Jagged-induced notch signaling promotes endogenous and bone marrow-mediated drug resistance in multiple myeloma

BACKGROUND Multiple myeloma (MM) represents 10% of all hematological malignancies. Malignant plasma cells accumulate in the bone marrow. Although in the last 10 years new drugs such as immunomodulators or proteasome inhibitors increased patients\u2019 survival, MM remains still incurable mainly due to the development of endogenous or BM mediated drug resistance. Therefore it is crucial to find new therapeutic targets. The deregulated expression of two Notch ligands, Jagged1 and 2, activates Notch pathway both in MM cells and in bone marrow stromal cell (BMSC) which express Notch receptors. Several Notch downstream effectors are involved in MM cell growth, survival and proliferation, i.e. IL-6, SDF-1alfa, CXCR4, NF-kB, VEGF and IGF. AIMS The aim of this study was to evaluate the role of Notch signaling in endogenous and BMSC-promoted drug resistance in MM, focusing on the contribution of the chemokine axis CXCR4/SDF-1alfa. MATERIAL AND METHODS The human MM cell lines, U266 and OPM2, were cultured alone in complete RPMI-1640 medium or co-cultured with murine (NIH3T3) or human (HS5) BMSC cell lines in DMEM medium supplemented with 10% V/V FBS. U266 and OPM2 cells were either kept in suspension for 24 hours at 3 x 105/mL or plated on BMSC monolayer for 24h, then treatments with drugs (1-2 mM Mitoxantrone, 5-8 nM Bortezomib or 100-30 mM Melphalan ) or 50\u3bcM AMD3100 were applied for additional 24 hours. Apoptosis assay: HS5 cells were colored with PKH26 red fluorescent dye (Sigma-Aldrich) before co-culturing to allowed flow-cytometric detection of MM cells co-cultured with BMSCs. At the end of the treatment, cells were stained with Annexin V-FITC (ImmunoTools) and processed with Cytomics FC500 software (Beckman Coulter). RNA interference: specific knock-down of Jagged1 and 2 was obtained by transient expression of specific siRNAs for Jagged1-2 (Stealth Select RNAiTM siRNA system, Life Technologies). MM cell lines were seeded at 350.000 cells/ml and, after 24h, Jagged1 and 2 genes were simultaneously silenced. Every 48h cells were diluted and transfected again. Quantitative PCR reactions were carried out on a 7500 Fast Real-time PCR system (Applied Biosystems) using the Maxima\u2122 SYBR Green/ROX qPCR Master Mix (Dasit). RESULTS RNA interference for Jagged-1 and 2 in OPM-2 and U266 cells resulted in the reduced expression of anti-apoptotic genes such as SDF-1alfa, CXCR4, Bcl-XL, Bcl-2, Survivin and ABCC1. At the same time, MM cells with reduced levels of Jagged-1 and 2 showed an increased sensivity to different drugs commonly used in MM therapy such as Bortezomib, Mitoxantrone and Melphalan. By co-culturing MM cell lines and BMSCs in the presence or the absence of chemotherapeutic agents we observed that BMSCs were able to protect MM cells from apoptosis. We investigated the underlying mechanism showing that MM cells and BMSC interaction resulted in the activation of Notch signaling in both cell types. MM cells-driven Notch signaling activation in BMSCs resulted in the increased expression of soluble growth factors relevant for MM cell growth and survival, such as SDF-1alfa and VEGF. On the other side, BMSCs increased in MM cells the expression of several anti-apoptotic genes, i.e. Bcl-XL, Bcl-2, Survivin and ABCC1. Interestingly, Jagged-1 and 2 silencing in MM cells could reverse all gene expression changes and BMSC protective effect. Finally, the CXCR4 antagonist AMD3100 could partially reverse the protective effect of BMSCs to drugs-induced apoptosis in MM cells, suggesting that Jagged ligands deregulation observed in MM is necessary to BM-promoted drug resistance by activating the SDF1alfa/CXCR4 chemokine signaling. CONCLUSION/DISCUSSION The evidence that anti-Jagged-1 and 2 siRNAs affect endogenous and BMSC-induced drug resistance in MM cells suggests that a Jagged-directed approach could be effective in MM therapy alone or in a combined treatment with commonly used drugs