Acute myeloid leukemia (AML) is a heterogeneous disease with diverse gene mutations and chromosomal abnormalities. Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for approximately 24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia. The encoded proteins, RUNX1 and CBFbeta, form a heterodimer to regulate gene expression, and they are both required for hematopoiesis in vertebrate animals from zebrafish to man. Extensive clinical studies have demonstrated that CBFB-MYH11 and RUNX1-ETO, the two common fusion genes in CBF leukemia, are the best biomarkers for diagnosis, prognosis, and residual disease monitoring of CBF leukemia patients. Over the years we have used mouse models and a variety of research tools to characterize the CBFB-MYH11 fusion gene, determine the effect of the encoded protein, CBFbeta-SMMHC, on normal hematopoiesis, and understand the leukemogenesis process associated with the fusion gene. We have generated both conventional and conditional knock-in mouse models to study CBFB-MYH11. Using these models we showed that CBFB-MYH11 is necessary but not sufficient for leukemia, and we were able to identify cooperating genetic events in the mouse models. We have generated knock-in mouse models expressing truncated CBFB-MYH11 to determine the importance of functional domains of CBFbeta-SMMHC. Overall our lab has been recognized in the field as the major contributor to the understanding of CBFB-MYH11 leukemia. Recently, it was shown that chromodomain helicase DNA-binding protein-7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 is also critical for CBFB-MYH11-induced leukemogenesis. To test this hypothesis, we generated Chd7f/fMx1-CreCbfb+/56M mice, which expressed the Cbfb-MYH11 fusion gene and deactivated Chd7 in hematopoietic cells upon inducing Crewith polyinosinic-polycytidylic acid. The Lin-Sca1-c-Kit+ (LK) population was significantly lower in Chd7f/fMx1-CreCbfb+/56M mice than in Mx1-CreCbfb+/56Mmice. In addition, there were fewer 5-bromo-2-deoxyuridine-positive cells in the LK population in Chd7f/fMx1-CreCbfb+/56M mice, and genes associated with cell cycle, cell growth, and proliferation were differentially expressed between Chd7f/fMx1-CreCbfb+/56Mand Mx1-CreCbfb+/56M leukemic cells. In vitro studies showed that CHD7 interacted with CBF-SMMHC through RUNX1 and that CHD7 enhanced transcriptional activity of RUNX1 and CBF-SMMHC on Csf1r, a RUNX1 target gene. Moreover, RNA sequencing of c-Kit+ cells showed that CHD7 functions mostly through altering the expression of RUNX1 target genes. Most importantly, Chd7 deficiency delayed Cbfb-MYH11-induced leukemia in both primary and transplanted mice. These data indicate that Chd7 is important for Cbfb-MYH11-induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation (Zhen et al, Blood 2017). The C-terminus of CBFbeta-SMMHC contains domains for self-multimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBFbeta-SMMHC. To test the role of the fusion proteins C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBFbeta-SMMHC (CBFbeta-SMMHCdC95). Embryos with a single copy of CBFbeta-SMMHCdC95 were viable and showed no defects in hematopoiesis, whereas embryos homozygous for the CBFbeta-SMMHCdC95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBFbeta-SMMHC. Importantly, unlike mice expressing full-length CBFbeta-SMMHC, none of the mice expressing CBFbeta-SMMHCdC95 developed leukemia, even after treatment with ENU, although some of the older mice developed a non-transplantable myeloproliferative disease. Our data indicate that the CBFbeta-SMMHCs C-terminus is essential to induce embryonic hematopoietic defects and leukemogenesis (Kamikubo et al. Blood 121:638, 2013). In a more recent study , we generated a new Cbfb-MYH11 knock-in mouse model to dissect the role of the multimerization domain at the C terminus of CBFbeta-SMMHC. Specifically, we mutated six amino acids in the helices D and E (mDE) of the assembly competent domain, which is important for SMMHC multimerization. We found that the embryos with the mDE mutation did not develop hematopoietic defects seen in embryos with full-length CBF-SMMHC. More importantly leukemia development was abolished in the adult mice with CBFbeta-SMMHCdC95 even after mutagenesis treatment. In addition, the gene expression profile of the hematopoietic cells from the CBFbeta-SMMHCdC95 mice was more similar to that of wildtype mice than the CBFbeta-SMMHC mice. Our data suggest that the C terminal multimerization domain is required for the defects in primitive and definitive hematopoiesis caused by CBFbeta-SMMHC, and it is also essential for leukemogenesis caused by CBFbeta-SMMHC (Zhao et al., Leukemia 2017). The conditional Cbfb-MYH11 knock-in mouse model (Kuo et. al, 2006; PMID: 16413472) allows us to study the earliest molecular events in leukemogenesis, since it takes 3-4 months to develop leukemia after the expression of CBF-SMMHC is induced in the adult mice. Previous work indicated that CBF-SMMHC expression leads to the formation of pre-leukemic hematopoietic cell populations in the bone marrow. In an ongoing study we are using single cell RNA-sequencing to profile dynamic changes of hematopoietic cell populations based on gene expression analysis in individual cells, in order to understand the function of the CBF-SMMHC fusion protein in hematopoietic cells and leukemogenesis. c-Kit+ stem and progenitor cells from wild-type mice. pre-leukemic CBF-SMMHC expressing mice, and leukemic CBF-SMMHC expressing mice were used for single cell capture and RNA-seq with the 10X Genomics Chromium platform. In total, cDNAs from more than 14,000 cells were sequenced, each expressing between 1500-4000 genes. This high dimensional data was reduced using t-distributed stochastic neighbor embedding (t-SNE), a process that clusters the cells that are the most related to one another based on their transcriptional profile. In wild-type mice, tSNE clustering revealed at least 14 unique cell identities among the c-Kit+ cells comprising stem cells, multipotent progenitors, and cells of myeloid, erythroid, and lymphoid lineages. Similar heterogeneity was observed for the c-Kit+ cells from CBF-SMMHC expressing mice that were pre-leukemic. The c-Kit+ cells from leukemic CBF-SMMHC expressing mice were more homogeneous, indicating a dominant leukemia clone. In the two pre-leukemic mice examined, a small cluster of c-Kit+ cells express some markers of megakaryocyte-erythroid progenitors (MEP), which were reported previously as abnormal myeloid progenitors (AMPs, Kuo et. al, 2006; PMID: 16413472). We have identified novel markers of this population, which expands as leukemia progresses. We also noted numerous other alterations when comparing the data from wild-type to pre-leukemic c-Kit+ cells, both in the number of cells occupying a cluster (cell type) as well as gene expression differences within a cell type. These data truly underscore the large impact CBF-SMMHC has on the hematopoietic landscape and, because these data were obtained on the single cell level, we will be able to examine the gene expression alterations the fusion protein drives in specific cell subtypes.

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24
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2018
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Human Genome Research
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Mitsuda, Yoshihide; Morita, Ken; Kashiwazaki, Gengo et al. (2018) RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells. Sci Rep 8:6423
Zhen, Tao; Kwon, Erika M; Zhao, Ling et al. (2017) Chd7 deficiency delays leukemogenesis in mice induced by Cbfb-MYH11. Blood 130:2431-2442
Jiang, Xi; Hu, Chao; Ferchen, Kyle et al. (2017) Targeted inhibition of STAT/TET1 axis as a therapeutic strategy for acute myeloid leukemia. Nat Commun 8:2099
Morita, Ken; Maeda, Shintaro; Suzuki, Kensho et al. (2017) Paradoxical enhancement of leukemogenesis in acute myeloid leukemia with moderately attenuated RUNX1 expressions. Blood Adv 1:1440-1451
Morita, Ken; Noura, Mina; Tokushige, Chieko et al. (2017) Autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells. Sci Rep 7:16604
Sood, Raman; Kamikubo, Yasuhiko; Liu, Paul (2017) Role of RUNX1 in hematological malignancies. Blood 129:2070-2082
Morita, Ken; Suzuki, Kensho; Maeda, Shintaro et al. (2017) Genetic regulation of the RUNX transcription factor family has antitumor effects. J Clin Invest 127:2815-2828
Zhao, L; Alkadi, H; Kwon, E M et al. (2017) The C-terminal multimerization domain is essential for leukemia development by CBF?-SMMHC in a mouse knockin model. Leukemia 31:2841-2844
Hyde, R Katherine; Liu, Paul; Friedman, Alan D (2017) RUNX1 and CBF? Mutations and Activities of Their Wild-Type Alleles in AML. Adv Exp Med Biol 962:265-282
Jiang, Xi; Hu, Chao; Arnovitz, Stephen et al. (2016) miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia. Nat Commun 7:11452

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