Abstract S4-8: Promoter CpG Methylation of <i>BRCA1</i> Predicts Sensitivity to PARP Inhibitors in Breast Cancer

Abstract Background: Poly(ADP)-ribose polymerase (PARP) inhibitors were shown to selectively kill BRCA1/2 mutated cancer cells due to conferring synthetic lethality, leading to first clinical trials in BRCA1 germline mutated breast and ovarian cancer with promising results. However, inherited breast and ovarian cancers are relatively rare. In sporadic breast cancer aberrant promoter methylation of BRCA1 is a more frequent event, intriguingly contributing to a common “BRCA phenotype”, as determined by high similarity of gene expression patterns between BRCA1 inherited and sporadic breast tumors. Currently, it is unknown whether BRCA1 methylated breast cancer cells are comparably sensitive to PARP inhibition like BRCA1 mutated breast cancer cells. Methods: We screened a panel of 7 breast cancer cell lines for BRCA1/2 promoter methylation by bisulfite genomic sequencing, none of which harbored methylation in BRCA2. One cell line (UACC3199) revealed dense methylation in the BRCA1 gene promoter. Diminished BRCA1 protein expression in these cells was re-established after treatment with 1 µM of the DNA demethylating agent 5-aza-2'-deoxycytidin. For further analysis, UACC3199 cells were compared with MDA-MB-231 cells (BRCA1 wildtype) and MDA-MB-436 cells (BRCA1 homozygous mutant). Results: In XTT assays the PARP inhibitors 3-ABA, DPQ and NU1025 revealed a similar toxicity in BRCA1 deficient UACC3199 and MDA-MB-436 cells, whereas BRCA1 proficient MDA-MB-231 cells were more resistant (IC50 values for Mda-MB-231, MDA-MB-436, and UACC3199 cells for 3-ABA: 8775 µM, 37 µM, 35 µM; for DPQ: 26 µM, 12 µM, 17 µM; for NU1025: 746 µM, 162 µM, 301 µM, respectively). Confocal immunofluorescent microscopy showed that after PARP inhibition, Y-H2AX focalization was strongly increased but not significantly different among all cell lines, indicating that the amount of DNA damage conferred by inhibition of PARP is independent of BRCA1 status. By comet assays after one week of PARP inhibition, however, we found that the amount of persistent DNA damage was significantly enhanced in both BRCA1 deficient lines, whereas it was low and similar to controls in MDA-MB-231 cells. This argues for defects in DNA damage repair in both BRCA1 deficient cell lines, most likely implicating disrupted homologous recombination integrity. Since BRCA1 mutations are associated with the triple-negative breast cancer subtype, we also determined the frequency of BRCA1 promoter methylation in non-inherited triple-negative breast cancers by methylation-specific PCR. Of the analyzed samples, a high fraction showed hypermethylation in BRCA1 (n=25/68; 37%). Conclusions: In conclusion, our results show for the first time that in addition to BRCA1 or BRCA2 mutation, also BRCA1 methylated breast cancer cell lines are considerably more sensitive to PARP inhibitors than BRCA1 wildtype cells. We therefore suggest to include BRCA1 methylation as a potential biomarker of drug sensitivity against these compounds in current and future prospective clinical trials of breast and ovarian cancer. Further in vitro experiments on the effects of PARP inhibition in BRCA1 methylated breast cancer cells are underway. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S4-8.