Runx1 Is Strictly Required for Cbfb-MYH11 Induced Leukemia Development

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. The prevailing hypothesis for the mechanism of leukemia development by CBFbeta-SMMHC, the fusion protein encoded by CBFB-MYH11, is that CBFbeta-SMMHC is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFbeta and CBFbeta-SMMHC. If this hypothesis is correct, reducing RUNX1 activity should facilitate leukemogenesis by CBFB-MYH11. In fact, loss-of-function mutations in RUNX1 are common in human AML, but not in inv(16) AML. However, we previously demonstrated that CBFB-MYH11 has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010). Moreover, we recently showed that a dominant negative allele of Runx1, Runx1-lz, delayed leukemogenesis by CBFB-MYH11 in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFbeta-SMMHC mediated leukemogenesis. However, our previous findings are not conclusive since the Runx1+/lz mice used in the previous study have one wild-type Runx1 allele, and still retain some Runx1 function.To definitively address this question, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) and Mx1-Cre mice to generate Runx1f/f, Mx1-Cre, Cbfb+/56M mice, which express CBFbeta-SMMHC but not Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre, Cbfb+/56M mice had more severe platelet deficiencies and higher numbers of Lin-/Sca1-/C-kit+ progenitors and Lin-/Sca1+/C-kit+ hematopoietic stem cells in the bone marrow when comapred with Runx1f/f, Mx1-Cre mice. Unexpectedly Runx1f/f, Mx1-Cre, Cbfb+/56M mice also developed severe macrocytic anemia within two weeks after pIpC induction, which was lethal in about 1/3 of the mice. However, none of the Runx1f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia up to one year after pIpC treatment. In contrast, all Mx1-Cre, Cbfb+/56M mice developed leukemia with an average survival of 4 months, as reported previously. These results suggest that Runx1 is strictly required for Cbfb-MYH11 induced leukemogenesis.To further study the mechanism of leukemogenesis, we performed RNA-Seq on C-kit+ bone marrow cells isolated from mice two weeks after pIpC treatment, to explore the global gene expression changes caused by Runx1 knockout on Cbfb-MYH11 expressing mice. Our preliminary data analysis showed that 1688 genes were differential expressed (Padj ≤0.05, FC ≥ 2) between Runx1f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M mice. Interestingly, many of these genes (48%) are Runx1 target genes. The above results suggest that mis-regulating the expression of Runx1 target genes contributes to leukemogenesis by CBFbeta-SMMHC. DisclosuresNo relevant conflicts of interest to declare.