Abstract Introduction We previously observed an increase of prostate CAFs (PCAFs) in prostate cancers that metastasize, consistent with the known role of fibroblasts in inducing growth, confer castration-resistance and increasing metastatic potential. While aberrant choline metabolism has been identified in prostate cancer cells, choline metabolism in fibroblasts and PCAFs is unexplored and may identify a treatment strategy through downregulation of enzymes such as choline kinase alpha (Chk-α), the enzyme that converts choline to phosphocholine (PC). Chk-α is typically increased in cancer cells. Here we characterized the metabolic profile of normal prostate fibroblasts and PCAFs as well as determined the effect of Chk-α downregulation with small interfering RNA (siRNA). Methods Experiments were performed using the human prostate myofibroblasts (WPMY-1, derived from stromal cells from the peripheral zone of the histologically normal adult prostate) and PCAFs (obtained from an adenocarcinoma of the prostate gland). Cell extracts were obtained using a dual-phase extraction method. High-resolution 1H MR spectra were recorded on a Bruker Biospin Avance-III 750 MHz NMR (Bruker Biospin) spectrometer operating at a 1H frequency of 750.21 MHz using a 5-mm broad band inverse (BBI) probe head equipped with z-gradient accessories. 1H MR spectra were manually phased and automated baseline corrected using TOPSPIN 3.2 software. Integrals of the metabolites of interest were determined and normalized to the TSP reference and the number of cells. Metabolites were estimated from at least three experimental samples. Statistical significance was evaluated using the Student’s t-test. To target Chk-α, cells were transiently transfected with small interfering RNA (siRNA) against Chk-α at a concentration of 100 nM for 48h using standard protocol. Total RNA was isolated, complementary cDNA synthesized and quantitative real-time PCR performed using IQ SYBR Green supermix and gene specific primers. Viability studies were performed using CCK8 assay as previously reported. Results and Discussion Metabolic profiles were significantly different between WPMY-1 and PCAFs, with significant differences in choline, phosphocholine, glutamate and alanine, among others. siRNA directed against Chk-α reduced mRNA levels in both WPMY-1 and PCAFs. We observed a significant decrease of cell viability in PCAFs but not in normal prostate fibroblasts. These data support investigating Chk-α as a target to eliminate CAFs in tumors. Increased glutamate observed in PCAFs also support targeting enzymes and transporters in glutamine/glutamate metabolism as potential therapeutic strategies against PCAFs. Future studies with CAFs from different cancers should further validate the metabolic differences between normal fibroblasts and CAFs identified in this study. Supported by NIH R35CA209960. JPT is supported by Martin-Escudero Foundation. Citation Format: Jesus Pacheco-Torres, Tariq Shah, Flonne Wildes, Balaji Krishnamachary, Zaver M. Bhujwalla. Choline kinase downregulation decreases prostate cancer associated fibroblast viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5261.