ROCK INHIBITION INDUCES SYNTHETIC LETHALITY IN BRCA2-DEFICIENT CELL LINES

MARTINO, JULIETA; CARBAJOSA S; GOTTIFREDI, VANESA; PANSA, MARÍA FLORENCIA; SORIA, GASTÓN; PAVIOLO, NATALIA; GARRO, CINTIA

Salta

Workshop; 1st Workshop on Drug Discovery - SAIB PABMB 2019; 2019

SAIB PABMB

BRCA2 (Breast cancer susceptibility protein 2) is involved in homologous recombination repair, a pathway that repairs DNA double strandbreaks, one of the most lethal DNA lesions. Hereditary and somatic loss of function mutations in BRCA2 are correlated with breast and ovariancancers. These tumors are highly invasive, do not respond well to conventional chemotherapy and have a poor prognosis. In recent years, it wasshown that BRCA2 deficient cells can be selectively killed using PARP (Poly-ADP Ribose Polymerase) inhibition and PARP inhibitors werequickly approved to be used in the clinic. Unfortunately, these treatments often develop resistance and there is an urgent need to find alternativetherapies for BRCA2 patients. To find novel alternatives, we screened a library of kinase inhibitors and identified that inhibition of ROCKkinases kills cells deficient in BRCA2. ROCK kinases are key regulators of cytoskeleton functions and few data exist regarding crosstalk withHR. 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 thepercent 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 Mphase. Interestingly, we found that BRCA2 cells treated with Fasudil display aberrant metaphases, chromosome bridges and have a decreasedpercent of cells in late mitotic stages such as anaphase and telophase. Moreover, abnormal mitotic figures were often accompanied by multipolarspindle poles and supernumerary centrosomes. Altogether, our data suggest that ROCK inhibition induces mitotic abnormalities and polyploidyin BRCA2 cells which are the likely cause of cell death. Intriguingly, these phenotypes are different than what is observed with PARP inhibitionsuggesting a new Achilles heel for BRCA2 cells. Future experiments aim at connecting cell death with the observed mitotic defects, as well asusing pre-clinical model to test if Fasudil can affect BRCA2 tumors.