Intact IBM2 is necessary for leukemogenic transformation by MLL-AF9. (A) Co-IP demonstrates that mutation FLAA in MLL-AF9 abolished the interaction with LEDGF but not with menin. HEK293 cells were transduced with Myc-menin, LEDGF-HA, and Flag-MLL-AF9 or Flag-MLL-AF9 FLAA and coimmunoprecipitated using anti-Flag antibody. (B) Growth curves for bone marrow cells (BMCs) transformed with MSCV vector, MLL-AF9, and the F129A and FLAA mutants. (C) Activity of MLL-AF9 and the mutants in colony formation assay in transformed BMCs. Colony counts after 3 rounds of replating (C) and representative colonies (D) are shown. (E) qRT-PCR in BMCs transformed with MLL-AF9 and the 2 mutants for 3 replating rounds showing expression of Hoxa9. Expression of Hoxa9 has been normalized to β-actin and is referenced to the MSCV vector. (F) Wright-Giemsa-stained cytospins demonstrating differentiation of BMCs transformed with MSCV, MLL-AF9, and the 2 mutants. (G) Survival of mice transplanted with MSCV-MLL-AF9 and F129 and FLAA mutants. NRAS has been included to accelerate development of leukemia; n represents number of animals in each group. (H) Schematic diagram showing the endogenous Hoxa9 locus and probes used in the ChIP assay. TSS, transcription start site. (I) ChIP assays performed in HEK293 cells transduced with MLL-AF9 and mutants F129A and FLAA to determine the recruitment of MLL-AF9, menin, and LEDGF to the Hoxa9 locus with 4 different probes. Antibodies against AF9, menin, and LEDGF were used.