Jagged1/2 inhibition promotes tumor cells response to Bortezomib in a zebrafish model of multiple myeloma

Background: Multiple myeloma (MM) is the second most frequently diagnosed hematological malignancy and today is still incurable, mainly due to the development of drug resistance that causes relapse and contributes to the fatal outcome of this disease. MM cells accumulate in the bone marrow (BM) and establish complex interactions with the surrounding normal cells, forcing them to assume a pro-tumor behavior. In this process, a key role is played by the two Notch ligands Jagged1 and 2, whose dysregulated expression causes an aberrant activation of the Notch pathway both in MM cells and in the BM niche cells. Aims: We aimed to validate the effect of Jagged1/2 silencing on MM cells resistance to the standard-of-care drug Bortezomib by: i) in vitro, on co-culture of MM cell lines and BM stromal cells (BNSC); ii) ex vivo, on co-culture of primary cells from MM patients and BMSC; iii) in vivo, using a zebrafish xenograft model of MM that allows a rapid and reliable screening of MM cells response to chemotherapics. Methods: Jagged1/2 expression was inhibited transiently in MM cell lines using specific siRNAs and constitutively in primary MM cells using a lentiviral vector that encodes specific shRNAs. Cells were cultured alone or co-cultured with BMSC and treated with 8nM Bortezomib. Apoptosis was evaluated by Annexin V staining and flow cytometry. For in vivo experiments cells were stained with the CM-Dil vital dye, resuspended in PBS + 3% polyvinyl pyrrolidone and injected in the yolk area of 2dpf (days post fertilization) zebrafish embryos. Injected embryos were treated with 10μM Bortezomib or DMSO for 48h. MM cells growth in zebrafish was evaluated by fluorescence microscopy and tumor area were calculated using ImageJ and normalized on tumor volume at the time of injection. Results: Jagged1/2 blockade reduces MM cells ability to induce Notch activation in BMSC, causing a decrease in their capacity to sustain MM resistance to Bortezomib. Results obtained in vitro on MM cells lines were further validated on co-culture of primary CD138+ cells and BMSC from newly diagnosed MM patients. The analysis performed on xenotransplanted embryos showed that the treatment with 10μM Bortezomib caused a decrease of about the 50% in tumor growth in comparison to DMSO-treated controls, with no effect on embryos viability. Jagged1/2 knockdown alone has a comparable effect to Bortezomib, while the combination of Bortezomib and Jagged1/2 inhibition results in a stronger decrease in tumor growth of about the 75% in comparison to the vehicle-controls. Summary/Conclusion: Our findings demonstrate that Jagged1/2 inhibition represents a suitable strategy to promote MM response to the standard of care drug Bortezomib, contrasting BM-induced drug resistance.