JAK/STAT molecular status influences efficacy of ruxolitinib and PRN694, as well as levels of STAT5b phosphorylation. (A) Induction of apoptosis shown as fold change in viability. Each point is one of the 24 T-PLL samples: 5 without any mutation in the JAK/STAT pathway, and 19 with at least 1 mutation in the pathway. The values presented are the ratio of the viability (percentage vs DMSO), with a combination treatment (ruxolitinib 1 μM for 24 hours and venetoclax 100 nM for 4 hours), vs the viability with monotherapy (venetoclax 100 nM for 4 hours). Unpaired Student t test; means ± standard error of the mean [SEM]). (B) Induction of apoptosis shown as a percentage of viable (annexin V negative/Hoechst positive vs control) cells after a 24-hour exposure to DMSO or ruxolitinib 1 μM and a 4-hour exposure to venetoclax 100 nM, showing individual paired values. The values in the left column of data are those of the 8 samples presenting at least a mutation of STAT5B, the values on the right are those of the 11 samples presenting an isolated mutation of JAK3 or JAK1. The mean reduction of viability (measured viability of DMSO vs ruxolitinib) is significantly larger in the 11 samples without STAT5B mutation (unpaired Student t test). (C) Densitometry analysis of the ratio of expression of phospho-STAT5 to total STAT5, in 9 T-PLL samples (each point is labeled with the T-PLL sample number). The samples were divided into 2 groups: 2 T-PLL samples without mutations in the JAK/STAT pathway genes and 7 T-PLL samples with at least 1 activating mutation in that pathway (unpaired Student t test; means ± SEM). (D) Representative examples of western blot analysis of phospho-STAT5 and total STAT5 in 4 T-PLL samples at baseline. β-Actin was used as the loading control. (E) Induction of apoptosis shown as fold change in viability, in the 24 T-PLL samples. The values presented are the ratio between the viability (percentage vs DMSO) with a combination treatment (PRN694 1 μM for 24 hours and venetoclax 100 nM for 4 hours) vs the viability with monotherapy (venetoclax 100 nM for 4 hours; unpaired Student t test; means ± SEM). *P < .05; **P < .01; ****P < .0001.