Fig. 6.

Synergistic effects after combined inhibition of HSP90 and JAK2. a Increased sensitivity to JAK2 inhibition. HSP90i-resistant clones CR2 and CR3 were treated with increasing concentrations of a JAK2 inhibitor, LY2784544 alone or in combination with 10 nM ganetespib for 72 h and subjected to resazurin-based cell viability assay. Cell viability (%) for each treatment was expressed relative to either DMSO-treated or ganetespib only-treated control for combination treatment. Representative graph from three independent experiments each performed in triplicates and error bars indicate SEM. b Increased sensitivity to HSP90i. HSP90i-resistant clones CR2 and CR3 were treated with increasing concentrations of ganetespib alone or in combination with 1 μM LY2784544 for 72 h and subjected to resazurin-based cell viability assay. Cell viability (%) for each treatment was expressed relative to either DMSO-treated or LY2784544 only-treated control for combination treatment. Representative graph from three independent experiments each performed in triplicates and error bars indicate SEM. c Inhibition of JAK-STAT signalling pathway and induction of apoptosis after combined treatment. HSP90i-resistant clones CR2 and CR3 were treated with ganetespib (10 nM), LY2784544 (1 μM) or in combination for 24 h. Protein lysates were analysed by western blotting and blotted with pSTAT3 (Y705), which is a downstream protein of the JAK signalling pathway. Levels of cleaved PARP and HSP70 were also determined to assess apoptosis and pharmacodynamic marker for HSP90i respectively. PPIB was used as loading control. d Increased cytotoxicity after combined treatment. HSP90i-resistant clones CR2 and CR3 were also treated with another JAK2 inhibitor, AZD1480 (2 μM) alone or in combination with ganetespib (10 nM) for 72 h and subjected to resazurin-based cell viability assay. The graph represents the overall mean cell viability (%) relative to DMSO-treated or ganetesib-treated control from two independent experiments each performed in triplicates. Error bars indicate SEM. The sensitivity to AZD1480; in the absence or presence of ganetespib were compared in the HSP90i-resistant clones. ** and *** indicate p values ≤0.01 and ≤ 0.001 respectively; by Student’s t-test