Abstract
Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. We recently showed that RUNX1 is indispensable for Cbfb-MYH11-induced leukemogenesis in a mouse model. We found that RUNX1 interacted with CBFβ-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 CBFβ-SMMHC and RUNX1 does not provide adequate explanation on how the RUNX1-CBFβ-SMMHC complex forms and how the complex interacts with DNA for leukemogenesis as CBFβ-SMMHC without the RUNX1 high-affinity-binding-domain (CBFβ-SMMHC-ΔHABD) is also able to induce leukemia while CBFβ-SMMHC with mutations in the C-terminal multimerization domain (CBFβ-SMMHC-mDE) is not able to induce leukemia in mice.
To address this question, we used RHD domain of RUNX1, CBFβ, CBFβ-SMMHC, CBFβ-SMMHC-ΔHABD and CBFβ-SMMHC-mDE proteins, which were purified from E. coli overexpressing these proteins, to explore how the HABD and DE domains affect the interactions between CBFβ-SMMHC, RHD and RUNX1-target DNA Bio-Layer Interferometry (BLI) and negative staining. As expected, deletion of the HABD domain significantly reduced CBFβ-SMMHC's binding affinity to RHD by BLI assay. Interestingly, differences in binding affinity between RHD and different versions of CBFβ-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 CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD that can induce leukemia than CBFβ-SMMHC-DE, which cannot. We also found that both CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD, but not CBFβ-SMMHC-mDE, could form a filament structure by negative staining, suggesting the filament formation ability is important for leukemogenesis by CBFβ-SMMHC. In addition, RHD reduces filament formation by CBFβ-SMMHC, which was overcome when target oligo was added. In contrast, RHD could not inhibit filament formation by CBFβ-SMMHC-ΔHABD, suggesting that HABD interaction is required for RHD to disrupt filament formation by CBFβ-SMMHC.
Overall, we found that leukemogenic capability of CBFβ-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 CBFβ-SMMHC are required for the formation of the RUNX1-CBFβ-SMMHC complex with higher binding affinity to target DNA.
Disclosures
No relevant conflicts of interest to declare.
Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli