One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. More and more evidence showed that not only the MM tumor cells should be targeted but also the bone marrow (BM) micro-environment. Interactions of MM cells with the BM micro-environment have a pivotal role in MM cell proliferation, survival, migration, angiogenesis as well as drug resistance. Many pathways are involved including the conserved Notch signaling pathway. The interaction of Notch receptors and ligands between adjacent cells induces proteolytic cleavage and release of the intracellular domain of the Notch receptor, also called Notch intracellular domains (NICD). NICD will then enter the nucleus and modify the expression of downstream target genes. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. We investigated whether Notch activation in myeloma cells by the interaction with Dll1 on stromal cells contributes to bortezomib resistance. We analyzed Notch1 and Notch2 surface expression by flow cytometry on MM cells after Dll1 interaction using a stromal cell line modified to overexpress Dll1. Notch1 surface expression was not disturbed on mouse 5T33MMvt and human MMS1 and LP-1 cells while Notch2 expression on MM cells was significantly decreased after Dll1 interaction for 2 days. Next, we investigated NICD1 and NICD2 expression by western blot after Dll1/Notch interaction. NICD1 did not change in murine 5T33MMvt and human LP-1 and MMS-1 cells, while NICD2 is increased after Dll1 interaction. These results suggest that Dll1 can activate Notch signaling likely through the Notch2 receptor. We investigated whether Dll1/Notch activation could contribute to MM bortezomib resistance. MM cells were cocultured on immobilized recombinant Dll1 ligand and treated with 5 nM bortezomib for 48h. Compared to control, MM cells cocultured with Dll1 ligand were less sensitive to bortezomib. Furthermore, blocking the Notch pathway by DAPT (a gamma secretase inhibitor, GSI) could reverse this effect and increased the sensitivity to bortezomib. To delineate the molecular mechanism of Dll1-induced bortezomib resistance, we performed a drug resistance and metabolism gene array and found that CYP1A1 was significantly upregulated by Dll1/Notch interaction. CYP1A1 is a member of the cytochrome P450 family and regulates drug metabolism. We further demonstrated that inhibiting CYP1A1 by either α-Naphthoflavone (inhibitor) or CYP1A1-siRNA increases the sensitivity of MM cells to bortezomib, suggesting that CYP1A1 is involved in bortezomib resistance. As also previously demonstrated, CD138- 5T33MM cells are less sensitive to bortezomib than CD138+ 5T33MM cells. We analyzed CYP1A1 expression and activity and observed a higher CYP1A1 amount in CD138- cells compared to CD138+ MM cells. The higher CYP1A1 expression in CD138- cells might be a possible mechanism for their decreased bortezomib sensitivity compared to CD138+ cells. In addition, an in vivo experiment was performed. Combination treatment of DAPT with bortezomib was able to increase bortezomib sensitivity and prolonged overall survival in the 5T33MM mouse model.
In conclusion, our results suggest that Dll1/Notch activation contributes to bortezomib resistance by upregulating CYP1A1, a molecule involved in drug metabolism. Our data provide a potential strategy to overcome bortezomib resistance by combination with a Notch pathway inhibitor.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.