3113 - Inhibition of de novo Lipogenesis through Acly and FASN Sensitizes PDAC to RT and Ferroptosis

Purpose/Objective(s): Pancreatic adenocarcinoma (PDAC) is a lethal disease with extensive metabolic adaptations rendering it resistant to many therapies. We previously found lipid-responsive gene Apolipoprotein E (APOE), and lipogenesis genes ATP-Citrate Lyase (ACLY) and fatty acid synthetase (FASN), to be highly expressed in human and mouse PDAC compared to normal tissues. We also identified an increase in tumor lipid-binding protein ApoE following radiation therapy (RT). Ferroptosis has recently been shown to be an important cell death mechanism in response to oxidative stress, and pathways of clearing lipid peroxides are emerging as resistance drivers. We hypothesize that elevated de novo lipogenesis protects against oxidative stress, promoting cell growth, survival, and resistance to RT.

Materials/Methods: We performed LC-MS/MS lipidomics on KPC mouse derived PDAC cells(PK5L1940 (PK)) after RT (4 Gy) to examine the effect of RT on lipid synthesis. We tested the effect of ACLY and FASN inhibition on mouse(PK, 7160c2) and human(Panc-01, ASPC-1) PDAC cell growth via MTT assay. We performed clonogenic assays in PK cells to determine whether ACLY and FASN inhibition could sensitize to RT. We added back acetate, the metabolite downstream of ACLY, but upstream of FASN, to determine if it could restore resistance to RT. We then examined whether ACLY and FASN inhibition could sensitize PK cells to ferroptosis by GPX4 inhibition.

Results: We identified significant upregulation in 25 categories of lipid species at 72 hr following 4 Gy in PK cells. We found the most significant upregulation of cholesterol esters and O-acyl-gamma hydroxyl fatty acids. 72 hours treatment with ACLY inhibitor BMS-303141(ACLYi; PK-EC50 14.0 uM, 7160c2-EC50 58uM, Panc-01-EC50 18uM, AsPC-1-EC50 40uM) or FASN inhibitor TVB-2640(FASNi; PK-EC50 24.4uM) resulted in decreased cell viability. ACLYi and FASNi resulted in radiation sensitivity enhancement ratios (SER) of 1.98 (ACLYi) and 1.67 (FASNi) respectively. We found an SER of 1.1 for ACLYi + acetate, indicating rescue, and 1.77 for FASNi + acetate, indicating acetate does not reverse sensitization by TVB. Finally, we examined the ability of ACLY and FASN inhibition to sensitize to ferroptosis induction with GPX4 inhibitor ML-210. Maximal cytotoxicity was 21.3% with ML alone, but increased to 44.2% combined with ACLYi, and 53.5% with FASNi.

Conclusion: Lipid synthesis is stimulated in PDAC cells following RT, resulting in altered expression of numerous lipid species. Inhibition of lipogenesis enzymes ACLY and FASN reduces cell viability and can sensitize to RT. This sensitivity to upstream ACLY inhibition, but not downstream FASN inhibition, can be reversed by adding back acetate. Inhibition of ACLY or FASN can sensitize PDAC cells to GPX4 inhibition, suggesting lipid synthesis is an alternate mechanism of resisting oxidative stress induced ferroptosis by providing a source of non-peroxidized lipids.