Abstract C15: Predicting response to PARP inhibitors through quantitative measurements of PARP activity in live BRCA1 mutated cells with a radio-iodinated PARP inhibitor

Abstract

Abstract Background: The breast cancer type 1 susceptibility protein (BRCA1) carries out a primary function in the DNA homologous recombination repair (HRR) pathway and mutations in the BRCA1 gene have been linked to a dramatic incidence in breast and ovarian cancer. BRCA1 associated cancers also lack HRR and are reliant on other DNA repair pathways such as base excision repair (BER) and non-homologous end joining (NHEJ). Three primary proteins involved in BER and NHEJ are Poly(ADP-ribose) Polymerases(PARP)1, 2 and 3, and inhibition of these proteins lead to a synthetic lethality in BRCA1 mutated tumors. Despite unveiling structural and mechanistic properties of BRCA1 and the PARP enzyme super family, PARP inhibition is only effective in relatively low percentages of patients who possess BRCA1 mutations. Therefore, the need for biomarkers to predict patient response to PARP inhibition is highly important. Our lab has developed a radio-iodinated-PARP inhibitor (iodine-125-KX1) capable of quantitative measurements of active PARP enzymes in whole cell assays that can predict the in vitro response to PARP inhibitors. Methods: [125I]KX1 was synthesized through radio-iododestannylation and purified by high-performance liquid chromatography. BRCA1 mutated and non-mutated ovarian and breast cancer cell lines: SNU-251, SKOV3, HCC1937, and MDA-MB-231 were investigated in a live cellular assay with [125I]KX1. Saturation, competitive inhibition, and kinetic assays were performed in all cell lines using [125I]KX1. Western analysis was performed to measure baseline PARP1, Poly(ADP-ribose(PAR), and BRCA1 protein expression. PARP inhibitor efficacy of talazoparib and olaparib was assessed through modified clonogenic assays. [125I]KX1 biodistribution experiments were carried out in mice bearing HCC1937 and MDA-MB-231 tumors to examine PARP in vivo in BRCA1 mutated and non-mutated cancer cell lines. Results: [125I]KX1 was synthesized in high radiochemical purity. Saturation experiments revealed that BRCA1 mutated cancer cell lines had a higher PARP binding potential measured by [125I]KX1. Subtle differences in PARP inhibitor affinity was noticed in the different cell lines through competitive inhibition assays. Kinetic analysis revealed that the SNU-251 had the slowest dissociation kinetics of [125I]KX1. Western analysis confirmed PARP activity measured immunohistochemically by PAR correlated with PARP binding potential measured with [125I]KX1. In vitro PARP inhibitor potency was strongly correlated with PARP binding potential. Biodistribution studies revealed that PARP can be measured quantitatively in vivo. Conclusion: With the utilization of [125I]KX1 we have been able to explore the PARP enzyme family in relation to BRCA1 mutations using in vitro and in vivo breast and ovarian cancer models. Quantitative measurements of PARP enzymes in live cancer cells has not been reported, and provides new insight into understanding the molecular target for PARP inhibitor therapy. Our data shows there is a positive correlation with PARP enzyme binding potential and response to PARP inhibitor therapy in vitro. Differences in PARP binding potential can also be measured in vivo and offer a prognostic biomarker marker for patient's who may receive PARP inhibitor therapy. Citation Format: Mehran Makvandi, Brian P. Lieberman, Kuiying Xu, Redmond-Craig Anderson, Chenbo Zeng, David A. Mankoff, Daniel A. Pryma, Roger A. Greenberg, Robert H. Mach. Predicting response to PARP inhibitors through quantitative measurements of PARP activity in live BRCA1 mutated cells with a radio-iodinated PARP inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C15.