Treatment with a RUNX-CBFβ inhibitor impairs the growth of human T-ALL cell lines and primary pediatric T-ALL samples. (A) Structures of inactive (AI-4-88) and active (AI-10-104) inhibitors are shown. (B) Protein lysates from the human T-ALL cell line KOPTK1 treated with DMSO or increasing concentrations of AI-10-104 for 6 hours were immunoprecipitated with RUNX1 or RUNX3 antibodies and immunoblotted with CBFβ, RUNX1, and RUNX3 antibodies. (C) Eight human T-ALL cell lines were treated with increasing concentrations of AI-10-104 for 3 days, and cell growth/metabolism was analyzed by MTS assay. (D) The human T-ALL cell line Jurkat was treated with vehicle, 10 μM of the inactive analog AI-4-88, or with increasing concentrations of AI-10-104 for 4 days. Cells were stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry. A representative flow profile of 3 independent experiments is shown. (E) Eleven pediatric T-ALL patient samples were treated with vehicle or increasing concentrations of AI-10-104 (1-15 μM) for 3 days, and cell growth/metabolism was analyzed by CellTiterGlo assay. Absorbance values were normalized to those obtained with vehicle control. (F) The sensitivity of patient samples to AI-10-104 (GI₅₀) correlates with RUNX1 and RUNX3 expression levels (Pearson’s r = 0.8781, P = .0093, sample TALL-X-5 excluded). (G) Patient sample TALL-X-15 was treated with 10 μM of AI-4-88 or with 5 or 10 μM of AI-10-104 for 6 days. Cells were stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry.