Chromosomal rearrangements involving the two subunits of the heterodimeric transcription factor, core-binding factor (CBF), are the most commonly observed cytogenetic abnormalities in adult acute myeloid leukemia (AML). CBF comprises one of three potential DNA-binding α-subunits (RUNX1, RUNX2 or RUNX3) 1 that interact with a non-DNA-binding subunit, CBFβ. The major rearrangement affecting CBFβ generates the inv (16)(p13. 1q22) that fuses the first 165 amino acids of CBFβ with the coiled-coil rod domain of the smooth muscle myosin heavy chain (SMMHC) gene, MYH11. 2, 3 The resulting CBFβ–SMMHC fusion protein retains the ability to interact with RUNX1, and functions by dominantly inhibiting normal CBF activity. This view is supported by studies where CBFβ-MYH11 was knocked into the endogenous CBFβ locus, resulting in an early embryonic lethality that phenocopied many of the developmental abnormalities observed in mice with homozygous deletions of either RUNX1 or CBFβ. 4, 5 CBFβ–SMMHC may dominantly inhibit CBF function by recruiting nuclear corepressor molecules, including mSin3A and HDAC8, to a C-terminal region of SMMHC that includes the 28-amino-acid assembly competence domain (ACD) that mediates skeletal or smooth muscle myosin oligomerization and filament formation. 6–9 To examine the role of the ACD in promotion of AML in vivo, we generated retroviral constructs that coexpressed a green fluorescent protein (GFP) reporter and either CBFβ–SMMHC or a CBFβ–SMMHC mutant lacking the 28-amino-acid ACD (Figure 1a). Bone marrow (BM) isolated from C57BL/6-CD45. 2 animals treated with 5-fluorouracil to enrich for hematopoietic stem/progenitor cells (HSPCs) was transduced with each retroviral vector and then transplanted into lethally irradiated, congenic C57BL/6-CD45. 1 mice. Western blot analysis of GFP+ splenocytes isolated from 4–5-month post-transplant (PT) animals indicated that each fusion protein was expressed at equivalent levels (Figure 1b). Analysis of transplant recipients showed that mice reconstituted with CBFβ–SMMHC-expressing cells (CBFβ–SMMHC mice) died of AML between 4 and 7 months PT (Figure 1c). Disease penetrance was 100% when GFP+ cells exceeded 5% in peripheral blood (PB) at 3–4 weeks PT (GFP+ chimerism ranged between 5.7 and 11.6%, mean= 9.1%, n= 16). GFP+ chimerism in MIG control and ΔACD mice was stable over time (Figure 1d), with no MIG or ΔACD animal showing outward signs of sickness at least 12 months PT. The comparable expression levels of the ΔACD mutant and CBFβ–SMMHC indicated that this does not account for their differing potencies in promoting AML.

Moribund CBFβ–SMMHC animals displayed splenomegaly (Figure 1e) and hepatomegaly (data not shown) and showed effacement of normal splenic architecture with expanded splenic red pulp and infiltrating blasts (Figure 1f). Leukemic infiltrate was also noted in the sinusoidal space of the liver and alveolar septae of the lung (Figure 1f). Transfer of one million BM cells from two independent moribund CBFβ–SMMHC animals with AML into each of 5 lethally irradiated secondary recipient mice showed that leukemia was transplantable, with secondary recipients becoming moribund more rapidly than primary recipients at 8–12 weeks PT (Figure 1g). All CBFβ–SMMHC animals exhibited an initial pre-leukemic period between 1 and 4 months PT where stable, low-level expression of CBFβ–SMMHC (based on the GFP surrogate marker) in PB was followed by rapid emergence of a GFP+ blast population that uniformly expressed high CBFβ–SMMHC (Figure 1d …