Abstract A23: BRCA1 mutations in the BRCT domain can be removed through alternative splicing and induce PARP inhibitor resistance

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Abstract Introduction: The BRCA1 protein harbors two C-terminal tandem BRCT domains. The first is encoded by BRCA1 gene sequences located toward the end of exon 16, and the second BRCT domain initiated at exon 21. BRCA1 BRCT domains bind to proteins containing a phosphorylated serine-proline-x-phenylalanine (pSPxF) motif, including Abraxas, BACH1 and CtIP. Protein–protein interactions mediated by BRCT domains regulate DNA damage repair and cell cycle checkpoint signaling. Many cancer-associated mutations located in the BRCT domains of BRCA1 result in protein structural defects, and consequently misfolding and proteasomal degradation. Experimental Procedures: In this study, we examined PARP inhibitor (PARPi) resistance mechanisms utilizing MDA-MBA-436, HCC-1395 and SNU-251 human breast cancer and ovarian cancer cell lines that harbor frameshift mutations in BRCA1 exon 20, exon 20 and exon 23, respectively; predicted to generate BRCT domain disruptions. Cells were cultured in the presence of increasing concentrations of the PARPi rucaparib until resistant clones emerged. Quantitative RT-PCR and Western blotting were used to measure BRCA1 mRNA and protein levels. Results: Despite BRCA1 mRNA being abundant in all parental cell lines, BRCA1 protein was low or undetectable. However, 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 proteins were markedly lower than the mutation-induced stop codon expected size. To identify potential protein products, we carried out immunoprecipitation and mass spectrometry to analyze BRCA1 peptide sequences. BRCA1 peptides encoded by exons 2-16 were readily detected; however, no peptides encoded by exon 17-24 were present in any cell line. qRT-PCR analyses suggested that BRCA1 mRNA was subject to alternative splicing and removal of exons 16-24. To understand why PARPi selection pressure generates BRCT-less BRCA1 proteins, we ectopically overexpressed BRCA1 cDNA that harbored stop codons located in BRCT domains or prior to the first BRCT domain. BRCT mutation containing constructs had undetectable protein levels, presumably due to protein misfolding, and cells were highly PARPi sensitive. In contrast, cells expressing BRCA1 constructs with stop codons prior to the BRCT domains had robust protein expression and demonstrated residual RAD51 foci and PARPi resistance. Conclusions: Our findings indicate that alternative splicing can remove deleterious mutations that disrupt BRCT peptide folding, generating more truncated but functional proteins capable of restoring residual DNA repair and PARPi resistance. Citation Format: Yifan Wang, Andrea J. Bernhardy, Neil Johnson. BRCA1 mutations in the BRCT domain can be removed through alternative splicing and induce PARP inhibitor resistance [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A23.