Mechanistic and translational studies of CBF leukemia

National Human Genome Research Institute

Investigators

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Abstract

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 such as zebrafish and mice. 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 leukemia development 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 development, 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 a major contributor to the understanding of CBFB-MYH11 leukemia. We recently showed that RUNX1 is indispensable for Cbfb-MYH11induced leukemogenesis in a mouse model. We further demonstrated that RUNX1 worked together with CBF-SMMHC, the fusion protein encoded by CBFB-MYH11, to directly regulate critical genes for leukemogenesis (Zhen et al., Blood, 2020). However, our current understanding of the interaction between CBF-SMMHC and RUNX1 does not provide adequate explanation on how this interaction contributes to leukemogenesis as CBF-SMMHC without the RUNX1 high-affinity-binding-domain (CBF-SMMHC-HABD) is still able to induce leukemia (Kamikubo et al., 2010) while CBF-SMMHC with mutations in the C-terminal multimerization domain (CBF-SMMHC-mDE), which does not affect RUNX1 binding, is not able to induce leukemia in mice (Zhao et al., 2017). The data from the mouse models indicate that RUNX1 is indispensable for Cbfb-MYH11 induced leukemogenesis by working together with CBFbeta-SMMHC to regulate critical genes associated with the generation of a functional AMP population. However, molecularly we know very little on how RUNX1 and CBFbeta-SMMHC work together. To address these questions, we used purified RUNX1 runt homology domain (RHD), CBF, CBF-SMMHC, CBF-SMMHC-HABD and CBF-SMMHC-mDE proteins to explore how the HABD and DE domains of CBF-SMMHC affect the interactions between CBF-SMMHC, RHD and RUNX1-target DNA, with Bio-Layer Interferometry (BLI). The data suggest that RUNX1 target sequence interaction and filament formation by CBF-SMMHC are important for leukemogenesis. In the coming year, we will focus on identification of target genes of RUNX1/CBF-SMMHC which are important for leukemia and illustrate the high-resolution structure of RUNX1/CBF-SMMHC complex through additional structural biological tools. Our recent data also showed that transcription factors such as GATA2 (Saida et al., 2020) and chromatin modulators such as CHD7 (Zhen et al., 2017) are important for leukemogenesis by RUNX1/CBF-SMMHC. In addition, we observed that CBF-SMMHC co-localized with RUNX1 near many activated genes in the preleukemic Cbfb-MYH11 knock-in mice (Zhen et al., 2020). We hypothesize that transition from normal hematopoiesis to leukemic state is accompanied by global re-localization of RUNX1 genomic binding sites via interaction with CBF-SMMHC, leading to reorganization of promoter-enhancer interactions. These chromatin structural changes will then induce gene expression alterations, leading to leukemia development. To test these hypotheses, we generated epitope-tagged Runx1b, Runx1c and Cbfb-MYH11 knock-in mice using CRISPR genome editing to overcome antibody problems and to precisely detect their binding sites in the genome. The two isoforms of the RUNX1 protein, RUNX1b and RUNX1c, were tagged with 3xMyc and 3xFLAG, respectively. CBF-SMMHC was tagged with HA. We are now generating mouse models incorporating these knock-in tagged genes. We will perform epigenomic and gene expression experiments with these mice to assess changes in super-enhancers, chromatin interactions and chromatin modification.