Abstract PR3: Patient derived BRCA1-deficient triple-negative breast cancer xenografts develop resistance to DNA damaging agents via genetic and epigenetic mechanisms

Abstract Background: BRCA1 mutated breast tumor cells are defective in DNA repair by homologous recombination and therefore especially sensitive to treatment with DNA double-strand break (DSB) inducing agents, such as alkylators and PARP inhibitors. However, such tumors can eventually develop therapy resistance. Understanding the underlying mechanisms may help in designing strategies to avoid or overcome acquired therapy resistance. Methods: We have developed patient derived BRCA1-deficient triple-negative breast cancer (TNBC) xenograft models by implantation of fresh human breast tumor pieces and subsequent serial passaging. These models show epigenetic loss of BRCA1 expression due to promoter hypermethylation or genetic inactivation of BRCA1 due to a frameshift mutation (c.2210delC) resulting in a premature stop codon (p.Thr737LeufsX15). We have used these 3 BRCA1-deficient TNBC models to study response and acquisition of resistance to alkylating therapy (cisplatin, melphalan, nimustine) and the clinical PARP inhibitor olaparib. Results: Treated tumors responded well to the alkylators cisplatin, melphalan and nimustine or the PARP inhibitor olaparib, in some cases resulting in complete remission with no palpable tumor left. However, relapses did occur in most cases and repeated treatment of recurrent tumors eventually led to acquired resistance. Since restoration of BRCA1 function has been suggested as a mechanism of therapy resistance (Swisher et al., Cancer Res 2008; 68: 2581), we determined BRCA1 expression in therapy-sensitive and -resistant tumors by Western blot analysis. While no full length BRCA1 protein could be detected in the therapy-sensitive tumors, expression of full length BRCA1 protein was found in the majority of alkylator resistant and olaparib resistant tumors. BRCA1 re-expression in the therapy-resistant BRCA1-c.2210delC tumors was caused in most cases by genetic restoration of the reading frame due to additional deletions near the c.2210delC mutation. In therapy-resistant TNBC xenografts that showed epigenetic loss of BRCA1 before treatment, resistance was also often associated with expression of BRCA1. In many of these tumors, loss of BRCA1 promoter hypermethylation was detected. Some of the therapy resistant tumors that showed BRCA1 expression did not show loss of BRCA1 promoter hypermethylation. In these tumors, complex rearrangements at the BRCA1 locus were found. These rearrangements placed the BRCA1 gene behind an alternative promoter, which led to expression of BRCA1 in these tumors. Conclusion: Although BRCA1-deficient TNBC xenografts are initially very sensitive to alkylating agents and olaparib, resistance to treatment develops in almost all treated tumors. This acquired resistance is frequently associated with re-expression of BRCA1 due to secondary mutations in BRCA1 mutated tumors. In breast tumors that showed epigenetic loss of BRCA1, acquired resistance is associated with loss of promoter methylation or complex rearrangements at the BRCA1 locus. This proffered talk is also presented as Poster A40.