Abstract RF01-05: Functional assessment of RAD51 foci and replication fork dynamics in PARPi resistant BRCA1/2 mutated breast cancer

Abstract

Abstract Background: Although platinum salts (Pt) or Poly (ADP-Ribose) Polymerase inhibitors (PARPi) are effective in treating homologous recombination defective (HRD) breast cancer, resistance often emerges, especially in advanced disease. Predicting response and relapse is complex, even in patients with germline BRCA1/2 mutations (gBRCA1/2m). Clinically approved HRD detection methods are limited to identification of pathogenic mutations in HR genes or mutational signatures in the genome of tumors caused by HRD. In PDX models derived from HRD breast cancer, the restoration of nuclear RAD51 foci formation, a key feature of functional HR, can predict resistance to HRD-targeted treatment.1 In addition, the restoration of replication fork stability, despite PARPi or induction of Pt adducts that induce replication fork arrest and collapse, confers resistance in pre-clinical models2; to date this resistance mechanism has not been clinically validated. Here we analyse RAD51 foci in FFPE samples and DNA replication fork dynamics, using DNA fibre combing assays, in patient derived organoids (PDO) relating these to clinical response to illustrate how such assays might predict clinical HRD-targeted therapy resistance in metastatic breast cancer (MBC). Patients and Methods: We used immunofluorescent detection of RAD51 foci as a marker for HR proficiency (HRP) in tumors from 29 patients with gBRCA1/2m with MBC treated with HRD-targeted treatment (n=6 PARPi, n=2 Pt and n=21 both agents in sequence). All patients developed resistance that was either de novo or acquired. RAD51 and BRCA1 foci were scored in a minimum of 50 geminin (a marker of S/G2 phase) positive tumor cells; cells with ≥5 foci were classified as positive, and tumours where ≤10% or >10% of cells were positive were considered HRD or HRP, respectively. DNA replication fork dynamics were assessed in with or without PARPi using thymidine analogue labelling. DNA fibre analysis was performed in PDOs developed from fresh tumor sampling of HRD-targeted treatment sensitive or resistant tumors, to determine the relationship between replication fork dynamics, stability and PARPi sensitivity. Results: RAD51 analysis was performed on 9 treatment naïve samples (n=9 patients), 8 samples obtained after HRD-targeted treatment resistance (n=8 patients) and 27 samples obtained pre and post HRD-targeted treatment resistance (n=12 patients). Functional HRD by RAD51 in treatment naive samples was seen in 100% (n=17) of patients with acquired resistance and 66% of patients with de novo resistance. All patients, whether with de novo or acquired resistance, exhibited high RAD51 scores in post-resistance tumour samples, suggesting restoration of HR function is the dominant mechanism of PARPi resistance. As such, RAD51 analysis shows potential as a biomarker of clinical PARPi resistance. Replication fork stability fibre was analysed after exposure to potent PARP1 trapping inhibitors or an ATRi control in 3 PDOs (2 gBRCA1m, 1 BRCAwt). As controls we used an isogenic 2D cell line with and without a CRISPR engineered BRCA1 reversion mutation (SUM149 parental BRCA1m and SUM149BS*1 revertant) or with a PARP1 mutation (SUM149 TR2 clone) that prevents PARP1 being trapped on DNA. These experiments indicate that replication fork dynamics can be assessed in “patient derived” models of breast cancer and that PARPi sensitivity was associated with PARPi induced replication fork instability (median ratio IdU:CldU in resistant lines: 0.98, sensitive lines: 0.60). Conclusion: We show that HRP restoration and RAD51 foci in advanced BRCA1/2m breast cancers is the dominant form resistance to HRD-targeted treatment. We also demonstrate for the first time that analysis of DNA replication fork dynamics can be carried out in breast cancer PDOs and could be further explored as a functional predictive biomarker of PARPi resistance. 1.Pellegrino et al, 2022 (PMID: 35425960) 2.Chaudhuri et al, 2016 (PMID: 27443740) Citation Format: Elizabeth Harvey-Jones, Maya Raghunandan, Luisa Robbez-Masson, Thanussuyah Alaguthurai, Alba Llop-Guevara, Jennifer Trendell, Olga Rodriguez, Marta Guzman, Daniel Weekes, Christelle deRenty, Eleanor Knight, Rebecca Marlow, Christopher Starling, Ruifang Liu, Nivedita Ravindran, Ioannis Roxanis, Natalia Lukashchuk, Violeta Serra, Stephen Pettitt, Christopher Lord, Andrew NJ Tutt. Functional assessment of RAD51 foci and replication fork dynamics in PARPi resistant BRCA1/2 mutated breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-05.