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. We previously showed that RUNX1 is indispensable for Cbfb-MYH11–induced leukemogenesis, but the underlying mechanism is still unclear. To address this question, we studied interactions between CBFβ-SMMHC, RUNX1, and RUNX1-target DNA using Bio-Layer Interferometry. We found that CBFβ-SMMHC could enhance the binding affinity between RUNX1 and its target DNA. Furthermore, a CBFβ-SMMHC variant with deletion of the RUNX1-high affinity binding domain (CBFβ-SMMHC-ΔHABD) retained the ability to enhance RUNX1-DNA binding, but not the variant with mutation in the C-terminal multimerization domain (CBFβ-SMMHC-mDE). We also found that the ability to enhance RUNX1’s DNA binding affinity correlated with leukemogenic capability of CBFβ-SMMHC variants in mouse models. We further demonstrated enhanced RUNX1-DNA binding in the myeloid progenitor cells from mice expressing the full-length CBFβ-SMMHC or CBFβ-SMMHC-ΔHABD, but not in cells from mice expressing CBFβ-SMMHC-mDE or wildtype mice. Moreover, we observed significant transcriptomic changes associated with leukemogenic CBFβ-SMMHC variants. Our data suggest that enhancing DNA binding by RUNX1 is an important mechanism of leukemogenesis by CBFβ-SMMHC. A manuscript reporting these findings have been accepted for publication in the Journal of Clinical investigation. DNA methylation is known to attenuate RUNX1 binding at target sites. Considering that RUNX1 function can be modulated by DNA methylation, we also aimed to investigate how the DNA methylation landscape in inv(16) AML affects disease development. To do this, we compared the methylomes and transcriptomes of Lin-Sca1-c-Kit+ bone marrow cells obtained from pre-leukemic inv(16) mice to those of controls. Enzymatic methyl sequencing data from these cells revealed 14,183 differentially methylated regions (DMRs, p-adj < 0.05), with 95% of these DMRs showing hypermethylation in pre-leukemic mice. Gene ontology (GO) term enrichment analysis of the genes found in proximity to these DMRs revealed biological processes of relevance to AML, including myeloid cell homeostasis (GO:0002262) and erythrocyte differentiation (GO:0030218). RNA-seq data obtained from the same cells revealed 1,019 differentially expressed genes (DEGs, absolute log2[fold change] ≥ 1, p-adj < 0.05). Included among these DEGs were RUNX1 targets that had both hypermethylated promoters and downregulated gene expression. Findings from this study indicate that DNA methylation can modulate the RUNX1 function of regulating the expression of target genes in inv(16) AML. A manuscript reporting these findings will be prepared soon for submission.