Jagged blockade promotes tumor cells response to Bortezomib in a zebrafish model of multiple myeloma

Multiple myeloma (MM) is the second most frequently diagnosed hematologic tumor and remains still incurable, mainly due to the development of drug resistance that leads to relapse and contributes to the fatal outcome of this disease. MM cells accumulate in the bone marrow (BM) and establish a complex interplay with the neighboring normal cells, inducing 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 promotes an aberrant activation of the Notch pathway both in MM cells and the normal cells of the BM milieu. Our preliminary results indicate that Jagged1 and 2 blockade reduces MM cell ability to induce Notch activation in BM stromal cells, causing a decrease in their capability to sustain MM resistance to Bortezomib. Results obtained in vitro on MM cell lines were further validated on co-cultures of primary CD138+ cells and BMSCs from newly diagnosed MM patients. The zebrafish embryo was recently validated as a complementary in vivo model for MM and allows the rapid screening of the outcome of different pharmacologic treatments. Moreover, key growth factor such as IL6 and SDF1 produced by zebrafish are biologically active also for human cells and support MM cells growth and survival. Thus, we aimed to validate our in vitro findings using a zebrafish xenograft model of MM that allows a rapid and reliable screening of the outcome of Jagged1 and 2 knockdown on MM cells response to Bortezomib. Jagged1 and 2 expression was inhibited transiently in MM cell lines using specific siRNAs. MM cells were stained with the CM-Dil vital dye, resuspended in PBS + 3% polyvinyl pyrrolidone and injected in the yolk area of 2dpf zebrafish embryos. Injected embryos were treated with 10µM Bortezomib or DMSO for 48h and MM cells growth in zebrafish was evaluated by fluorescence microscopy. The analysis performed on xenotransplanted embryos showed that the treatment with 10nM Bortezomib caused a decrease of about the 50% in tumor growth in comparison to DMSO-treated controls, with no effect on embryos viability. Jagged inhibition alone has a comparable effect to Bortezomib, while the combination of Bortezomib and Jagged knockdown results in a stronger decrease in tumor growth of approximately 80% in comparison to the vehicle-controls. In conclusion, our findings demonstrate that Jagged inhibition represents a suitable strategy to increase MM cell sensitivity to Bortezomib and support the use of zebrafish embryos as an innovative and feasible tool to assess tumor cell sensitivity to standard of care drugs.