Fig. 2. Effects of pexmetinib on human CML cells.
A Proliferation of human Ph+ KCL22 cells carrying the T315I mutation treated with the indicated inhibitors. Dose-response curves (upper panels) and time-course experiments (lower panels) are shown. KCL22-DasR and KCL22-BosR indicate KCL22 cells selected for resistance to dasatinib and bosutinib, respectively. B Inhibition of BCR::ABL1T315I autophosphorylation in drug-resistant KCL22 cells, shown by western blot using phospho-specific (pABL1) and total ABL1 antibodies. C Summary of drug-resistant KCL22 cells growth inhibition data, shown as IC50 fold-change values relative to parental KCL22, highlighting the loss of potency in resistant cells; Ph− cells (SUPM2) are shown as a reference of unspecific toxicity. D Tumor growth of KCL-DasR xenografts in nude mice treated daily with vehicle (n = 6), dasatinib (20 mg/kg p.o.; n = 5), or pexmetinib (40 mg/kg i.p.; n = 5). *p < 0.05 versus vehicle and dasatinib. Mice were injected subcutaneously with 8 × 106 cells in right flank; treatments started when tumors were measurable (30 mm3). E Inhibition of colony formation of BCR::ABL1-negative, WT and T315I patient-derived bone marrow myeloid cells by pexmetinib (3–10 μM), dasatinib (3–10 nM) and imatinib (0.3–1 μM); 2 × 105 cells were seeded in methylcellulose and colonies were counted after 14 days. The data represent the normalized mean ± SD of triplicate experiments. The average of two T315I patients is reported. *p < 0.05; **p < 0.01; ***p < 0.001 versus vehicle. Statistical significance is calculated by unpaired, two-tailed Student’s t-test. F Molecular docking of pexmetinib (gray sticks) in T315I-mutated ABL1 kinase shown by ribbon representation. The DFG motif is in pink color. G Superposition of pexmetinib (gray) and axitinib (green) bound to ABL1T315I kinase. Docking studies were performed using the Maestro suite from Schrodinger. IM imatinib; NIL nilotinib; DAS dasatinib; BOS bosutinib; PON ponatinib; PEX pexmetinib.