FLT3-Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis.
Authors: Desterke Christophe , Bilhou-Nabera Chrystele , Guerton Bernadette , Tonetti Carole , Clay Denis , Pierre-Louis Olivier , Vannucchi Alessandro M , Gisslinger Heinz , Bordessoule Dominique , Hasselbalch Hans C , Lazar Vladimir , Vainchenker William , Bennaceur-Griscelli Annelise , Giraudier Stéphane , Le Bousse-Kerdilès Marie-Caroline 
Published: October 2019

Abstract
Abstract 963

Introduction:
Flt3, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis. Primary myelofibrosis (PMF) is a Ph-negative (Ph−) myeloproliferative neoplasm (MPN) characterized by a myeloproliferation with increased hematopoietic progenitors (HPs) and a prominent proliferation of “dystrophic” megakaryocytes (MK). The JAK2V617F mutation is present in about 50% of PMF patients. Previous results from our group have revealed a MAPK pathway gene deregulation associated with an Flt3 transcript modulation in PMF patients. Since activation of Flt3 receptor is known to activate MAPK pathway, which plays a role in megakaryopoiesis, we studied the functional impact of MAPK and Flt3 abnormalities on PMF dysmegakaryopoiesis.

Patients and Methods:
The study included a group of 106 PMF patients. Transcriptome and QRT-PCR studies were performed on MACS selected CD34+ HPs, megakaryocytes (MK)-derived from CD34+ cell cultures and peripheral blood mononuclear cells (PBMNC) from PMF patients and healthy donors. Cell phenotype associated with Flt3 expression as well as phosphorylation levels of Flt3 and MAPK effectors were analyzed by flow cytometry. Functional studies (FL-induced stimulation and migration) were performed on MK-precursors at day 6 of CD34+ culture. Effect of i) MAPK inhibitors: PD98059, targeting ERK1/2; SB202190, SB203580, PD169316, targeting p38 and, SP600129, targeting JNK, ii) Flt3 inhibitors and iii) Flt3 monoclonal antibody was tested on PMF MK cell cultures. MAPK-induced transcripts were quantified by QRT-PCR in MK-precursors during an 18-hour FL-stimulation kinetic. FL mRNA level was evaluated by using QRT-PCR in bone marrow stromal cells and FL protein was quantified in plasma by using ELISA.

Results:
Comparative transcriptome analysis of CD34+ HPs and MK cells from PMF patients (with or without JAK2 mutation) and healthy donors showed that the MAPK pathway gene deregulation was independent of the presence of the JAK2V617F mutation. This alteration was associated with a modulation of mRNA Flt3 level in both types of cells. PMF patients also had a higher proportion of circulating Flt3+CD34+CD41+ cells as compared to healthy donors. This population demonstrated an increased phosphorylation of Flt3 on tyr591 and of MAPK (p38, p42/p44, JNK). MAPK effector phosphorylation was also increased in PMF CD34+ cells and MK-derived from CD34+ cell cultures, independently of JAK2 mutational status. In contrast to healthy donors, Flt3 membrane expression was maintained at all stages of in vitro megakaryocyte differentiation in PMF patients. The FL level was increased in the plasma of patients and was mainly expressed by bone marrow stromal cells. In contrast to healthy donors, in MK-derived from PMF CD34+ cell cultures, activation of Flt3/FL axis by addition of exogenous FL induced a MAPK hyper-phosphorylation, especially of p38 and p42/p44 as well as an up-regulation of downstream p38 transcripts (ATF-2, NFATC4, p53, AP-1, IL-8). Addition of chemical inhibitors targeting either MAPK or Flt3 and of an antibody directed against Flt3 reduced the phosphorylation of p38 and of its pathway effectors (MKK3/MKK6, MSK1, ATF2, HSP27 and MAPKAPK2) and normalized the PMF altered megakaryopoiesis. Lastly, in contrast to healthy donors, MK-derived from PMF CD34+ cells showed a FL-induced migration that was reversed by addition of p38αβ inhibitors.

Conclusion:
Our results demonstrated an increase in the FL circulating level in PMF patients that was mainly secreted by stromal cells. This was associated with an aberrant expression of Flt3 in CD34+ and MK cells and an alteration of the MAPK pathway activation in patients, independent of their JAK2 mutational status. The persistence of Flt3-mediated MAPK activation that participates in the PMF dysmegakaryopoiesis, suggests that drugs targeting “FL/Flt3-MAPK” axis could be promising agents for rescuing the altered megakaryopoiesis observed in patients. Our demonstration that FL, a cytokine mainly produced by stromal cells, participates in the altered megakaryopoiesis in PMF patients strengthens the hypothesis highlighting the crucial role of stroma cells in the hematopoietic deregulation that characterizes the disease.

Disclosures:
No relevant conflicts of interest to declare.