The Multimerization Domain of Cbfß-SMMHC Is Required for Leukemogenesis
Authors: Zhao Ling , Alemu Lemlem , Cheng Jun , Zhen Tao , Friedman Alan D. , Liu Pu Paul 
Published: October 2019

Abstract
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 (CbfbmDE/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 CbfbmDE/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 CbfbmDE/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+/MYH11 micedied 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.

Disclosures
No relevant conflicts of interest to declare.