Dysregulated Expression of Myeloid Nuclear Differentiation Antigen (MNDA) in MDS: Possible Role in TRAIL-Induced Apoptosis.

A reduced level of MNDA expression has been reported with gene expression profiling in both sporadic and familial MDS. We undertook studies to evaluate MNDA protein expression in MDS, and its possible functional significance. Patients (n=7) clinical histories, lab data, cytogenetics, and morphology were reviewed blinded to study results to confirm diagnoses. Patients had Refractory Cytopenia with Multilineage Dysplasia (n=6) or Refractory Anemia with Excess Blasts-1 (n=1)(WHO). Patients lacking primary marrow disease (n=5) were used as controls. Samples were assessed with multiparametric flow cytometry (MPFC) for CD45 antigen density, log side scatter, CD34, CD71, and MNDA (using a specific rat monoclonal antibody) with an FC500 (Beckman-Coulter) flow cytometer, and analyzed with Winlist 5.0 and ModFitLT 3.0 (Verity Software) using published methods and ECFI-generated algorithms. Analyses were blinded to clinical results. Immunohistochemistry (IHC) for MNDA was performed using the same antibody and published methods. Both MPFC and IHC demonstrated MNDA expression in MDS predominantly in granulocyte-macrophage (G-M) progenitors, similar to controls. G-M progenitors in MDS demonstrated reduced MNDA protein expression compared to controls, with 3 patterns: uniform reduced MNDA protein, a mix of normal and absent MNDA protein, or a mix of reduced and absent MNDA protein. We interpret the normal expression in some cases as consistent with presence of a mixture of normal and MDS cells, while the mix of reduced and absent expression may represent clonal evolution of MDS. To evaluate possible functional significance of MNDA expression we used stable K562 clones that express ectopic MNDA protein via retroviral transduction. Parental K562 cells lack MNDA expression. Compared to parental K562, ectopic expression of MNDA failed to protect cells from intrinsic path apoptosis induced by genotoxic agents (mechlorethamine, etoposide, cisplatin) or H₂O₂. Apoptosis was evaluated by morphology, TUNEL assay, trypan blue exclusion, and phosphorylation of Histone H2B at serine 14 (a specific marker of apoptosis). In sharp contrast, ectopic MNDA expression provided marked protection of cells from extrinsic path apoptosis induced by TRAIL (TNF-related apoptosis inducing ligand) exposure. Disordered apoptosis is hypothesized to be a factor in MDS pathogenesis. Our results indicate MNDA promotes viability in myeloid progenitors, and its down-regulation in MDS might relate to the G-M progenitor sensitivity to TRAIL-induced apoptosis that has been reported in MDS.