NAR Cancer

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.

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.

Background: Metabolic adaptation is a recognized feature of therapeutic resistance in hepatocellular carcinoma (HCC), but it is unclear whether transcriptional states exposed during acquired resistance are restricted to drug adaptation or reflect broader aggressive tumor biology. We tested whether metabolic programs derived from a lenvatinib-resistance model identify a clinically adverse transcriptional state in an independent HCC patient cohort. Methods: The discovery framework was based on GSE186191, comprising parental and acquired lenvatinib-resistant Hep3B and Huh7 cells. A pre-specified 33-gene lipid-source ledger served as a biological anchor, and three discovery-derived programs, MYC Targets V2, mTORC1 Signaling, and Fatty Acid Metabolism, were frozen before patient-level evaluation. In TCGA-LIHC, single-sample enrichment scores for the three programs were population-standardized and summed to generate an integrated metabolic score. Overall survival was assessed by Kaplan-Meier and Cox analyses. Whole-transcriptome differences between high- and low-score tumors were characterized by preranked gene set enrichment analysis (GSEA). Results: The survival cohort comprised 282 patients (118 deaths), with 141 patients in each median-defined score group. High-score patients had shorter overall survival (log-rank P=0.000419). The continuous score was associated with mortality in univariable analysis (HR 1.86, 95% CI 1.33-2.61; P=0.000293) and in the frozen model adjusted for age, sex, and stage indicators (HR 1.93, 95% CI 1.35-2.76; P=0.000350; n=277). In 327 primary tumors, Fatty Acid Metabolism was strongly depleted in high-score tumors (NES -2.06; FDR<0.001). MYC Targets V2 (NES 1.18; FDR=0.232) and mTORC1 Signaling (NES 1.11; FDR=0.229) showed positive directional enrichment without FDR significance. Conclusions: A lenvatinib-resistance-derived transcriptional program is associated with an adverse-survival state in HCC. The strongest patient-level pathway feature is depletion of canonical fatty-acid metabolism, accompanied by directional MYC/mTORC1 features rather than statistically established pathway activation. These findings support a testable model of metabolic identity remodeling but do not establish causality or clinical prediction of lenvatinib response.

Early- and average-onset colorectal cancer (CRC) are separated at age 50, but whether this defines a biological threshold remains unclear. To clarify this, we identified molecular profiles in nine harmonised cBioPortal CRC cohorts (4,609 patients) by fitting Bernoulli mixture models to 31 repair-state, genomic-burden and gene-alteration features, excluding age, sex and tumour site, and compared their prevalence using <50/[&ge;]50 and decade-resolved groups. Four profiles captured conventional/CIN-like (P1), intermediate MSS (P2), KRAS/PI3K/APC-rich (P3) and hypermutated/MSI-high (P4) states along a left-to-right gradient. Although molecular identities remained stable, profile prevalence followed non-linear P1/P4 and opposing linear P2/P3 age trajectories. Profile-prevalence patterns did not track chronological proximity: profile composition at 30-39 differed from 50-59 but not clearly from 60-69. The age-50 threshold captured only 17.8% of decade-resolved deviance, whereas the optimal age-70 cut-off retained only 51.3%. Validation in 2,579 non-overlapping MSK-IMPACT patients (2,476 age-evaluable) reproduced molecular-feature patterns (r=0.97-0.98), age trajectories (r=0.92) and limited binary-threshold performance: age 50 and the optimal age-66 cut-off retained 12.7% and 45.1%, respectively. Thus, age reorganizes the prevalence of shared CRC states rather than defining a biological threshold at age 50.

Aberrant DNA methylation is a hallmark of cancer, but its clinical interpretation remains debated. UHRF1, a key epigenetic adaptor for DNA methylation maintenance and chromatin bivalency regulation in embryonic stem cells, is frequently overexpressed yet shows context-dependent prognostic behaviour. By integrating bulk and single-cell transcriptomics, CpG-resolution methylation, developmental chromatin states, immune profiling and clinical outcomes across gastric (STAD), clear-cell renal (KIRC) and adrenal (ACC) carcinomas, we identified a four-class UHRF1-embryonic morphogenesis (UHRF1-EM) framework resolving this paradox. This axis revealed an inverse prognostic pattern: whilst across all three tumours EM-low and EM-high states mark better or worse prognosis, respectively, UHRF1-high levels associate with favourable outcome in STAD (UH-EML), and unfavourable in KIRC and ACC (UH-EMH). The classification proved reproducible and independently prognostic after adjustment for stage and molecular subtypes, outperforming existing classifiers and exceeding pathological stage in KIRC and ACC. Multivariable models incorporating UHRF1-EM yielded uniformly positive {Delta}C-indices. Hypermethylation associated with the UHRF1-EM axis was enriched at ESC bivalent developmental loci (EM and oncofoetal genes), but not at housekeeping cell-cycle sites. In STAD, this pattern was related to oncofoetal gene downregulation and best prognosis, whereas in KIRC and ACC it matched with gene-body/enhancer methylation, higher EM expression, immunosuppressive microenvironments and worst prognosis. Together, these findings establish the UHRF1-EM axis as a clinically robust molecular classifier and support a mechanistic model in which tumour-specific epigenetic engagement of developmental loci may contribute to the prognostic inversion, providing a foundation for further mechanistic experimental validation.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression primarily in the cytoplasm. However, emerging evidence highlights their additional roles in the nucleus. In particular, spliceosomal miRNAs have been implicated in novel regulatory functions, including the modulation of gene expression. Here, we investigate the nuclear role of spliceosomal miR-99b in breast cancer cells, focusing on its interaction with the long non-coding RNA (lncRNA) SPACA6-AS1. Using non-tumorigenic (MCF-10A) and breast cancer cell lines (MCF-7 and MDA-MB-231), we demonstrate that spliceosomal miR-99b expression increases with malignancy and correlates with elevated SPACA6-AS1 pre-mRNA levels. Notably, miR-99b exhibits full complementarity to the 5-prime splice junction of SPACA6-AS1, suggesting a direct role in splicing regulation. Functional assays reveal that inhibition of miR-99b reduces SPACA6-AS1 pre-mRNA levels, whereas its overexpression enhances pre-mRNA accumulation, indicating that miR-99b promotes the formation or stabilization of the unspliced transcript. Furthermore, increased miR-99b expression is associated with altered ratios of SPACA6 isoforms, supporting a broader role in RNA-level regulation of gene expression. Phenotypically, miR-99b enhances breast cancer cell migration and is required for efficient invasion, particularly in highly aggressive cancerous cells. Our findings uncover a novel nuclear function of miR-99b in modulating lncRNA splicing and gene expression. This spliceosomal miR-99b-SPACA6-AS1 axis represents a previously unrecognized regulatory pathway that contributes to breast cancer progression and may provide a potential target for diagnostic and therapeutic strategies.

Ewing Sarcoma (ES) is an aggressive neoplasm arising in bones and soft tissues driven by the oncogenic fusion EWSR1::FLI1. Through epigenetic deregulation, EWSR1::FLI1 generates de novo super-enhancers that control the expression of key genes for tumor cell maintenance. By an integrative in silico analysis, we identified the subunit of the Mediator complex MED13L and RERE, a member of the atrophin family of arginine-glutamic acid dipeptide repeat-containing proteins, as genes regulated by EWSR1::FLI1-bound super-enhancers. We confirmed that EWSR1::FLI1 regulates MED13L and RERE expression in ES cell lines and showed that these proteins are highly expressed in Ewing primary tumors. Besides the well-established role of the Mediator complex in transcriptional regulation given its association with the RNA polymerase II, in ES cells the DNA binding sites of MED13L overlap with those of RERE and EWSR1::FLI1 in genes that control protein translation and alternative splicing (AS). Accordingly, the expression of various spliceosome components is co-regulated by MED13L, RERE and the oncogene, leading to AS in ES cells. We identified RBM39, a splicing factor downregulated after MED13L and RERE depletion, as a direct transcriptional target of EWSR1::FLI1. Consistently, in vitro viability experiments using indisulam, which induces selective DCAF15-dependent proteosome degradation of RBM39, demonstrate ES cells highly and specifically sensitive to RBM39 inhibition. In vivo experiments with mice xenografted with ES cells show complete tumor regression with indisulam, highlighting the potential of this approach as a novel and promising therapeutic strategy for Ewing sarcoma. STATEMENT OF SIGNIFICANCEEwing sarcoma (ES) is characterized by FET::ETS oncoproteins that act as pioneer transcription factors. Here, we identified two genes controlled by EWSR1::FLI1-bound super-enhancers, MED13L and RERE, and characterized the mechanism by which these proteins cooperate with the oncogene to regulate RNA metabolism and ribosomal processes in ES cells. These findings have led to the identification of the splicing factor RBM39 as a vulnerability in ES, as supported by the extraordinary sensitivity of these tumors to monotherapy with RBM39 degrader indisulam.

BRAF mutant colorectal cancer (CRC) remains difficult to treat despite the clinical use of combined BRAF and EGFR inhibition, highlighting a need to define tumour-intrinsic mechanisms that limit therapeutic response. Here, using genome-wide CRISPR-Cas9 screening in BRAF-mutant CRC cells, we identify MEN1, encoding the chromatin-associated protein Menin, as a selective determinant of sensitivity to combined encorafenib and cetuximab (EC). MEN1 loss markedly enhanced EC-mediated inhibition of cell proliferation and ERK activity while having comparatively little effect in untreated cells, and re-expression of Menin restored resistance. Transcriptomic and chromatin profiling revealed that Menin supports the transcriptional response associated with MAPK signalling. Menin occupied promoters of MAPK/BRAF-responsive genes and EC treatment caused widespread displacement of Menin from chromatin. Phosphoproteomic analysis demonstrated extensive remodelling of MAPK signalling following EC treatment, whereas proximity proteomics showed that the Menin-associated protein complexes remained largely intact despite loss of Menin chromatin occupancy. Importantly, MLL1 loss did not reproduce the sensitising effect of MEN1 deletion, and pharmacological Menin inhibition with revumenib failed to phenocopy either genetic MEN1 loss or acute Menin degradation, indicating that this phenotype is independent of Menin-MLL activity. Together, these findings identify a previously unrecognised, MLL-independent role for Menin in buffering the response of BRAF-mutant CRC cells to MAPK pathway inhibition and suggest targeting Menin, rather than disruption of its interaction with MLL, may provide a strategy for enhancing the response to BRAF-targeted therapy for CRC.

Circular RNAs (circRNAs) are an emerging class of RNAs with biomarker potential, but their detection in liquid biopsies is challenging due to low abundance. We developed Ouro-seq, a novel long-read sequencing protocol optimized for full-length circRNA recovery. Applied to urine, cervico-vaginal self-samples from cervical cancer patients, and plasma from lung cancer patients and controls, Ouro-seq recovered 2-5 times more and substantially longer circRNA molecules than conventional methods. Plasma contained predominantly exonic circRNAs, while urine and cervico-vaginal samples were dominated by previously undercharacterized intergenic circRNAs. We also identified extensive alternative circularization and splicing events. Functional analysis revealed distinct specialization patterns: exonic circRNAs showed enhanced miRNA sponging potential, while circRNAs from unplaced genomic scaffolds demonstrated greater peptide-coding capacity. This study establishes Ouro-seq as a valuable tool for comprehensive circRNA characterization in low-yield clinical samples and advances circRNA biology understanding with potential biomarker discovery and disease monitoring applications. MotivationWhile circular RNAs (circRNAs) constitute a minor fraction of total RNA, they may play critical roles in cancer development. CircRNA concentrations are typically too low for detection by Oxford Nanopore Long-Read Sequencing (LRS), particularly in samples with limited RNA content, such as liquid biopsies. Consequently, LRS-based circRNA analysis from liquid biopsies remains unexplored. To overcome these technical limitations, we developed an optimized circRNA enrichment method utilizing short-amplicon suppression, enabling circRNA profiling from urine, plasma, and cervico-vaginal samples.

Despite advances in screening and treatment, breast cancer remains a leading cause of cancer-related mortality. APOBEC enzymes, particularly APOBEC3B (A3B), are upregulated in many cancers, contributing to a characteristic C-to-T mutational signature found in 30-50% of breast cancers. However, the relationship between A3B mutational signatures and A3B expression across subtypes, and the resulting potential biologic consequences, have not been fully defined. Using TCGA and ICGC datasets, we analyzed DNA and RNA expression data to assess the relationship between A3B mRNA expression and APOBEC enrichment scores. Pathway enrichment analyses (KEGG, GO, Reactome) were performed to identify biological processes associated with high A3B expression, specifically stratifying by breast cancer intrinsic subtypes (HR+/HER2-, HR+/HER2+, HR-/HER2+, and TNBC). Over 64% of tumors with enriched A3B mutational genomic signatures demonstrated above-median A3B mRNA expression (p < 0.001). High A3B-expressing tumors exhibited specific alterations in drug metabolism pathways. Notably, we observed reduced expression of CYP2D6 and CYP3A isoforms which is required for the conversion of tamoxifen to its active metabolites. Conversely, genes involved in pyrimidine metabolism, including IMPDH1, NME1, TK1, and DPYS, were downregulated in high A3B tumors. Elevated A3B expression correlates with mutational signatures and may contribute to impaired tamoxifen activation and endocrine resistance, while concurrently creating metabolic vulnerabilities to pyrimidine-based chemotherapies. Targeting A3B or exploiting these metabolic dependencies may improve therapeutic response in selected patient subsets.

Urothelial cancer (UC) cells of the luminal subtype exhibit partial, incomplete differentiation towards umbrella cells that line bladder lumen, seen by morphology and gene expression. Differentiation is stalled even though the cells express master transcription factors (MTFs) that drive luminal urothelial differentiation, e.g., FOXA1 and CEBPB, at levels seen in normal differentiated urothelium. We therefore analyzed the FOXA1/CEBPB MTF hub by mass spectrometry. SWI/SNF coactivator complex (CoA) components, e.g., SMARCA4, ARID1A, that read the epigenetic activation mark histone 3 lysine 27 acetylation (H3K27ac) and use ATP-hydrolysis to open chromatin, were the most abundant proteins pulled-down with FOXA1/CEBPB. However, genes for these and other CoA, e.g., CREBBP, EP300 that write H3K27ac, were mutated/deleted in >95% of UCs in clinical series. Also contained in the hub were corepressors (CoR) that erase H3K27ac and close chromatin, e.g., HDAC1, CHD4 - genes for these CoR were recurrently gained in UCs. Chromatin analyses showed H3K27ac-centered remodeling was needed to activate umbrella but not constitutively accessible cell growth/division/housekeeping genes. Restoring ARID1A into ARID1A-mutated UC cells using lentiviral transduction, or inhibiting CoR with siRNA or small molecules, activated umbrella genes and terminated replications. In summary, UC-genesis selects for loss- and gain-of-function of CoA and CoR respectively in the urothelial-lineage MTF hub; small molecule CoR-inhibitors are candidate remedies to renew maturation towards terminal differentiated-fates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/744501v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@19e853org.highwire.dtl.DTLVardef@e3c934org.highwire.dtl.DTLVardef@ae7874org.highwire.dtl.DTLVardef@66331b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cancer cell dormancy and the resultant resistance to conventional therapies present significant challenges for the successful treatment of high-grade serous ovarian cancer (HGSC). We used genome wide, and specialized sgRNA, libraries in CRISPR-based screens to identify critical cell survival mechanisms in dormancy and metastasis. Our findings demonstrate that low expression Wnt ligands WNT8B and WNT9B are essential for sustaining cell survival during prolonged dormant spheroid culture conditions. These Wnt ligands utilize non-canonical signaling to activate expression of stem cell genes such as ALDH1A1, CD44 and others during spheroid dormancy. The loss of WNT8B and WNT9B reduced survival of xenografted ovarian cancer cells during early dissemination of disease that extended survival. Furthermore, treatment of WNT8B/9B deficient xenografts with carboplatin demonstrated increased sensitivity that further reduced dissemination and extended survival. These findings reveal that rare Wnt ligands can possess outsized functions in cancer pathogenesis and offer new avenues for improving treatment outcomes for HGSC through their inhibition.

Head and neck squamous cell carcinoma (HNSCC) remains clinically challenging, with limited molecularly targeted options and a strong dependence on the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are major stromal regulators that shape tumor progression, extracellular matrix remodeling, invasion, and therapeutic response. However, how CAF heterogeneity and plasticity translate into distinct tumor-promoting functions and targetable vulnerabilities remains insufficiently defined. Here, we integrated patient-matched primary CAFs and normal fibroblasts with 3D functional assays, tumor-stroma co-culture models, quantitative extracellular matrix analysis, whole-proteome profiling, and pharmacological perturbation. Primary fibroblast populations displayed marked interpatient heterogeneity and context-dependent plasticity in invasion, contractility, and responsiveness to tumor-derived signals, whereas enhanced fibronectin-rich matrix deposition and disorganization emerged as a conserved CAF-associated feature. Both normal fibroblasts and CAFs promoted HNSCC cell invasion in a population-dependent manner, whereas CAFs consistently induced less compact and more dispersed tumor nest architectures. Integrative functional analyses identified distinct CAF phenotypes characterized by either invasive and matrix-remodeling activity or high responsiveness to tumor-derived cues. Proteomic profiling revealed recurrent enrichment of adhesion, cytoskeletal, and extracellular matrix programs and guided the selection of pharmacological inhibitors aimed at modulating specific CAF-mediated pro-tumoral functions. Pharmacological targeting selectively altered these functions: CHI3L1 inhibition disrupted fibronectin matrix deposition, broad phosphodiesterase inhibition increased matrix alignment, and FZD7 inhibition consistently blocked tumor-induced CAF invasion across all tested populations. These findings define functionally distinct and pharmacologically targetable CAF programs in HNSCC and support stromal-directed interventions as a rational component of future combination treatment strategies.

Background: LGR5 marks colorectal cancer stem cells and is associated with poor outcome, but its expression on normal intestinal stem cells has constrained direct therapeutic targeting, and the molecular landscape of LGR5-high tumors remains incompletely defined. A transcriptional signature is not itself a set of drug targets: its constituent genes differ in whether and how they can be engaged pharmacologically, a distinction rarely applied systematically to a tumor-defined gene set. Methods: We stratified 396 colorectal tumors from The Cancer Genome Atlas by LGR5 expression and compared transcriptional, somatic mutation, and copy number profiles between LGR5-high and LGR5-low groups using non-parametric testing with combined significance and effect-size thresholds. Genome-wide CRISPR knockout data were interrogated to test genetic dependency. Each signature gene was then triaged by pharmacological tractability rather than essentiality, along three axes: surface accessibility, from surfaceome annotation and membrane topology; cavity ligandability, from pocket detection on predicted structures using three independent algorithms; and condensate propensity, from saturation concentration prediction and coarse-grained molecular dynamics simulation. Results: LGR5-high tumors displayed a coordinated program spanning Wnt signaling, stemness, and matrix remodeling, arising on an APC-mutant background with co-occurring IGF2 amplification. No constituent gene scored as a selective dependency. The three axes partitioned the signature with minimal overlap and nominated three candidates engaged by orthogonal modalities: ENPP3, a single-pass ectoenzyme presenting an accessible ectodomain and carrying clinical antibody-drug conjugate precedent; PLCB4, combining a well-defined catalytic pocket with additional predicted ligandable sites; and NKD1, accessible by neither route but undergoing RNA-stabilized homotypic phase separation, unlike SATB1 and MEX3A. Simulations further indicated that NKD1 partitions into DVL2-containing condensates and reduces DVL2-Wnt contacts, suggesting a biophysical basis for its negative-feedback role. Conclusions: LGR5 expression defines a colorectal cancer subset that is pharmacologically tractable despite the absence of genetic dependency. Triaging by modality rather than essentiality converts descriptive tumor signatures into stratified, experimentally testable therapeutic hypotheses, including condensate-directed modulation of NKD1 as a route to targets inaccessible by antibody- or pocket-based approaches.

Chemoradiotherapy (CRT) is the standard-of-care therapy for many solid malignancies, yet predictive biomarkers of treatment response remain limited. We identified a germline single nucleotide polymorphism (SNP) in an intrinsically disordered region of the lysine demethylase KDM3C/JMJD1C (p.S464T) that is associated with CRT outcomes in locally advanced rectal cancers (LARC) and head and neck squamous cell carcinoma (LA-HNSCC). In silico modeling with AlphaFold predicted S464T substitution influenced interaction between phosphorylated KDM3C and RNF8 FHA domain. In cellular models, conversion of S464 to T464 increased sensitivity to DNA-damaging agents. S464T substitution impaired damage-induced MDC1-RAP80 signaling and downstream RAP80-BRCA1 colocalization. SNP carrying cells impaired DNA repair causing genotoxic stress that is associated with increased cGAS-cGAMP innate immune signaling and increased apoptosis. Population analyses with the SNP highlighted an increase incidence of UV-induced skin and other cancers, linking inherited variation in the chromatin regulatory gene KDM3C to genome instability, cancer risk, and therapeutic vulnerability.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

Carriers of germline BRCA1 or BRCA2 alterations have a substantially increased lifetime risk of high-grade serous ovarian carcinoma (HGSOC), which originates from the secretory cells of the fallopian tube. However, comparative multi-omic analyses of bulk fallopian tube tissue from BRCA1/2 carriers and the general population have, to date, revealed only limited differences. New molecular biomarkers of early malignant transformation in the FT are needed to enable non-surgical cancer interception in high-risk individuals through window-of-opportunity trials prior to risk-reducing surgery. We performed a comprehensive single-cell, multi-regional analysis of fallopian tubes from 34 women, including 15 carriers of germline BRCA1/2 alterations. Using a metacell-based approach applied to single-cell transcriptomic data, we identify both established and previously unrecognised cellular populations, and characterise phenotypic variation associated with menopausal status, menstrual cycle phase, hormonal contraception use, and anatomical region of the fallopian tube. Menopause was associated with depletion of ciliated cells, whilst both secretory (SEC) and ciliated epithelial cells (CEC) shifted to a glandular phenotype in the luteal phase. Previous hormonal contraception usage had lasting effects including depletion of CD163-positive tissue resident macrophages and progesterone-specific increase of MHC-II expression in SECs. Metacell analysis further identified distinct subpopulations of SECs, most frequently in BRCA1/2 carriers, characterised by high TP53 expression and markedly elevated histone levels. This phenotype is consistent with replication stress, cell-cycle arrest, and activation of innate immune signalling pathways. Protein-level validation in matched samples showed enrichment of cells with increased {gamma}H2AX expression and persistent 53BP1 foci in BRCA1/2 carriers. Together our data supports the role of BRCA1/2 in maintaining genomic integrity and a BRCA1/2 haploinsufficient phenotype characterised by increased replication stress in the fallopian tube epithelium. Our findings provide evidence for distinct immune responses in users of hormonal contraception and demonstrate early events in malignant transformation. They establish potential biomarkers in microscopically normal FT and a framework for measurement of cancer risk with the goal of enabling molecularly informed cancer interception in high-risk individuals.

Transfer RNA (tRNA) modifications are essential for translational accuracy and cellular adaptation to environmental changes. While short-term modification dynamics are well documented, the impact of prolonged stress exposure on the global tRNA landscape remains largely unexplored. This study provides the first systematic profiling of tRNA modifications in Saccharomyces cerevisiae following long-term exposure to distinct stress types: heat, suboptimal pH, oxidative stress (paraquat and diamide), osmotic stress (NaCl and KCl), and genotoxic stress (MMS). To this end, we used a broad-range UPLC-MS protocol to quantify global changes in tRNA modification and identify stress-specific signatures. The results revealed that long-term stress triggers a global reprogramming of the tRNA epitranscriptome in a stress-specific and time-dependent manner. Importantly, while our findings confirm the previously reported temperature-sensitivity of wobble uridine thiolation, we also identified a complete or partial loss of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) modification upon exposure to paraquat and pH stress. Furthermore, this loss of thiolation is accompanied by an accumulation of the non-thiolated 5-methoxycarbonylmethyl (mcm5U) precursor, suggesting a stress-dependent impairment of the thiolation pathway. Next, we applied MarathonRT-based tRNA sequencing and showed that these modification changes occur independently of tRNA isoacceptor abundance. To further integrate these results, we devised a modification deviation (MDm) index, which indicates that the observed reprogramming is primarily linked to events that are independent from changes in tRNA abundance. Together, this study provides a comprehensive atlas of tRNA modification dynamics under prolonged stress, addressing a critical gap in our understanding of RNA-based translational control and establishes the MDm index as a robust quantitative framework to decouple the influence of tRNA abundance from global modification signals, providing a necessary metric for the field to interpret epitranscriptomic reprogramming. TABLE OF CONTENTS GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/745200v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2cf571org.highwire.dtl.DTLVardef@1a5f3c2org.highwire.dtl.DTLVardef@266d61org.highwire.dtl.DTLVardef@d7214f_HPS_FORMAT_FIGEXP M_FIG C_FIG

We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3{gamma} and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3{gamma}-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3{gamma}-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.