Mice with AML1 (Runx1) Haploinsufficiency Have Significant Platelet Abnormalities That Accurately Mimic Those Seen in Human Patients with Germ Line Inactivating Mutations in AML1.

Abstract

The AML1/CBFβ transcription complex, a critical regulator of the formation of definitive hematopoietic stem cells (HSC), is one of the most frequent targets of genetic alterations in acute leukemia. In addition to somatic alterations of AML1 and CBFβ in acute leukemia, germ-line loss-of-function mutations of AML1 are the underlying cause of an autosomal dominant familial platelet disorder with a predisposition to acute myeloid leukemia (FPD/AML). Importantly, a subset of the mutations identified in families with FPD/AML result in AML1 null allele, suggesting that AML1 haploinsufficiency is the underlying molecular abnormality. To explore the functional consequences of AML1 halpoinsufficiency on megakaryocyte development and platelet function, we analyzed the hematopoietic system of AML1+/- mice. Loss of a single AML1 allele resulted in a 15% reduction in the number of circulating platelets and a significant impairment in platelet function including a decrease in dense granule content and an impaired ability to aggregate in response to collagen stimulation. Further analysis indentified a left shift in the DNA ploidy of megakaryocytes and a reduction in GPV expression, consistent with impaired megakaryocyte maturation. In addition, electron microscopy indicated a reduction in platelet demarcation channels within the cytoplasm of megakaryocytes. Importantly, however, we did not observe a reduction in the total number of megakaryoctyes or a decrease in megakaryocyte colony forming units. These data suggest that the haploinsufficiency of AML1+/− does not alter the initial formation of megakaryocytes, but instead impairs the ability of these cells to efficiently mature and produce functional platelets. To explore the underlying mechanism responsible for the observed impairment in megakaryocyte maturation, we analyzed the pattern of expression of several putative AML1 transcriptional targets. Although AML1 binding sites have been identified within the promoter of c-mpl, the gene encoding the receptor for thrombopoietin (TPO), we did not observe any difference in c-mpl expression levels or in circulating TPO concentration between AML1+/− and +/+ mice. In addition, in vivo TPO stimulation induced a similar magnitude of megakaryocyte maturation and platelet production in both AML1+/+ and +/− mice. By contrast, analysis of members of the protein kinase C (PKC) family of gene, several which have been identified as transcriptional targets of AML1, revealed a reduction in PKCδ levels in platelets from AML1+/− mice. Taken together, our data suggest that AML1 haploinsufficiency leads to abnormalities in platelet that are identical to those observed in patients with FPD/AML. Thus, these mice should prove useful for exploring the molecular mechanisms through which AML regulates the normal maturation of megakaryocytes. Our early analysis suggests altered PKCδ signaling is a possible contributing factor to the observed phenotypic abnormalities.