RUNX1 and CBFB-MYH11 Are Required for the Maintenance of Inv(16) AML
Authors: Wang Yiqian , Hannon Kira , Richter Lisa , Becker Michelle , Garett Lisa , Rivas Cecilia , Alemu Lemlem , Zhao Ling , Liu Pu Paul , Hyde R. Katherine 
Published: October 2019

Abstract
The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML.
To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites (CbfbflMYH11), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the CbfbflMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb+/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb+/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in Csf2rb- cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced CbfbflMYH11leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation.
To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the Csf2rb-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs.
Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML.

Disclosures
No relevant conflicts of interest to declare.