Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells Running Title: PKC mediates N-Ras-induced dyserythropoiesis

RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukaemia and preleukaemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (EPO-independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective sub-population of CD34+ erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Though early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues which regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukaemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukaemia. only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From