Second-generation inhibitor MI-2-2 exhibits strongly enhanced cellular activities compared with MI-2. (A) Growth curves for MLL-AF9 transduced BMC grown in liquid culture treated with DMSO, MI-2, and MI-2-2. The experiment was performed 2 times. (B-C) Quantitative real-time PCR showing the expression of Hoxa9 (B) and Meis1 (C) in MLL-AF9–transduced BMCs over the 6 days of treatment with MI-2 and MI-2-2. Expression of Hoxa9 and Meis1 has been normalized to β-actin and is referenced to the DMSO-treated cells. Data represent the mean values for duplicates ± SD. The experiment was performed 3 times. (D) Colony counts for methylcellulose colony assay performed with MLL-AF9–transduced BMCs treated for 7 days with MI-2-2 and MI-2-2. Error bars indicate SD from duplicate experiments; experiments were performed 2 times. (E) Representative colonies shown for DMSO-, MI-2-, and MI-2-2–treated MLL-AF9–transduced BMCs plated on methylcellulose. Black lines represent the scale bars (500 μm). (F) Quantification of CD11b expression in MLL-AF9 transduced BMC treated for 6 days with the menin-MLL inhibitors as detected by flow cytometry. Data represent the mean values for duplicates ± SD. The experiment was performed 2 times. (G) Wright-Giemsa–stained cytospins for MLL-AF9–transformed BMCs after 7 days of treatment with DMSO, MI-2 (12μM), and MI-2-2 (12μM). Black lines represent the scale bars (50 μm). Statistical analysis and calculation of P values was performed using 2-way ANOVA.