NAR Cancer

APOBEC3B (A3B) is a single-stranded DNA cytosine deaminase overexpressed in cancer, where it causes genomic DNA damage and mutations associated with tumor evolution. Enforced A3B overexpression triggers a dependency on the replication-stress response in different cellular models. However, whether endogenous A3B in cancer cells might yield a similar vulnerability is unclear. Here, we investigate how endogenous A3B expression and catalytic activity affect sensitivity to CHK1 inhibition, using two cancer cell lines, JHOC5 and U2OS. A3B-expressing cancer cells are sensitive to two chemically distinct CHK1 inhibitors, GDC-0575 and Prexasertib. CHK1 inhibitor sensitivity is reduced by A3B CRISPR knockout and restored by re-expressing wildtype A3B in knockout cells. Moreover, an endogenous A3B-E255A catalytic mutant generated by homology-directed repair phenocopies the reduced CHK1 inhibitor sensitivity of A3B-null cells, demonstrating a DNA deamination-dependent mechanism. CHK1 inhibition induces replication-associated DNA damage and cell-cycle perturbation dependent on A3B expression. As a result, A3B-expressing cells accumulate more pan-nuclear {gamma}H2AX, aberrant DNA-content profiles, and an expanded EdU-negative S-phase population, which are hallmarks of stalled DNA replication. In comparison, A3B-null and A3B-E255A cells retain defined cell-cycle distributions and are less sensitive to CHK1 inhibition. Together, these findings identify endogenous A3B-catalyzed deamination as a therapeutically actionable source of replication-associated DNA damage that renders tumor cells selectively dependent on CHK1 function. Statement of significanceAPOBEC3B causes mutations in cancer cells and simultaneously imposes DNA replication stress. This combines to sensitize tumor cells to chemical inhibitors of the DNA damage response kinase CHK1.

Oncogenes and tumor-suppressor genes play opposing roles in cancer biology to promote and restrict growth, respectively. Codon usage patterns interface with tRNA modifications to control translation, leading to gene-specific codon signatures with regulatory potential. As such, codon-biased translational regulation has been identified as a driver of proliferation and drug resistance in multiple cancers. We used advanced codon analytics methods to characterize and compare codon usage bias in oncogenes and tumor suppressor genes (TSGs) from humans and mice at group and gene-specific levels. We demonstrate that human oncogenes exhibit a distinct and opposing codon usage pattern to TSGs. This phenomenon is also present in mice but with less distinct oncogene bias relative to humans. Further comparison to 447 gene ontology groups demonstrated that human oncogenes have the most distinct codon usage patterns in the genome, while also highlighting that codon bias can separate functionally related genes and pathways from other biological processes. Using gene-specific codon analytics, we determined that human oncogenes have two types of extreme codon bias: a large group (N = 43) over-using G/C ending (GC3) codons and a smaller group (N = 12) over-using A/U (AU3) ending codons. While GC3 bias has been linked to increased translation in general, the AU3 finding suggests that genetic, environmental, or stress-related signals could drive the translation of this small group of oncogenes. The less extreme bias observed in mouse oncogenes and tumor suppressors likely underscores species-specific differences in oncogenic translation programs. Together, our findings highlight codon usage bias as a potential determinant of oncogene expression, provide a framework for ontology-based codon analysis, and uncover on species-specific differences in oncogene translation and codon usage biases.

Background: Colorectal cancer (CRC) is a major cause of cancer-related mortality, with distant metastasis strongly associated with poor clinical outcomes. Integrating transcriptomic and epigenomic data through machine learning may improve the molecular characterization of metastatic CRC. Methods: We analyzed 518 primary tumors from the TCGA-COAD/READ cohort (436 non-metastatic [M0] and 82 metastatic [M1]) with matched RNA-seq and DNA methylation data. Five machine learning classifiers were evaluated for discrimination of metastatic status using stratified nested cross-validation. Model explainability was assessed using model coefficients for linear classifiers and SHAP-based feature importance for tree-based models. Differential expression, functional enrichment, methylation-expression correlation, immune-related transcriptional scoring, statistical mediation, and survival analyses were subsequently performed. Results: Integrated RNA-seq and DNA methylation showed the strongest discrimination between M0 and M1 tumors, with SVM reaching a ROC-AUC of 0.787 +/- 0.047. Six features - ARC, ASPDH, C13orf15, C4orf23, GPATCH3, and cg12040555 - ranked among the top 20 predictors across four models with consistent directions. cg10057218 methylation was inversely correlated with GSDMB expression (Spearman rho = -0.589) and increased in M1 tumors. Statistical mediation indicated a significant indirect association between M1 status and reduced GSDMB expression through cg10057218 methylation (indirect effect = -0.370; 95% CI [-0.519, -0.224]; proportion mediated = 82.1%). M1 tumors also exhibited reduced immune-related transcriptional activity. A 20-feature canonical molecular score separated M1 from M0 tumors (in-sample ROC-AUC = 0.918), while the corresponding Logistic Regression model achieved a nested cross-validated ROC-AUC of 0.781 +/- 0.046. Higher scores were associated with shorter overall survival (log-rank p = 1.67 x 10^-7). Conclusions: Transcriptomic and DNA methylation integration identified a consistently prioritized multiomics feature set associated with metastatic CRC. The findings highlight coordinated molecular and immune-related differences between M0 and M1 tumors and identify cg10057218-associated GSDMB repression as a candidate epigenetic feature of metastatic disease.

The mutational landscape of neural innervation axes in cancer has garnered increasing attention due to their significant influence on tumorigenesis, metastasis, and treatment resistance. This study provides an overview of the alterations of neural signaling pathways based on multi-omics data analysis. In particular, we conducted a comprehensive analysis of mutations in key genes associated with neurotrophin signaling, norepinephrine and cholinergic pathways, alongside paracrine and synaptogenic factors. Our findings reveal that 65% of patients in a pancancer cohort exhibit at least one somatic alteration in these pathways, with neurotrophin genes being the most frequently altered. Notably, alterations in these genes (e.g., NGF) correlate with poor patient survival across various cancer types, suggesting their oncogenic potential. We also identified significant co-occurrence of mutations and differential expression patterns from both bulk and single-cell RNAseq data, indicating complex interactions among these pathways that contribute to tumor innervation and neuroplasticity. This study underscores the necessity of understanding the crosstalk between neural signaling and cancer biology to improve patient stratification based on mutational profiles and identify potential therapeutic targets. Further research utilizing advanced genomic techniques is essential to elucidate the mechanistic roles of these pathways in tumor innervation.

This study proposes a novel therapeutic strategy to suppress cancer growth by modulating the MDM2-p53 axis via Alternative Polyadenylation (APA). MDM2 normally promotes tumorigenesis by ubiquitinating and degrading the tumor suppressor p53. In cancer cells, preferential use of proximal polyadenylation signals (PAS) results in shortened 3'UTRs, allowing oncogenic transcripts like MDM2 to evade nuclear sequestration mediated by Inverted Alu (IRAlu) double-stranded RNA structures. We hypothesized that forcing distal PAS usage would elongate the MDM2 mRNA, promoting its nuclear retention and reducing protein translation, thereby restoring p53 activity. Using CRISPR-Cas9, we targeted and deleted the most frequent proximal PAS in the MDM2 3'UTR of A549 cells. Successful genome editing was confirmed via PCR. As expected, Western blot analysis showed a significant reduction in MDM2 expression in PAS-edited cells. However, experimental outcomes contradicted our initial hypothesis: edited cells exhibited higher viability under doxorubicin treatment compared to wild-type cells. Furthermore, despite decreased MDM2 levels, a concurrent reduction in phosphorylated p53 (p-p53) was observed. These unexpected results suggest that MDM2 3'UTR elongation may trigger a non-canonical regulatory mechanism that bypasses the traditional MDM2-p53 interaction. This study highlights the complexity of post-transcriptional regulation and suggests that APA-mediated gene modulation can induce unforeseen compensatory survival pathways in cancer cells, necessitating further investigation into the broader functional landscape of elongated 3'UTRs.

Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted a PE of a tumor suppressor (EZH2) from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, escaped AS-NMD, and restored the protein expression. This subsequently reinstated H3K27 histone methylation and rescued defective chromatin regulation associated with impaired hematopoietic stem cell differentiation. Finally, we showed the preferential advantages of CRISPR over the antisense technology we recently developed targeting the PE of EZH2. Therefore, the CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases.

Background: Biopsy-measurable biomarkers that stratify colorectal cancer (CRC) by therapeutic responsiveness remain scarce. We asked whether dual MDM2 promoter (P1/P2) usage acts as a molecular switch separating two opposed CRC phenotypes: a chromosomal instability type and an environment-adaptive type (microsatellite instability-like/serrated pathway with gastric metaplasia). Methods: Sixty three organoid samples from 22 CRC patients were classified by deep-learning morphology (VGG16) and an array based MDM2 Splicing Index. P1 and P2 signatures were evaluated in TCGA-COAD/READ (n = 624) and GSE39582 (n = 519), and drug responses were examined in cell lines (GDSC2, DepMap) and 65 independent CRC organoid lines. Statistics used Wilcoxon, Kruskal Wallis and Spearman tests (Benjamini Hochberg-corrected) and patient-level mixed models. Results: Deep earning classification reached 98.5% test accuracy; morphology corresponded to isoform usage: Type1 (compact glandular) morphology predominated in P1-dominant samples (median Type1 fraction 0.826 versus

PI3K/AKT signalling is a key driver of ovarian cancer progression and an important therapeutic target. Its deregulation is usually explained by genetic changes and altered signalling at the cell surface. Here, we identify an additional layer of pathway control, in which a change in the level of a cancer-associated ribosomal RNA modification alters the translation of proteins that regulate PI3K/AKT activity. SNORA81 is a high-grade serous ovarian carcinoma-associated H/ACA small nucleolar RNA that directs pseudouridylation of 28S ribosomal RNA at position U4636. Integration of tumour transcriptomes with independent SNORA81-depletion datasets identified 22 SNORA81-linked transcripts with reversed disease-associated expression, 19 of them linked to PI3K/AKT signalling. SNORA81 depletion reduced AKT phosphorylation, impaired cell growth, migration, and altered cell-cycle distribution. Molecularly, polysome profiling following SNORA81 depletion identified 524 mRNAs with altered ribosome association, 485 of which changed independently of RNA abundance and included 29 PI3K/AKT regulators. SNORA81 depletion reduced ribosome association of ITGB4, RASGRP2, IGF1 and FGF18 transcripts, and reduced ITGB4 and RASGRP2 protein abundance. IRAK1 and SPC24 acted as downstream RNA-response genes contributing to selected SNORA81-associated phenotypes. ITGB4 expression partially restored cell growth and restored downstream RNA responses after SNORA81 depletion. These findings identify rRNA modification as a translational regulator of PI3K/AKT signalling in ovarian cancer.

Purpose Long non-coding RNAs (lncRNAs) are important regulators of tumor biology, but their dependence on major oncogenic drivers and cancer specificity remain poorly characterized. We aimed to identify lncRNAs regulated by MYC, mutant KRAS, and mutant TP53 across colorectal, lung, and pancreatic cancer and to determine their functional relevance and molecular mechanism. Methods We performed an integrative analysis of lncRNA expression profiles driven by MYC, mutant KRAS, and mutant TP53 across colorectal, lung, and pancreatic cancer cell lines. Candidate lncRNAs were functionally characterized using assays of cancer cell viability, migration, and clonogenicity. Expression in patient-derived tumor and normal tissues and downstream molecular pathways were investigated. Results MYC was associated with the most extensive lncRNA regulation, with expression patterns largely independent of tissue origin. MYC-dependent LINC00997 and the previously uncharacterized AC104447.1, as well as specifically KRAS-dependent RAKRAR (RASSF3 antisense KRAS regulated lncRNA) promoted cancer cell viability, migration, and clonogenicity. LINC00997 and AC104447.1 expression was elevated in specific tumor types compared to normal tissues. We identified CAMOR (CARNMT1 Antisense Multiple-Oncoprotein Regulated lncRNA), a pan-cancer lncRNA upregulated by all three oncogenic drivers. CAMOR promoted viability and migration specifically in cancer cells across analyzed cell lines and tumor types. Mechanistically, CAMOR and neighboring CARNMT1 form a negative feedback regulatory loop affecting CAMOR oncogenic activity. Distinct downstream gene expression signatures suggested that the studied lncRNAs act through different regulatory processes. Conclusion Our findings expand the repertoire of oncogenic lncRNAs and demonstrate that integrating multiple oncogenic contexts enables identification of both driver-specific and broadly acting lncRNAs, providing a framework for identifying lncRNAs that may contribute to cancer development and represent potential diagnostic or therapeutic targets.

Homologous recombination deficiency (HRD) can force cancer cells to rely on alternative DNA repair pathways, including microhomology-mediated end joining (MMEJ), an error-prone mechanism that can generate deletions with microhomology at repair junctions. Because such patterns may reflect DNA repair defects with clinical relevance, this study aimed to assess the biological and prognostic significance of microhomology-associated deletions and optimize their detection parameters to improve patient stratification and inform targeted therapeutic strategies, including PARP inhibition. We optimized the microhomology length threshold (Mlt parameter) using signal-to-noise ratios and survival models. We evaluated the utility of whole-exome (WES) versus whole-genome sequencing (WGS). We then investigated how Loss of Function (LoF) alterations affect microhomology-associated deletion burden and assessed their prognostic significance across an ovarian cancer (OV) cohort and a TCGA Pan-Cancer dataset. Applying an Mlt = 2 threshold maximized the precision and statistical reliability of microhomology-associated deletion identification. WGS yielded higher reproducibility, whereas WES proved insufficient for accurate estimation. Inactivation of tumor suppressor genes, including RB1 and CCDC122, was associated with increased microhomology-associated deletion burden. Importantly, higher burden correlated with extended overall survival in ovarian cancer. Furthermore, while baseline microhomology-associated deletion burden varies by tumor type, Pan-Cancer analysis identified candidate gene-level alterations, including FDX1 and PDE8B, whose LoF increased microhomology-associated deletion burden across tumors. Precise parameterization combined with WGS resolution highlights the extent to which specific gene losses affect deletion burden consistent with MMEJ-mediated repair. Identifying these genetic vulnerabilities and the resulting deletion burden may provide a candidate prognostic biomarker and foundation for personalized therapies.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian and breast cancers with homologous recombination deficiency (HRD). Despite substantial initial efficacy, the frequent development of acquired resistance poses a critical clinical challenge, necessitating a deeper understanding of the underlying molecular mechanisms. While genomic profiling provides information on baseline HRD status, it is insufficient to capture the dynamic and functional adaptations accompanying resistance during treatment. In this study, we investigated the role of alternative splicing in PARPi resistance using longitudinal pre- and post-treatment RNA-sequencing data from an ovarian cancer patient cohort (n=40). We demonstrated that profiling alternative splicing dynamics and differentially used transcripts (DUTs) provides transcriptomic insights distinct from conventional differential gene expression analysis. Notably, we observed significant differences between acquired resistance (AR) and innate resistance (IR) groups. Specifically, longitudinal analysis revealed that AR tumors exhibit a distinct transcript usage pattern, characterized by an increase in canonical protein-coding transcripts involved in cell-cycle processes and a decrease in non-canonical non-coding transcripts within the homologous recombination pathway following treatment. Extending this comparison across AR and IR tumors at both pre- and post-treatment stages, these transcriptomic features were most pronounced in the AR post-treatment group. Further analysis of subsequent treatment outcomes showed that, among AR patients, greater resistance-associated transcriptomic remodeling was associated with poorer response and shorter progression-free survival. The resistance-associated transcriptomic signatures identified through longitudinal profiling were further recapitulated in an independent pre-treatment cohort (n=71; 43 AR and 28 IR), with more pronounced differences among BRCA wild-type patients. Taken together, our findings identify differential transcript usage as dynamic transcriptomic features associated with acquired PARPi resistance and establish a framework for utilizing longitudinal transcriptome dynamics to investigate the molecular basis of clinical drug resistance.

Decades of research have shown that tumor hypoxia is associated with resistance to anti-cancer treatments. Analysis of TCGA gene expression profiles indicates that NSCLC is among the most hypoxic of cancers despite the high levels of oxygen in the surrounding lung tissue. Several groups have shown that extrinsic factors such as poorly formed tumor vascular contributes to tumor hypoxia. Here, we have investigated the possibility that genetic abnormalities within the tumor also contribute to the development of hypoxia. Our analysis of NSCLC patient datasets in the Cancer Genome Atlas (TCGA) PanCancer and ORIEN datasets revealed a strong correlation between tumor hypoxia and amplification of chromosome 3q which is found in up to 40% of NSCLC. Several oncogenic driver genes have been identified in 3q, and we identified a passenger gene encoding mitochondrial complex I subunit NDUFB5 at 3q26.33. To provide experimental evidence that NDUFB5 amplification can drive tumor hypoxia, we have used CRISPR activation technology to generate murine cells overexpressing the endogenous NDUFB5 gene. We found that cells overexpressing NDUFB5 have elevated rates of oxygen consumption, and tumors grown from these cells have increased amounts of hypoxia with associated treatment resistance. Here, we investigate the impact of manipulating NDUFB5 gene expression on mitochondrial complex I activity and experimentally validate the clinical observations that NDUFB5 overexpression leads to increased levels of intratumoral hypoxia and increased resistance to radiation therapy and immunotherapy.

BackgroundThe KMT2D histone 3-lysine 4 methyltransferase (also known as MLL4) is a critical chromatin regulator, which is frequently inactivated by gene mutations in various types of cancer including colorectal cancer (CRC). Several lines of evidence suggest a strong association between chromatin regulation, tumor immunity and drug sensitivity. To explore the role of KMT2D in tumor immunity, we analyzed genomic and clinical datasets from The Cancer Genome Atlas and Memorial Sloan Kettering Cancer Center for various cancer entities. ResultThe results showed that KMT2D-mutated CRCs displayed a significantly higher expression of immune checkpoint regulators including PD-L1, CTLA4 and CD8 when compared to KMT2D wild-type CRCs. Mutations in KMT2D correlate with elevated T-effector and interferon-{gamma} gene signatures indicating infiltration with active immune cells. By performing immune cell deconvolution from transcriptomic data, CRCs harboring KMT2D mutations are associated with increased infiltration of CD8+ T cells, NK cells and macrophages, but they display low amounts of Treg cells suggesting that KMT2D loss-of-function mutations correlate with an immunologically "hot tumor" phenotype. Furthermore, patients with KMT2D mutant CRC displayed better clinical responses to immune checkpoint inhibitor (ICI) therapy with an improved overall patient survival compared to patients with KMT2D wild-type CRC. Importantly, this effect did not exist in cohorts of CRC patients, which have not been treated with immunotherapies. To further understand the differential drug response effect related to KMT2D, we treated wild-type and KMT2D inactive epithelial cells with various cancer drugs. KMT2D mutant cells exhibited increased DNA damage and higher sensitivity to cisplatin in comparison to KMT2D wild-type cells. ConclusionsKMT2D loss-of-function mutations are associated with a positive outcome of immunotherapy efficacy in CRC and responses to cisplatin treatments. The stratification of CRC patients by KMT2D gene status may enable personalized approaches by identifying CRC patient populations that benefit from ICI therapy and chemotherapy.

Background: DNA damage response (DDR) pathways are central regulators of genome maintenance and major determinants of cancer cell survival. The extensive genomic instability and high replicative stress that characterize glioblastoma render tumor cells highly dependent on DDR pathways to preserve genome integrity and sustain proliferation. This reliance creates potential therapeutic vulnerabilities, making the systematic identification of essential DDR genes a promising strategy for uncovering novel therapeutic targets. Methods: We developed DNA Damage Response KnockOut Library (DDRKOL), a custom CRISPR/Cas9 sgRNA library targeting 819 DDR genes with approximately 10 sgRNAs per gene, together with positive (essential), negative (non-essential) and non-targeting controls. Parallel depletion screens were performed in Cas9-expressing U87-MG and A172 cells cultured for 15 population doublings. Hits were prioritized utilizing TCGA and DepMap databases and validated by viability, clonogenic, apoptosis and GFP competition assays. Clinically relevant patient-derived glioblastoma spheroids and an orthotopic xenograft model was employed to characterize the effects of hit genes. Results: Sequencing confirmed near-complete recovery of the designed sgRNAs from the plasmid pool, with uniform representation across the library and complexity preserved through transduction and selection. Essential-gene controls depleted strongly while non-targeting controls remained neutral, confirming screen performance in both cell lines. The screens identified DDR dependencies in each line and defined a shared core composed of 20 genes belonging to homologous recombination, nucleotide excision repair and ATM/DSB signaling pathways. This shared dependency landscape highlighted four high-confidence candidate genes (TOP2A, CDK1, XRCC6, and RAD21), which were successfully validated across multiple orthogonal assays. These genes displayed grade-associated expression and their expressions were positively correlated with proliferation markers in TCGA. Individual knockouts reduced viability, colony formation and competitive fitness, induced apoptosis, and impaired growth of patient-derived glioblastoma spheroids. Both genetic depletion and pharmacological inhibition of TOP2A induced S/G2-M cell cycle arrest. In orthotopic xenografts, TOP2A depletion prevented tumor progression, and led to significantly prolonged survival. Conclusion: DDRKOL represents a robust and versatile focused CRISPR platform for systematic functional interrogation of the DDR associated genes. Using glioblastoma, we demonstrate that the library reliably identifies biologically significant and clinically relevant genetic dependencies through multiple orthogonal validation approaches. As a reusable platform rather than a disease-specific tool, DDRKOL can be broadly applied across diverse biological contexts to discover context-dependent DDR vulnerabilities, therapeutic targets, and mechanisms of treatment resistance.

BRCA1 promoter methylation predicts sensitivity to PARP inhibitors in high-grade serous ovarian cancer, yet therapeutic resistance is common and mechanistically unresolved. Using long-read direct DNA sequencing of patient-derived xenografts and cell lines, we resolved BRCA1 methylation at single-molecule resolution with structural and transcriptomic analyses. We revealed two convergent PARP inhibitor and platinum resistance mechanisms, validated in patient tumors. First, focal, allele-specific loss of BRCA1 methylation arose through local cis-acting genomic alterations, instead of global epigenetic reprogramming. Engineered in cis sequence alterations near the methylated BRCA1 promoter were sufficient to induce methylation loss, restore homologous recombination, and confer resistance. Similar associations were observed across the genome, suggesting this mechanism extends beyond BRCA1. Second, BRCA1 expression was restored despite intact promoter methylation via structural variant-mediated promoter bypass or alternative transcription initiation. Together, these findings redefine BRCA1 methylation loss as a locus-restricted process and reveal multiple routes by which tumors escape PARP inhibitor therapy. Statement of SignificanceWe show that high-grade serous ovarian cancers can restore BRCA1 expression after therapy through multiple genomic mechanisms, including local methylation loss and promoter bypass, thereby re-establishing homologous recombination and driving PARP inhibitor resistance. These findings challenge reliance on BRCA1 methylation alone as a predictive biomarker and support rational combination therapies for more durable responses.

Retinoblastoma (RB), the most common pediatric intraocular malignancy, is initiated by biallelic inactivation of the RB1 tumor suppressor gene. Although LINE-1 (L1) retrotransposon activation has been linked to genomic instability and tumor evolution in several adult cancers, its contribution to pediatric malignancies remains poorly understood. Here, we investigated L1 expression in retinoblastoma samples from an Indian pediatric cohort by integrating transcriptomic, locus-specific, and immunohistochemistry. Transcriptomic profiling revealed increased L1 expression in RB tumors compared with control retinal tissues, which was validated by ORF1p immunostaining in tumor sections. Locus-specific analysis identified a subset of transcriptionally active L1 loci, suggesting selective activation of young L1 elements. We also observed altered expression of genes associated with proliferation, chromatin regulation, and oncogenic signaling, including MYCN, MDM2, E2F3, SOX4 and CREBBP. Our findings identify previously underexplored L1 activation as a molecular feature associated with retinoblastoma and suggest that disruption of transposon silencing accompanies the RB transcriptional landscape. Validation in larger patient cohorts will be important; however, this study provides a foundation for exploring L1 activity as a potential molecular marker and future therapeutic vulnerability in retinoblastoma. HighlightsO_LITranscriptomic profiling reveals increased LINE-1 expression in retinoblastoma. C_LIO_LIORF1p protein is robustly expressed in retinoblastoma. C_LIO_LIA subset of LINE-1 loci exhibits transcriptional activation, suggesting locus-specific derepression. C_LI In BriefRetinoblastoma is initiated by RB1 loss, but the contribution of transposable elements to its molecular landscape remains poorly understood. Here, we show that retinoblastoma is associated with LINE-1 promoter hypomethylation, increased LINE-1 transcript expression, ORF1p accumulation, and preferential activation of selected LINE-1 loci. These changes occur together with altered expression of oncogenic signalling pathways and host factors involved in transposable element repression, identifying dysregulated LINE-1 control as a molecular feature of this paediatric cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/740688v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@10dcf8forg.highwire.dtl.DTLVardef@1b5d07forg.highwire.dtl.DTLVardef@18b5116org.highwire.dtl.DTLVardef@1788145_HPS_FORMAT_FIGEXP M_FIG C_FIG Retinoblastoma exhibits epigenetic activation of LINE-1 characterized by promoter hypomethylation, increased expression of young LINE-1 elements, and ORF1p accumulation. This is accompanied by coordinated transcriptomic remodeling and disruption of host mechanisms that normally restrain transposable elements, linking LINE-1 activation to the molecular landscape of retinoblastoma.

Approximately 10% of bladder, upper urinary tract, and endometrial cancers harbor deleterious somatic alterations in nucleotide excision repair (NER) pathway genes. Although platinum-based chemotherapy is the standard treatment for NER-deficient tumors, its utility is limited by nephrotoxicity, motivating alternative strategies that avoid direct DNA targeting. Here, we employed a genome-wide CRISPR-Cas9 screen to identify synthetic lethal vulnerabilities in NER-deficient cancer cells. An ERCC4-null RT112/84 bladder cancer cell line was generated and validated by Sanger sequencing and Western blotting, and functional NER deficiency was confirmed using a host cell reactivation assay. Parallel screens in ERCC4-deficient and isogenic wild-type cells. nominated SLC25A28 as the top-ranked synthetic lethal candidate in ERCC4-deficient cells, an interaction further supported by DepMap dependency. Clonogenic assays confirmed that loss of SLC25A28 selectively impaired cell viability in cells deficient for ERCC2, ERCC3, ERCC4, or ERCC5 and inducible knockout selectively suppressed ERCC4-deficient xenograft growth in-vivo. Mechanistically, RNA sequencing followed by gene set enrichment analysis revealed enrichment of oxidative stress-related pathways, including cellular responses to reactive oxygen species (ROS) and DNA damage signatures, upon SLC25A28 loss. These transcriptional changes are consistent with increased oxidative stress and DNA damage, placing heightened demand on NER-mediated repair. Direct measurement confirmed increased intracellular ROS. Collectively, our nominates SLC25A28 as a previously unrecognized synthetic lethal vulnerability in NER-deficient cancers and implicate mitochondrial iron homeostasis as a candidate metabolic dependency that may be targeted independently of platinum-based chemotherapy.

PurposeSWI/SNF (BAF) chromatin remodeling complex alterations are common in urothelial carcinoma, yet no biomarker-directed therapeutic strategies have been established for this population. We investigated whether BAF alterations delineate a biologically distinct, therapeutically actionable urothelial carcinoma subtype. Experimental DesignWe performed integrative genomic and transcriptomic analyses of 792 urothelial carcinoma tumors from the Oncology Research Information Exchange Network (ORIEN) and validated findings in the TCGA-BLCA cohort. Mechanistic studies incorporated RNA sequencing and ATAC-seq following histone deacetylase (HDAC) inhibition. Functional dependencies were assessed using patient-derived xenograft organoids and cell line models. Clinical relevance was explored in a biomarker-enriched investigator-initiated trial. ResultsApproximately half of urothelial carcinoma tumors exhibited BAF alterations, defining a previously unrecognized chromatin-altered molecular subtype characterized by activation of proliferative programs, loss of lineage identity, and altered metabolic signaling. This subtype was enriched for transcriptomic programs associated with HDAC inhibitor sensitivity and depleted of HDAC inhibitor resistance signatures. Mechanistically, HDAC inhibition induced widespread chromatin remodeling with reduced accessibility at AP-1 and TEAD-associated regions, and downregulation of E2F- and MYC-driven transcriptional networks. Functional studies confirmed enhanced HDAC inhibition sensitivity in ARID1A-mutated cell lines and a patient-derived organoid model. Early clinical observations demonstrated a durable responder treated with HDAC inhibitors and immunotherapy. ConclusionsBAF alterations define a chromatin-dependent tumor state in urothelial carcinoma that is selectively vulnerable to HDAC inhibition. Integrating genomic, epigenomic, functional, and early clinical evidence, these findings provide a rationale for biomarker-enriched clinical trials and HDAC inhibitor-based combination strategies in urothelial carcinoma.

Schlafen 11 (SLFN11) is a key determinant of sensitivity to DNA-damaging therapies in multiple cancers including leukemia, Ewing sarcoma, head and neck cancers, mesothelioma and small cell lung cancer (SCLC). Here, utilizing immunofluorescence microscopy and single-cell RNA sequencing we characterized SLFN11 expression across eight different human cancer cell lines and found cell-to-cell heterogenous expression both at the protein and RNA levels. Gene set enrichment and pathway analyses show that SLFN11 is coexpressed with a distinct stress-response state and enhanced genome maintenance programs while being inversely correlated with protein translation pathways. Publicly available single-cell RNA-sequencing datasets of human SCLC tumors confirmed the heterogeneous distribution of SLFN11 expression and confirmed its association with replication-related transcriptional programs. These findings indicate that heterogeneous SLFN11 expression reflects dynamic regulation at the single-cell level. They also underscore the limitations of bulk proteomic and transcriptome profiling and point to a potential reservoir of intrinsic chemotherapy resistance.

Glyoxal (GO) is a small, highly reactive molecule that is produced naturally in cells during normal metabolism and can also come from processed foods and oxidative stress. Because of its high reactivity, glyoxal can modify DNA and proteins to form harmful products called advanced glycation end-products (AGEs), which have been linked to diseases such as diabetes, cancer, and aging. Although glyoxal is known to modify DNA and proteins, it is not well understood whether it can form DNA-protein crosslinks (DPCs), a type of DNA damage in which proteins become permanently attached to DNA. In this study, we investigated glyoxal induced DPC formation in HeLa cells using biochemical assays and mass spectrometry-based proteomics experiments. We observed that glyoxal exposure elevated cellular DPC formation in a concentration- and time-dependent manner. Cells with reduced SPRTN expression accumulated higher levels of DPCs, suggesting that SPRTN plays an important role in repairing glyoxal induced DNA damage. Proteomics experiments revealed 469 proteins exhibited elevated DNA association in glyoxal-treated samples, including histones and other proteins involved in chromatin organization, DNA replication, DNA repair, and gene expression. In-vitro experiments confirmed that glyoxal can directly crosslink DNA with histone proteins. Overall, this study provides the first evidence that glyoxal forms DNA-protein crosslinks in human cells. These findings provide a foundation for future studies on the chemical structure, biological effects and repair of glyoxal induced DNA-protein crosslinks and their possible role in human disease.