(A) Enrichment analysis showing increased sensitivity of cancer lines with mutations in RB1 to an mTOR inhibitor, sirolimus. (B) Synergy scores of the combination of CDK4/6 and mTOR inhibition in two GIC lines calculated with both the Bliss and the Chou-Talalay methods. (C) 10 and 100 GICs were cultured in 24-well plates over two weeks to compare sphere formation upon treatment with vehicle, palbociclib (1 μM), everolimus (4 μM), and the combination of palbociclib and everolimus (*P < 0.05; **P < 0.001; ***P < 0.0001; one-way analysis of variance (ANOVA) with post-hoc Tukey analysis). (D) The combination of a different mTOR inhibitor temsirolimus (4 μM) and a different CDK4/6 inhibitor ribociclib (4 μM) is also synergistic against GICs (***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis). (E) The combinations of palbociclib (4 μM) with mTOR siRNA and everolimus (5 μM) with CDK4/6 siRNA are also synergistic against GICs (each treatment line received either DMSO or the indicated drug and either control siRNA or a specific siRNA) (**P < 0.001; ***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis). (F) Constitutively-active CDK4 and mTOR plasmids partially rescued from the effects of the combination treatment (each treatment line received either a control plasmid or the indicated active plasmid and either DMSO or the combination treatment) (*P < 0.05; two-tailed t-test). (G and H) Combined CDK4/6 and mTOR inhibition induces significant apoptosis. Shown is an immunoblot using antibodies specific for Bcl-2 and PARP. Caspase-3/7 level is increased with the three days of combined treatment. (*P < 0.05; ***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis).
Eve: Everolimus, Palbo: Palbociclib, Ribo: Ribociclib, Tem: Temsirolimus, NS: Non-Significant. All values are mean ± SEM of triplicates. Cell viabilities were determined via cell counts following 3 days of treatment. Each experiment was performed 3 times using separate samples.