The last twenty years have seen significant progress in ovarian cancer treatment, in particular for the women carrying pathogenic variants in BRCA1 or BRCA2. The foundation for this progress was the identification and cloning of BRCA1 and BRCA2, which allowed for the development of genetic tests to identify individuals at elevated risk who could benefit from increased surveillance, prophylactic surgery, and chemoprevention. A dissection of the biological role of BRCA1 and BRCA2 proteins led to the realization that synthetic lethal approaches could be successful in treating tumors with inactivated BRCA1/2 using PARP1 inhibitors. Despite significant response rates upon PARPi therapy in BRCA-linked advanced ovarian cancer, some challenges, most notably drug resistance, persist. A sizable percentage of patients display primary or intrinsic resistance despite predictions based on their BRCA1/2 status. Our long term goal is to extend the benefits of PARPi therapy to a larger proportion of patients. We hypothesize that BRCA-linked ovarian tumors that do not respond to PARPi display significant changes in PARP1 protein complexes and the cellular signaling network that can be detected using integrated functional proteomics. We have recently shown that mass spectrometry (MS)-based affinity proteomics with DDR proteins or with PARPis as baits is a powerful method to characterize multiprotein complexes, and that phosphoproteomics is ideally suited to capture complementary proteome- wide phosphorylation changes. We propose to define PARP1 interacting complex proteins and compensatory signaling in BRCA-linked ovarian cancers. We propose the following Specific Aims:
Aim 1. To determine PARP1 multiprotein complexes in PARPi-sensitive and ?intrinsically resistant BRCA-mutant ovarian cancer cells. Using quantitative PARPi affinity proteomics in tumor cells, we will identify PARP1 protein complex changes that correlate with PARPi response in BRCA-deficient cancer cells. We will utilize a unique resource of established and primary ovarian cancer cell lines and CRISPR-based isogenic cell lines.
Aim 2. To determine basal and drug adaptive signaling in PARPi-sensitive and ?intrinsically resistant BRCA-mutant ovarian cancer cells. We will harness the panel of tumor models introduced in Aim 1, including primary and isogenic cancer cell lines, and patient-derived organoids. Time-resolved quantitative MS-based global (pSTY) and tyrosine (pY) phosphoproteomics will measure proteome-wide signaling changes in untreated and PARPi- treated sensitive and intrinsically resistant cancer cells. We will interrogate and validate specific signals using a panel of known DDR signaling pathway inhibitors by immunoblotting and cellular assays applied in Aim 1. This project will transform our understanding of the complexity and dynamics of proximal (i.e. PARP1 complex- associated) and network-wide signaling events that, individually or in conjunction, lead to primary PARPi resistance in BRCA-linked ovarian cancer.
Significant progress has been achieved in the fight against ovarian cancer with the recent clinical use of PARP1 inhibitors which has shown promising clinical results. However, in order for PARP1 to achieve its full potential in the clinic primary resistance to PARP1 inhibitors needs to be circumvented. The results of this project are expected to improve patient stratification and better predict responses to PARP1 inhibitors.
