Mechanistic and translational studies of CBF leukemia

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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. It is generally considered that CBFbeta-SMMHC is a dominant negative repressor of RUNX1. However, recent findings challenge the RUNX1-repression model for CBFbeta-SMMHC mediated leukemogenesis. To definitively address the role of Runx1 in CBFB-MYH11 induced leukemia, we crossed conditional Runx1 knockout mice with conditional Cbfb-MYH11 knockin mice. We found that all conditional Cbfb-MYH11 knockin mice developed leukemia in 5 months while no leukemia developed in conditional Cbfb-MYH11 knockin mice that also lost Runx1, and this effect was cell autonomous. Importantly, the abnormal myeloid progenitors (AMPs), a leukemia initiating cell population induced by Cbfb-MYH11 in the bone marrow, decreased and disappeared in conditional Cbfb-MYH11 knockin mice that also lost Runx1. RNA-seq analysis of AMP cells showed that genes associated with proliferation, differentiation blockage and leukemia initiation, were differentially expressed between conditional Cbfb-MYH11 knockin mice and conditional Cbfb-MYH11 knockin mice that also lost Runx1. In addition, with chromatin immunocleavage sequencing (ChIC-seq) assay, we observed a significant enrichment of RUNX1/CBFbeta-SMMHC target genes in cells from conditional Cbfb-MYH11 knockin mice that also lost Runx1, especially among down-regulated genes, suggesting that RUNX1 and CBFbeta-SMMHC mainly function together as activators of gene expression through direct target gene binding. 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. Therefore, in this fiscal year, we have started to collaborate with Dr. Jiansen Jiangs group in NHLBI to study the interaction between RUNX1 and CBFbeta-SMMHC with structural biological tools. In addition, we are generating new mouse models using CRISPR-mediated targeting strategies to tag the endogenous RUNX1, CBFbeta, and CBFbeta-SMMHC proteins. We hope such models will facilitate functional studies of these proteins in hematopoietic and leukemia cells in the mice.