Mechanistic and translational studies of CBF leukemia

National Human Genome Research Institute

Investigators

Linked publications, trials & patents

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 found that RUNX1 interacted 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 the RUNX1-CBF-SMMHC complex forms and how the complex interacts with DNA for leukemogenesis as CBF-SMMHC without the RUNX1 high-affinity-binding-domain (CBF-SMMHC-HABD) is also able to induce leukemia while CBF-SMMHC with mutations in the C-terminal multimerization domain (CBF-SMMHC-mDE) is not able to induce leukemia in mice. To address this question, we used purified RHD domain of RUNX1, CBF, CBF-SMMHC, CBF-SMMHC-HABD and CBF-SMMHC-mDE proteins to explore how the HABD and DE domains affect the interactions between CBF-SMMHC, RHD and RUNX1-target DNA with fluorescence polarization (FP), Bio-Layer Interferometry (BLI), and Cryogenic Electron Microscopy (Cryo-EM). As expected, deletion of the HABD domain significantly reduced CBF-SMMHCs binding affinity to RHD. Interestingly, differences in binding affinity between RHD and different versions of CBF-SMMHC did not correlate with their leukemogenic capability. On the other hand, the binding affinity between RHD and its target oligo was more significantly enhanced by CBF-SMMHC and CBF-SMMHC-HABD that can induce leukemia than CBF-SMMHC-DE, which cannot. We also found that both CBF-SMMHC and CBF-SMMHC-HABD, but not CBF-SMMHC-mDE, could form a filament structure, suggesting the filament formation ability is important for leukemogenesis by CBF-SMMHC. In addition, RHD reduces filament formation by CBF-SMMHC, which was overcome when target oligo was added. In contrast, RHD could not inhibit filament formation by CBF-SMMHC-HABD, suggesting that HABD interaction is required for RHD to disrupt filament formation by CBF-SMMHC. Overall, we found that leukemogenic capability of CBF-SMMHC correlates with its ability to enhance binding between RHD and its target DNA and to form multimerized filaments. The results also suggest that HABD and DE domains of CBF-SMMHC are required for the formation of the RUNX1-CBF-SMMHC complex with higher binding affinity to target DNA. 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 CBFb-SMMHC. In addition, we observed that CBFb-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 CBFb-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. CBFb-SMMHC was tagged with HA. We have generated mouse models incorporating these knock-in tagged genes, and induced CBFb-SMMHC expression in the mouse hematopoietic cells through Cre-lox excision. Through collaboration with Keji Zhaos lab in NHLBI, we are performing ChIC-seq experiments to determine and analyze binding sites for RUNX1 and CBFb-SMMHC. We will also evaluate chromatin accessibility and interactions with Hi-TrAC, which is recently developed by the Zhao lab based on the Trac-looping method (Lai et al., 2018). We will also perform RNA-seq to probe transcriptome changes. Integrated analyses of the data above will lead us to a better understanding of the mechanism of leukemogenesis associated with CBFB-SMMHC and RUNX1. References: Zhen T, Kwon E, Zhao L, Hsu J, Hyde RK, Alemu L, Speck NA, Liu PP. CHD7 deficiency delays leukemogenesis in mice induced by CBFB-MYH11. Blood 130:2431-2442, 2017 Lai, B. et al. Trac-looping measures genome structure and chromatin accessibility. Nat Methods 15, 741-747, 2018. Saida S, Zhen T, Kim E, Yu K, Lopez G, McReynolds LJ, and Liu PP. Gata2 deficiency delays leukemogenesis while contributing to aggressive leukemia phenotype in Cbfb-MYH11 knockin mice. Leukemia 2019 Zhen T, Cao Y, Ren G, Zhao L, Hyde RK, Lopez G, Feng D, Alemu L, Zhao K, Liu PP. RUNX1 and CBFb-SMMHC transactivate target genes together in abnormal myeloid progenitors for leukemia development. Blood 2020