The C-Terminus of the CBFβ-SMMHC Fusion Protein Is Required for the Myeloblastic Transformation of inv16 Leukemia

Inv(16)(p13q22) is found in almost all human acute myeloid leukemia (AML) subtype M4Eo cases and forms a fusion gene CBFB-MYH11, which encodes a fusion protein CBFβ–SMMHC. CBFβ forms a heterodimeric transcription factor with RUNX1 and both are required for embryonic hematopoiesis, while SMMHC is the smooth muscle myosin heavy chain. Using knock-in mouse strategy, we previously demonstrated that Cbfb_+/MYH11 F1 embryos have severe blockage of definitive hematopoiesis and die from CNS hemorrhage. The phenotype was similar to that of embryos with homozygous deletion of Cbfb or Runx1, suggesting that Cbfb-MYH11 dominantly represses Runx1/CBFb function. We further demonstrated that Cbfb-MYH11 is necessary but not sufficient for leukemia to develop, as the Cbfb^+/MYH11 mice required additional mutations for leukemogenesis. Several hypotheses have been proposed, based on in vitro studies, to explain how CBFβ-SMMHC dominantly inhibits RUNX1/CBFβ. The C-terminal region of CBFβ-SMMHC is responsible for multimerization and also interacts with corepressors; thus this region might be critical for RUNX1 repression. To determine the importance of this multimerization domain in vivo, we generated knock-in mice expressing CBFβ-SMMHC with a 95 aa C-terminal deletion (CBFβ-SMMHC_dC95), which truncates this multimerization/repression domain. CBFβ-SMMHC_dC95 expressing F1 heterozygous embryos (Cbfb^+/MYH11dC95) developed normally with no hematopoietic defects and no hemorrhage. Hematopoiesis was normal in the adult Cbfb^+/MYH11dC95 mice except for mild increase of mature neutrophils and minor T cell developmental defects in the first year. However, the mice proceeded to a lethal myeloproliferative disorder (MPD) during their second year of life. There was a significant increase of Mac1/Gr1 double positive cells in the peripheral blood and the spleen, which were negative for the stem cell/progenitor cell marker, c-kit. Morphologically the erythrocytes and neutrophils were dysplastic, and the mice developed severe splenomegaly. ENU treatment of the Cbfb^+/MYH11dC95 mice accelerated the development of the MPD phenotype but could not induce overt, transplantable, myeloid leukemia. These data suggest that the multimerizatin domain of SMMHC is important for both hematopoiesis blockage and leukemogenesis, especially the blastic transformation of inv16 leukemia.