Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment

Abstract

To find new anti-cancer drug therapies, we wanted to exploit homeostatic vulnerabilities within Tuberous Sclerosis Complex 2 (TSC2)-deficient cells with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivity. We show that nelfinavir and mefloquine synergize to selectively evoke a cytotoxic response in TSC2-deficient cell lines with mTORC1 hyperactivity. We optimize the concentrations of nelfinavir and mefloquine to a clinically viable range that kill cells that lack TSC2, while wild-type cells tolerate treatment. This new clinically viable drug combination causes a significant level of cell death in TSC2-deficient tumor spheroids. Furthermore, no cell recovery was apparent after drug withdrawal, revealing potent cytotoxicity. Transcriptional profiling by RNA sequencing of drug treated TSC2-deficient cells compared to wild-type cells suggested the cytotoxic mechanism of action, involving initial ER stress and an imbalance in energy homeostatic pathways. Further characterization revealed that supplementation with methyl pyruvate alleviated energy stress and reduced the cytotoxic effect, implicating energy deprivation as the trigger of cell death. This work underpins a critical vulnerability with cancer cells with aberrant signaling through the TSC2-mTORC1 pathway that lack flexibility in homeostatic pathways, which could be exploited with combined nelfinavir and mefloquine treatment.

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis

“…Highly interestingly, many proteins were involved in energy metabolism (GO terms: ATP synthesis coupled electron transport, respirasome, NADH dehydrogenase complex). This is in line with previous studies that found the energy metabolism of Schistosomes, Streptococcus and mammalian cells to be affected by MEF ( Martín-Galiano et al, 2002 ; Manneck et al, 2012 ; Li et al, 2017 ; McCann et al, 2018 ). An important consideration is that binding to a drug does not necessarily lead to functional inhibition.…”

The Anti-Cancer Properties of the HIV Protease Inhibitor Nelfinavir

“…Dual therapy by nelfinavir and bortezomib resulted in increased expression of CHOP and ATF4 in the tumors derived from xenograft models of mTOR hyperactive cells [ 70 ]. Moreover, combined therapy of nelfinavir and mefloquine (an analogue of chloroquine) or salinomycin (an anti-cancer antibiotic) resulted in activation of the ATF4-CHOP-GADD34 arm of the ER stress pathway in Tsc2 −/− mTOR-hyperactive cells [ 71 , 72 ]. Tian et al also reported phosphorylation of eIF2α and increased protein levels of ATF4 and CHOP in response to nelfinavir in glioblastoma cells [ 52 ].…”

“…AMPK leads to inhibition of mTOR and activates autophagy at the downstream level, facilitating synergism between nelfinavir and autophagy inhibitors, such as chloroquine [ 69 ]. Activation of AMPK and downregulation of mTOR also occurred following dual treatment of nelfinavir and salinomycin or mefloquine [ 71 , 72 ]. The addition of energy substrate methyl pyruvate inhibited nelfinavir- and mefloquine-mediated AMPK activation and rescued from cell death [ 72 ].…”

“…Activation of AMPK and downregulation of mTOR also occurred following dual treatment of nelfinavir and salinomycin or mefloquine [ 71 , 72 ]. The addition of energy substrate methyl pyruvate inhibited nelfinavir- and mefloquine-mediated AMPK activation and rescued from cell death [ 72 ]. Nelfinavir promotes inhibitory phosphorylation of eEF2 through eEF2K, which leads to the arrest of protein synthesis [ 66 , 67 ].…”

Drug Resistance and Novel Therapies in Cancers

“…Anther research aimed to indirectly target rapamycin complex 1 (mTORC1) hyperactivity in cancers by exploiting homeostatic vulnerabilities within Tuberous Sclerosis Complex 2 (TSC2)-deficient cells [ 4 ]. Both chloroquine (an autophagy inhibitor) and nelfinavir (an ER stress inducer) selectively enhance cell death in mTORC1 hyperactive cells.…”