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.