Targeting Leucine and Valine Catabolism to Disrupt Prostate Cancer Progression and Resistance Charles Bidgood1, Lisa Philp1, Anja Rockstroh1, Melanie Lehman1,2, Colleen Nelson1, Martin Sadowski3, Jennifer Gunter1 1. Australian Prostate Cancer Research Centre – Queensland, Institute of Health and Biomedical Innovation, school of Biomedical Sciences, Faculty of Health, Translational Research Institute, 37 Kent St, Brisbane, 4102, Qld, Australia. 2. Vancouver Prostate Centre, Vancouver General Hospital, 2660 Oak St, Vancouver, BC V6H 3Z6, Canada. 3. Universität Bern, Institut für Pathologie, Raum L420, Murtenstrasse 31, CH-3008 Bern, Switzerland In advanced prostate cancer (PCa), the therapeutic efficacy of androgen targeted therapies (ATTs) such as enzalutamide is limited by adaptive reprogramming to androgen receptor (AR) inhibition resulting in therapy resistance. These resistance pathways include metabolic remodelling which facilitates tumour survival. As a result, there is an ever-growing need for novel strategies which can disrupt these metabolic adaptations and in turn, inhibit the progression of advanced PCa. We have analysed the transcriptome of androgen-sensitive PCa cell lines and PDX models to identify the metabolic pathways that predictably change in response to ATTs. This project aims to target the catabolism of two branched-chain amino acids: leucine and valine, which have been shown to be largely upregulated in both patient metastatic castrate resistant prostate cancer (mCRPC) biopsies and enzalutamide-treated prostate cancer cells (LNCaPs). Through modification of the extracellular amino acid environment, utilisation of RNAi technologies to suppress catabolism and strategic application of known metabolic inhibitors, we have identified key targets of leucine and valine catabolism affecting PCa cell growth, proliferation, and survival. These include two enzymes, HIBCH and MCCC2 which are specific for valine and leucine metabolites, respectively. Additionally, we have implemented a multiparametric quantitative single-cell imaging (mqSCI) approach which shows that disruption of these pathways induces fundamental changes to mitochondrial dynamics, lipid synthesis and storage, redox potential, and metabolic viability to a range of androgen-sensitive and insensitive PCa cell lines. These findings suggest the catabolism of leucine and valine are critical in the adaptive metabolic rewiring by PCa cells in response to ATTs and targeting these enzymes may serve as an effective therapeutic strategy in order to combat the progression of advanced PCa and therapeutic resistance to ATTs.