Abstract
BACKGROUND: Glioblastoma (GBM) has dysregulated signaling of the mTOR pathway. Rapalink-1, a bivalent inhibitor created by linking rapamycin and sapanisertib, can bind to both drug binding pockets, potently inhibits mTORC1 in vitro and in vivo. Although Rapalink-1 represents a novel path towards mTOR inhibition, cellular responses in glioblastoma are mostly cytostatic. B cell lymphoma 2 (Bcl-2) family members play a central role in programmed cell death, orchestrating pro- and anti- apoptotic signals. Pro-survival Bcl-2 family member Mcl-1 is overexpressed in GBM. We hypothesize that blockade of Bcl-2 family members in combination with RapaLink-1 will drive apoptosis in glioblastoma. METHODS: We screened a panel of prototype and clinical Bcl-2 family inhibitors, namely ABT-737, ABT-199 (Venetoclax), ABT-263(Navitoclax), and GX15-070(Obatoclax) in combination with Rapalink-1, against pediatric (SF188) and adult (LN229, SF767, U373) and GBM cell lines. We assayed cell viability, annexin V FITC flow cytometry and western blot. RESULTS: Rapalink-1 in combination with Bcl-2 family inhibitors inhibited cell viability significantly more than single drug. ABT-199, a selective inhibitor of Bcl-2, did not induce apoptosis when combined with Rapalink-1. GX15-070, a pan pro-survival Bcl-2 family inhibitor and inducer of autophagy actually blocked apoptosis when combined with RapaLink-1. ABT-737 and its clinical derivative ABT-263, both of which inhibit Bcl-2, Bcl-xl and Bcl-w, synergized with Rapalink-1 to induce apoptosis in multiple GBM cell lines, evidenced by PARP cleavage and annexin V staining. We found that Rapalink-1 potently downregulated expression of Mcl-1 and induced pro-survival members Bcl-2 and Bcl-xl, suggesting that blockade of mTORC1, Bcl-2 and Bcl-xl, in combination cooperate in driving apoptosis. These results present a preclinical strategy to drive tumor cells towards mTOR inhibition-induced cytotoxicity, offering a translatable strategy for treatment of pediatric GBM.