ROCK inhibition induces synthetic llethality in BRCA2-deficient cell lines

MARTINO J, PAVIOLO NS, SIRI S, PANSA MF, CARBAJOSA S, GARRO CA, SORIA G, GOTTIFREDI V

Congreso; SAIB-PABMB; 2019

SAIB-PABMB

BRCA2 (Breast cancer susceptibility protein 2) is involved in homologous recombination repair, a pathway that repairs DNA double-strand breaks, one of the most lethal DNA lesions. Hereditary and somatic loss of function mutations in BRCA2 are correlated with breast and ovarian cancers. These tumors are highly invasive, do not respond well to conventional chemotherapy and have a poor prognosis. In recent years, it was shown that BRCA2 deficient cells can be selectively killed using PARP (Poly-ADP Ribose Polymerase) inhibition and PARP inhibitors were quickly approved to be used in the clinic. Unfortunately, these treatments often develop resistance and there is an urgent need to find alternative therapies for BRCA2 patients. To find novel alternatives, we screened a library of kinase inhibitors and identified that inhibition of ROCK kinases kills cells deficient in BRCA2. ROCK kinases are key regulators of cytoskeleton functions and few data exist regarding crosstalk with HR. Using Fasudil, a commercially available ROCK inhibitor, we validated our screen hit in multiple BRCA2 deficient cell lines. Additionally, we found that Fasudil treatment in BRCA2 cells induces abnormal cell cycle distributions characterized by a population of polyploid cells. Consistent with this, we observed an increase in multinucleated cells. The observed changes were not due to an abnormal S phase since both the percent of EdU+ cells and EdU intensity were similar in wild-type vs. BRCA2 cells. The replication stress marker γH2AX was also unaffected. We also did not observe increased genome instability, measured by micronuclei and chromosome aberrations. Additionally, mitotic entry, measured by phosphorylation of H3, was also normal. This suggests the observed phenotypes could be due to an abnormal transition through the M phase. Interestingly, we found that BRCA2 cells treated with Fasudil display aberrant metaphases, chromosome bridges and have a decreased percentage of cells in late mitotic stages such as anaphase and telophase. Moreover, abnormal mitotic figures were often accompanied by multipolar spindle poles and supernumerary centrosomes. Altogether, our data suggest that ROCK inhibition induces mitotic abnormalities and polyploidy in BRCA2 cells which are the likely cause of cell death. Intriguingly, these phenotypes are different than what is observed with PARP inhibition suggesting a new Achilles heel for BRCA2 cells. Future experiments aim at connecting cell death with the observed mitotic defects, as well as using a pre-clinical model to test if Fasudil can affect BRCA2 tumors.