD51. In OC clinical specimens and cell lines, miR-221-5p downregulation inversely correlates with RAD18/RAD51 expression. Functionally, miR-221-5p restoration suppressed platinum-induced PCNA mono-ubiquitination and HR, inducing a "functional BRCAness" that sensitized both established and patient-derived primary OC cells to carboplatin and PARP inhibition. Furthermore, in vivo disseminated xenograft models demonstrated that stable miR-221-5p expression significantly reduced tumor burden. Collectively, our results delineate a novel regulatory mechanism where loss of miR-221-5p drives chemoresistance by derepressing the RAD18/RAD51 axis, identifying this axis as a promising therapeutic target.

Endometrial cancer (EC) exhibits one of the most striking racial disparities in oncology with black women disproportionately affected by aggressive high-grade subtypes that have poorer outcomes. While social and environmental factors undoubtedly contribute, the molecular underpinnings of these disparities remain critically understudied. To bridge this knowledge gap, we performed matched tumor-normal whole-genome sequencing and tumor transcriptome sequencing on 71 predominantly high-grade EC patient samples from an ancestrally diverse cohort of women recruited at a large hospital system in the New York metropolitan area. Our analysis characterized the germline and somatic mutation landscape, identifying ancestry-associated molecular differences. Notably, focal amplification of the EVI1 transcription factor (encoded at the MECOM locus) was significantly more frequent in African ancestry patients and associated with poorer clinical outcomes in an external validation cohort. Additionally transcriptome analysis revealed decreased CD8+ T cell infiltration with increasing African ancestry, suggesting tumor immune microenvironment differences with potential therapeutic implications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/721962v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@12125b9org.highwire.dtl.DTLVardef@133c787org.highwire.dtl.DTLVardef@707af0org.highwire.dtl.DTLVardef@97615c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThis study represents the most ancestrally diverse whole-genome sequencing characterization of high-grade endometrial cancer, with 62% of patients of African ancestry. C_LIO_LIMECOM focal amplification preferentially targets the oncogenic short isoform (EVI1) and is more frequent in patients of African ancestry. C_LIO_LIAfrican ancestry is associated with reduced CD8+ T cell infiltration and differential activation of immune and metabolic pathways in copy-number high endometrial tumors. C_LI

The RNA polymerase-associated factor 1 complex (PAF1C) is an evolutionarily conserved transcription elongation complex that regulates RNA polymerase II-mediated transcription and chromatin modification. LEO1, a core subunit of PAF1C, has been implicated in developmental gene regulation, WNT signaling, and leukemogenesis; however, its role in solid tumor progression remains poorly understood. In this study, we found that although LEO1 expression is generally elevated in colorectal cancer (CRC), its expression is reduced in stage IV tumors and is associated with poor clinical outcomes. To investigate its function, we established LEO1 -deficient HCT116 cell line and performed transcriptomic analyses. Loss of LEO1 suppressed epithelial differentiation and developmental gene programs while inducing cell cycle delay. Despite these changes, LEO1-deficient cells exhibited aggressive phenotypes, including enlarged nuclei and increased expression of migration-associated genes, which were further enhanced under glucose deprivation. Motif analysis identified FOXM1 as a key regulator of these migration-related genes. Mechanistically, LEO1 deficiency promoted accelerated transcriptional activation of GRP78, a central regulator of endoplasmic reticulum (ER) stress adaptation. GRP78 was required for survival under ER stress conditions, and its inhibition suppressed both migration and migration-associated gene expression. In addition, transcriptomic analyses revealed upregulation of cholesterol metabolism-related genes in LEO1-deficient cells. Consistently, treatment with the HMG-CoA reductase inhibitor atorvastatin selectively impaired their survival, indicating cholesterol metabolic dependency. Collectively, these findings demonstrate that LEO1 loss promotes ER stress-adapted migration and cholesterol metabolic dependency in CRC, suggesting that these pathways may represent therapeutic vulnerabilities in metastatic LEO1-low CRC.