Exploring the endonuclease MUS81 as a new factor impacting on cancer chemosensitivity

The MUS81 complex is a crucial factor for genome stability maintenance. It is often overexpressed in breast and ovarian cancer and has been proposed as cancer predisposition gene. The complex is regulated by Casein Kinase 2 (CK2) through phosphorylation at serine 87 (S87), which is essential for resolving branched intermediates during mitosis. Our previous findings show that the phosphomimetic MUS81S87D mutant confers PARP inhibitor (PARPi) resistance in BRCA2-deficient backgrounds, possibly because replication-related double-strand breaks (DSBs), induced by deregulated MUS81 function during the S-phase, can be completely resolved through alternative end-joining (Alt-EJ). Thus, deregulated function of the MUS81 complex in S-phase emerged as a potential modifier of the response to PARPi when BRCA2 is mutated or absent. In this study, we investigate why the MUS81S87D mutant aberrantly targets DNA replication forks. We found that MUS81S87D is hyper-recruited at ongoing replication forks and that the characteristic phenotype of S87D-MUS81 mutant cells is reversed by depletion of the protein scaffold SLX4 by siRNA transfection. This indicates that the unscheduled cleavage function conferred to MUS81 by the S87D mutation and excessive recruitment at DNA replication forks depend on SLX4. We confirm these findings also using a set of mutations that abrogate the binding of MUS81 to SLX4. Finally, we assessed if counteracting the association between SLX4 and MUS81 in S-phase affects PARPi resistance in BRCA2-deficient cells. Consistent with the DNA damage and localization data, abrogation or reduction of the S87D-MUS81/SLX4 association was sufficient to largely restore normal PARPi sensitivity of BRCA2-deficient cells. These findings could lead to a better understanding of the relationship between MUS81 status, PARPi chemosensitivity and a possible MUS81 exploitation in target therapy. Moreover, this study investigates the hypothesis that MUS81 loss creates a synthetic lethal vulnerability with PARP inhibition, particularly in the context of oncogene-induced replication stress. Using CRISPR/Cas9, MUS81 was knocked out in a panel of human cell lines. We demonstrate that MUS81 loss induces a clear sensitivity to the PARPi Olaparib in HR-proficient breast (MCF7) and ovarian (OVCAR-3) cancer cells, but not in a non-cancerous mammary epithelial model cell line (HMEC). This sensitivity directly correlates with a significant accumulation of DNA double-strand breaks (DSBs). Interestingly, the triple-negative breast cancer cell line MDA-MB-231 remained resistant to Olaparib even after MUS81 knockout. We identified MYC oncogene amplification, which is present in the sensitive MCF7 and OVCAR-3 lines but not in the resistant MDA-MB-231 line, as the key differentiating factor. Moreover, we found that the increased PARPi sensitivity is also linked with a metabolic shift occurring upon MUS81 loss. These findings reveal a novel, cancer-specific synthetic lethality between MUS81 and PARP that seems to be conditional upon MYC overexpression. Our results suggest that high MYC levels create an intense state of RS, making cancer cells critically dependent on MUS81 for survival. We propose that MYC amplification could serve as a key biomarker to identify patients with HR-proficient tumours who may benefit from a combination therapy involving PARP inhibitors and potential future MUS81-targeting agents, thereby expanding the utility of PARPis beyond their current application. Furthermore, the metabolic rewiring conferred by MUS81 KO could be studied to identify novel vulnerabilities and to find novel therapeutic targets.