Abstract 3834


CD9, a four transmembrane glycoprotein belonging to the tetraspanin family, is suggested to regulate cell motility and adhesion and to play a role in megakaryopoiesis. It has been reported to be a molecular marker of primary myelofibrosis (PMF) being characterized by myeloproliferation, dysmegakaryopoiesis, alterated bone marrow/spleen stroma and extramedullary haematopoiesis. CD9 mRNA has been shown to be overexpressed in CD34+ PMF HPs and its membrane expression level was correlated with platelet counts. Our recent data evidencing an alteration of CD9 expression in PMF megakaryocytes (MK) have encouraged us to investigate whether CD9 participates in the dysmegakaryopoiesis and whether it is involved in the dialogue between MK and stromal cells in PMF patients.

Patients and Methods:

CD34+ cells were MACS selected from the peripheral blood of PMF patients (n=67) and of unmobilized healthy donors (n=61). Functional studies were performed on MK precursor-derived from CD34+ cells cultured in MK medium with ou without monoclonal antibodies (Syb mAb) or siRNAs targeting CD9. CXCL12-induced MK migration was performed in Boyden chambers. Bone marrow mesenchymal stromal cells (MSC) from healthy donors and PMF patients were cultured in DMEM+10%FCS.


Our results showed that CD9 membrane expression was altered on CD34+ cells and on MK precursor-derived from PMF CD34+ cells. Binding of CD9 with Syb mAb restored the in vitro megakaryocyte differentiation process that was altered in patients as shown by an increase in: i) megakaryocytic colony formation in semisolid medium, ii) CD41 and CD62p MK differentiation marker and GATA-1 expression, iii) MK cytoplasmic maturation, iv) apoptotic MK number (reduced AKT phosphorylation and Bcl-XL expression and increased percentage of Annexin+ cells). Activation of CD9 was also associated with regulation of MAPK and AKT-GSK3β pathways whose balance is involved in MK differentiation. Treatment of PMF MK precursors by Syb modulated activation of the MAPK pathway as shown by an increased of p38, JNK and GSK3β phosphorylation and of AP-1 mRNA expression. Taking into account the structure of the tetraspanin molecular network, binding with Syb mAb might also impact the effects associated to the multimolecular complex in which CD9 is involved. This prompted us to study the effects of a molecular silencing of CD9 on the PMF MK differentiation. We showed that, in contrast to the Syb mAb, addition of CD9 siRNA to PMF megakaryocytes reduced their transcriptional program including c-Myb, a transcription factor that is involved in CD9 regulation during megakaryopoiesis. Given the role of CD9 in cell migration, we further investigated whether it could be involved in the megakaryocytic precursor migration observed in patients. We showed that silencing CD9 reduced the CXCL12-dependent megakaryocytic precursor migration as well as the CXCR4 and CXCL12 transcription and that this migration involved actin polymerization. c-Myb siRNA restored CXCR4 and CXCL12 expression and reduced actin polymerization suggesting that CD9 was involved, via c-Myb, in the CXCL12-dependent megakaryocytic precursor migration. Effect of CD9 on cell migration is often interpreted as related to modulation of integrins participating in the integrin/tetraspanin network and of their interaction with mesenchymal stromal cells (MSC). We showed that several genes involving the CD9 partner interactome were over-expressed in MSC from PMF bone marrow as compared to MSC from healthy donors. Preliminary results showing that PMF MK precursors display different behaviour in terms of cell survival and adhesion when co-cultured on bone marrow MSC from PMF patients as compared to healthy donors suggest that interactions between MKs and bone marrow MSC is involved in PMF dysmegakaryopoiesis. Addition of Syb reverses these alterations suggesting the participation of CD9 in the abnormal dialogue between MK and MSC.


Our results show a deregulation of CD9 expression in megakaryocytes from PMF patients. They also suggest that CD9 i) participates in PMF dysmegakaryopoieis in terms of MK differentiation and survival and ii) is involved in the increased MK precursor migration through alterations of the CXCL12/CXCR4 axis. Our data further support the role of bone marrow stroma in PMF dysmegakaryopoeisis through CD9 interactions.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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