Figure 3. The effect of TSLP stimulation on the efficacy of JAK inhibitors.

Cells (PDX or primary ALL) were pre-incubated for 1 hour with or without 25 ng/ml TSLP, after which cells were exposed for four days to indicated concentrations of momelotinib or ruxolitinib. Cell viability was measured using an MTT assay. Sensitivity was calculated relative to vehicle treated controls. Individual samples were tested in duplicate. (A-B) Efficacy of momelotinib and ruxolitinib on JAK2 mutated cells with or without TSLP pre-incubation. Mean±SEM of six independent samples is shown. (C-D) Efficacy of momelotinib and ruxolitinib on cells with JAK2 translocations. Mean±SEM of two independent samples is shown. (E-F) Efficacy of momelotinib and ruxolitinib on JAK2 wildtype PDX cells. Mean±SEM of three independent samples is shown. (G-H) Combined graph of the efficacy of momelotinib (G) and ruxolitinib (H) on TSLP stimulated cells with JAK2 mutations (n=6), JAK2 translocations (n=2), or JAK2 wildtype cells (n=3). Mean±SEM of independent samples is shown. Cell viability of samples was compared using the independent sample T-test. ^**p≤0.01, ^*p≤0.05. ¹JAK2 translocations versus JAK2 wildtype,²JAK2 wildtype versus JAK2 mutations. (I-K) Western blot of JAK2^R683S, TERF2-JAK2 and JAK2^wt PDX cells with or without TSLP stimulation (25 ng/ml for 1 hour). (L) JAK2^R683G cells were pre-incubated for 1 hour with or without 25 ng/ml TSLP, after which cells were exposed for four hours to vehicle control medium, 1.5 μM momelotinib or 0.75 μM ruxolitinib. Levels of (phosphorylated) JAK2, STAT1, STAT5, MEK1/2 and ERK1/2 were analyzed using western blot.