Bafilomycin A1 Targets B-cell Acute Lymphocytic Leukemia Stem Cells

Objective:To explore whether bafilomycin Al targets B-cell acute lymphocytic leukemia stem cells or not.Methods:(1)BM cells of B-ALL patients and normal healthy control werecollected and the percentage of CD34+cells,CD34+CD19+ cells were measured by flow cytometer.(2)B-ALL CD34+CD19+ cells and NBM CD34+ cells were sorted by flow cytometer.Check whether bafilomycin Al will affect cell proliferation of B-ALL stem cells and normal stem cells in vitro.(3)Annexin-V/Propidium Iodide staining method was performed to detect apoptosis in B-ALL CD34+CD19+ cells or NBM CD34+ cells.(4)Check whether B-ALL stem cells will differentiate after bafilomycin Al treatment.(5)B-ALL CD34+CD19+ cells and NBM CD34+ cells were sorted by flow cytometer,and then check whether the cell cycle of B-ALL stem cells and normal stem cells will be affected by bafilomycin A1 treatment.(6)B-ALL CD34+CD19+ cells(1-5x 106 cells/mous)were transplanted into 8-week-old NSG mice by tail vein injection.Mice were euthanized 7 weeks after B-ALL stem cells injection.PB cells,BM cells and spleen cells were harvested,the engraftment of human cells was analyzed by flow cytometer.Results:(1)Bafilomycin Al treatment conspicuously decreased the percentage of B-ALL CD34+cells,while the percentage of NBM CD34+cells remain unchanged by treatment with bafilomycin Al.Bafilomycin Al reduces the percentage of primary B-ALL stem cells while sparing normal hematopoietic cells.(2)Dose response studies show that 1 nM bafilomycin Al can induced a clear cytotoxicity in B-ALL stem cells compared with untreated controls,while bafilomycin A1 had no toxic effect on bone marrow cells isolated from healthy donors.(3)Bafilomycin A1 of 1 nM caused apoptotic death in the primary B-ALL stem cells at 48 h treatment,confirming that low concentration bafilomycin A1 truly kills pediatric B-ALL stem cells while sparing normal hematopoietic stem cells.(4)A small number of B-ALL stem cells differentiate after treatment with bafilomycin Al.(5)Bafilomycin A1 induced B-ALL stem cells out of GO phase.(6)In contrast to untreated mice,in vivo bafilomycin Al treatment after transplantation significantly reduced leukemic burden.Bafilomycin Al significantly prolonged survival in the bafilomycin Al-treated mice with advanced disease compared with control mice.Bafilomycin Al treatment significantly reduced the number of leukemia stem cells in the bone marrow and the peripheral blod.In the disease model group,the mice had seriously hepatosplenomegaly compared with the control group.However,the size and weight of livers and spleens in the bafilomycin Al-treated group were significantly normalized compared with the disease model group.The livers from the disease nice were significantly infiltrated.In contrast,considerably fewer ALL cells showed infiltration in the livers of bafilomycin Al-treated mice compared with the model disease mice.These data suggest that bafilomycin Al dramatically inhibits pediatric B-ALL stem cell engraftment by targeting leukemia stem cells in vivo.Conclusion:(l)In vitro treatment with bafilomycin Al of low concentration kills primary B-ALL stem cells derived from patients.(2)In vivo treatment with bafilomycin Al of tow concentration prolongs the life of mice engrafted with primary B-ALL stem cells.(3)In vivo treatment with bafilomycin Al of low concentration improves the pathological conditions of mice ergrafted with primary B-ALL stem cells.