Figure 6.
Sensitivity of xenografted ponatinib-resistant cells and primary chronic phase TKI-resistant chronic myeloid leukemia cells to NVP-BEZ235 and hydroxychloroquine (HCQ)-mediated autophagy inhibition. A) KCL22Pon-Res cells were labeled with firefly luciferase and treated ex vivo with 100 nM NVP-BEZ235, alone and in combination with 10 µM HCQ. Seventy-two hours following drug treatment, cells were transplanted intravenously into sublethally irradiated NSG mice (four mice per group, two independent experiments). Thirty minutes after the transplant, the mice were injected with D-luciferin substrate to ensure the success of the transplantation and the cell viability. Leukemic progression was measured weekly by luciferase bio-imaging (left). Overall survival was monitored by Kaplan-Meier analysis (right). A table showing the number of mice at risk is shown below the graph. B) Firefly luciferase labeled KCL22Pon-Res cells were transplanted intravenously into NSG mice (five to six mice per group, two independent experiments). Mice were then treated with NVP-BEZ235 (45 mg/kg, oral gavage), HCQ (60 mg/kg, intraperitoneal injection), and the combination for up to five weeks. Leukemia progression and overall survival were measured by luciferase bio-imaging (left) and Kaplan-Meier analysis (right), respectively. A table showing the number of mice at risk is shown below the graph. Untr = untreated. C) Bone marrow (BM)–derived mononuclear cells (MNCs) from four TKI-resistant patients (Pts 1–4) were cultured in SFM supplemented with PGF and treated with 100 nM ponatinib, 100 nM NVP-BEZ-235 alone, or in combination with HCQ-mediated autophagy inhibition for 72 hours. Survival of progenitor cells was measured by colony forming cell (CFC) assay. Each dot represents average of two to three technical replicates. D) BM cells were collected on four occasions from patient No. 1 (Pt 1) during the period of 2013–2016. Ph-negative CD34+ cells (n = 4) were cultured in SFM supplemented with PGF and treated with 100 nM ponatinib, 100 nM NVP-BEZ-235, 10 µM HCQ, 10 nM omacetaxine, and the combination of NVP-BEZ-235 and HCQ. Survival of progenitor cells was measured by CFC assay following 72 hours of drug treatment. Error bars = SD. Statistical analyses were performed using the Gehan-Breslow Wilcoxon test (A and B) or the two-tailed Student’s t test (C and D). CML = chronic myeloid leukemia; HCQ = hydroxychloroquine; Untr = untreated.