Bone marrow-mediated drug resistance is promoted by Jagged-induced notch pathway in multiple myeloma

Background: Multiple myeloma (MM) rapresents 10% of all hematological malignancies and is caused by the accumulation of malignant plasma cells in the bone marrow (BM). Although treatments with new drugs (immunomodulators and proteasome inhibitors) are increasing patients’ survival, MM is still incurable because of the development of endogenous or BM mediated drug resistance. Therefore it is crucial to find new therapeutic targets. The dysregulated expression of two Notch ligands, Jagged1 and 2, hyperactivates Notch pathway both in MM cells and in bone marrow stromal cell (BMSC). Several Notch downstream effectors support 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 investigate the role of Notch signaling in endogenous and BMSC-promoted drug resistance in MM. Methods: U266 and OPM2 MM cell lines 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. MM cells were either kept in suspension for 24 hours or plated on BMSC monolayer for 24h, then the MM cell lines were treated with drugs (Mitoxantrone, Bortezomib or Melphalan) for additional 24 hours. Apoptosis assay: HS5 cells were stained with PKH26 red fluorescent dye (Sigma-Aldrich) before co-culturing to allowed flow- cytometric detection of MM cells co-cultured with BMSCs. Cells were stained with Annexin V-FITC and processed with Cytomics FC500 software (Beckman Coulter). RNA interference: Silencing of Jagged1 and Jagged2 was obtained by transient expression of two specific siRNAs (Stealth Select RNAiTM siRNA system, Life Technologies). Quantitative PCR reactions were carried out on a 7500 Fast Real-time PCR system (Applied Biosystems) using the MaximaTM SYBR Green/ROX qPCR Master Mix (Dasit). Anti-apoptotic protein such as SDF-1alfa, CXCR4, Bcl-2, Survivin and ABCC1 were analyzed by flow cytometry using appropriate antibodies. Results: Jagged1 and 2 silencing in OPM-2 and U266 cells is able to reduce the expression of anti-apoptotic genes i.e. SDF-1alfa, CXCR4, Bcl-XL, Bcl-2, Survivin and ABCC1. At the same time, MM cells with reduced levels of Jagged1 and 2 showed an increased sensivity to different drugs commonly used in MM therapy such as Bortezomib, Mitoxantrone and Melphalan. We investigated the underlying mechanism showing that MM cells and BMSC interaction resulted in the activation of Notch signaling in both cell types. When co-coltured with human or murine BMSCs, we observed that MM cells showed increased drug resistance due to: first of all, an increased expression of anti-apoptotic genes in MM cells among which SDF-1alfa, CXCR4, Bcl-XL, Bcl-2, Survivin and ABCC1; secondary, BMSC release of soluble factors, i.e. SDF-1alfa and VEGF, relevant for MM cell growth and survival. Interestingly, Jagged1 and 2 silencing in MM cells could reverse all gene and protein expression changes and BMSC protective effect. All these results were confirmed both at gene expression and protein level. Summary/Conclusion: The evidence that Jagged-1 and 2 silencing affect endogenous and BMSC-induced drug resistance in MM cells supports the use of a Jagged-targeted approach in MM therapy alone or in a combination with common drugs