Figure 2.
Reduction of the HoxA9-eIF4E interaction impairs leukemogenesis. (A) Colonies of primary lin–Sca-1+c-Kit+ (LSK) cells transduced with bicistronic vectors for HoxA9-IRES-GFP or HoxA9AA-IRES-GFP and Meis-IRES-YFP and corresponding vector controls after 4 rounds of serial replating in cytokine supplemented semisolid medium. The mean number of colonies and the standard deviation are shown below done in 3 replicates. Right, western blot of HoxA9 proteins from leukemic mouse cells, using the FLAG antibody, Hsp90 is provided as a loading control. (B) Schematic of murine transplantation model. (C) Kaplan-Meyer plot of mice transplanted with HoxA9/Meis1 (n = 10) or HoxA9AA/Meis1 (n = 14) transduced LSK cells. (D) Kaplan-Meyer plot after secondary transplants of HoxA9wt/Meis1 or HoxA9AA/Meis1 leukemic bone marrow cells into secondary recipient mice (n = 5 per group). Cells were analyzed by flow cytometry (supplemental Figure 2A-D). Serial replating assays were as in.21 (E) Response curve of murine leukemic bone marrow subjected to pharmacological inhibition of eIF4E with ribavirin. Clinically achievable ribavirin concentration (10 uM) after 168 hours in technical triplicates and biological duplicates. 2 cell lines generated from mouse primary cell lines (HoxA9wt and HoxA9AA) compared to untreated cells. (F) Impact of genetic reduction of eIF4E on proliferation in human AML cell lines based on HoxA9 levels. Cells with HoxA9-dependency (MV411 and SHI-1) were more sensitive to 2 different short hairpin RNAs (shRNAs) to eIF4E than those with low levels of HoxA9 (Kasumi-1 and HL-60) (left panels). eIF4E western blots confirm shRNA mediated knockdown in each cell type and actin is provided for loading (right panels). Experiments were carried out 3 times for each hairpin. Means and standard deviations are shown. For panels E-F, means are provided, error bars indicate standard deviation, P values shown (Student t test). (G) Sensitivity to pharmacological targeting of eIF4E with high-HoxA9 cells (blue) were more sensitive to ribavirin than low-HoxA9 cells (red). IC50s provided in inset. Experiments were carried out in 3 replicates. (H) Microarray analysis of fold change in gene expression of HoxA9AA/Meis1 over HoxA9wt/Meis1 leukemic mouse cells demonstrated highly similar transcriptional signatures. Bone marrow cells from 3 HoxA9wt/Meis1and 3 HoxA9AA/Meis1 mice were harvested with ≥97% GFP+ cells in the leukemic bone marrow. The expression levels of HoxA9-regulated gene sets, selected from Gene Set Enrichment Analysis (GSEA) database, were analyzed for overlapping genes in our microarray data set, and mean log2 fold change was determined for each gene set. EV, empty vector; IC50, 50% inhibitory concentration.

Reduction of the HoxA9-eIF4E interaction impairs leukemogenesis. (A) Colonies of primary lin–Sca-1+c-Kit+ (LSK) cells transduced with bicistronic vectors for HoxA9-IRES-GFP or HoxA9AA-IRES-GFP and Meis-IRES-YFP and corresponding vector controls after 4 rounds of serial replating in cytokine supplemented semisolid medium. The mean number of colonies and the standard deviation are shown below done in 3 replicates. Right, western blot of HoxA9 proteins from leukemic mouse cells, using the FLAG antibody, Hsp90 is provided as a loading control. (B) Schematic of murine transplantation model. (C) Kaplan-Meyer plot of mice transplanted with HoxA9/Meis1 (n = 10) or HoxA9AA/Meis1 (n = 14) transduced LSK cells. (D) Kaplan-Meyer plot after secondary transplants of HoxA9wt/Meis1 or HoxA9AA/Meis1 leukemic bone marrow cells into secondary recipient mice (n = 5 per group). Cells were analyzed by flow cytometry (supplemental Figure 2A-D). Serial replating assays were as in.21 (E) Response curve of murine leukemic bone marrow subjected to pharmacological inhibition of eIF4E with ribavirin. Clinically achievable ribavirin concentration (10 uM) after 168 hours in technical triplicates and biological duplicates. 2 cell lines generated from mouse primary cell lines (HoxA9wt and HoxA9AA) compared to untreated cells. (F) Impact of genetic reduction of eIF4E on proliferation in human AML cell lines based on HoxA9 levels. Cells with HoxA9-dependency (MV411 and SHI-1) were more sensitive to 2 different short hairpin RNAs (shRNAs) to eIF4E than those with low levels of HoxA9 (Kasumi-1 and HL-60) (left panels). eIF4E western blots confirm shRNA mediated knockdown in each cell type and actin is provided for loading (right panels). Experiments were carried out 3 times for each hairpin. Means and standard deviations are shown. For panels E-F, means are provided, error bars indicate standard deviation, P values shown (Student t test). (G) Sensitivity to pharmacological targeting of eIF4E with high-HoxA9 cells (blue) were more sensitive to ribavirin than low-HoxA9 cells (red). IC50s provided in inset. Experiments were carried out in 3 replicates. (H) Microarray analysis of fold change in gene expression of HoxA9AA/Meis1 over HoxA9wt/Meis1 leukemic mouse cells demonstrated highly similar transcriptional signatures. Bone marrow cells from 3 HoxA9wt/Meis1and 3 HoxA9AA/Meis1 mice were harvested with ≥97% GFP+ cells in the leukemic bone marrow. The expression levels of HoxA9-regulated gene sets, selected from Gene Set Enrichment Analysis (GSEA) database, were analyzed for overlapping genes in our microarray data set, and mean log2 fold change was determined for each gene set. EV, empty vector; IC50, 50% inhibitory concentration.

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