Fig. 5: CDK4/6 inhibition in combination with HSP90 inhibitors impairs CRC cell viability independent of the p53 status.

A–C Cell viability matrices of p53-proficient HCT116 cells treated with Palbociclib combined with Ganetespib for A 72 h or B 48 h; or C the HSP90 inhibitor Onalespib combined with Ganetespib for 48 h. n ≥ 3 biological replicates. D Relative confluence of HCT116 cells after 72 h with the indicated treatments. Cell confluence was analyzed by Celigo imaging cytometry. Confluence relative to DMSO control. Palbo: Palbocliclib, Ganet: Ganetespib. E Determination of dead cells. PI/Hoechst/Annexin V staining of HCT116 cells treated for 72 h with Ganetespib and two different Palbociclib concentrations alone or in combination. Percent dead cells (PI+ only, annexin V+ only, and PI+ Annexin V+ cells) were analyzed by Celigo imaging cytometry. D, E Mean ± SEM from ≥3 biological replicates. Student’s t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns not significant. F PARP-1 cleavage in HCT116 cells treated with 10 µM Palbociclib, 50 nM Ganetespib alone or in combination for 48 h. Representative immunoblot from 2 biological replicates. G Cell viability matrix of PDO #2 culture, treated as small organoids with the indicated concentrations of Ganetespib, Palbociclib alone or in combination for 72 h. Three replicates (different passages) with 2 in-plate technical replicates each. Right, Representative brightfield images after the indicated treatments. Scale bars, 200 μm. H Cell viability matrices of p53-deficient SW480 (left) and SW620 (right) human CRC cells treated with Ganetespib - Palbociclib alone or in combination for 48 h. n = 3 biological replicates each. I Determination of dead cells as in (E) for p53-deficient SW480 and SW620 cells treated with Ganetespib and Palbociclib alone or in combination for 72 h. Dead cells were examined by Annexin and PI staining. Mean ± SEM from ≥2 biological replicates. Student’s t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns not significant. A–C, G, and H Color scheme represents changes in cell viability. Numbers in the matrix are HSA synergy scores. Synergy scores: <−10 antagonistic; −10 to 10 additive, >10 synergistic.