Abstract B107: Metabolic reprogramming enhances the efficacy of mTOR inhibition in colorectal cancer
Authors: Klauck Peter J , Hawkins Hayley J , Weber Madison , Bagby Stacey M , Lieu Christopher H , Hartman Sarah J , Yacob Betelehem W , Sullivan Kelly D , Brown Monica , Reisz Julie A , D’Alessandro Angelo , Messersmith Wells A , Eckhardt S Gail , Pitts Todd M 
Published: March 2020

Abstract
Background: PI3K/mTOR pathway is mutated in 10-20% of colorectal cancer (CRC) specimens and has been associated with poor survival. Phosphatidic acid (PA) is a central lipid membrane metabolite and lipid second messenger which has been shown to target mTOR. It is thought that PA lipid signaling to mTOR in part promotes mTOR mediated cancer cell growth, proliferation and survival. Diacylglycerol kinases (DGKs) are one of several mechanisms of PA generation. In this study, we found diacylglycerol kinases to be synthetically lethal in mTOR inhibitor resistant CRC. We evaluated the anti-proliferative, pharmacodynamic and metabolic effects of dual inhibition with mTOR (TAK-228) and DGK (ritanserin and R59022) inhibitors. Methods: A synthetic lethal screen was performed with two TAK-228 resistant colorectal cancer cell lines (HCT116 and SW620). Subsequent experiments were performed with one TAK-228 sensitive (DLD1) and one resistant (HCT116) CRC cell lines. Efficacy of TAK-228/ritanserin and TAK-228/R59022 combination therapy was evaluated with CellTiter-Glo cell viability and clonogenic colony formation assays. Global metabolomics profiling of DLD1 and HCT116 cells upon treatment with TAK-228, R59022, and in combination was performed using ultra high pressure liquid chromatography coupled to mass spectrometry. Pharmacologic DGK inhibition was phenocopied using lentiviral shRNA knockdown of DGKα. Immunoblotting was performed to evaluate mechanism of action of TAK-228 combination therapy. Results: TAK-228 combined with ritanserin and R59022 demonstrated decreased cell viability and colony formation as compared to either single agent. Immunoblotting confirmed TAK-228 abrogates PI3K/mTOR pathway activity. DGK inhibition alone resulted in a compensatory activation of mTOR signaling. DGK inhibition disrupted the phosphatidic acid pathway in DLD1 and HCT116 as evidenced by a decrease in PA synthesis and elevation of glycerol 3-phosphate levels, respectively: altering energy metabolism. Specifically, in HCT116, glucose utilization, glutaminolysis, and Krebs cycle anaplerosis were elevated; while one carbon metabolism was decreased. Lentiviral shRNA transduction resulted in DGKα knockdown as evaluated by RT-PCR and immunoblot. Phenocopy combination therapy with TAK-228 and DGKα knockdown resulted in an increased sensitivity to mTOR inhibition compared to mock transduced control. Conclusions: Pharmacologic and shRNA knockdown inhibition of DGK in combination with mTOR inhibition resulted in decreased cancer cell viability and decreased colony formation. Pharmacologic inhibition of mTOR and DGK, alone or in combination, alter metabolic wiring in crucial pathways such as energy metabolism, nucleotide biosynthesis, and the generation of lipid precursors. Impaired phosphatidic acid production may sensitize cells to mTOR inhibition. These results suggest a therapeutic anti-cancer advantage of simultaneously targeting lipid signaling/metabolism via diacylglycerol kinases and mTOR.
Citation Format: Peter J Klauck, Hayley J Hawkins, Madison Weber, Stacey M Bagby, Christopher H Lieu, Sarah J Hartman, Betelehem W Yacob, Kelly D Sullivan, Monica Brown, Julie A Reisz, Angelo D’Alessandro, Wells A Messersmith, S Gail Eckhardt, Todd M Pitts. Metabolic reprogramming enhances the efficacy of mTOR inhibition in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B107. doi:10.1158/1535-7163.TARG-19-B107