R-Loop-Associated Genomic Instability and Implication of WRN and WRNIP1.

Maintenance of genome stability is crucial for cell survival and relies on accurate DNA replication. However, replication fork progression is under constant attack from different exogenous and endogenous factors that can give rise to replication stress, a source of genomic instability and a notable hallmark of pre-cancerous and cancerous cells. Notably, one of the major natural threats for DNA replication is transcription. Encounters or conflicts between replication and transcription are unavoidable, as they compete for the same DNA template, so that collisions occur quite frequently. The main harmful transcription-associated structures are R-loops. These are DNA structures consisting of a DNA-RNA hybrid and a displaced single-stranded DNA, which play important physiological roles. However, if their homeostasis is altered, they become a potent source of replication stress and genome instability giving rise to several human diseases, including cancer. To combat the deleterious consequences of pathological R-loop persistence, cells have evolved multiple mechanisms, and an ever growing number of replication fork protection factors have been implicated in preventing/removing these harmful structures; however, many others are perhaps still unknown. In this review, we report the current knowledge on how aberrant R-loops affect genome integrity and how they are handled, and we discuss our recent findings on the role played by two fork protection factors, the Werner syndrome protein (WRN) and the Werner helicase-interacting protein 1 (WRNIP1) in response to R-loop-induced genome instability.

ATM pathway activation limits R-loop-associated genomic instability in Werner syndrome cells.

Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after various forms of replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit other repair pathway. We demonstrate that loss of WRN leads to enhanced ATM phosphorylation upon prolonged exposure to aphidicolin, a specific inhibitor of DNA polymerases, resulting in CHK1 activation. Moreover, we find that loss of WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation. Together, these findings suggest a potential role of WRN as regulator of R-loop-associated genomic instability, strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer.

Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Checkpoint Defects Elicit a WRNIP1-Mediated Response to Counteract R-Loop-Associated Genomic Instability.

Conflicts between replication and transcription are a common source of genomic instability, a characteristic of almost all human cancers. Aberrant R-loops can cause a block to replication fork progression. A growing number of factors are involved in the resolution of these harmful structures and many perhaps are still unknown. Here, we reveal that the Werner interacting protein 1 (WRNIP1)-mediated response is implicated in counteracting aberrant R-loop accumulation. Using human cellular models with compromised Ataxia-Telangiectasia and Rad3-Related (ATR)-dependent checkpoint activation, we show that WRNIP1 is stabilized in chromatin and is needed for maintaining genome integrity by mediating the Ataxia Telangiectasia Mutated (ATM)-dependent phosphorylation of Checkpoint kinase 1 (CHK1). Furthermore, we demonstrated that loss of Werner Syndrome protein (WRN) or ATR signaling leads to formation of R-loop-dependent parental ssDNA upon mild replication stress, which is covered by Radiorestistance protein 51 (RAD51). We prove that Werner helicase-interacting protein 1 (WRNIP1) chromatin retention is also required to stabilize the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall, our findings suggest a novel function for WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication-transcription conflicts are handled.