Data from Amplification of the Mutation-Carrying BRCA2 Allele Promotes RAD51 Loading and PARP Inhibitor Resistance in the Absence of Reversion Mutations

Abstract

<div>Abstract<p>Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in <i>BRCA2</i> confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the <i>BRCA2</i> gene. In this study, we generated olaparib- and rucaparib-resistant <i>BRCA2</i>-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant <i>BRCA2</i> allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of <i>BRCA2</i> or <i>DOT1L</i> was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a <i>BRCA2</i> reversion, mutation amplification of a mutant-carrying <i>BRCA2</i> contributes to PARP inhibitor resistance.</p></div>