Abstract A07: 53BP1-driven homologous recombination and PARP inhibitor resistance requires intact BRCA1-PALB2 association
Authors: Nacson Joseph , Bernhardy Andrea , Hua Xiang , Wang Yifan , Krais John , Johnson Neil 
Published: August 2018

Introduction: BRCA1 mutant cancers are defective for homologous recombination (HR) and as a result are highly sensitive to PARP inhibitor (PARPi) therapy. HR is initiated by DNA end resection, followed by the loading of RAD51 onto resected DNA. 53BP1 blocks DNA end resection and consequently prevents HR. In contrast, BRCA1 promotes DNA end resection, possibly by displacing 53BP1 from double-stranded DNA breaks. BRCA1 also contributes to HR downstream of DNA end resection by directly interacting with PALB2 and forming a larger BRCA1-PALB2-BRCA2-RAD51 complex that promotes RAD51 loading. In the absence of BRCA1, genetic disruption of 53BP1 has been shown to activate DNA end resection and restore HR, resulting in PARPi resistance. Despite clear evidence of activated DNA end resection, the efficiency of RAD51 loading and overall HR in the absence of BRCA1 and 53BP1 is unclear, and may be dependent on specific BRCA1 mutations that are present in a given cell line or mouse model.

Experimental Procedures: We investigated the impact of BRCA1 mutations on 53BP1-mediated restoration of HR and PARPi resistance. SUM149PT, UWB1.289, L56-BRC1 cell lines have BRCA1 exon 11 mutations and express the hypomorphic BRCA1-Δ11q splice variant; whereas MDA-MB-436, HCC1395 and SUM1315MO2 harbor RING or BRCT domain mutations and have undetectable truncated BRCA1 protein expression. We used CRISPR/Cas9 to generate a Brca11361fsX allele that was crossed with 53bp1-/- mice.

Results: 53BP1-targeting shRNA provided dramatic PARPi rescue in SUM149PT, UWB1.289, L56-BRC1 cells, with rucaparib IC50 values ranging from 100 to 500 nM. In contrast, 53BP1 shRNA scarcely impacted MDA-MB-436, HCC1395, and SUM1315MO2 cells, with IC50 values ranging from 0.5 to 2 nM. A closer inspection of HR steps revealed that 53BP1 loss activated DNA end resection in all cell lines, measured using RPA32 foci. However, downstream of resection, RAD51 foci increased in SUM149PT, UWB1.289, L56-BRC1 cells, but remained low in MDA-MB-436, HCC1395, and SUM1315MO2 cells. Because the former cell lines express the BRCA1-Δ11q splice variant that retains the ability to interact with PALB2, we examined the impact of hypomorphic BRCA1 protein expression and the BRCA1-PALB2 interaction on 53BP1-mediated PARPi rescue. Exogenous expression of BRCA1-Δ11q, BRCA1-ΔRING, or BRCA1-ΔBRCT all dramatically enhanced 53BP1-mediated RAD51 foci and PARPi resistance in an isogenic MDA-MB-436 complementation system. However, when a BRCA1L1407P missense mutation was introduced that abrogates the BRCA1-PALB2 interaction, 53BP1-induced RAD51 loading and PARPi resistance was completely abolished. The Brca11361fsX allele has a stop codon prior to critical amino acids required for Brca1-Palb2 association and homozygosity results in embryonic lethality. In contrast to previous studies using hypomorphic Brca1 alleles, matings with 53bp1-/- mice did not rescue the embryonic viability of Brca11361fsX mice.

Conclusions: These findings suggest that although 53BP1 loss of function is capable of promoting DNA end resection, efficient RAD51 loading remains dependent on BRCA1 and intact BRCA1-PALB2 complexes. Patients with BRCA1 mutations that prevent BRCA1-PALB2 association may have prolonged responses to PARPi therapy and are unlikely to succumb to 53BP1-loss of function-driven resistance.

Citation Format: Joseph Nacson, Andrea Bernhardy, Xiang Hua, Yifan Wang, John Krais, Neil Johnson. 53BP1-driven homologous recombination and PARP inhibitor resistance requires intact BRCA1-PALB2 association. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A07.