TOP1 and R-loops facilitate transcriptional DSBs at hypertranscribed cancer driver genes.

DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employ several techniques to map DSBs, R-loops, and topoisomerase 1 cleavage complex (TOP1cc) to comprehensively investigate the interplay between transcription, DSBs, topoisomerase 1 (TOP1), and R-loops. Our findings reveal the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops. Remarkably, transcription-associated DSBs at these loci are significantly reduced upon depletion of R-loops and TOP1, uncovering the pivotal roles of TOP1 and R-loops in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development, highlighting the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations.

Protocol for mapping physiological DSBs using in-suspension break labeling in situ and sequencing.

Physiological double-stranded breaks (DSBs) are a major source of genomic instability. Here, we present a protocol for mapping physiological DSBs by in-suspension break labeling in situ and sequencing (sBLISS) in a single-nucleotide resolution. We describe steps for cell fixation, labeling of DSBs, DNA isolation followed by in vitro transcription (IVT), reverse transcription, and library preparation. sBLISS provides a map of DSBs over the genome and can be used to study the role of different factors in DSB formation. For complete details on the use and execution of this protocol, please refer to Hidmi et al.1.

Unveiling the relationship between WWOX and BRCA1 in mammary tumorigenicity and in DNA repair pathway selection.

Breast cancer is the leading cause of cancer-related deaths in women worldwide, with the basal-like or triple-negative breast cancer (TNBC) subtype being particularly aggressive and challenging to treat. Understanding the molecular mechanisms driving the development and progression of TNBC is essential. We previously showed that WW domain-containing oxidoreductase (WWOX) is commonly inactivated in TNBC and is implicated in the DNA damage response (DDR) through ATM and ATR activation. In this study, we investigated the interplay between WWOX and BRCA1, both frequently inactivated in TNBC, on mammary tumor development and on DNA double-strand break (DSB) repair choice. We generated and characterized a transgenic mouse model (K14-Cre;Brca1fl/fl;Wwoxfl/fl) and observed that mice lacking both WWOX and BRCA1 developed basal-like mammary tumors and exhibited a decrease in 53BP1 foci and an increase in RAD51 foci, suggesting impaired DSB repair. We examined human TNBC cell lines harboring wild-type and mutant BRCA1 and found that WWOX expression promoted NHEJ repair in cells with wild-type BRCA1. Our findings suggest that WWOX and BRCA1 play an important role in DSB repair pathway choice in mammary epithelial cells, underscoring their functional interaction and significance in breast carcinogenesis.

WWOX promotes osteosarcoma development via upregulation of Myc.

Osteosarcoma is an aggressive bone tumor that primarily affects children and adolescents. This malignancy is highly aggressive, associated with poor clinical outcomes, and primarily metastasizes to the lungs. Due to its rarity and biological heterogeneity, limited studies on its molecular basis exist, hindering the development of effective therapies. The WW domain-containing oxidoreductase (WWOX) is frequently altered in human osteosarcoma. Combined deletion of Wwox and Trp53 using Osterix1-Cre transgenic mice has been shown to accelerate osteosarcoma development. In this study, we generated a traceable osteosarcoma mouse model harboring the deletion of Trp53 alone (single-knockout) or combined deletion of Wwox/Trp53 (double-knockout) and expressing a tdTomato reporter. By tracking Tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow mesenchymal stem cells of non-osteosarcoma-bearing mice (young BM). We found that double-knockout young BM cells, but not single-knockout young BM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these double-knockout young BM cells revealed their resemblance to osteosarcoma tumor cells. Interestingly, one of the observed significant transcriptomic changes in double-knockout young BM cells was the upregulation of Myc and its target genes compared to single-knockout young BM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing revealed its increased enrichment on Myc targets, which were upregulated in double-knockout young BM cells. Restoration of WWOX in double-knockout young BM cells reduced Myc protein levels. As a prototype target, we demonstrated the upregulation of MCM7, a known Myc target, in double-knockout young BM relative to single-knockout young BM cells. Inhibition of MCM7 expression using simvastatin resulted in reduced proliferation and tumor cell growth of double-knockout young BM cells. Our findings reveal BM mesenchymal stem cells as a platform to study osteosarcoma and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.