4EGI-1 dramatically reduces the clonogenic growth of AML progenitors with a moderate impairment of normal CD34⁺ hematopoietic progenitor clonogenicity. (A) AML blast cells from 5 patients were plated in H4230 methylcellulose medium without or with 25, 50, or 100 μM 4EGI-1 (4E25, 4E50, and 4E100, respectively), 10 nmol/L RAD001 (R), or the association of RAD001 with 25 μM or 50 μM 4EGI-1. Cells were then incubated for 7 days, and the leukemic colonies (> 20 cells), referred to as CFU-L, were scored under an inverted microscope. In the control condition, the numbers of CFU-L were 66 to 258 in 5 independent experiments. (B) Normal CD34⁺ cells were purified from 5 healthy donors and then plated as described in “Colony assays,” without or with 50 or 100 μM 4EGI-1. The BFU-E, CFU-GM, and CFU-GEMM colony-forming units were counted under an inverted microscope. In the control condition, the numbers of colonies were 82 to 118, 43 to 58, and 11 to 15 for BFU-E, CFU-GM, and CFU-GEMM, respectively, in 5 experiments. Each histogram represents the mean of 5 independent experiments, performed in duplicates. Results are expressed as a ratio between each condition and the control condition. Vertical bars represent SD. ***P < .001. ns indicates not significant.