NAR Cancer

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.

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.

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.

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.

Bulk-tumor expression signatures can reflect cellular composition as well as tumor-cell programs. We evaluated a frozen, equal-weight 87-gene score in colorectal cancer using 11 Gene Expression Omnibus (GEO) cohorts, TCGA COAD/READ, and E-MTAB-12862. In GEO (1,783 tumors; 441 progression-related events), the hazard ratio per score standard deviation was 1.33 (95% confidence interval, 1.19-1.49). The estimate was similar in seven disease-free-survival cohorts. In 376 TCGA tumors, the hazard ratio was 1.25 (1.03-1.52) for progression-free interval. The association did not reproduce in E-MTAB-12862: among 948 stage I-III tumors with 436 recurrence-free-survival events, the hazard ratio was 1.05 (0.95-1.16). The frozen score correlated with CMS4, cancer-associated fibroblast, and stromal measures. The stromal-remodeling module produced a GEO estimate similar to the full score, whereas removing that module attenuated the association. Composition residualization did not detect additional prognostic information after accounting for the measured CMS4 and stromal components. Single-cell and spatial analyses localized the gene program to fibroblast-rich compartments. The 87-gene score mainly reflects a fibroblast-rich, matrix-remodeling component of CMS4 rather than an independent prognostic subtype. Its prognostic association varied across cohorts, and current evidence does not support clinical use.

Oral squamous cell carcinoma (OSCC), a highly aggressive subtype of head and neck cancer, remains a major clinical challenge with limited therapeutic options and poor survival outcomes. The Protein Inhibitor of Activated STAT1 (PIAS1), a SUMO E3 ligase involved in transcriptional regulation, DNA repair, and epithelial-mesenchymal transition (EMT), exhibits cancer type-specific roles, but its function in OSCC has not been well characterized. Here, we investigated the molecular and functional role of PIAS1 in OSCC progression and prognosis. All patient cohorts analysed here comprise HPV-negative disease. Analysis across multiple cohorts revealed that PIAS1 mRNA expression is significantly lower in tumor tissues than in normal oral cavity squamous epithelium, including in a cohort of 60 patient-matched tumor and normal pairs. Within the tumor compartment, protein abundance of PIAS1 correlated positively with patient survival. Functional assays using OSCC cell lines demonstrated that PIAS1 acts in a SUMO E3-dependent manner to suppress TGF{beta}-induced migration and invasion. Transcriptomic profiling of PIAS1-depleted cells revealed alterations in expression of genes with relevance in TGF{beta} signaling and pro-invasive cellular responses. Further analyses showed that 46 of these differentially altered hits intersected with The Cancer Genome Atlas (TCGA)-OSCC datasets, where four of these genes, including INHBA, associated with cell stemness-pathway. Spatial analysis of the mRNA abundance of INHBA within OSCC-derived surgical specimens showed increased abundance at the invasive front, and positively associated with aggressive tumor phenotypes, poor patients' outcomes, and resistance to immunotherapy. Single-cell RNA-seq analyses of INHBA revealed increased levels within tumor cells and cancer-associated fibroblasts (CAFs), suggesting potential tumor-intrinsic and extrinsic (tumor microenvironment) sources and hence actions. Collectively, our findings establish PIAS1 as a tumor suppressor in OSCC that restrains TGF{beta}-driven aggressive behaviour. Furthermore, we identify INHBA as a potential therapeutic target, among other genes characterized by spatially restricted, invasive tumor phenotypes.

Germ cell tumors (GCTs) are highly sensitized to cell death in response to DNA damaging agents, a property that underlies the success of current chemotherapeutic regimens. To address the molecular basis for this, known as apoptotic priming, we evaluated how different BCL2 family members modulate the heightened sensitivity of GCTs to therapy. Our analysis of human GCTs finds consistently high expression of the pro-survival factors MCL1 and BCL2L1 (BCLX) in a cohort of primary tumors and in their embryonic precursor cells, frequently accompanied by copy number gains of these loci and reciprocal losses of their pro-apoptotic interaction partners and inhibitors, PMAIP1 (NOXA) and BAD. We find that co-inhibition of MCL1 and BCLX using selective BH3 mimetics results in a potent synthetic lethality in multiple GCT embryonal carcinoma cell lines. When these cell lines were cultured with the DNA damaging agents cisplatin or etoposide, inhibition of MCL1 or BCLX potentiated their apoptotic effect in undifferentiated embryonal carcinoma cell lines, but not in retinoic acid-differentiated cells. The inhibition of MCL1 also heightened cisplatin sensitivity in p53-deficient or -mutant cell lines, which is associated with resistance to therapy. Employing an in ovo human xenograft model, we validate that the combination of cisplatin and MCL1 inhibition enhanced the therapeutic response by eliminating tumor cells. Our findings identify MCL1 and BCLX as critical factors to maintain GCT viability and as putative therapeutic targets to further augment GCT responsiveness to DNA damaging agents.

Background: Archival biobanks of formalin-fixed paraffin-embedded (FFPE) specimens represent a vast and underused resource for retrospective molecular studies linked to clinical follow-up. However, fixation-induced chemical modification and nucleic acid fragmentation limit scalable genomic and transcriptomic profiling. FFPE-CUTAC (Cleavage Under Targeted Accessible Chromatin) is an RNA polymerase II-targeted DNA assay specifically designed to profile regulatory activity in FFPE tissue. Here, we assess its archival robustness, information content, compatibility with RNA-seq, and ability to recover copy-number alterations. Results: In meningioma and breast cancer cohorts, we benchmarked FFPE-CUTAC against matched fresh-frozen RNA-seq, FFPE RNA-seq and FFPE whole-genome sequencing. For FFPE specimens spanning up to 25 years, FFPE-CUTAC showed no systematic collection-year-associated decline in library yield or signal-quality metrics. In contrast, FFPE RNA-seq is more affected by the FFPE tissue degradation and specimen age on read composition, gene-body coverage, and gene detection. By measuring RNA polymerase II occupancy on chromatin rather than mature RNA abundance, FFPE-CUTAC is less constrained by transcript half-life, polyadenylation or transcript-capture design. FFPE-CUTAC extends beyond gene regions to non-coding regulatory elements, while the shared gene-level signals supported its integration with existing RNA-seq cohorts for large-scale, long-term clinical association study. FFPE-CUTAC libraries also retain DNA dosage information and can be used to recover chromosome-arm gain, loss and intact states with 96% concordance to matched whole-genome sequencing. Conclusions: By converting routinely preserved pathology sections into integrated regulatory and copy-number profiles, FFPE-CUTAC provides a practical foundation for constructing clinically annotated disease maps to support molecular stratification, prognostic modeling and treatment-association studies.

Introduction: Sporadic colorectal cancers (CRCs) form following initiating APC mutations, inducing constitutive {beta}-catenin/TCF signaling. Similarly, the guanylyl cyclase C (GUCY2C) tumor suppressor axis is universally silenced in CRC. While the GUCY2C receptor is retained, loss of its paracrine hormones guanylin (GUCA2A) and uroguanylin (GUCA2B) is among the earliest events in transformation. Mutant APC/{beta}-catenin/TCF signaling driving CRC transcriptionally silences GUCY2C hormone expression while suppressing that oncogenic pathway reconstitutes hormone production opposing tumorigenesis. p38 opposes tumor initiation, and its loss promotes transformation in chemical and genetic mouse models of CRC. In healthy intestine, p38 supports a gradient of epithelial differentiation along the crypt-surface axis. Here, we reveal that p38 links regulation of oncogenic APC/{beta}-catenin/TCF signaling and GUCY2C hormone expression. Methods: Regulation of p38 by APC/{beta}-catenin/TCF signaling was defined in four genetically engineered human CRC cell lines with conditional oncogenic APC/{beta}-catenin/TCF signaling and healthy human colon organoids. Expression of p38 was eliminated from conditional human CRC cell lines by pharmacological inhibition, RNA knockdown, and CRISPR/Cas9 gene editing. Transcriptomic analysis of these cells by RNA sequencing defined the set of genes regulated by APC/{beta}-catenin/TCF signaling controlled through p38. Results: In CRC cell lines, APC/{beta}-catenin/TCF signaling suppressed p38 phosphorylation and activity, whereas inhibition of APC/{beta}-catenin/TCF restored p38 signaling. In the absence of APC/{beta}-catenin/TCF signaling, inhibiting p38 repressed GUCY2C hormone expression. Inhibiting APC/{beta}-catenin/TCF signaling enriched phosphorylated p38 at the GUCA2A promoter. In that context, analysis of the CPTAC proteomic database revealed that phosphorylation of p38 and its upstream kinases MKK3 and MKK4 was reduced in human CRC. Finally, transcriptomic analysis revealed that p38 is a critical signaling node controlling the expression of a substantial subset of APC/{beta}-catenin/TCF-regulated genes in human CRC cells and patient tumors. Conclusions: APC/{beta}-catenin/TCF signaling inhibits p38 phosphorylation and transcriptional activity, suppressing GUCA2A and GUCA2B expression and functionally silencing the retained GUCY2C receptor. These findings identify p38 as a signaling intermediary integrating oncogenic APC/{beta}-catenin/TCF activity to extinction of the GUCY2C tumor-suppressor axis and reveal a broader p38-dependent component of the APC/{beta}-catenin/TCF-regulated transcriptome.

Therapeutic resistance remains a barrier to durable treatment of colorectal cancer (CRC), highlighting the need to identify vulnerabilities in resistant tumor cells. REV7 (MAD2L2) is a multifunctional genome-maintenance protein with established roles in REV3-dependent translesion synthesis (TLS) as well as mitotic progression, spindle organization, and chromosome segregation. Although perturbing REV7 dependent DNA damage tolerance has emerged as a strategy to overcome chemoresistance, whether pharmacologic targeting of REV7 exposes vulnerabilities to mitotic disruption remains poorly understood. Here, we investigated the REV7 targeting small molecule inhibitor C10, which disrupts the REV7 and REV3 interaction, and found that C10 sensitized HCT116 and HT29 CRC cells to the microtubule-disrupting agent colchicine, resulting in markedly reduced clonogenic survival. In contrast, C10 did not substantially enhance sensitivity to cisplatin. Genetic loss of REV7 similarly increased sensitivity to colchicine, independently supporting a REV7 dependent vulnerability to microtubule disruption. Combined C10 and colchicine treatment was associated with pronounced alterations in mitotic and post-mitotic nuclear morphology, including condensed/mitotic-like and multinucleated configurations, and increased micronucleus formation. In HT29 cells, combined treatment also increased the frequency of cells containing multiple gamma tubulin positive foci, consistent with disrupted centrosome-associated organization. Together, these findings support a model in which REV7 dependent functions contribute to cellular tolerance and recovery from microtubule mediated mitotic stress, whereas REV7 and REV3 disruption renders CRC cells vulnerable to mitotic perturbation. This study broadens the potential consequences of targeting REV7 beyond its canonical role in TLS and suggests that combining REV7 directed inhibition with mitotic perturbation warrants further investigation as a therapeutic strategy in CRC and potentially other cancers.

PTEN-deficient tumors are widely assumed to be glutamine-dependent, a view that has helped motivate the clinical development of glutaminase inhibitors. Whether glutaminolytic transcription scales with PTEN copy-number loss in human tumors, and by what mechanism, has not been established. We classified TCGA PanCancer Atlas tumors by GISTIC copy number as PTEN intact, hemizygous, or homozygous deletion, and scored a five-gene glutaminolysis signature (GLS, SLC1A5, GOT1, GLUD1, GPT2) against loss severity across fourteen tumor types, testing MYC mediation and chromosome-10 co-deletion as alternative mechanisms, with GLS dependence assessed in DepMap CRISPR data. The signature decreased with PTEN loss in all fourteen types, significantly in twelve, but was not MYC-mediated. The decline tracked chromosomal position rather than pathway membership: GLUD1 and GOT1 flank PTEN on 10q, and thirty-seven neighboring genes carrying no glutaminolysis annotation tracked PTEN copy number just as closely (mean rho 0.843 compared with 0.842), with co-deletion fidelity falling monotonically with distance from PTEN (rho = -0.993). Adjustment for genome-wide aneuploidy left the association intact, whereas conditioning on each gene's own copy number abolished it for the chromosome-10 genes and left the others unchanged. In a natural experiment contrasting PTEN point-mutant, copy-neutral tumors with copy-neutral wild-type tumors, GLUD1 was lower in the mutant group when all cohorts were pooled (P = 1.0 x 10^-8), an effect attributable to endometrial carcinoma, the one lineage in which mutation alone lowered GLUD1; excluding that lineage in a post hoc sensitivity analysis left no detectable difference (P = 0.22, equivalence P = 0.015), whereas hemizygous and homozygous deletion lowered GLUD1 dose-dependently in every analysis. GLS dependence did not differ across dosage tiers. Prostate carcinoma was the sole departure in the negative direction, its off-chromosome-10 decline surviving adjustment for tumor purity, stage, ERG status and proliferation, opposite in direction to the signaling prediction; breast and stomach showed small positive associations. The inverse PTEN-glutaminolysis relationship in this transcript signature is therefore explained predominantly by passenger co-deletion rather than by a PTEN-linked metabolic program, making PTEN copy number unreliable as a stand-alone glutaminolysis biomarker and illustrating a general hazard: any signature scored against a frequently co-deleted locus can be confounded by where its genes sit. We provide a four-step screening procedure: decompose the signature by chromosomal position relative to the driver; test each constituent gene's own copy number against the driver's; compare driver-mutant copy-neutral tumors with copy-neutral wild-type tumors; and check the inferred pathway activity against an independent functional readout.

Although MSI-H/dMMR colorectal cancers and the rarer subset with pathogenic POLE exonuclease domain mutations can benefit from immune checkpoint blockade, MSS colorectal cancer remains largely refractory, raising the question of whether other DNA damage response defects may also confer sensitivity to immunotherapy. Here, we performed a systematic in vivo screen of recurrently altered DNA damage response (DDR) genes to determine whether distinct repair defects modulate response to PD-1 blockade in MSS colorectal cancer. Using a genome-edited murine model in immunocompetent mice, we found that DDR perturbations produced heterogeneous outcomes, indicating that the immunologic consequences of DNA repair deficiency may be pathway-specific. Loss of Brca1, Brca2, Atm, and Fanca enhanced response to anti-PD-1 therapy, whereas Atr and Msh6 deficiency were associated with poor benefit. Brca1/2-deficient tumors showed the strongest regressions, supporting a link between core homologous recombination deficiency and increased immune sensitivity. In an independent cohort of colorectal cancer patients treated with ICB from the MSK-IMPACT Clinical Sequencing Cohort, BRCA1 alterations were associated with improved survival status, while FANCA alterations showed a similar trend. Because ATM is therapeutically targetable, we evaluated pharmacologic ATM inhibition in MSS colorectal cancer. Sustained ATM inhibition induced immune-related transcriptional programs in vitro and, when administered as a pre-treatment in vivo, enhanced the antitumor activity of PD-1 blockade. Together, these findings suggest that selected DDR alterations may represent candidate biomarkers of immunotherapy sensitivity, while ATM inhibition may provide a rational combination strategy to enhance PD-1 blockade in MSS colorectal cancer.

Detecting somatic mutations is a cornerstone of cancer genomics and clinical genotyping, traditionally relying on whole exome (WES) or whole genome sequencing (WGS) data. While RNA sequencing (RNA-seq) is primarily used to assess gene expression, it can also be utilized for somatic variant detection. RNA-seq inherently captures expressed variants, thereby reducing the identification of passenger mutations and mitigating the annotation biases associated with WES. We performed somatic variant calling and cancer driver detection from tumor RNA-seq data from 1,359 RNA-seq samples with matched WGS from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Our approach using RNA-seq data, alone, identified at least one putative driver gene in 15 of 16 cancer types, consistent with driver predictions from WGS. We further observed that the variant allele frequency (VAF) of RNA-seq-derived variants reflects their predicted functional impact, providing information specific to transcriptomic data. Because RNA-seq data can be generated and analyzed at lower computational cost and time than WGS or WES, it represents a resource-efficient alternative for cancer driver detection. This study provides an extensive pan-cancer resource of RNA-seq-derived variants from the PCAWG consortium and supports the utility of RNA-seq as a complementary approach for characterizing expressed somatic variation in cancer.

8-Oxoguanine (8-oxoG) is a common oxidative DNA lesion that can alter coding output during transcription without changing the underlying DNA sequence. When present on the template strand, 8-oxoG can direct RNA polymerase II to insert adenine, generating a transient coding-sense C>A error restricted by the genetic code to 18 directional amino-acid substitutions. Mapping these substitutions onto ClinVar identified 3,910 disease-associated protein states across 1,333 genes, representing 5.7% of evaluable pathogenic or likely pathogenic missense states. In 211 cases, transcriptional miscoding and the corresponding genomic disease variant affect different nucleotides within the same codon yet converge on the same amino acid substitution, allowing the disease-associated protein state to arise while the implicated genomic nucleotide remains wild type. Accessible states were enriched among genes with post-mitotic expression and neurological disease annotation, particularly in neuronal signaling pathways. Published oxidized-guanine maps supported 2,146 accessible genomic coordinates, while human proteogenomic data contained 78 exact codon-compatible protein states not explained by sample-specific transcript sequence. Together, these analyses define a chemically constrained set of disease-associated protein states accessible through 8-oxoG transcriptional miscoding without permanent genomic mutation.

Abstract: Background. Class IA PI3K isoforms are candidate therapeutic targets in small-cell lung cancer (SCLC). The transcriptional consequences of isoform-directed inhibitors in SCLC cells have not been compared genome-wide. Methods. We re-analysed GEO series GSE40564: PIK3CA-altered NCI-H69 cells treated for 24 h with vehicle, PIK-75 (p110-directed) or TGX-221 (p110{beta}-directed), three arrays per group (Affymetrix HG-U133 Plus 2.0). Differential expression used limma, with re-processing from raw files and spike-in-anchored normalisation as sensitivity analyses. Gene sets were assessed by effect size, competitive and rotation tests, and size-matched random-set nulls. Isoform dependency was examined in DepMap CRISPR screens. A PIK-75 response score was tested in 81 primary SCLCs against random-signature nulls. Results. PIK-75 reduced SREBP-controlled sterol- and lipid-synthesis genes (SQLE, HMGCS1, HMGCR, LDLR, SCD; log2 fold-change -1.1 to -1.6). Cholesterol, mTORC1, hypoxia and glycolysis gene sets fell far more than size-matched random sets. E2F-target and homologous-repair genes fell more modestly, including BRCA1 (-0.91). The response was broad: 11,645 of 17,458 genes changed at FDR < 0.05, but only 316 decreased and 20 increased significantly beyond two-fold, an asymmetry that persisted under spike-in-anchored normalisation. TGX-221 changed 358 genes, none two-fold. Its set-level effects were four- to nine-fold smaller and depended on two of three replicates. PIK-75 did not induce canonical FOXO targets, and it inhibits kinases other than p110, so the response cannot be attributed to p110 alone. In DepMap, SCLC lines depended more on PIK3CA than on PIK3CB (median Chronos -0.38 vs -0.15), as did other lineages. In patients, the response score was not associated with survival or subtype beyond what random gene signatures achieve (empirical p = 0.14 and 0.11). Conclusion. PIK-75, but not TGX-221, suppresses an SREBP-centred anabolic transcriptional programme in NCI-H69 cells. Attributing this to p110 requires confirmation with selective inhibitors or genetic perturbation.

A minority of asbestos-exposed individuals develop Pleural mesothelioma (PM), suggesting a role for inherited genetic factors. We performed germline whole-exome sequencing of blood from 127 EPIC participants who developed PM and controls. Rare pathogenic/likely pathogenic variants were assessed in 193 cancer-associated genes and were evaluated for association with PM risk and overall survival. Exome-wide gene-based rare-variant aggregation analyses were performed using SKAT-O and Firth logistic regression. A PM polygenic risk score (PRS) was integrated with PTV status and asbestos exposure. Protein Truncating Variants (PTVs) were enriched in cases and associated with increased PM risk (OR=3.5, 95% CI 1.3-11.2, p-value = 0.01). PTV carriers had improved survival and lower mortality after adjustment for age at diagnosis and sex (HR=0.54, 95% CI 0.32-0.92, p-value = 0.023). PTV carriers had lower PRS values, and among individuals with low asbestos exposure, they were associated with increased PM risk (OR=5.75, 95% CI 1.09-58.2, p-value = 0.023). Gene-based analyses identified four novel loci. Rare variants and polygenic background may jointly influence PM risk, however validation in larger cohorts is warranted.

Micro-RNAs (miRNAs) are key post-transcriptional regulators of gene expression that influence cancer progression and outcome. Whereas transcriptional dysregulation of miRNA abundance underlies abnormal functionality of some miRNAs in cancer, emerging data points to RNA-binding proteins (RBPs) as prominent post-transcriptional regulators of miRNA activity. Here, by modelling the regulon activities of well-characterized miRNA families in RNA-seq data of a comprehensive collection of RBP inhibition experiments, human cell lines and tissues, we report a map of regulatory connections between 34 RBPs and 28 miRNAs. Furthermore, through a global analysis of the abundance as well as the activity of miRNAs in thousands of tumor and control samples from 17 human cancers, we show that RBP-driven regulation of miRNA activity is common in tumors and contributes to the heterogeneity in miRNA activity profiles. We uncover a set of RBPs that regulate the activity of tumor-suppressor and oncogenic miRNAs, including miR-29-3p, miR-17-5p and miR-19-3p. Of particular interest, we describe a pan-cancer negative regulatory role for DDX55 on the activity of the miR-29-3p family, a critical miRNA family with tumor suppressor functions that is involved in most human cancers. Our functional studies confirm that DDX55 inhibition restores the gene-silencing activity of miR-29-3p and reduces cancer cell viability. Overall, our study provides the first pan-cancer investigation of functional regulatory connections between RBPs and miRNAs, and highlights RBPs whose interventional modulation may serve as a new approach to reprogram abnormal miRNA activities in cancer.

Multiple lines of evidence suggest that RNase H1-sensitive RNA:DNA hybrids (thereafter referred to as hybrids) could disrupt the stability and restart of stalled replication forks. The prolonged over-expression of RNase H1 is the most popular method to manipulate hybrids in situ but it cannot discriminate between direct and indirect effects resulting from their loss. To overcome this limitation, we developed iCoRD (in Cellulo RNase H Delivery) to rapidly deliver comparable amounts of either Escherichia coli RNase H1 (RnhA) or control proteins in live cells, with a negligible impact on the transcriptome. iCoRD-delivered RnhA recognises hybrids in situ, is enriched in the vicinity of active replication forks and rescues replication fork progression in different stress conditions. While iCoRD-delivered RnhA had a clear impact on stress-induced, fork-proximal hybrids and R-ChIP signals, it had no effect on DRIP or S9.6 Cut&Tag signals, even when these were induced by stress. Our results strongly suggest the existence of distinct populations of hybrids that are best mapped by different methods and are differently sensitive to RNase H1 activity in situ.

Glioblastoma (GBM) exhibits extensive loss of circular RNAs (circRNAs), yet the mechanisms driving this depletion and its functional consequences remain unclear. Here, we identify the splicing co-factor SON as a previously unrecognized suppressor of circRNA biogenesis in GBM. CircRNA sequencing revealed widespread increases in circRNA abundance following SON knockdown. Conversely, elevated SON expression in GBM, with further increases in radiation-resistant models, was associated with widespread circRNA loss, including reduced expression of circUSP1 and circSUCO. SON knockdown increased circRNA abundance and attenuated activation of the dsRNA-responsive kinase PKR and downstream NF{kappa}B signaling, whereas radiation-resistant GBM cells exhibited enhanced PKR/NF{kappa}B activation that was attenuated by SON depletion. Importantly, overexpression of the SON-suppressed circRNAs circUSP1 and circSUCO reduced PKR activation and selectively decreased clonogenic survival following irradiation in radiation-resistant GBM cells, with little effect on parental cells. Together, these findings identify a previously unrecognized SON/circRNA/PKR regulatory pathway linking RNA processing to acquired radiation resistance in GBM and highlight specific PKR-inhibitory circRNAs as potential therapeutic tools for resensitizing radiation-resistant GBM to radiotherapy.