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1337 Title: Runx1 Is Indispensable for Leukemia Development Induced By Cbfb-MYH11

Program: Oral and Poster Abstracts
Session: 603. Oncogenes and Tumor Suppressors: Poster I
Hematology Disease Topics & Pathways:
Diseases, AML, Myeloid Malignancies
Saturday, December 1, 2018, 6:15 PM-8:15 PM
Hall GH (San Diego Convention Center)

Tao Zhen, PhD1,2*, Erika Mijin Kwon, PhD2*, R. Katherine Hyde, PhD3, Ling Zhao, MD, PhD4*, Guadalupe Lopez1*, Lemlem Alemu, BS5* and Paul P. Liu, MD6

1Translational and Functional Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, MD
2Oncogenesis and Development Section, National Human Genome Research Institute/ NIH, Bethesda, MD
3Department of Biochemistry and Molecular Biology, and Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, NE
4Oncogenesis and Development Section/NHGRI, Bethesda, MD
5Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
6National Human Genome Research Institute, National Institutes of Health, Bethesda, MD

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 (Runx1f/f, Mx1-Cre, Cbfb+/56M), which express Cbfb-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre, Cbfb+/56M mice 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 Runx1f/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 Runx1f/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 Runx1f/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 Runx1f/f, Mx1-Cre, Cbfb+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre, Cbfb+/56M and Runx1f/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; Padj ≤0.05, absolute fold change ≥ 2) between Runx1f/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 Runx1f/f, Mx1-Cre, Cbfb+/56M cells, suggesting that the AMP population from Runx1f/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.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH