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. The murine conditional knock-in model allows us to study the pre-leukemic changes in the hematopoietic cells. Gene expression profiling suggests that Gata2, a hematopoietic transcription factor, is a top upregulated genes in preleukemic Cbfb-MYH11 knockin mice and is expressed in human inv(16) AML. On the other hand, we have also identified recurrent monoallelic deletions of GATA2 in relapsed human CBF-AML patients. To clarify the role of Gata2 in leukemogenesis by Cbfb-MYH11, we generated conditional Cbfb-MYH11 knockin mice with Gata2 heterozygous knockout. Gata2 heterozygous knockout reduced abnormal myeloid progenitors, which are capable of inducing leukemia in the Cbfb-MYH11 mice. Consequently, Cbfb-MYH11 mice with Gata2 heterozygous knockout developed leukemia with longer latencies than those with intact Gata2. Interestingly, leukemic cells with Gata2 heterozygous knockout gained higher number of mutations and showed more aggressive phenotype in both primary and transplanted mice. Moreover, leukemic cells with Gata2 heterozygous knockout showed higher repopulating capacity in competitive transplantation experiments. In summary, reduction of Gata2 activity affects mutational dynamics of leukemia with delayed leukemia onset in Cbfb-MYH11 knockin mice, but paradoxically results in a more aggressive leukemia phenotype, which may be correlated with leukemia relapse or poor prognosis in human patients. It is generally considered that CBFbeta-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, which physically interacts with CBFbeta and CBFbeta-SMMHC. However, the exact role of RUNX1 in leukemogenesis induced by CBFbeta-SMMHC is not clear. To address this question, we generated mice with both Cre-based conditional Runx1 knockout and Cbfb-MYH11 knockin, which express Cbfb-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. None of such Runx1-deficient, Cbfb-MYH11 expressing mice developed leukemia up to one year after pIpC treatment, while all Cbfb-MYH11 expressing mice with normal Runx1 developed leukemia with an median survival time of 4 months. The finding indicated that Runx1 is indispensable for Cbfb-MYH11 induced leukemogenesis. To understand the mechanism of Runx1 contribution to Cbfb-MYH11 induced leukemogenesis, we studied the abnormal myeloid progenitors (AMPs) from which the leukemia cells arise. The AMPs started to decrease and disappear 4 weeks after pIpC in Runx1-deficient, Cbfb-MYH11 expressing mice, suggesting that this is a critical stage for the failure of leukemogenesis in these mice. We performed RNA-seq on the AMP population and we found that more than 1600 genes were differential expressed between Cbfb-MYH11 expressing mice that were Runx1-deficient and those that were Runx1-proficient. Many of these differentially expressed genes were RUNX1 target genes. Among the significantly enriched gene sets were those related to leukemic stem cells, suggesting these genes are important for the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFbeta-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFbeta-SMMHC.