Chromosome 16 inversion, inv (16), is the signature chromosome abnormality in M4Eo subtype of acute myeloid leukemia (AML), which produces a fusion gene CBFB-MYH11. 1 We previously generated a knockin mouse model for CBFB-MYH11 (Cbfb+/MYH11). 2 Heterozygous Cbfb-MYH11 knockin mice have definitive hematopoiesis blockage and die at mid-gestation, which is similar to the phenotypes of Runx1−/− and Cbfb−/− mice, 3, 4 indicating that CBFβ-SMMHC, the fusion protein encoded by Cbfb-MYH11, dominantly suppresses RUNX1 and CBFβ. Chimeric and conditional Cbfb-MYH11 knockin mice develop AML when they acquire additional mutations. 5

An important domain of CBFβ-SMMHC is the C-terminal region of SMMHC, which catalyzes homo-dimerization and multimerization of the fusion protein that may be functionally important. 6 To test the function of this region, we previously generated knockin mice that expressed a truncated CBFβ-SMMHC missing C-terminal 95 amino acids. These mice would not develop leukemia, indicating the importance of C-terminal region for leukemogenesis. 7 The CBFβ-SMMHC C-terminal region contains an assembly competence domain (ACD), which is important for SMMHC multimerization, 8, 9 as well as a transcriptional repression domain. 8–10 Specific amino acid residues in the helices D and E of ACD are important for multimerization of CBFβ-SMMHC but not for transcription repression. 8 To distinguish which domain is critical for the role of CBFβ-SMMHC in leukemia development, we generated the current mouse model with mutations in helices D and E of CBFβ-SMMHC to impair multimerization but leave the transcriptional repression domain intact. Specifically, we mutated six charged residues of helices D and E to threonine, serine or alanine residues: D (NANRRKL to NSNRASL), E (QRELDEA to QAELTSA), as published previously. 8 We incorporated these mutations into the full length Cbfb-MYH11 knockin construct2 (Supplementary Figure S1a). Correct knockin was confirmed by Southern blot hybridization (Supplementary Figure S1b) and the expression of the fusion protein CBFβ-SMMHCmDE was detectable in BM cells, at a level similar to CBFβ-SMMHC when compared to CBFβ (Supplementary Figure S1c).