Figure 3
CML SC express functional DPPIV activity. (A) DPPIV enzyme activity of recombinant human (rh) DPPIV/CD26 (upper panel) and cell-derived CD26 (lower panel) measured as described in the supplemental material. Upper right: rhCD26 (200 ng/mL) was preincubated with various concentrations of sitagliptin or vildagliptin (as indicated) at 37°C for 30 minutes. Lower left: DPPIV activity in lysates of CD26-transfected KU812 cells (red bars) and control-vector–transfected CD26− KU812 cells (blue bars) (each 50 × 103). Before being analyzed, cells were incubated with various concentrations of sitagliptin or vildagliptin for 30 minutes. Lower right: Primary sorted CD26+ SC (red bars) and CD26− SC (blue bars) (each 25 × 103 cells) from a patient with CP CML were incubated with or without vildagliptin at 37°C for 30 minutes. DPPIV enzyme activity was expressed as relative luminescence units (RLU) per 50 × 103 cells. Results represent the mean ± SD of triplicates. (B) For chemotactic migration, CXCR4+ U937 cells (1.5 × 105/well) were applied in a double-chamber chemotaxis assay. Cells were allowed to migrate through 5-µm filters against various concentrations of SDF-1 (upper left) at 37°C for 4 hours. Transmigrated cells were counted by flow cytometry. In a next step, SDF-1 (25 ng/mL) was incubated with various concentrations of rhCD26 overnight and then used to induce migration of U937 cells (upper right subpanel). Finally, 200 ng/mL of rhCD26 were preincubated with various concentrations of sitagliptin (lower left) or vildagliptin (lower right) for 30 minutes, before SDF-1 (25 ng/mL) was added (overnight). Results represent the mean ± SD from 4 experiments. *P < .05 compared with untreated cells. (C) Left: CML MNC were plated in methylcellulose supplemented with cytokines in the absence (control) or presence of various concentrations of vildagliptin at 37°C for 16 days. Then CFU-GM colonies (black bars) were counted. Thereafter, colonies were picked and BCR/ABL1 mRNA expression was determined by qPCR (white bars). Right: CML MNC were incubated on irradiated M2-10B4 feeder cells for 3 weeks in the absence (control) or presence of various concentrations of vildagliptin. Then cells were harvested and plated in a CFU assay for 16 days. CFU-GM colonies (black bars) and BCR/ABL1+ colonies (white bars) are shown. *P < .05 compared with untreated cells. (D) CML MNC were plated on a feeder layer of irradiated M2-10B4 cells in various concentrations of imatinib (left panel) or imatinib in combination with 10 µM vildagliptin (right panel) for 48 hours. Thereafter, nonadherent cells of each well were harvested and plated in a CFU assay. After 2 weeks, colonies were counted. (E) The percentage of CD26+ and CD26− SC in the BM (left bars) and PB (right bars) of patients with CML determined by flow cytometry. (F) NSG mouse BM sections stained with an antibody against human CD45. NSG mice were injected with CD26+ SC, CD26− SC, lineage-positive (Lin+; CD14+, CD15+ sorted) cells (negative control), or CD3–depleted MNC (positive control) obtained from patients with CP CML. CD26+ LSC produced diffuse engraftment in the NSG BM, whereas normal CD26− SC produced myeloid engraftment at the endosteal surface. Original magnification ×40/0.85. (F) Lower right: Engraftment of CD26+ CML LSC after preincubation with control medium (−) or vildagliptin, 10 µM (+) at 37°C for 30 minutes. Engraftment in NSG mice (3 mice per group) was determined by measuring the percentage of CD45+ cells in the BM (after 18 weeks) by flow cytometry. Results represent the mean ± SD of 3 mice (each group). (G) BCR/ABL1 transcript levels, quantified by qPCR, in 2 patients with imatinib-resistant CML who received nilotinib (left panel patient: 200 mg/day; right panel patient: 800 mg/day), and later, because of uncontrolled diabetes mellitus, saxagliptin, 5 mg daily per os (left panel) or sitagliptin, 50 mg daily per os (right panel). (H) BCR/ABL1 mRNA levels in 8 CML patients (4 with high BCR/ABL1 burden and 4 with low BCR/ABL1 burden) who lost MMR or CMR during therapy with nilotinib and did not receive a gliptin. BCR/ABL1 transcript levels were quantified by qPCR according to the international scale.

CML SC express functional DPPIV activity. (A) DPPIV enzyme activity of recombinant human (rh) DPPIV/CD26 (upper panel) and cell-derived CD26 (lower panel) measured as described in the supplemental material. Upper right: rhCD26 (200 ng/mL) was preincubated with various concentrations of sitagliptin or vildagliptin (as indicated) at 37°C for 30 minutes. Lower left: DPPIV activity in lysates of CD26-transfected KU812 cells (red bars) and control-vector–transfected CD26 KU812 cells (blue bars) (each 50 × 103). Before being analyzed, cells were incubated with various concentrations of sitagliptin or vildagliptin for 30 minutes. Lower right: Primary sorted CD26+ SC (red bars) and CD26 SC (blue bars) (each 25 × 103 cells) from a patient with CP CML were incubated with or without vildagliptin at 37°C for 30 minutes. DPPIV enzyme activity was expressed as relative luminescence units (RLU) per 50 × 103 cells. Results represent the mean ± SD of triplicates. (B) For chemotactic migration, CXCR4+ U937 cells (1.5 × 105/well) were applied in a double-chamber chemotaxis assay. Cells were allowed to migrate through 5-µm filters against various concentrations of SDF-1 (upper left) at 37°C for 4 hours. Transmigrated cells were counted by flow cytometry. In a next step, SDF-1 (25 ng/mL) was incubated with various concentrations of rhCD26 overnight and then used to induce migration of U937 cells (upper right subpanel). Finally, 200 ng/mL of rhCD26 were preincubated with various concentrations of sitagliptin (lower left) or vildagliptin (lower right) for 30 minutes, before SDF-1 (25 ng/mL) was added (overnight). Results represent the mean ± SD from 4 experiments. *P < .05 compared with untreated cells. (C) Left: CML MNC were plated in methylcellulose supplemented with cytokines in the absence (control) or presence of various concentrations of vildagliptin at 37°C for 16 days. Then CFU-GM colonies (black bars) were counted. Thereafter, colonies were picked and BCR/ABL1 mRNA expression was determined by qPCR (white bars). Right: CML MNC were incubated on irradiated M2-10B4 feeder cells for 3 weeks in the absence (control) or presence of various concentrations of vildagliptin. Then cells were harvested and plated in a CFU assay for 16 days. CFU-GM colonies (black bars) and BCR/ABL1+ colonies (white bars) are shown. *P < .05 compared with untreated cells. (D) CML MNC were plated on a feeder layer of irradiated M2-10B4 cells in various concentrations of imatinib (left panel) or imatinib in combination with 10 µM vildagliptin (right panel) for 48 hours. Thereafter, nonadherent cells of each well were harvested and plated in a CFU assay. After 2 weeks, colonies were counted. (E) The percentage of CD26+ and CD26 SC in the BM (left bars) and PB (right bars) of patients with CML determined by flow cytometry. (F) NSG mouse BM sections stained with an antibody against human CD45. NSG mice were injected with CD26+ SC, CD26 SC, lineage-positive (Lin+; CD14+, CD15+ sorted) cells (negative control), or CD3–depleted MNC (positive control) obtained from patients with CP CML. CD26+ LSC produced diffuse engraftment in the NSG BM, whereas normal CD26 SC produced myeloid engraftment at the endosteal surface. Original magnification ×40/0.85. (F) Lower right: Engraftment of CD26+ CML LSC after preincubation with control medium (−) or vildagliptin, 10 µM (+) at 37°C for 30 minutes. Engraftment in NSG mice (3 mice per group) was determined by measuring the percentage of CD45+ cells in the BM (after 18 weeks) by flow cytometry. Results represent the mean ± SD of 3 mice (each group). (G) BCR/ABL1 transcript levels, quantified by qPCR, in 2 patients with imatinib-resistant CML who received nilotinib (left panel patient: 200 mg/day; right panel patient: 800 mg/day), and later, because of uncontrolled diabetes mellitus, saxagliptin, 5 mg daily per os (left panel) or sitagliptin, 50 mg daily per os (right panel). (H) BCR/ABL1 mRNA levels in 8 CML patients (4 with high BCR/ABL1 burden and 4 with low BCR/ABL1 burden) who lost MMR or CMR during therapy with nilotinib and did not receive a gliptin. BCR/ABL1 transcript levels were quantified by qPCR according to the international scale.

Close Modal

or Create an Account

Close Modal
Close Modal