THE ROLE OF NOTCH PATHWAY IN MULTIPLE MYELOMA-ASSOCIATED DRUG RESISTANCE

BACKGROUND: Multiple myeloma (MM) is an incurable hematological malignancy characterized by drug resistance, intrinsic or induced by bone marrow stromal cells (BMSCs). In MM cells, Notch pathway may be aberrantly activated due to the hyperexpression of Notch1, Notch2 or Jagged1 and 2 ligands. This effect may be attributed to genetic mutation only in part (i.e. translocations involving the MAF transcription factors may increase the transcriptional activity on their target gene Notch2). MM cells settle in the bone marrow (BM) and the BM microenvironment may be another player contributing to Notch signaling activation by triggering Notch receptors through BMSC-derived ligands or other stimuli including hypoxia. Indeed, recent evidences indicate that both hypoxic stimuli and Notch signaling activation are involved in cancer stem cell maintenance and self-renewal, thereby contributing to drug resistance due to the resilience of this cancer subpopulation. MM stem cells (MM-SCs) have been characterized as a CD138- subpopulation. AIM: The aim of this study was to investigate the outcome of Notch signaling hyper-activation in intrinsic and BMSC-mediated drug resistance in MM cells and MM-SCs. METHODS: I assessed the effect of Jagged ligands by an inhibitory approach on MM cells. This was carried out by silencing Jagged1 and 2 through specific siRNAs or lentivirally expressed shRNAs. The study got advantage of OPM2 and U266 cell lines and MM primary cells from 10 patients. MM cells were cultured alone, to assess the effect of Jagged silencing on intrinsic drug resistance, or co-cultured with BMSCs, to investigate the effect of Jagged inhibition on BMSC-mediated drug resistance. The BMSC models used were: i) the human HS5GFP+ cell line that, when cultured with MM cell lines, enabled a flow cytometric analysis of variations in drug resistance and anti-apoptotic proteins expressed by MM cells, along with changes in BMSC-production of pro-tumor cytokines (i.e. IL-6 and SDF-1); ii) the murine fibroblasts NIH3T3. These cell lines mimic BMSCs and, when cultured with MM cell lines, enabled to confirm changes in key proteins by gene expression analysis through RT-PCR using species-specific primers to distinguish the contribution of MM cells (human) or NIH3T3 cells (murine); iii) experiments on primary CD138- MM cells were carried out using primary BMSCs stained with PKH26 as feeder cells. Intrinsic and BMSC-induced drug resistance was analyzed by challenging MM cells cultured alone or in co-culture systems with standard-of care drugs, i.e. Bortezomib. Apoptosis was assessed by detection of the percentage of AnnexinV+ cells through flow cytometry. Hypoxic BM microenvironment was mimicked by using cobalt chloride (CoCl2), while Notch pathway activation was inhibited using DAPT (a γ-secretase inhibitor). MM-SCs were analyzed in H929 cell line by flow cytometric analysis of the CD138- subpopulation. The effect of hypoxia on protein expression changes of Notch pathway members (i.e. Notch2 and Jagged1) was assessed by Western blot assay, while changes of Notch transactivation activity were assessed by dual luciferase Notch reporter assay in OPM2 cells and HEK293 cells. The high transfectability level of HEK293 cell line also enabled its transfection with multiple plasmids to assess the specific effect of CoCl2 treatment on the transcriptional activity of Notch1 and Notch2. RESULTS: The results of this work demonstrate that Jagged1 and 2 increased expression levels affect MM cell biology maintaining high levels of intrinsic drug resistance through the expression of anti-apoptotic genes, i.e. BCL2, Survivin and ABCC1, BCLXL, SDF-1α, CXCR4, with the consequent increase of MM cell sensitivity to standard-of-care drugs. Concerning the interaction of MM cells and BMSCs, MM cells stimulate the protective behavior of BMSCs, by inducing Notch activation through tumor-derived Jagged1 and 2, with a consequent increase of drug resistance due to: i) release of pro-tumor soluble factors by BMSCs, i.e. SDF-1α and VEGF; ii) the induction of an elevated anti-apoptotic background in MM cells due to an increased expression of anti-apoptotic genes such as BCL2, Survivin and ABCC1. In vitro results were confirmed by co-cultures of primary MM cells. Finally, I verified that an hypoxic stimulus, mimicked by CoCl2, may be a cause of Notch activation in MM cells by increasing the transcriptional activity of Notch1 and Notch2, supposedly through interaction with HIF-1α that prevents ICN proteosomal degradation. Notch signaling activated by CoCl2 positively regulates the MM-SC population. The resilience that characterizes MM-SCs suggests that hypoxia-mediated activation of Notch signaling may be a further mechanism by which the BM microenvironment may induce the acquisition of drug resistance in MM. CONCLUSION: The evidences that Jagged1 and 2 silencing affects the intrinsic and acquired drug resistance in MM cells support the rationale for a Notch-tailored approach to overcome MM patients relapse.