Effect of anagrelide on megakaryocytic cytoskeleton during platelet production. Possible genes involved

Abstract

We previously reported changes in maximum platelet diameter (MPD) in patients with Phi-negative chronic myeloproliferative neoplasms treated with different drugs. Anagrelide (ANA), alfa Interferon (IFN) and ruxolitinib (Ruxo) increased MPD, while hydroxyurea (HU) decreased MPD. Evaluation of proplatelet (PP) morphology in normal megakaryocyte (MK) culture showed that ANA and IFN increased the size of tips and swellings (structures that give rise to platelets), while Ruxo and HU did not.To deepen into possible causes of these changes during ANA treatment, two approaches were carried outA) Morphological analysis of MK cytoskeleton by indirect immunofluorescence: a trend toward decreased actin polarization was observed (phalloidin-FITC), suggesting that demarcation membrane development necessary for thrombopoiesis is abnormal. Additionally, microtubular disorganization was observed (anti-alpha tubulin-FITC) (Wilcoxon test p<0.05), which would interfere with PP elongation.B) To identify molecular targets responsible for ANA-induced cytoskeleton abnormalities, gene expression data obtained by Ahluwalia (GEO database:GSE60621) was analyzed. 81 genes were found down-regulated (Log-fold change ≤3). This group was further analyzed with Cytoscape v3.8.1 (ClueGo plugin) to build a visualization network of associated biological processes. From three groups detected, the one corresponding to Positive regulation of phospholipase activity showed topological characteristics that suggest high number of processes with potential relevance in MK. Within this group, CCL5, an inflammatory cytokine that stimulates PP formation, is of particular interest. We confirmed decreased CCL5 expression by RT-PCR in our culture conditions (Wilcoxon test, p<0.05). Our results suggest that ANA alters both, actin and tubulin cytoskeleton. In addition, we confirm decreased CCL5 expression, which could be related to cytoskeletal alterations during thrombopoiesis under ANA treatment.