Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells

Abstract

Uncontrolled cell growth in Tuberous Sclerosis Complex occurs due to inappropriate activation of mechanistic (mammalian) target of rapamycin complex 1 (mTORC1). The current therapy, rapamycin, produced promising clinical trial results, but patient tumours regrow if treatment is discontinued, revealing rapamycin has cytostatic properties rather than a cytotoxic effect. Taking advantage of the enhanced levels of endoplasmic reticulum (ER) stress present in TSC2-null cells, we investigated drug combinations producing a cytotoxic response. We found a nelfinavir and salinomycin combination specifically killed TSC2-deficient, mTORC1 hyperactive cells. Cytotoxicity was rescued by reducing protein synthesis, either through mTORC1 inhibition or cycloheximide treatment. This indicates that the drug combination targets the cells by tipping the protein homeostasis balance of the already metabolically stressed TSC2-deficient cells in favour of cell death. Furthermore, this drug combination also inhibited tumour formation in TSC2-deficient cell models and caused tumour spheroid death in 3D culture. Importantly, the 3D assay could differentiate the cytostatic agent, rapamycin, from the cytotoxic nelfinavir/salinomycin combination. Sporadic cancer cell lines with hyperactive mTORC1 signalling were also susceptible to this nelfinavir/salinomycin drug combination. This work indicates that the protein homeostasis pathway is an attractive therapeutic target in both Tuberous Sclerosis Complex and mTORC1-driven sporadic cancers.

Keywords: TSC; cell death; mTORC1; nelfinavir; therapy.

Figure 1. ER stress is elevated by nelfinavir and salinomycin treatment

Tsc2^+/+ and Tsc2^−/− MEFs were treated with either DMSO vehicle, 20 μM nelfinavir (NFV) or 5 μM salinomycin (Sal) as single agents or in combination for 6 h. Gene expression of CHOP A., HSP70 B. and EDEM1 C. was determined by real time PCR and relative mRNA expression was standardised to β-actin. Cells which had undergone treatment for 24 h were tested for the protein expression of IRE1α and AFT4, with β-actin and total TSC2 as controls D.. PCR products of spliced and unspliced XBP1 following 6 h drug treatment were resolved on agarose gels (unspliced = 480 bp upper band, spliced = 454 bp lower band) E. and quantified in F.. * p<0.05, **p<0.01, ***p<0.001.

Figure 2. Dual nelfinavir/salinomycin treatment kills TSC2^−/− MEFs synergistically

Tsc2^+/+ and Tsc2^−/− MEFs were treated with either DMSO vehicle, 20 μM nelfinavir (NFV) or 5 μM salinomycin (Sal) as single agents or in combination for 24 h and cell death was assessed by flow cytometry using DRAQ7 A., quantified in B. ELT3-V3 and TSC2-re-expressing ELT3-T3 cells were similarly treated for 48 h and analysed for cell death C., D. Synergy was assessed by examining cell death across a range of salinomycin concentrations, with or without 20 μM nelfinavir E., F. and calculated using CompuSyn software G.. Graphs show an average of three independent replicates, mean +/− S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3. Dual treatment kills cells in a mTORC1-dependent manner

Tsc2^+/+ and Tsc2^−/− MEFs were treated with DMSO or a 20 μM nelfinavir (NFV) plus 5 μM salinomycin (Sal) combination for 24 h, with or without the mTOR inhibitor, rapamycin (Rap). Cell death was assessed by flow cytometry and mTORC1 inhibition was determined by western blotting for phospho-S6K1 (T389) and phospho-4E-BP1 (Ser65) A. and B. Similar analysis of ELT3 cells was performed following 48 h drug treatment, with phospho-S6K1 used as a readout of mTORC1 activity C. and D. Graphs show an average of three independent replicates, mean +/− S.E.M. **p<0.01, ***p<0.001.

Figure 4. Mechanisms of nelfinavir/salinomycin-induced cell death

Following a 24 h incubation with the indicated treatments, Tsc2^+/+ and Tsc2^−/− MEFs were lysed and protein levels of phosphorylated S6K1, rpS6, 4E-BP1 and ACC were determined by western blot. Total TSC2, S6K1, rpS6, 4E-BP1, ACC and β-actin are shown as controls A.. The proteasome activity of drug treated samples was determined by monitoring the turnover of the fluorescent chymotrypsin-like substrate B.. Samples prepared as in A were further analysed for total GADD34, phospho-FOXO3a, phospho-PRAS40 and phospho-Bad. Tsc2^+/+ and Tsc2^−/− samples were run on the same gel and are shown at the same exposure C.. Tsc2^+/+ and Tsc2^−/− MEFs were pre-treated with cycloheximide (CHX) for 1 h, then treated with the indicated combinations for 24 h. Cell death was assessed by flow cytometry D.. *** p < 0.001.

Figure 5. Dual nelfinavir/salinomycin treatment is effective in 3D models

Tsc2^−/− MEFs A. or TSC-null AML cells B. were grown in soft agar and treated with DMSO, 10 μM nelfinavir (NFV), 2 μM salinomycin (Sal) or a NFV/Sal combination for 11 days. Images of the colonies were taken and the diameters measured using Image J. The scale bar represents 50 μm. Tsc2^−/− MEFs were allowed to self-aggregate into spheroids under non-adherent conditions and were treated with DMSO, 10 μM nelfinavir (NFV) plus 2 μM salinomycin (Sal) in combination or 25 nM rapamycin (Rap), as indicated, for 96 h. DRAQ7 was added for the final 36 h to monitor cell death. DRAQ7 images were taken and quantified C.. Spheroid diameter was determined from phase contrast images taken after 96 h drug treatment and plotted against DRAQ7 staining intensity D.. Spheroids were then replated onto standard tissue culture plates and allowed to grow under drug free conditions. Images were taken every 24 h and the area of outgrowth calculated using Image J E.. The scale bar represents 200 μm. Spheroid experiments were repeated in ELT3-V3 cells (F-H). ***p<0.001, NS = not significant.

Figure 6. Sporadic cancer cell lines with hyperactive mTORC1 are also sensitive to nelfinavir and salinomycin

Cell death of NCI-H460 cells was determined by flow cytometry following 48 h incubation with 20 μM nelfinavir (NFV) and/or 5 μM salinomycin (Sal), with or without 1 h rapamycin (Rap) pre-treatment A.. Cell lysates collected after 24 h treatment were analysed for mTORC1 activation (phosphorylated S6K1 and 4E-BP1) and total GADD34 levels A.. Experiments were repeated in HCT116 cells B.. *** p < 0.001.