Jagged1/2 inhibition promotes multiple myeloma cell sensitivity to Bortezomib in vitro, ex-vivo and in vivo in a novel zebrafish model

Background: Multiple myeloma (MM) is the second most diffuse hematological malignancy and nowadays is still incurable, despite the development of innovative therapies. MM cells accumulate in the bone marrow (BM) and establish vicious interactions with the surrounding normal cells, inducing them to promote tumor progression and the development of drug resistance. In this process, a crucial role is played by the dyregulated Notch ligands Jagged1 and 2, whose overexpression boosts Notch both in MM cells and in the BM cells. Here, we investigated how Jagged1/2 inhibition affects MM cells resistance to the standard-of-care drug Bortezomib. Methods: Jagged1/2 expression was inhibited in MM cell lines using specific siRNAs and in primary MM cells using a lentiviral vector that encodes two shRNAs. Cells were cultured alone or with BMSCs and treated with 6nM Bortezomib. Apoptosis was evaluated by Annexin V staining in flow cytometry. For in vivo experiments cells were stained with the CM-Dil dye, resuspended in PBS+3% polyvinyl pyrrolidone and injected in the yolk of 2dpf (days post fertilization) zebrafish embryos. Injected embryos were treated with 10nM Bortezomib or DMSO for 48h. Tumor burden was evaluated by fluorescence microscopy and tumor area was calculated using ImageJ and normalized on tumor volume at the time of injection. RESULTS: Jagged1/2 silencing hamper MM cell capacity to trigger Notch activation in BMSCs, reducing in their ability to promote MM resistance to Bortezomib. Results obtained in vitro on MM cell lines were further validated on co-cultures of primary BMSCs and CD138+ cells siolated from newly diagnosed patients. Data on zebrafish xenotransplanted embryos showed that the treatment with 10nM Bortezomib decrease tumor growth of about the 57% in comparison to DMSO-treated controls, with no effect on embryos viability. Jagged1/2 knockdown alone shown a similar effect, while the combination of Jagged1/2 inhibition and Bortezomib causes a stronger decrease in MM growth of approximately 82% in comparison to the controls. CONCLUSION: Our findings provide the first proof-of-principle that Jagged1/2 withdrawal represents a suitable strategy to increase MM cells response to the commonly-used drug Bortezomib, restoring response to therapy.