Abstract A01: Vps34 promotes macropinocytosis in Tsc2-deficient cells

Abstract Purpose: The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is constitutively active in many human cancers and in tuberous sclerosis complex (TSC). mTORC1 hyperactivation drives extensive metabolic reprogramming via several interconnected mechanisms, including glucose and glutamine utilization, nucleic acid synthesis, and lipid synthesis. We recently found that treatment with chloroquine (CQ), a lysosomal/autophagy inhibitor, upregulates the uptake and endosomal processing of cholesterol selectively in TSC2-deficient cells, creating a TSC2-specific dependence on exogenous cholesterol. This dependence on exogenous nutrients and the mechanisms of nutrient uptake may be therapeutically targetable. Our central hypothesis is that Tsc2-deficient cells depend on the uptake of exogenous nutrients via macropinocytosis to maintain cellular metabolic homeostasis and proliferation. Methods and Results: To investigate the role of macropinocytosis-mediated nutrient uptake in Tsc2-deficient cells, we measured the uptake of dextran, a polysaccharide taken up via macropinocytosis. Tsc2-deficient cells showed a striking increase in both dextran (3.6-fold, p<0.001) and exogenous protein uptake (~2-fold, p<0.001) relative to Tsc2-expressing cells. Interestingly, mTOR inhibition using Torin1 decreased dextran uptake (3-fold, p<0.001). Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of dextran in Tsc2-deficient cells (~50%, p<0.0001). Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms (25%, p<0.001), suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Additionally, exogenous protein activates mTORC1 signaling of Tsc2-deficient cells grown in amino acid-free media. Treatment with ethylisopropylamiloride (EIPA) resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p<0.0001). Genetic inhibition of autophagy (Atg5-/- MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p<0.0001). Finally, pharmacologic inhibition of lysosomal function with CQ and macropinocytosis with SAR405 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Conclusions: These data suggest that macropinocytosis is upregulated in Tsc2-deficient cells via a Vps34-dependent mechanism to support tumorigenesis. Macropinocytosis-mediated nutrient uptake represents an unexplored opportunity for therapeutic intervention in TSC and may also be relevant to other diseases characterized by mTORC1 hyperactivation. Citation Format: Charilaos Filippakis, Amine Belaid, Brian Siroky, Constance Wu, Nicola Alesi, Thomas Alesi, Julie Nijmeh, Hilaire Colletta Lam, Ellizabeth Henske. Vps34 promotes macropinocytosis in Tsc2-deficient cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A01.