Disruption of CD9 Expression Affects Adhesion, Migration, and Actin Polymerization through RAC1 Signalling Pathway in ETV6/RUNX1 Pre-B Lymphocytes
Authors: Arnaud Marie-Pierre , Vallée Audrey , Robert Guillaume , Bonneau Jacinthe , Dondi Elisabetta , Le Roy Christine , Varin-Blank Nadine , Galibert Marie-Dominique , Troadec Marie-Bérengère , Gandemer Virginie 
Published: October 2019

Abstract
Introduction and hypothesis : B-acute lymphoblastic leukemia (B-ALL) expressing the fusion transcript ETV6/RUNX1 is one of the most frequent subtype of childhood leukemia. This disease is associated with a good survival rate but 5 to 10% of patients relapse more than five years after treatment most notably in extra-hematopoietic sanctuary sites like testis.
CD9, a protein member of the tetraspanin family, has been shown to be under-expressed in ETV6/RUNX1 B-ALL. This protein is involved in hematopoietic stem cell engraftment. Its expression has also been correlated with the risk of metastasis and is associated with a poor clinical outcome in various types of cancer.
Our hypothesis is that CD9, through its functional properties on migration and homing, could be a key actor of ETV6/RUNX1 leukemia relapse. The purpose of our study was to investigate the effect of CD9 expression on motility and engrafment of pre-B lymphocytes.
Methods and results : We used the human pre-B lymphoblastic REH cell line which express CD9. By lentiviral transduction of small hairpin RNAs targeting CD9 mRNA, we generated REH cells depleted in CD9 protein. We performed engraftment assays in vivo using Nod Scid Gamma immunodeficient mice. Mice injected with REH cells depleted in CD9 had a longer survival rate than mice injected with REH control cells. In vitro analysis of adhesion on fibronectin demonstrated that cellular adhesion is dependent of membrane expression of CD9. As well, the more CD9 is expressed, the higher migration rate in response to CXCL12 chemokine is. F‐actin labelling after CXCL12 stimulation showed also an increased F‐actin polymerization in CD9‐positive cells and the formation of actin extensions where CD9 and actin are colocalized. No modifications in CXCR4 (receptor of CXCL12) internalization has been found in CD9 depleted cell lines, which suggests that CD9 does not affect the receptor itself but the signalling pathway downstream the receptor. We investigated the activation of RAC1 protein, a Rho GTPase known to induce the formation of actin-rich lamellipodia in diverse cellular functions including migration. We demonstrated that cells depleted in CD9 have lost their ability to activate RAC1 in response to CXCL12. The inhibition and the depletion of RAC1 respectively have the same effect as CD9 depletion on the migration in response to CXCL12 in vitro and on the survival in vivo.
Finally, we tested the ability of REH cells and REH depleted in CD9 cells to migrate to testis in vivo after xenograft in mouse model. We showed that only CD9 positive-REH cells were found in testis tissue after xenograft. Moreover we tested in vitro, the ability of testis tissue to induce the migration of REH cells in a Boyden chamber model. We showed that testis tissue induced the migration of REH cells and this migration was strongly decreased using either an inhibitor of CXCR4, an inhibitor of RAC1 or an antibody targeting specifically CD9.
Conclusion : Our data show that CD9 is a key actor in pre‐B lymphoblasts adhesion, CXCL12 dependant migration and actin remodelling. We show for the first time in pre-B cells a link between CD9, RAC1 pathway and homing in testis which is a preferential site of late relapse.
This work is supported by CNRS, University of Rennes 1, University Hospital of Rennes, Ligue Régionale contre le Cancer (sections 22, 35 and 56) (MPA, VG, MBT), Région Bretagne (MPA, MBT, VG), SFR Biosit UMS 3480 (VG, MBT), Association Laurette Fugain (VG).

Disclosures
No relevant conflicts of interest to declare.