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A.A. Cortés et al.
AB
Figure 1. Effect of each drug in combination with 100 nM ruxolitinib in BA/F3 JAK2-V617F3 cell line (A) or in patients' peripheral blood mononuclear cells (B).
y-axis: the increment between the EC50 for each drug in the presence of ruxolitinib minus its EC50 in monotherapy. Results are expressed as the mean±Standard Deviation (SD) of 2 independent experiments in cell lines (A) and median and interquartile range in patients' samples (B).
regression was performed for the correlation of time of response with ex vivo activity of ruxolitinib. For the statistical analysis of phospho-kinase array, an ANOVA test was performed. A t-test was used to assess whether the CI of each combination was sig- nificantly synergistic. For collagen expression assays and phospho- proteomics studies, Student t-test was used when the populations were normal and the non-parametric Wilcocox t-test when they were not. P<0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism v.6.00 for Windows (GraphPad Software, La Jolla, CA, USA) or STATA v.13 (StataCorp., College Station, TX, USA).
Results
Ruxolitinib activity in cell lines and patients’ samples
We first evaluated the activity of ruxolitinib in JAK2-mutated cell lines. Ruxolitinib efficiently inhibited the viability of BA/F3 and SET2 V617F JAK2 cells with an EC50 of 35 nM and 25 nM, respectively (Online Supplementary Figure S1A). The EC50 for ruxolitinib in BA/F3 WT cells was 212 nM, indicating the importance of the JAK2-V617F mutation for the activity of ruxolitinib. Nonetheless, when we compared the activity of ruxoli- tinib in patients' PBMCs with or without a JAK2 muta- tion, using ex vivo model A, we found that its activity was not significantly different, with an EC50 of 55 nM. For this reason, subgroups based on the mutation in JAK2 were not studied further.
To determine the best cell model to screen drugs in com- bination with ruxolititinb, its activity was tested in the two different ex vivo culture models. The only method that provided a sufficient number of cells for screening was model B, although the EC50 for ruxolititinb using this model was 0.747 μM. Greater ruxolitinib activity was found when PBMCs were seeded in methylcellulose in the presence of ruxolitinib (model A: EC50 = 43 nM) (Online Supplementary Table S4 and Online Supplementary Figure S1B). Moreover, if ex vivo activity of ruxolitinib was com-
pared with the time of response to ruxolitinib of each patient sample, it was found that both models A and B distinguished patients' samples with responses >6 months (Online Supplementary Figure S1C).
BCR/ABL or ABL kinase inhibitors and PDGFR and TGFβR inhibitors are effective combinations with ruxolitinib in cell lines and patients' samples
To examine the best combination with ruxolitinib, dose-response curves of all tested drugs in monotherapy or in combination with ruxolitinib were first analyzed in BA/F3 V617F JAK2 cells using an automated flow cytom- etry platform. Drugs exhibiting the best behavior in the presence of ruxolitinib were then selected to perform the same assay using PBMCs of MF patients in ex vivo model B. Drugs with more activity in the screening were also included in dose-response assays in monotherapy with patients' samples.
Results showed that the BCR/ABL or SRC/ABL tyrosine kinase inhibitors (TKI) nilotinib and bosutinib, respective- ly, together with danazol, a synthetic androgen reported to reverse anemia,23 and SB432542, an inhibitor of the TGF-β receptor related to the fibrogenic processes, were among the four best combinations in BA/F3 JAK2 V617F cells (Figure 1A). Accordingly, they presented the lowest increments between their EC50 in the presence or absence of ruxolitinib (ΔEC50 nilotinib = -92.4%; ΔEC50 bosutinib = -87.7%; ΔEC50 danazol = -80.1%; ΔEC50 SB432542 = -77.1%). When tested in patients' samples, of the two BCR/ABL inhibitors, only nilotinib showed a lower EC50 in the presence of ruxolitinib than in its absence (ΔEC50 nilotinib = -21.6%), together with SB432542 (ΔEC50 = -11.7%) (Figure 1B).
Online Supplementary Table S5 shows the drugs listed by their Emax and EC50. The most active drugs in BA/F3 JAK2 V617F cells were the histone deacetylase HDAC6 inhibitor panobinostat (EC50 = 0.041mM), the proteasome inhibitor bortezomib (EC50 = 0.041 mM), and the immunomodulatory HSP90 inhibitor HSP990 (EC50 =
940
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