Abstract NT-092: FUNCTIONAL HETEROGENEITY OF ACQUIRED PARP INHIBITOR RESISTANCE IN BRCA1-DEFICIENT CELLS
Authors: Färkkilä Anniina , Rodriquez Alfredo , Olkkonen Jaana , Sambel Larissa , Dominiquez Julieta , Joshi Niraj , Clairmont Connor , Zhou Jia , Lim Kah-Suan , Hautaniemi Sampsa , D'Andrea Alan 
Published: June 2021

Abstract
INTRODUCTION: More than 50% of high-grade serous ovarian cancers (HGSOC) are deficient in Homologous Recombination (HR) DNA repair, predominantly due to inactivation of BRCA1 or BRCA2 genes. HR-deficient cancers are sensitive to inhibitors of Poly-ADP Ribose Polymerase (PARP), and PARP inhibitors (PARPi's) have shown promising efficacy in the treatment of HGSOC. However, the majority of HGSOC patients will eventually develop resistance to PARPi's, and no overall survival benefit has been reported. Clinically, best characterized mechanism of PARPi resistance is restoration of BRCA1/2 protein expression accounting for 20-50% of clinical cases. Recently, emerging preclinical evidence has emphasized the role of replication fork protection in PARPi resistance. In addition, transcriptional regulation of the DNA damage response (DDR) proteins, including 53BP1, have been shown to contribute to PARPi resistance. However, the lack of understanding on the functional heterogeneity of the different PARP inhibitor resistance mechanisms has hampered the clinical targeting and development of biomarkers for acquired PARPi resistance in HGSOC.
RESULTS: To understand the dynamics and origin of PARPi resistance, we generated a TP53-/- and BRCA1-/- deficient epithelial (RPE) cell line using the CRISPR/Cas9 system. These cells are deficient in HR, and therefore are hypersensitive to PARPi in vitro. Using cyclic exposure of increasing concentrations of the PARPi Niraparib, we selected a cell population that were resistant to high concentrations (µM range) of the drug. We next isolated single cell clones from the resistant pool and performed deep functional profiling. As expected, the clones were resistant to multiple PARPi, including Niraparib, Olaparib and Talazoparib, and showed decreased DNA damage after PARPi treatment. However, none of the clones has restored BRCA1 protein expression. In functional assays, some clones had different levels of restored HR, and others showed a predominantly fork-stable, non HR-restored phenotype. Upon cytogenetic analyses, the parental RPETP53-/-BRCA1-/- cells exhibited high clonal heterogeneity in terms of ploidy, whereas the PARPi resistant clones are mostly triploid. Interestingly, the HR-restored clones have lower levels of baseline chromosomal aberrations; however, mitomycin C induced chromosomal breaks in all the clones. In flow cytometry-based cell cycle profiling, the clones retained the G2/M accumulation upon PARPi treatment, similar to the parental RPETP53-/-BRCA1-/- cells. Interestingly, all PARPi resistant clones show decreased levels of the DDR protein KAP1, and divergent expression levels of other DDR proteins, such as 53BP1. Importantly, the clones show significant heterogeneity in terms of sensitivity to cisplatin, as well as to DDR targeting agents, such as inhibitors of ATR and CHK1.
CONCLUSIONS: We have engineered BRCA1- deficient cells that are resistant to PARPi, and show that subclones of from these cells have significant functional heterogeneity in DNA repair dynamics, and are differentially vulnerable to DDR targeting agents. Importantly, our model system suggests that acquired PARP inhibitor resistance involves the adoption of several distinct resistance mechanisms that are potentially linked to DDR regulation. To enable clinical translation of our findings, we are in the process of performing genomic and transcriptomic profiling to discover the genomic evolution, common vulnerabilities, and novel biomarkers for acquired PARPi resistance in HGSOC.
Citation Format: Anniina Färkkilä, Alfredo Rodriquez, Jaana Olkkonen, Larissa Sambel, Julieta Dominiquez, Niraj Joshi, Connor Clairmont, Jia Zhou, Kah-Suan Lim, Sampsa Hautaniemi, and Alan D'Andrea. FUNCTIONAL HETEROGENEITY OF ACQUIRED PARP INHIBITOR RESISTANCE IN BRCA1-DEFICIENT CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-092.