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Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52's ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.

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BACKGROUND: Pathogenic BRCA1 or BRCA2 germline mutations contribute to hereditary breast, ovarian, prostate, and pancreatic cancer. Paradoxically, bi-allelic inactivation of BRCA1 or BRCA2 (bBRCA1/2) is embryonically lethal and decreases cellular proliferation. The compensatory mechanisms that facilitate oncogenesis in bBRCA1/2 tumors remain unclear. METHODS: We identified recurrent genetic alterations enriched in human bBRCA1/2 tumors and experimentally validated if these improved proliferation in cellular models. We analyzed mutations and copy number alterations (CNAs) in bBRCA1/2 breast and ovarian cancer from the TCGA and ICGC. We used Fisher's exact test to identify CNAs enriched in bBRCA1/2 tumors compared to control tumors that lacked evidence of homologous recombination deficiency. Genes located in CNA regions enriched in bBRCA1/2 tumors were further screened by gene expression and their effects on proliferation in genome-wide CRISPR/Cas9 screens. A set of candidate genes was functionally validated with in vitro clonogenic survival and functional assays to validate their influence on proliferation in the setting of bBRCA1/2 mutations. RESULTS: We found that bBRCA1/2 tumors harbor recurrent large-scale genomic deletions significantly more frequently than histologically matched controls (n = 238 cytobands in breast and ovarian cancers). Within the deleted regions, we identified 277 BRCA1-related genes and 218 BRCA2-related genes that had reduced expression and increased proliferation in bBRCA1/2 but not in wild-type cells in genome-wide CRISPR screens. In vitro validation of 20 candidate genes with clonogenic proliferation assays validated 9 genes, including RIC8A and ATMIN (ATM-Interacting protein). We identified loss of RIC8A, which occurs frequently in both bBRCA1/2 tumors and is synthetically viable with loss of both BRCA1 and BRCA2. Furthermore, we found that metastatic homologous recombination deficient cancers acquire loss-of-function mutations in RIC8A. Lastly, we identified that RIC8A does not rescue homologous recombination deficiency but may influence mitosis in bBRCA1/2 tumors, potentially leading to increased micronuclei formation. CONCLUSIONS: This study provides a means to solve the tumor suppressor paradox by identifying synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers.

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PURPOSE: The importance of the DNA damage response in mediating effects of radiotherapy (RT) has galvanized efforts to target this pathway with radiosensitizers. Yet early clinical trials of this approach have failed to yield a benefit in unselected populations. We hypothesized that ataxia-telangiectasia mutated (Atm)-null tumors would demonstrate genotype-specific synergy between RT and an inhibitor of the DNA damage response protein ataxia-telangiectasia and Rad3-related (ATR) kinase. EXPERIMENTAL DESIGN: We investigated the synergistic potential of the ATR inhibitor (ATRi) RP-3500 and RT in two Atm-null and isogenic murine models, both in vitro and in vivo. Staining of γ-H2AX foci, characterization of the immune response via flow cytometry, and tumor rechallenge experiments were performed to elucidate the mechanism of interaction. To examine genotype specificity, we tested the interaction of ATRi and RT in a Brca1-null model. Finally, patients with advanced cancer with ATM alterations were enrolled in a phase I/II clinical trial to validate preclinical findings. RESULTS: Synergy between RP-3500 and RT was confirmed in Atm-null lines in vitro, characterized by an accumulation of DNA double-strand breaks. In vivo, Atm-null tumor models had higher rates of durable control with RT and ATRi than controls. In contrast, there was no synergy in tumors lacking Brca1. Analysis of the immunologic response indicated that efficacy is largely mediated by cell-intrinsic mechanisms. Lastly, early results from our clinical trial showed complete responses in patients. CONCLUSIONS: Genotype-directed radiosensitization with ATRi and RT can unleash significant therapeutic benefit and could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments.

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PURPOSE: Homologous recombination deficient (HRD) tumors are exquisitely sensitive to platinum-based chemotherapy and when combined with radiation therapy (RT), leads to improved overall survival in multiple cancer types. Whether a subset of tumors with distinct molecular characteristics demonstrate increased benefit from cisplatin and RT (c-RT) is unclear. We hypothesized that HRD tumors, whether associated with BRCA mutations or genomic scars of HRD, exhibit exquisite sensitivity to c-RT, and that HRD may be a significant driver of c-RT benefit. METHODS AND MATERIALS: Sensitivity to c-RT was examined using isogenic and sporadic breast cancer cell lines. HRD was assessed using 4 assays: RT-induced Rad51 foci, a DR-GFP reporter assay, a genomic scar score (large-scale state transitions [LST]), and clonogenic survival assays. Whole-genome sequencing of 4 breast tumors from a phase 2 clinical trial of neoadjuvant c-RT in triple-negative breast cancer was performed and HRD was defined using HRDetect. RESULTS: BRCA1/2 deficient cell lines displayed functional HRD based on the Rad51 functional assay, with c-RT to RT or cisplatin interaction ratios (IR) of 1.11 and 26.84 for the BRCA1 isogenic pair at 2 µM cisplatin and 6 Gy, respectively. The highest LST lines demonstrated HRD and synthetic cytotoxicity to c-RT with IR at 2 Gy and cisplatin 20 µM of 7.50, and the lowest LST line with IR of 0.65. Of 4 evaluable patients in the phase 2 trial, one achieved a pathologic complete response with corresponding HRD based on multiple genomic scar scores including HRDetect and LST scores, compared with patients without a pathologic complete response. CONCLUSIONS: HRD breast cancers, whether identified by BRCA1/2 mutation status, functional tests, or mutational signatures, appear to be significantly more sensitive to c-RT compared with isogenic controls or tumors without HRD mutational signatures. HRD tumors may be exquisitely sensitive to c-RT which warrants further clinical investigation to guide a precision oncology approach.

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Y-box-binding protein 1 (YB-1) is a DNA/RNA binding protein increasingly implicated in the regulation of cancer cell biology. Normally located in the cytoplasm, nuclear localisation in prostate cancer is associated with more aggressive, potentially treatment-resistant disease. This is attributed to the ability of YB-1 to act as a transcription factor for various target genes associated with androgen receptor signalling, survival, DNA repair, proliferation, invasion, differentiation, angiogenesis and hypoxia. This review aims to examine the clinical potential of YB-1 in the detection and therapeutic management of prostate cancer.

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Tumors resistant to PARP inhibitors frequently show signs of replication stress, with hyper-activated PARP. In this issue of Cancer Cell, Pillay et al. demonstrate that inhibiting PAR-chain turnover results in cell-cycle arrest, which is cytotoxic when combined with cell-cycle checkpoint inhibition and constitutes a novel cancer therapy.

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The Notch pathway is a highly conserved pathway increasingly implicated with the progression of human cancers. Of the four existing receptors associated with the pathway, the deregulation in the expression of the Notch-3 receptor is associated with more aggressive disease and poor prognosis. Selective targeting of this receptor has the potential to enhance current anti-cancer treatments. Molecular profiling strategies are increasingly incorporated into clinical decision making. This review aims to evaluate the clinical potential of Notch-3 within this new era of personalised medicine.

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BACKGROUND: miRNAs are increasingly associated with the aggressive phenotype of prostate tumours. Their ability to control radiobiologically-relevant cellular processes strengthens their potential as novel markers of response to radiation therapy. PURPOSE: To identify miRNAs associated with increased clonogenic survival following radiation exposure. MATERIAL AND METHODS: The miRNA expression profiles of a panel of 22RV1 cells with varying levels of radiosensitivities (hypoxic H-22Rv1 cells, RR-22Rv1 cells derived from WT-22Rv1 cells through 2-Gy fractionated repeated exposure, the associated aged matched cells (AMC-22Rv1) and the WT-22Rv1 cell lines) were generated and cross-analysed to identify common miRNAs associated with a radioresistant phenotype. RESULTS: Increased clonogenic survival following irradiation was associated with significant modifications in miRNA expression pattern. miR-221 (up) and miR-4284 (down) in RR-22Rv1 and MiR-31 and miR-200c in AMC-22Rv1 were the most uniquely significantly deregulated miRNAs when compared to WT-22Rv1 cells. miR-200c ranked as the most downregulated miRNAs in hypoxic, when compared to RR-22Rv1 cells. miR-200a was the only differentially expressed miRNA between RR-22Rv1 and AMC-22Rv1 cells. miR-210 yielded the highest fold change in expression in H-22Rv1, when compared to WT-22RV1 cells. CONCLUSION: This study identifies candidate miRNAs for the development of novel prognostic biomarkers for radiotherapy prostate cancer patients.

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The Notch-3 receptor is a recognized key regulator of vascular responses and is increasingly associated with tumorigenesis. Hypoxia-inducible factors activate specific signaling pathways such as Notch in a number of cellular models. This study aimed to evaluate the regulation of Notch-3 by hypoxia in prostate cancer cells. Notch-3 gene and protein expression was established in a panel of aerobic and hypoxic prostate cell lines in vitro, the CWR22 xenograft model and RNA extracted from low grade (Gleason score < = 6); high grade (Gleason score > = 7); non-hypoxic (low HIF, low VEGF); hypoxic (high HIF, high VEGF) patient FFPE specimens. NOTCH-3 was upregulated in PC3 (3-fold), 22Rv1 (4.1-fold) and DU145 (3.8-fold) but downregulated in LnCaP (12-fold) compared to the normal cell lines. NOTCH-3 expression was modified following hypoxic exposure in these cells. NOTCH-3 was upregulated (2.2-fold) in higher grade and hypoxic tumors, when compared to benign and aerobic pools. In the CWR22 xenograft model, Notch-3 expression was restored in castrate resistant tumors. Nuclear translocation of the Notch-3 intracellular domain was no longer detected following exposure of cells to hypoxia but not associated with a change in expression of HES-1. Our data further identifies Notch-3 as a potentially key hypoxic-responsive member of the Notch pathway in prostate tumorigenesis.

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Prostate cancer is among the most prevalent life-threatening cancers diagnosed in the male population today. Various methods have been exploited in an attempt to treat this disease but these treatments, alongside preventative tactics, have been insufficient to control mortality rates and have usually resulted in detrimental adverse events. An opportunity to devise more-specific and potentially more-effective approaches for the eradication of prostate tumours can be found by targeting specific biological pathways. NUMB (protein numb homologue), a key regulator of cell fate, represents an attractive, actionable target in prostate cancer. NUMB participates in the observed deregulation of NOTCH (neurogenic locus notch homologue protein) signalling in prostate tumours, and the NUMB-NOTCH interaction regulates cell fate. NUMB has potential both as a target for control of prostate tumorigenesis and as a biomarker for identification of patients with prostate cancer who are likely to benefit from NOTCH inhibition.

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PURPOSE: The comparison of cell lines with differing radiosensitivities and their molecular response to radiation exposure has been used in a number of human cancer models to study the molecular response to radiation. This review proposes to analyze and compare the protocols used by investigators for the development and validation of these isogenic models of radioresistance. CONCLUSION: There is large variability in the strategies used to generate and validate isogenic models of radioresistance. Further characterization of these models is required.

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