BRCA1 INTRON RETENTION GENERATES TRUNCATED PROTEINS THAT AVOID BRCT MUTATION MISFOLDING AND PROMOTE PARP INHIBITOR RESISTANCE

Abstract INTRODUCTION: BRCA1 BRCT domain mutations result in protein structural defects, and consequently misfolded proteins are subject to proteasomal degradation. Loss of BRCA1 protein activity results in sensitivity to PARP inhibitor (PARPi) therapy. EXPERIMENTAL PROCEDURES: In this study, we examined PARPi resistance using the SNU-251 human endometrial ovarian cancer cell line. SNU-251 cells harbor a nonsense mutation c.5444G>A (E1815X) in BRCA1 exon 23; that disrupts BRCT domain folding. Cells were cultured in the presence of increasing concentrations of the PARPi rucaparib until resistant cells emerged. RT-PCR, Western blotting and mass spectrometry were used to measure BRCA1 mRNA and protein levels. RESULTS: PARPi resistant cell lines all demonstrated elevated BRCA1 protein levels that could be detected with N- but not C-terminal specific antibodies. Notably, the gel migration and molecular weight of BRCA1 was different from the mutation-induced stop codon expected size. Mass spectrometric analyses identified BRCA1 peptides encoded by exons 2-15; however, despite harboring a mutation in exon 23, no peptides encoded by exons 16-24 were present in SNU-251 cells. RT-PCR analyses showed that SNU-251 cells translated protein from exon 15 and into intron 15, terminating at an intron 15 generated stop codon. We overexpressed BRCA1 cDNA that harbored stop codons located in either the BRCT domain or in intron 15. BRCT mutation containing constructs had undetectable protein levels due to protein misfolding, and cells were highly PARPi sensitive. In contrast, cells expressing the BRCA1 intron 15 encoded stop codon had robust protein expression, demonstrated RAD51 foci and chemotherapy resistance in vitro and in vivo. CONCLUSIONS: In summary, we discovered that BRCA1 intron translation generates new stop codons resulting in loss of the entire BRCT domain. BRCTless proteins avoid mutant protein folding problems and promote residual DNA repair and chemotherapy resistance. Citation Format: Yifan Wang, Andrea J. Bernhardy, Joseph Nacson, John J. Krais, Yin-Fei Tan, Michael Slifker, Suraj Peri and Neil Johnson. BRCA1 INTRON RETENTION GENERATES TRUNCATED PROTEINS THAT AVOID BRCT MUTATION MISFOLDING AND PROMOTE PARP INHIBITOR RESISTANCE [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP30.