Aberrant Megakaryocyte Antigen Expression on Myelodysplastic Erythroid Cells: Role of Protein Kinase C?.

Erythroid lineage dysplasia is one of the most frequent findings in Myelodysplastic Syndrome (MDS) and in clinical terms contributes significantly to the morbidity in affected patients. There is currently no satisfactory treatment available for the majority of these patients and many rely on regular red cell transfusions for support. Previous work using an in vitro model system based on human CD34+ progenitors indicated that the developmental abnormalities affecting the erythroid cells may in part arise from inappropriate activation of protein kinase C (PKC). Evidence suggests that PKC acts as a lineage discriminator for bipotential erythro-megakaryocytic progenitor cells where low PKC activity promotes erythroid commitment and high PKC activity promotes megakaryocytic commitment. In the in vitro model system aberrant activation of PKC in erythroid cells led to developmental disruption and inappropriate megakaryocytic antigen expression such as GPIIb (CD41). Restoration of normal development was possible by inhibition of PKC. In this study we investigate the extent to which aberrant megakaryocyte antigen expression occurs in MDS, and whether PKC inhibition can normalise this and the associated erythroid lineage dysplasia. Bone marrow was obtained from 27 MDS patients and compared with 16 normal bone marrows. Fresh bone marrow mononuclear cells from both groups were analysed by 4- colour flow cytometry to establish the frequency of aberrant CD41 expression on nucleated erythroid (GlyA+ CD36+) cells. Cells bearing adherent platelets were excluded based on co-expression of CD42b. We have established that aberrant expression of CD41 occurred in 56% of the MDS patient marrows compared with normal bone marrow erythroid cells (n=27 and 16 respectively). These patients also showed clear morphological evidence of erythro-megakaryocytic lineage dysplasia. To determine the effect of PKC inhibition on this aberrant CD41 expression, some of these cases were examined in bulk liquid culture in the presence of PKC inhibitors. We assessed three PKC inhibitors in this way: GF109203X, a proprietary staurosporine derivative and Tamoxifen. In 42% of the cases examined in this way there was a phenotypic response to the highly selective PKC inhibitor GF109203X in terms of a reduction in CD41 expression. There was also morphological evidence of erythroid differentiation. The other two agents showed no effect on these parameters. The expression profile of individual PKC isoforms in the erythroid (GlyA+) cells was examined by western blotting, to determine if as in the model system the developmental abnormalites seen in the MDS marrow resulted from hyperactivation of PKC. We were able to evaluate 13 MDS and 6 normal controls in this way. The expression of 3 PKC isoforms (α, ε, ι) was determined as well as the level of phosphorylated PKC and the level of PDK1 kinase (which phosphorylates PKC). More than half of the patients examined demonstrated elevated levels of PKC expression. Aberrations in phosphorylation of PKC were less common. Patients with the highest expression of CD41 also had the highest expression of PKCα. These data suggest a role of inappropriate PKC activity in the erythroid lineage dysplasia and subsequent developmental failure seen in MDS, and, that PKC inhibitors may be valuable therapeutic agents in a subset of these patients.