Title: Runx1 Is Indispensable for Leukemia Development Induced By Cbfb-MYH11

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. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFβ and CBFβ-SMMHC. While loss-of-function mutations in RUNX1 are common in human AML, they have not been found in inv(16) AML. Moreover, we have demonstrated that CBFβ-SMMHC has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010), and Runx1 insufficiency (Runx1^+/lz) delays Cbfb-MYH11-induced leukemia in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFβ-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 generated mice with both Cre-based conditional Runx1 knockout and Cbfb-MYH11 knockin (Runx1^f/f, Mx1-Cre,Cbfb^+/56M), which express Cbfb-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1^f/f, Mx1-Cre,Cbfb^+/56Mmice had higher numbers of Lin^-/Sca1^-/c-Kit⁺ (LK) and Lin^-/Sca1⁺/c-Kit⁺ cells in the bone marrow when comapred with Mx1-Cre, Cbfb^+/56M mice at 3 weeks after pIpC. However, none of the Runx1^f/f, Mx1-Cre, Cbfb^+/56M mice developed leukemia up to one year after pIpC treatment. As reported previously, all Mx1-Cre,Cbfb^+/56M mice developed leukemia with an median survival time of 4 months. Moreoever, none of the recipients transplanted with bone marrow cells from Runx1^f/f, Mx1-Cre,Cbfb^+/56M mice and then treated with pIpC developed leukemia, while all of the recipients of the Mx1-Cre,Cbfb^+/56M bone marrow cells did, suggesting that the loss of leukemogenesis is cell autonomous. All together, these results indicate that Runx1 is indispensable for Cbfb-MYH11 induced leukemogenesis.

Even though at 3 weeks after pIpC the Runx1^f/f, Mx1-Cre,Cbfb^+/56M mice had more c-Kit⁺ cells, and more significantly the abnormal myeloid progenitors (AMPs) (LK/CD34^-/Fc□RII/III⁺) from which the leukemia cells arise, these cells started to decrease and disappear 4 weeks after pIpC, suggesting that this is a critical stage for the failure of leukemogenesis in the Runx1^f/f, Mx1-Cre,Cbfb^+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre,Cbfb^+/56M and Runx1^f/f, Mx1-Cre,Cbfb^+/56M mice three weeks after pIpC treatment, to explore the global gene expression changes between them. Our preliminary data analysis showed that many genes (1635) were differential expressed (DEGs; P_adj ≤0.05, absolute fold change ≥ 2) between Runx1^f/f, Mx1-Cre, Cbfb^+/56M and Mx1-Cre, Cbfb^+/56M mice. Many of these DEGs (37.6%) are RUNX1 target genes (Mandoli et al., Leukemia. 28:770, 2014). Among the significantly enriched gene sets by gene set enrichment analysis of the DEGs, the "GAL_LEUKEMIC_STEM_CELL_DN" gene set, which contains genes down-regulated in leukemic stem cells, is positively correlated with DEGs upregulated in Runx1^f/f, Mx1-Cre,Cbfb^+/56M cells, suggesting that the AMP population from Runx1^f/f, Mx1-Cre,Cbfb^+/56M mice lost the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFβ-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFβ-SMMHC.