A New Target in Multiple Myeloma: Inhibition of Notch Pathway Induces Apoptosis and Enhances Drug Sensitivity of Myeloma Cells In Vitro and In Vivo.

Abstract

Bone marrow (BM) microenvironment and particularly BM stromal cells play a critical role in de novo drug resistance of multiple myeloma (MM) cells. BM stromal cells express Notch ligands and activate Notch signaling in MM cells. We have previously demonstrated that Notch signaling is one of the major mechanisms of BM stroma mediated MM cell protection from chemotherapeutic drugs. Here we investigated whether the pharmacological inhibition of Notch signaling with γ-secretase inhibitor (GSI) could affect viability of MM cells and overcome BM stroma mediated resistance of MM cells to chemotherapy. GSI (6–8 μM) induced apoptosis of MM cells cultured in suspension or on monolayer of BM stroma via specific inhibition of Notch signaling. The effect of GSI was evaluated in four different MM cell lines and primary MM cells isolated from BM of four patients with MM. Treatment with GSI alone significantly reduced the viability of MM cells with IC50 almost 5-fold lower than that for peripheral blood mononuclear cells or BM cells from healthy donors. In addition, treatment of MM cells with GSI reversed BM stroma mediated protection of MM cells from drug-induced apoptosis. This effect of GSI was associated by dramatic up-regulation of pro-apoptotic protein NOXA and down-regulation of anti-apoptotic proteins bcl-xL, bcl-2, and Akt. Inhibition of NOXA with siRNA canceled the cytotoxic effect of GSI on MM cells indicating that NOXA could mediate the direct effect of GSI on MM cells. To test the effect of GSI on MM cells in vivo we used SCID/NOD mouse model. Mice were injected s.c. with human MM RPMI-8226 cells. These cells express both Notch ligands and Notch receptors so Notch signaling is activated by interaction between MM cells themselves. Tumor became visible in ~3 weeks after inoculation and grew as a solid tumor which allowed easily monitoring tumor size. Mice were treated with doxorubicin (1.5 mg/kg, i.p., once a 4 days, 3 times), GSI (5mg/kg/day i.p. for 14 days), combinations of doxorubicin and GSI, or vehicle control (PBS). Tumor size was constantly monitored during treatment and 3 weeks after the treatment. Treatment with doxorubicin and GSI alone resulted in moderate decreased in tumor burden as compared with control group. In contrast, combination of GSI and doxorubicin induced dramatic antitumor effect. Thus, our study, for the first time, demonstrates that inhibition of Notch signaling with GSI can be a new promising approach for therapeutic intervention in MM.