Abstract Bladder cancer is a major malignancy worldwide. Although cigarette smoking is the leading modifiable risk factor, the contribution of waterpipe smoking (WPS), which is increasingly popular globally and often misperceived as safer, remains unclear. In 2019, Lebanese males had the highest bladder cancer incidence in the Arab world (five times the global average), prompting investigation of WPS as a potential carcinogen. We performed a cross-sectional genomic analysis of patients with exclusive WPS and urothelial carcinoma treated at the American University of Beirut Medical Center (n=250 screened; 14 exclusively WPS), integrating targeted sequencing (MSK-IMPACT®) and whole-exome sequencing (TEMPO pipeline). Tumor mutational burden (TMB), microsatellite instability, mutational signatures, and oncogenic alterations were assessed. Previously profiled bladder cancer tumors from cigarette smokers and non-smokers at Memorial Sloan Kettering Cancer Center (n=751) served as comparators. MSK-IMPACT® successfully profiled 13 tumors. WPS-associated tumors exhibited a numerically higher median TMB than cigarette (20 vs. 10 mut/Mb; p=0.10, q=0.2), with 38.5% classified as MSS-hypermutated, a phenotype associated with elevated mutational load despite microsatellite stability. Common mutations included TERT promoter (62%), TP53 and FGFR3 (46%), and PIK3CA (38%). APOBEC mutational signatures predominated across all cohorts, suggesting shared mutagenic processes, while chromatin-modifying gene alterations were less frequent. PD-L1 expression was generally low. Implications: Exclusive WPS induces bladder tumors with overall genomic features resembling cigarette-associated disease, yet with numerically higher TMB and enrichment of MSS-hypermutated phenotypes. These findings provide novel mechanistic evidence of tobacco-related carcinogenesis beyond cigarettes and highlight potential biomarker relevance for immunotherapy in a globally rising exposure group.
Abstract Transcription factor fusions are recurrent drivers of sarcomas and other cancers, but their oncogenic reorganization of chromatin is difficult to target. One such fusion, PAX3::FOXO1, drives an aggressive subset of pediatric rhabdomyosarcoma (RMS) through chromatin remodeling, blocking terminal differentiation and sustaining proliferation. We hypothesized that identifying PAX3::FOXO1 genetic dependencies would reveal new facets of transcription factor fusion biology. We report an unexpected role for PAX3::FOXO1 in driving DNA replication fork progression, uncovered by its dependence on the INO80 chromatin remodeling complex (INO80c). Loss of the INO80c member UCHL5 reduces the competitive fitness of fusion positive (FP), but not fusion negative (FN) RMS cell lines, and is accompanied by reduced DNA replication fork speed. UCHL5 is required to maintain the N-terminal subdomain of INO80c, but a catalytically dead UCHL5 is sufficient to rescue complex formation, fork progression, and FP-RMS fitness. Similarly, loss of PAX3::FOXO1 rapidly leads to RPA32 foci and slows DNA fork progression. These findings demonstrate that PAX3::FOXO1 and INO80c are each critical to maintain replication fork progression in FP-RMS, a novel function for this transcription factor fusion. Implications: PAX3::FOXO1 and the N-terminal INO80 complex are both required to drive replication fork progression in FP-RMS, offering a counterpoint to models in which fusion oncogenes drive replication stress, and identifying INO80 complex members as a genetic dependency in FP-RMS cells.
Abstract Cancer-related somatic histone mutants are present in 4% of diverse tumor types and display a similar genomic prevalence to other somatic mutations in cancer, but they are not well characterized. We curated and characterized 17 of the most frequently recurring missense mutations in histone H2A, H2B, and H3 genes in breast cancers occurring at glutamate (E), aspartate (D), serine (S), and arginine (R) residues. Many of these mutants co-occur with PIK3CA mutations in breast cancers. We expressed these breast cancer-associated histone mutants in MCF-7 and MDA-MB-468 breast cancer cells, or MCF-10A mammary epithelial cells, and screened them for effects on oncogenic phenotypes in a variety of cell- and tumor-based assays (i.e., proliferation, migration, invasion, competitive outgrowth, transformation). In addition, we examined the effects of selected mutants on DNA damage, gene expression, and histone modifications. We observed varying phenotypic and functional effects. Some of the histone mutants promoted cancer-related phenotypes, with H2B-E76Q, H3-E97K, and H3-E105K eliciting strong oncogenic phenotypes, alterations in gene expression, and context-dependent growth enhancement in a PI3K-activated background. We further showed that the H2B-E76Q and H3-E105K mutants exhibit reduced ADP-ribosylation compared to their cognate wild-type histones, resulting in altered H3-S10 phosphorylation and H3-K27 acetylation in cells. Our results indicate that some histone mutants can promote oncogenic effects in breast cancers, contributing to cancer pathogenesis and tumor progression. Implications: These findings identify a subset of breast cancer-associated histone variants with oncogenic activity, highlighting the role of histone mutations in promoting tumor phenotypes through epigenomic alterations and signaling crosstalk.
Abstract tRNA-derived fragments (tRFs) are a relatively recently discovered class of small RNAs implicated in gene-regulation, particularly behaving as microRNA to degrade target mRNAs with seed-sequence matches, but there have been very few reports of a clear phenotypic role of these small RNAs in cancer cell growth and other properties. Here we report that in three different cancer cell lineages, tRF-3009a, tRF-3021a and tRF-3030a are required for cell invasion and migration. Of the three tRFs, tRF-3021a was uniquely required for cell proliferation and suppression of apoptosis. Interestingly, tRF-3021a knockdown decreases global protein synthesis prior to and independent of apoptosis in a number of cancer cell lines of different lineages. Mutation of tRF-3021a shows that the effect on protein synthesis is very dependent on specific sequences in the tRF. RNA-seq reveals that the results cannot be explained by tRF-3021a decreasing the levels of target mRNAs that carry seed sequence matches with the tRF. These data indicate that tRF-3021a supports cancer cell survival and particularly protein synthesis while promoting cellular invasion and migration. Implications: tRF-3021a promotes malignant cell phenotypes, sustains global protein synthesis and prevents spontaneous apoptosis.
