Figure 4. IKZF2 is required for human leukemia cell survival.
(A) Western blot analysis showing depletion of IKZF2 in human cord blood CD34+ (HSPCs) at day 4 post transduction with lentivirus expressing either Scramble control or two independent IKZF2 shRNAs. (B) Colony assay was performed with HSPCs in (A). 104 cells were plated at day 4 post transduction and different colonies were counted two weeks later. Result is from n=3 independent experiments. (C) Western blot of IKZF2 was performed in whole cell lysates from eight cell lines, using ACTIN as loading control. (D) Western blot analysis showing IKZF2 depletion in MOLM-13 cells after four days post-transduction with lentivirus expressing IKZF2 shRNAs. ACTIN was used as loading control. (E) Depletion of IKZF2 in MOLM-13 cells reduced colony formation. Colonies were measured a week after plating shRNA transduced-MOLM-13 cells. (F) IKZF2 knockdown increases apoptosis in MOLM-13 cells. Apoptosis was measured by flow cytometry using Annexin V-PE and 7-AAD staining at day 7 post-transduction. (G-H) IKZF2 knockdown leads to increased differentiation. Flow cytometry measuring myeloid markers (G) CD13/CD14 and (H) CD11b/CD33 in MOLM-13 cells at day 7 post-transduction. Results in (B-H) are from more than three independent experiments using triplicates are shown. Mean +/− S.E.M Student’s t test p value. *p<0.05, **p<0.01 and ***p<0.001. (I) IKZF2 knockdown prolongs leukemia-free survival in mice. MOLM-13 cells transduced with scramble or IKZF2 shRNA virus were counted at day 4 post-transduction and transplanted into sub-lethally irradiated NSG mice. Results from four combined transplants with total scram n=20, Ikzf2 shRNA-3 n=18, and Ikzf2 shRNA-5 n=20 mice are shown. log-rank test. **p<0.01 and ***p<0.001. (J) IKZF2 is highly expressed in CD34+CD38− population in AML patients. Left, IKZF2 intracellular flow cytometry data showing IKZF2 expression in CD34− and CD34+CD38− populations in nine AML patients. Right, representative flow plot showing gating of CD34− and CD34+CD38− populations. (K) IKZF2 Intracellular flow cytometry data in AML patients cells transduced with lentivirus expressing Scram control or IKZF2 shRNA at day 4 post transduction. Results are from experiments using cells from four AML patients. Mean +/− S.E.M Student’s t test p value. *p<0.05, (L) CD34+CD38− population is reduced when IKZF2 is knocked down in AML patient cells. Leukemic stem cell population was analyzed at day 4 post transduction with Scram or IKZF2 shRNA. Results are from four AML patients. Mean +/− S.E.M Student’s t test p value. *p<0.05. (M) IKZF2 depletion reduces colony formation in AML patient cells. Cells from Patient #1 were transduced with lentivirus expressing Scramble or IKZF2 shRNA and sorted for GFP at day 4 post transduction. Left, graph showing number of colonies scored 2 weeks after plating. Right, flow plot of intracellular IKZF2 staining in patient cells.