Myeloid Expansion In the Absence of RUNX1 Is Associated with Increased Monopoiesis, Reduced Granulopoiesis, and Diminished CEBPA Gene Expression, Effects of Potential Relevance to Myeloid Transformation

Abstract 3147

RUNX1 directs formation of definitive HSC during development. RUNX1 gene deletion in adult mice leads to multiple hematologic alterations, including expansion of the LSK stem cell population, markedly reduced B and T lymphoid and platelet formation and a myeloid expansion with a 3-fold increase in GMP, a 5-fold increase in myeloid CFUs, expansion of mature myeloid cells in bone marrow, and extra-medullary hematopoiesis with an abundance of maturing myeloid cells in the spleen and liver. The mechanisms underlying the myeloid expansion that occurs in the absence of RUNX1 may be relevant to the pathogenesis of AML cases associated with RUNX1 gene mutation or with expression of dominant-negative oncoproteins such as AML1-ETO or CBFβ-SMMHC. We further evaluated the myeloid compartment upon RUNX1 gene deletion. RUNX1(f/f);Mx1-CRE or littermate RUNX1(f/f) mice were exposed to 5 injections of pIpC at age 8–16 weeks and allowed to recover for 4 weeks. Marrow evaluation demonstrated an increase in Mac-1+Gr1- monocytes from 2% to 8% upon RUNX1 deletion, and liquid culture for 4 days in IL3/IL6/SCF, RUNX1 deletion increased the monocyte population from 23% to 50%. CFU assays demonstrated a 5-fold increase in CFU-M, with a statistically significant 1.8-fold decrease in CFU-G in three independent experiments. FACS analysis of pooled CFUs confirmed an increase in the ratio of monocytes to granulocytes. RUNX1(f/f);Mx1-CRE mice injected 4 weeks earlier with pIpC were exposed to 5-FU and 5 days later marrow cells were isolated and transduced with pBabePuro or with the same retroviral vector expressing RUNX1-ER. Cells were then cultured in methylcellulose with IL3/IL6/SCF in the absence or presence of 4HT. Activation of RUNX1 specifically increased the formation of CFU-G and reduced the formation of CFU-M in three independent experiments, rescuing the defect in myelopoiesis observed upon RUNX1 deletion. As C/EBPα and PU.1 are key regulators of myeloid lineage determination, we evaluated their expression by Western blotting and by Q-RTPCR in marrow mononuclear cells from RUNX1-deleted or control, pIpC-injected mice. A 3-fold decrease in C/EBPα protein and RNA was evident in three separate experiments; PU.1 RNA was reduced 2-fold, but PU.1 protein was not significantly reduced; RUNX1 RNA was reduced 20-fold, reflecting efficient gene deletion. Transduction of RUNX1-deleted cells with RUNX1-ER, selection of lineage-depleted cells, and addition of 4HT led to a 3-fold increase in C/EBPα protein expression, but no change in PU.1. The murine CEBPA promoter is activated 2-fold by RUNX1 in NIH 3T3 cells, and the promoter contains tandem cis elements that fit the RUNX1 consensus. We demonstrate that these sites bind RUNX1 in a gel shift assay. One of these sites is conserved at the same location in the human promoter, and the other site can be found at -139 in the human promoter. The ability of RUNX1 to directly regulate the CEBPA gene via these identified binding sites is currently under investigation. A dominant negative RUNX1 protein, KRAB-RUNX1-ER, markedly reduces endogenous C/EBPα protein expression within 8 hr of activation in the granulocytic 32Dcl3 cells line, consistent with direct repression of the CEBPA gene. In summary, absence or RUNX1 leads to reduced C/EBPα expression that potentially contributes to the observed myeloid expansion associated with increased monocyte and reduced granulocyte lineage commitment. In AML cases with reduced RUNX1 activity, consequent reduction in C/EBPα expression may contribute to transformation via inhibition of granulocytic differentiation, as well as via loss of C/EBPα-mediated cell cycle inhibition.