S1P Modulator FTY720 Targets Multiple Myeloma Cell Proliferation and Stromal Interactions Via CXCR4/CXCL12 and mTOR Pathways
Authors: Beider Katia , Rosenberg Evgenia , Bitner Hanna , Leiba Merav , Koren-Michowitz Maya , Abraham Michal , Klein Shiri , Wald Hanna , Shimoni Avichai , Peled Amnon , Nagler Arnon 
Published: October 2019

Abstract
Introduction: Multiple myeloma (MM) is an incurable hematological malignancy characterized by proliferation of malignant plasma cells in the bone marrow (BM). Interactions between MM cells and BM milieu facilitate disease progression and therapy resistance. Chemokine receptor CXCR4 and its cognate ligand CXCL12 are implicated in these processes and are associated with poor prognosis. Sphingosine-1-phosphate (S1P) pathway is involved in cancer progression, including oncogenesis, cell survival and cell migration, therefore representing an attractive target for anti-cancer therapy. FTY720 (fingolimod) is a modulator of S1P signaling system that exhibit immunosuppressive and anti-cancer properties. The role of S1P system and FTY720 modulator in MM is less defined. The aim of this study was to explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the S1P axes in MM cells and to evaluate the effect of S1P targeting with FTY720 as potential anti-MM therapeutic strategy.
Results: The partners of the S1P pathway (S1P receptor 1 and sphingosine kinase 1 (SPHK1)) and CXCL12 chemokine were found to be co-expressed in MM cell lines and primary BM samples from MM patients. Increased mRNA levels of SPHK1 and CXCL12 were detected in MM BM samples (n=24) comparing to BM from healthy donors (n=7) (p<0.01). In vitro treatment of MM cell lines (n=6) with FTY720 modulator resulted in time- and dose-dependent cell death (IC50 2.8 – 5.3 µM). Further characterization of cell death mechanisms revealed that FTY720 treatment induced MM cell apoptosis with mitochondrial involvement, cytochrome C release and caspase 3 activation.
Interestingly, suppressive potential of FTY720 negatively correlated with CXCR4 expression on MM cells. Enforced expression of CXCR4 reduced the sensitivity to FTY720, whereas silencing of endogenous CXCL12 increased the sensitivity of MM cells to FTY720-mediated cell death. These results suggested the CXCR4 axis to be directly regulated by S1P pathway. In support, we have found that FTY720 treatment significantly reduced CXCR4-dependent MM cell adhesion to fibronectin and abrogated MM migration toward CXCL12. Activation of signaling pathways, such as MAPK and Akt, in response to CXCL12 stimulation was also fully blocked by FTY720 pre-treatment. In addition to functional suppression, FTY720 directly and profoundly reduced CXCR4 cell-surface levels in a dose-dependent manner. Importantly, none of the suppressive effects of FTY720 (neither apoptosis, nor migration or adhesion inhibition) were dependent on protein phosphatase 2A (PP2A) activation, suggesting alternative mechanism of action.
To further investigate down-stream molecular machinery involved in FTY720-mediated CXCR4 targeting in MM cells, the intra-cellular levels of different signaling mediators were evaluated. We identified the mTOR pathway to be regulated by CXCR4 and targeted by FTY720. FTY720 treatment suppressed mTOR signaling in MM cells, as demonstrated by de-phosphorylation of p70S6K and S6. Forced expression of CXCR4 and interaction with BM stromal cells antagonized with FTY720-mediated apoptosis and prevented FTY720-induced S6 de-phosphorylation. While, combination of FTY720 with mTOR inhibitor RAD001 resulted in significantly increased cell death, effectively abrogating CXCR4- and stroma-dependent resistance to FTY720 and suppressing mTOR signaling in MM cells.
Finally, in a recently developed novel xenograft model of CXCR4-dependent systemic MM with BM involvement, in vivo FTY720 effectively reduced tumor burden in two third of the treated mice, decreasing both the levels of M protein in blood and the number of MM cells in BM.
Conclusions: Taken together, our findings demonstrate cross talk between S1P and CXCR4/CXCL12 signaling pathways that may be of importance for MM cell survival and localization of the MM cells in CXCL12-expressing protective niches in the BM. Moreover, this is, to our knowledge, the first evidence that CXCR4 can be directly targeted with FTY720 modulator, thus restricting the tumor-promoting activities of S1P and CXCR4/CXCL12 axes. In addition, mTOR pathway was recognized as down-stream molecular partner being involved in FTY720-mediated anti-myeloma activities. Combining FTY720 with mTOR inhibitors may thus serve as promising novel therapeutic strategy in MM.

Disclosures
Peled: BioLineRx: Research Funding.