Elucidating Synergy of Enzalutamide and Src Kinase Inhibitors in Castration-Resistant Prostate Cancer

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Project Summary Prostate Cancer (PCa) is the most commonly diagnosed non-skin cancer amongst the American male population. Therapeutics to treat PCa, which are known as Androgen Deprivation Therapies (ADTs), have been developed to target the androgen receptor (AR) or androgen synthesis pathways. PCa progresses into Castration-Resistant Prostate Cancer (CRPC) in the presence of ADTs due to upregulated tyrosine kinase activity and truncated versions of AR called AR splice variants. While targeting AR with newer second generation hormonal therapies is at the forefront of treatment of CRPC, combination therapy may be required to produce a more profound clinical benefit. Previous work from our laboratory has shown that kinase phosphorylation and activity are elevated in CRPC and have established SRC kinase as a key kinase target via phosphoproteome-guided multi-omic integration. The objective of the application is to evaluate the synergy between enzalutamide, a second generation anti-androgen, and SRC kinase inhibitors in prostate cancer models. The central hypothesis is that enzalutamide paired with SRC kinase inhibitors drives the downregulation of both AR full-length activity and AR splice variant activity on SRC, resulting in reduced AR- driven prostate cancer survival and hence greater prostate cancer cell death. Evidence will be provided to support the hypothesis in the following aims: 1) Elucidate the synergism between enzalutamide and SRC kinase inhibitors in vitro and in vivo. 2) Determine the binding or phosphorylation between AR and SRC that contribute to drug synergy. 3) Determine changes in cellular signaling altered by AR and SRC inhibition that contribute to drug synergy. In vitro experiments for Aim 1 will involve cell viability assays to determine IC50s of each therapeutic and to determine synergy between SRC kinase inhibitors and enzalutamide via the Combination Index Equation across a panel of prostate cancer cell lines. In vivo experiments will assess the synergy between SRC kinase inhibitors and enzalutamide in cell line derived xenografts and previously established mouse prostate tissue recombination cancer models expressing activated SRC with over-amplified AR. Experiments for Aim 2 involve immunoassays and targeted phosphoproteomics to analyze changes in binding or phosphorylation on residues of interest on AR in the presence or absence of SRC kinase inhibitors. This will involve mass spectrometry to identify key residues phosphorylated on AR in the presence of enzalutamide followed by site-directed mutagenesis of these residues to determine if the phosphorylation on AR splice variants contributes to ligand-independent activity of AR. Experiments for Aim 3 involve shotgun phosphoproteomics and RNA sequencing to assess changes in cellular signaling after administration of our synergistic combinations. The proposed research is significant because this will provide critical pre-clinical data that could influence how future clinical trials are designed via combining specific kinase inhibitors with enzalutamide, ultimately leading to the goal of increasing survival for men suffering from lethal CRPC.