Figure 5

The combination of ganetespib and ABT737 overcomes acquired resistance through exploitation of MCL1 downregulation. (a) STAR cells were treated with ganetespib 200 nm, ABT737 200 nm or a combination of both for 48 h. PARP cleavage and MCL1 expression were measured by western blot. The effect on colony formation was measured by clonogenic assay. STAR cells were treated for 24 h with ganetespib 200 nm, ABT737 200 nm or a combination of both. After being washed, colonies were let to grow for 12 days, then fixed in methanol and stained with crystal violet. (b) Mice bearing established STAR tumours (n=7 per group) were dosed with vehicle, 100 mg/kg ABT263 (5 × /week), 100 mg/kg ganetespib (1 × /week), or the combination of ABT263 and ganetespib. Tumour volumes were assessed at end of the study. The combination of ABT263 with ganetespib resulted in a statistically significant decrease in tumour volume compared with vehicle control (error bars ±s.e.m.). (2-way Anova repeated measurements results: 19 days, ABT 263 + Ganetespib vs Vehicle, **P=0.0095; 24 days, ABT 263 + Ganetespib vs Vehicle, **P=0.0017; 27 days, ABT 263 + Ganetespib vs Vehicle, ****P<0.0001, ABT 263 + Ganetespib vs Ganetespib P=0.0132, ABT 263 + Ganetespib vs ABT 263 P=0.0004). The average body weight loss at end of the study was −7.1% with vehicle, −9.1% with ganetespib, −12.1% with ABT263 and −9.6% for the combination. (c) STAR cells were treated with ganetespib 200 nm, ABT199 200 nm, or a combination of both for 48 h. A combination of ganetespib and ABT737 in STAR and ABT199 in DOHH2 were used as a positive control. PARP cleavage and MCL-1 expression were measured by western blot. (d) STAR cells have been transfected with siRNAs targeting BH3-only protein. Twenty-four hours after transfection, cells have been treated with ganetespib 200 nm and ABT737 200 nm for further 48 h and caspase 3 activity measured. Data were normalized to siNT control (*P=0.0146). PARP cleavage, BAX, BAK, BID and PUMA expression was assessed by western blot. (e) STAR cells were transfected with siRNA targeting MCL1. Twenty-four hours after transfection, cells were left untreated or treated with ABT737 both for further 48 h. PARP cleavage and MCL1 expression were measured by western blot. (f) NCI-H28 cells were treated with ganetespib 200 nm, ABT737 200 nm or a combination of both for 48 h. PARP cleavage and MCL1 expression were measured by western blot. (g) NCI-H28 cells were transfected with siRNA targeting MCL1. Twenty-four hours after transfection, cells were left untreated or treated with either ABT737 or ganetespib for further 48 h. PARP cleavage and MCL1 expression were measured by western blot.