Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.
Most cancers have lost a critical DNA damage response (DDR) pathway during tumor evolution. These alterations provide a useful explanation for the initial sensitivity of tumors to DNA-targeting chemotherapy. A striking example is dysfunctional homology-directed repair (HDR), e.g., due to inactivating mutations in BRCA1 and BRCA2 genes. Extensive efforts are being made to develop novel targeted therapies exploiting such an HDR defect. Inhibitors of poly(ADP-ribose) polymerase (PARP) are an instructive example of this approach. Despite the success of PARP inhibitors, the presence of primary or acquired therapy resistance remains a major challenge in clinical oncology. To move the field of precision medicine forward, we need to understand the precise mechanisms causing therapy resistance. Using preclinical models, various mechanisms underlying chemotherapy resistance have been identified. Restoration of HDR seems to be a prevalent mechanism but this does not explain resistance in all cases. Interestingly, some factors involved in DNA damage response (DDR) have independent functions in replication fork (RF) biology and their loss causes RF instability and therapy sensitivity. However, in BRCA-deficient tumors, loss of these factors leads to restored stability of RFs and acquired drug resistance. In this review we discuss the recent advances in the field of RF biology and its potential implications for chemotherapy response in DDR-defective cancers. Additionally, we review the role of DNA damage tolerance (DDT) pathways in maintenance of genome integrity and their alterations in cancer. Furthermore, we refer to novel tools that, combined with a better understanding of drug resistance mechanisms, may constitute a great advance in personalized diagnosis and therapeutic strategies for patients with HDR-deficient tumors.
MDC1 is a key protein in DNA damage signaling. When DNA double-strand breaks (DSBs) occur, MDC1 localizes to sites of damage to promote the recruitment of other factors, including the 53BP1-mediated DSB repair pathway. By studying mechanisms of poly(ADP-ribose) polymerase inhibitor (PARPi) resistance in BRCA2;p53-deficient mouse mammary tumors, we identified a thus far unknown role of MDC1 in replication fork biology. MDC1 localizes at active replication forks during normal fork replication and its loss reduces fork speed. We show that MDC1 contributes to the restart of replication forks and thereby promotes sensitivity to PARPi and cisplatin. Loss of MDC1 causes MRE11-mediated resection, resulting in delayed fork restart. This improves DNA damage tolerance and causes chemoresistance in BRCA1/2-deficient cells. Hence, our results show a role for MDC1 in replication fork progression that mediates PARPi- and cisplatin-induced DNA damage, in addition to its role in DSB repair.
The PSMC3IP-MND1 heterodimer promotes RAD51 and DMC1-dependent D-loop formation during meiosis in yeast and mammalian organisms. For this purpose, it catalyzes the DNA strand exchange activities of the recombinases. Interestingly, in a panel of genome-scale CRISPR-Cas9 mutagenesis and interference screens in mitotic cells, we found that depletion of either PSMC3IP or MND1 caused sensitivity to clinical Poly (ADP-Ribose) Polymerase inhibitors (PARPi). A retroviral mutagenesis screen in mitotic cells also identified PSMC3IP and MND1 as genetic determinants of ionizing radiation sensitivity. The role PSMC3IP and MND1 play in preventing PARPi sensitivity in mitotic cells appears to be independent of a previously described role in alternative lengthening of telomeres (ALT). PSMC3IP or MND1 depleted cells accumulate toxic RAD51 foci in response to DNA damage, show impaired homology-directed DNA repair, and become PARPi sensitive, even in cells lacking both BRCA1 and TP53BP1. Although replication fork reversal is also affected, the epistatic relationship between PSMC3IP-MND1 and BRCA1/BRCA2 suggests that the abrogated D-loop formation is the major cause of PARPi sensitivity. This is corroborated by the fact that a PSMC3IP p.Glu201del D-loop formation mutant associated with ovarian dysgenesis fails to reverse PARPi sensitivity. These observations suggest that meiotic proteins such as MND1 and PSMC3IP could have a greater role in mitotic cells in determining the response to therapeutic DNA damage.
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