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Ruxolitinib, nilotinib and prednisone for myelofibrosis
Figure 2. Screening for signaling proteins affected by 32 nM ruxolitinib (R), 1.6 μM nilotinib (N), 0.8 mM prednisone (P), and their combinations, using a phospho- protein array. C: Control. The SET2 cell line was incubated with R, N, P or their combination for 48 hours and phosphoprotein analysis was performed.
0.045 mM). Interestingly, these inhibitors were also the most active drugs in patients' samples (Table 2), showing an EC50 of 0.008 mM, 0.033 mM and 0.041 mM, respective- ly. Furthermore, prednisone, an immunosuppressant used to control symptoms of MF by decreasing the levels of cytokines and growth factors such as TGF-β,24 showed an EC50 of 0.144 mM. Other active drugs in the low micromo- lar range included the signaling pathway inhibitors BKM120 (EC50 = 3.331 mM) and ponatinib (EC50 = 5.530 mM) (Online Supplementary Table S5). Patients' PBMCs were even more sensitive to these drugs: EC50 BKM120 = 0.893 mM, EC50 ponatinib = 1.716 mM (Table 2).
Curiously, drugs used in treatment of MF such as dana- zol or prednisone did not have any effect on BA/F3 JAK2 V617F viability (Online Supplementary Table S5), but pred- nisone was effective in combination with ruxolitinib (ΔEC50 = -13.9%) (Figure 1A). Patients' samples showed the same response (ΔEC50 = -18.0%) (Figure 1B).
Ruxolitinib/nilotinb/prednisone combination has a synergistic effect on myeloid cell lines and patient peripheral blood mononuclear cells
Given the synergistic effect of nilotinib with ruxolitinib in BA/F3 JAK2 V617F cells, we next studied the effect of adding prednisone to this combination in myeloid cell lines and patients' PBMCs. As well as being a potent BCR/ABL TKI, nilotinib has been reported to inhibit the PDGF receptor, which is involved in fibrogenesis.20
A synergistic effect with all combinations of the three
drugs tested was found in BA/F3 JAK2 V617F and SET2 cells (Table 3). By contrast, only four of the eight doses tested were synergistic in BA/F3 wild-type (WT) cells, again suggesting an important role for the V617F mutation in the activity of the drug combination. We repeated this assay using monotherapy or combination regimens in model A cultures (PBMCs) to test the effect of the combi- nations in myeloid progenitors. Myelofibrosis patients' samples were sensitive to nilotinib and prednisone with an EC50 of 6.6 mM and 13.1 mM, respectively. Moreover, all combinations tested (ruxolitinib/nilotinib and ruxoli- tinib/niloitnib/prednisone) exhibited synergic behavior in at least two of the five patients' samples tested. In addi- tion, the combination of 160 nM ruxolitinib, 8 mM nilo- tinib and 0.8 mM prednisone was synergistic in all the patients' samples tested (Table 4).
Ruxolitinib/nilotinb/prednisone combination blocks JAK/STAT and MAPK signaling
The signaling pathways affected by the ruxolitinib/nilo- tinib/prednisone combination or monotherapy were next characterized in SET2 cells using the Proteome ProfilerTM phosphoprotein array after treatment for 30 min. Results showed that, among others, phosphorylation of STAT5 was significantly inhibited 88.94%, TOR 76.55%, ERK 87.55%, and SRC 76.88% (Figure 2) by drug combination (ANOVA ***P<0.005). Western blotting confirmed that the phosphorylation of STAT5 and ERK was inhibited by rux- olitinib in monotherapy by 69.8±8.1% and 87.7±2.3%,
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