Deletion Of Core Binding Factors Runx1 and Runx2 Leads To Perturbed Hematopoiesis In Multiple Lineages

The core binding factor (CBF) is a transcription factor that regulates key modulators of growth, survival and differentiation pathways. The CBF consists of a DNA binding α subunit (encoded by RUNX1, RUNX2, and RUNX3) and a common non-DNA binding β subunit (CBFB). RUNX1 and CBFB have been shown to be indispensible for embryo definitive hematopoiesis and to regulate adult hematopoiesis, and are targets of mutations in acute myeloid leukemia and myeloid dysplastic syndromes. We have shown that Runx2 is expressed in hematopoietic stem and early progenitor cells (HSPC: LSK⁺= Lin^-ckit⁺Sca1⁺) and that it modulates leukemia latency in mice. However, little is known of Runx2 role in hematopoiesis. In this study, we have used conditional knock out mice for Runx1, Runx2, Runx1 and Runx2, and Cbfb (namely: Rx1ko, Rx2ko, Rx12dko, and Cbfbko) and the Cre deletors Mx1Cre and Vav-Cre, to show that Runx1 and Runx2 regulate hematopoietic lineage differentiation.

Analysis of HSPCs 2 weeks post Mx1Cre induction, the HSCs (LSK⁺, FLT3^-) were increased 4 fold in Rx1ko and Rx12dko mice, while the multipotential progenitors (MPPs:LSK⁺, FLT3⁺) of Rx12dko mice were expanded 5 fold. These data indicate that Runx1 regulates HSCs while both Runx factors regulate MPPs. The cell-intrinsic role of CBF factors in hematopoiesis was studied by evaluating the multilineage repopulation in competitive repopulation assay. To this end, recipient mice were transplanted 1:1 ratio of test (Rx1^fl/fl, Rx2^fl/fl, Rx1^fl/flRx2^fl/fl, or Cbfb^fl/fl; each with Mx1Cre;CD45.2) and competitor (wt;CD45.1) bone marrow cells, treated with pIpC 4 weeks later, and analyzed every 4 weeks up to week 20 by flow cytometry. This analysis showed that Runx1 and Runx2 regulate differentiation in cell type specific manner. Runx1 and Runx2 have antagonistic functions in B cell lineage development, and Runx1 (but not Runx2) regulates T cell differentiation. The monocytes were not affected by the loss of Runx1 or Runx2, but were markedly reduced in the absence of both factors, suggesting that Runx1 and Runx2 may co-regulate monocyte development. The granulocytes (Mac1+Gr1+) were not affected in by Runx1 and/or Runx2, but were drastically reduced in Cbfb-null cells, suggesting that Runx3 could regulate granulocyte differentiation.

The mechanism of HSPC regulation by Runx factors was studied by expression analysis of genes associated with HSC function. We have found that expression of adhesion molecules Alcam, Cx43 and Cxcr4 were deregulated in Rx1ko and Rx2ko HSCs and MPPs, as well as self-renewal factors, including Cdkn1a, Gfi1 and Mpl. To assess whether these alterations would impair the retention of HSPCs in the niche, we tested the ability of HSPCs to recover from cytotoxic stress, using 5-fluorouracil. At day 7, the percentage of immature (c-kit⁺) cells in peripheral blood had returned to normal in Rx1ko, Rx2ko, and wt mice. However, Rx12dko mice showed a 15-20 fold increase in circulating immature (c-kit+) cells. In addition, the administration of a second 5-fluorouracil dose at day 14 induced hematopoietic exhaustion and death in wt, Rx1ko and Rx2ko mice, but Rx12dko mice survived and recovered. These experiments indicate that loss of both Runx factors impairs the adhesion of HSCs to the niche and re-establishment of HSPC homeostasis

To further study the role of CBF factors in hematopoiesis, we analyzed lineage contribution in Cbfb^fl/fl, Vav-Cre mice at week 8 after birth. The HSPCs (LSKs) were increased 10 fold in Cbfb-null mice. These mice presented pancytopenia, with a 2-fold reduction in white blood cell count and anemia. The erythroid lineage was affected, including reduction of megakaryocyte/erythroid progenitors and Ter119⁺ progenitor cells in bone marrow, and reduction of red blood cell count and hematocrit in peripheral blood. The peripheral blood T and B cells were also reduced 6 and 2 fold respectively. In the myeloid compartment, the granulocyte/monocyte progenitor cells were increased 2 fold in bone marrow, and granulocytes increased 3 fold in peripheral blood.

These studies reveal that Runx1 and Runx2 transcription factors regulate expression of adhesion and self-renewal genes in the HSPC compartment, modulating the homeostasis of HSCs in the bone marrow niche. In addition, Runx1 and Runx2 regulate hematopoiesis differentiation by synergistic and opposing effects in lineage specific manner.