FMNL1 Is Downregulated In Myelodysplastic Syndromes and Is Involved In Megakaryocytic Differentiation
Authors: Rodrigues Lopes Matheus , Machado-Neto João Agostinho , Traina Fabiola , Novais Pereira João Kleber , Lorand-Metze Irene , Costa Fernando Ferreira , Olalla Saad Sara Teresinha , Favaro Patricia 
Published: October 2019

Abstract

Background
Myelodysplastic syndromes (MDS) are disorders characterized by morphological dysplasia, impaired differentiation and defective cellular functions, resulting in peripheral cytopenias. FMNL1 belongs to a family of formin-related proteins, indispensable for many fundamental actin-dependent processes. Recently, FMNL1 has been described to be upregulated and play a role in the actin cytoskeleton dynamics during monocyte differentiation to macrophages.

Aims
The aim of this work was to characterize FMNL1 expression in total bone marrow cells of patients with MDS comparing to normal donors. We also analyzed FMNL1 expression in erythrocytic, granulocytic and megakaryocytic differentiation, using cell line models. Finally, we evaluated the impact of inhibition of FMNL1 during megakaryocytic differentiation.

Methods
A total of 49 patients with a diagnosis of MDS, receiving no treatment, and 18 samples from normal donors were included in the study, which was approved by the National Ethical Committee Board. Samples were submitted to RNA extraction after removal of erythrocytes by hemolysis. FMNL1 expression levels from cell lines or total bone marrow cells were determined by quantitative PCR (q-PCR) or Western blot. KU812 was treated 50 μM hemin and 100 μM hydroxyurea for erythrocytic differentiation. K562 was stimulated with 20nM of PMA for megakaryocytic differentiation. NB4 was treated with 10-6 M of ATRA for granulocytic differentiation. Megakaryocytic differentiation was followed by the increase in megakaryocytic marker (CD61) determined by flow cytometry and cells were also stained with May–Grunwald–Giemsa. K562 cells were transduced with lentivirus-mediated shRNA targeting LacZ or FMNL1. Apoptosis was assessed by Annexin-V/PI staining and cell cycle was evaluated by flow cytometry, both at 24 and 48 hours after induction with PHA. The statistical methods used were the age-adjusted multivariate linear regression analysis, Mann Whitney test or t test.

Results
FMNL1 expression in bone marrow samples was significantly lower in MDS when compared with normal donor cells (P=0.01), especially in the high risk group (P<0.02). Using cell line models for hematopoietic differentiation, there was a fifteen-fold increase and a five-fold increase in FMNL1 expression for megakaryocytic (P=0.002) and granulocytic differentiation (P=0.05) respectively. Western blot analysis corroborated these findings. There was no difference for erythrocytic differentiation. After PMA treatment, the level of the megakaryocytic markers CD61 was significantly lower in K562 shFMNL1 when compared with shLacZ (P=0.01). The level of CD41a (P=0.5) and CD42b (P=0.1) showed a trend toward a decrease in K562 shFMNL1 when compared with shLacZ, however not statistically significantly. Corroborating these data, K562 shFMNL1 showed abnormal megakaryocytic morphological features (larger cells with polylobulated or polysegmented nuclei and vacuolization) compared with cells shLacZ. There were no statistical differences in the apoptosis levels and cell cycle analysis between K562 shFMNL1 and shLacz cells.

Conclusions
The family of formin-related proteins has mainly been related to actin-dependent processes although little is known regarding their possible involvement in haematopoiesis. The lower FMNL1 expression in MDS BM could reflect the role of this protein in cell differentiation. We were therefore prompted to study this issue in depth using megakaryocytic differentiation as a system. In this study, we show increased expression of FMNL1 in PMA-induced megakaryocytic differentiation of K562 cells. Furthermore, knockdown of FMNL1 deregulates differentiation, suggesting that FMNL1 is required in order to maintain the effective megakaryocytopoiesis in MDS. Although FMNL1 silencing effectively down-regulated CD61 expression, CD41a and CD42b were reduced to a lesser extent. The effect of FMNL1 cannot be explained by modifications of the cell cycle or apoptosis during differentiation; and is probably due to the effect in changes in the dynamic remodeling of the cytoskeleton.

Disclosures:
No relevant conflicts of interest to declare.