Functional Dissection of the C Terminus of CBFβ-SMMHC Indicates a Critical Role of the Multimerization Domain during Hematopoiesis and Leukemogenesis

Abstract

The recurrent chromosome 16 inversion (inv(16)) in acute myeloid leukemia (AML) subtype M4Eo results in a fusion between CBFB and MYH11 genes, which encodes a chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain). We previously generated mouse CBFB-MYH11 knock-in models that mimic the human inv(16) AML and demonstrated that the CBFβ-SMMHC fusion protein blocks RUNX1 and CBFβ function during definitive hematopoiesis and plays a driving role in leukemogenesis. Our recent studies indicated that the C-terminus of CBFβ-SMMHC, which contains domains for multimerization and transcriptional repression, is important for leukemogenesis by CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). In this study we generated a new CBFB-MYH11 knock-in mouse model to determine the role of the multimerization domain of CBFβ-SMMHC during hematopoiesis and leukemogenesis.Previous studies have dissected the assembly competence domain (ACD) of the CBFβ-SMMHC C-terminus to identify the critical amino acid residuals for multimerization (Zhang et al., Oncogene 25:7289, 2006). Among them, mutations in helices D and E are the ones that affect multimerization the most. Importantly, the helices D and E mutations do not interfere with the repression function of CBFβ-SMMHC. Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D &E in the ACD of the C-terminus (mDE) to determine the role of multimerization for the in vivo function of CBFβ-SMMHC.The embryonic hematopoietic phenotype in the mDE knockin embryos is very similar to what we have observed in the knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013), i.e., heterozygous embryos (Cbfb+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (CbfbmDE/mDE) showed hemorrhage in the central nervous system and died around E12.5, as seen in the full length CBFβ-SMMHC heterozygous knockin mice and the Cbfb-/- and Runx1-/- mice. Analysis of peripheral blood from adult Cbfb+/mDE mice showed decreased B cell population and increased T cell population, while the myeloid compartment was unchanged. Preliminary findings suggest that leukemogenesis is at least delayed in the Cbfb+/mDE mice as compared to mice expressing full-length CBFβ-SMMHC. Therefore the multimerization function of CBFβ-SMMHC is critical for its ability to induce defects in embryonic hematopoiesis and for leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.