Abstract Altered metabolism displayed by cancer cells helps circumvent a plethora of microenvironmental stressors. We and others have identified enhanced lipid metabolism as an important metabolic pathway in tumors of different etiologies. This “gain of function” contributes to malignant transformation in various malignancies, including brain tumors. Paradoxical to these findings, a high-fat/low carbohydrate ketogenic diet (KD) has demonstrated anti-tumor activity, both alone and in combination with radiation therapy (RT). Therefore, using our novel patient-derived GBM stem cell models, we sought to define mechanistic underpinnings contributing to this disconnect from a metabolic perspective. As an initial investigation, we fed ad libitum standard diet (SD) or KD alone or in combination with hypofractionated RT (hfRT; 6 Gy x 3) to mice injected orthotopically with aggressive GBM tumor cells. Consistent with previous reports, mice fed a KD demonstrated independent anti-tumor activity and potent synergy with RT compared to mice fed a SD. To provide a window into the metabolic consequences of KD in GBM, we performed global metabolomic profiling on tumors and serum from mice fed a SD and KD. Although ketosis was confirmed, no change in glucose was observed in serum of KD mice, suggesting carbohydrates might not be contributing to the observed anti-tumor activity. Interestingly, profound intra-tumoral metabolic changes were observed in tumors from mice fed a KD, with an accumulation of unsaturated fatty acids emerging as a central metabolic node differentiating KD and SD, which was consistent with serum studies. To begin to understand the biologic consequence of these findings, we recapitulated these findings in culture conditions in vitro. Intriguingly, culturing GBM cells with the polyunsaturated fatty acid (PUFA) linoleic acid demonstrated anti-proliferative activity, decreased clonogenic capacity, and synergy with RT in, recapitulating in vivo findings. This was not observed when cultured with the monounsaturated fatty acid oleic acid. Through a series of investigations, we went on to identify lipid storage homeostasis played a contributory role in the differential activity of these different classes of fatty acids. Specifically, we discovered that long-term storage within lipid droplets was less efficient for PUFA, leading to an increase free fatty acid accumulation in cells and lipotoxicity. The anti-tumor activity of PUFAs were rescued following inhibition of lipolysis. These studies collectively reaffirm and provide mechanistic underpinnings of the antitumor activity of KD, suggesting enhanced FA metabolism in brain tumors may serve as an exploitable metabolic vulnerability through diet modification. In addition, it provides a framework to potentially modify this diet to both enhance its antitumor activity and improve tolerability to allow for clinical implementation. Citation Format: Shiva Kant, Pravin Kesarwani, Yi Zhao, Prakash Chinnaiyan. Enhanced lipid metabolism in brain tumors: An exploitable metabolic vulnerability through diet-induced ketosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 17.