LAUSR

Multiple myeloma (MM) is a hematologic malignancy characterized by the proliferation of malignant plasma cells in the bone marrow (BM). Despite recent advances in therapy, MM remains incurable, largely due to the emergence and maintenance of drug-resistant myeloma cells, whose interactions within the BM microenvironment, the BM myeloma niche, are critical. Because Notch signaling, a pathway activated only via cell-cell contacts, has been implicated as an integral part of the onset and progression of MM, we investigated how Notch activation in myeloma cells, specifically through interactions with ligand-expressing niche cells, affects the pathophysiology of MM. We first demonstrated the presence of at least one Notch receptor in 10 arbitrarily selected MM cell lines (Supplementary Figure 1a and b). The presence of these receptors indicates that Notch signaling can be activated and is functional in human myeloma cells. Of the two Notch ligands examined, Jagged1, but not Jagged2, is known to be abundantly expressed in many types of cells in the BM myeloma niche . Therefore, we investigated the effects of niche-induced Jagged1-Notch activation on myeloma cells. To distinguish niche-induced Notch activation from the homotypic activation of Notch in myeloma cells, in which Notch and its ligands can be simultaneously expressed, we selected five MM cell lines that expressed Jagged1 weakly or not at all for analyses. Contrary to previous reports, the presence of immobilized human Jagged1 did not significantly alter the proliferation of any of the five MM cell lines in vitro (Supplementary Figure 1c). To further investigate the roles of niche-induced Notch signaling, we established a clinically relevant animal model of human MM that would allow us to examine myeloma cell interactions within the BM niche (Supplementary Figure 1d), where malignant plasma cells primarily proliferate in patients with MM. The engraftment of human MM cells in the BM did not increase even when U266 cells were transplanted into non-obese diabetic/severe combined immunodeficient/IL2Rγnull (NOG) mice expressing human Jagged1 in osteoblasts (NOGJ), a cell type that constitutes the BM myeloma niche (Supplementary Figure 1e). The results indicate that niche-induced Jagged1-Notch signaling is not specifically associated with the proliferation of myeloma cells. We then assessed whether niche-induced Jagged1Notch activation affects the sensitivity of myeloma cells to drugs used clinically to treat MM patients. In co-culture experiments, transgenic expression of human Jagged1 in stromal cells (ST2J) increased the survival of U266 cells only when the cells were exposed to bortezomib (BTZ), which coincided with the upregulation of human Hey1 and Hes1 expression in culture (Supplementary Figure 2a-c), indicating a role of Notch signaling in BTZ resistance. Five other MM cell lines similarly demonstrated resistance to BTZ in the presence of ST2J cells (Supplementary Figure 2d). As expected, cells cultured on immobilized human Jagged1 demonstrated significant resistance to BTZ treatment (Fig. 1a) but not to melphalan treatment (Supplementary Figure 2e). In addition, a marked upregulation in the expression of Hey1 and Hes1 was detected in cells that survived BTZ treatment (Fig. 1b), confirming that the observed resistance to BTZ