Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: PHOSPHORYLATION STATUS OF MUS81 IS A MODIFIER OF SENSITIVITY TO THE PARP INHIBITOR OLAPARIB IN BRCA2-DEFICIENT CELLS

Genome instability is a hallmark of cancer and initiates from the replicative stress (RS) induced by oncogene activation. Genome instability, however, can arise through multiple mechanisms that may show inter-tumour heterogeneity and may differently operate during tumour development. Cells evolved several independent mechanisms to replicate and survive on pathological replication. Some of them involves the action of structure-specific endonucleases (SSEs), such as MUS81, which generate harmful DNA double strand breaks (DSBs) at replication forks. A subset of factors involved in the DNA repair and/or in response to RS, including SSEs, was found mutated or deregulated in human breast and ovarian cancers. MUS81 is crucial to overcome oncogene-induced RS or breast-cancer associated conditions, such as BRCA2-deficiency. In our lab, it was recently identified a mechanism that specifically regulates functionality of the MUS81 complex in M-phase. This mechanism involves phosphorylation of MUS81 at S87 by Casein Kinase 2 (CK2). Loss of phosphorylation at this site does not impact on function of MUS81 in S-phase but a mutant mimicking constitutive phosphorylation, S87D-MUS81, confers unscheduled ability to target DNA intermediated in S-phase. We observed, through viability assays, that S87D-MUS81 confers PARPi resistance in BRCA2-depleted cells. In a BRCA2-deficient background, a fundamental aspect of the sensibility to Olaparib is how the replication fork is processed by a subset of factors, like RAD51 and SMARCAL1. In our experiments, through analysis of chromatin recruitment, immunofluorescence and in situ PLA, we have observed that an unscheduled activity of MUS81 (S87D-MUS81) reduces the recruitment of SMARCAL1 and RAD51 at parental ssDNA early after fork arrest, and it could prevent engagement of fork reversal, a physiological response to perturbed replication. CK2 is often overexpressed in cancer and some cancer-associated mutations of MUS81 of unknown functional significance are located near the regulatory phosphorylation sites. Although, mutations of S87-MUS81 have not been reported in tumours yet. Overexpression of CK2 increase S87 phosphorylation proving that it might stimulate unscheduled S-phase function of this nuclease, phenocopying the S87D-MUS81 in vivo. Consistent with this, we observed that overexpression of CK2 in MRC5 MUS81 wild-type cells, depleted for BRCA2, can mimic resistance to Olaparib conferred by the presence of S87D-MUS81. Hence, this mutant forms of MUS81 can be very useful to model the effect of deregulated function of MUS81 on chemosensitivity of cancer.