The Multimerization Domain of Cbfß-SMMHC Is Required for Leukemogenesis

Among acute myeloid leukemia (AML) with cytogenetic abnormalities, core binding factor (CBF) leukemia acounts for 20-30% of adult AML, and 20-30% of pediatric AML. The chromosome 16 inversion (inv(16)), which results in a fusion gene CBFB-MYH11 and an encoded chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain), is observed primarily in AML subtype M4Eo. Using Cbfb-MYH11 knock-in mouse models we previously demonstrated that CBFβ-SMMHC needs its C terminal domains for leukemogenesis (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 at the C terminus of CBFβ-SMMHC for hematopoietic defects and leukemogenesis.

Previous studies have shown that the C-terminal 29-residue assembly competent domain (ACD) is essential for multimerization of SMMHC. Within ACD, clustered point mutations in helices D and E specifically disrupts multimerization of CBFβ-SMMHC without interfering with the repression function of CBFβ-SMMHC (Zhang et al., Oncogene 25:7289, 2006). Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D and E (mDE) to study the role of the multimerization domain in vivo.

Heterozygous embryos (Cbfb^+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (Cbfb^mDE/mDE) showed complete blockage of definitive hematopoiesis, hemorrhage in the central nervous system and midgestation lethality, similar to the phenotype in Cbfb^+/MYH11 mice and the Cbfb or Runx1 null mice. This phenotype is also similar to that in the homozygous knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). The fetal liver of E12.5 Cbfb^mDE/mDE embryos gave no colonies while the fetal liver of Cbfb^+/mDE mice generated similar number of colonies as the WT controls. We further looked at the peripheral blood of E10.5 Cbfb^mDE/mDE embryos and found that the primitive hematopoiesis was not affected, while E10.5 Cbfb^+/MYH11 embryos showed a developmental delay at this stage.

 Analysis of peripheral blood showed decreased B cell population in young adult Cbfb^+/mDE mice, while the myeloid compartment was unchanged. In aged mice (>12 months), however, there was an increase of immature myeloid cells in the peripheral blood. Importantly, there was no leukemia development in the Cbfb^+/mDE mice one year after ENU treatment (to induce cooperating mutations), while Cbfb^+/MYH11mice died of leukemia within 2 months of ENU treatment. Notably bone marrow cells in the Cbfb^+/mDE and Cbfb^+/MYH11 mice expressed their respective fusion proteins at similar levels.

Overall our data suggest that the C terminal multimerization domain is required for the defects in primitive and definitive hematopoiesis caused by CBFβ-SMMHC, and the domain is essential for leukemogenesis by CBFβ-SMMHC.  Further mechanistic studies of this domain may lead to new drug targets for treating inv(16) leukemia.  For this purpose we have performed gene expression profiling with microarray and RNA-seq technologies, comparing gene expression changes in adult bone marrow c-Kit+ cells as well as embryonic primitive blood cells from Cbfb^+/mDE and Cbfb^+/MYH11 mice. Preliminary analysis indicates that the gene expression profile of the hematopoietic cells from the Cbfb^+/mDE mice was much similar to that of Cbfb^+/+ than Cbfb^+/MYH11 mice.  Validation and pathway analysis of those differentially expressed genes are ongoing and the results will be presented at the annual meeting.