Notch signaling drives myeloma cells homing to the bone marrow by regulating the CXCR4/CXCL12 axis

Introduction The Notch pathway dysregulation has been reported in multiple myeloma (MM) and several evidences indicate it plays an oncogenic role in MM. Recently, γ-secretase inhibitors (GSIs) were studied as potential anti-cancer drugs for their ability to inhibit the Notch signaling. In this study, we analyzed the downstream effector of Notch signaling involved in MM cell growth, survival and migration, specifically focusing on the CXCR4/SDF1α chemokine axis. SDF1a is expressed at high levels in the bone marrow (BM) stroma and is widely recognized for playing a role in MM cells proliferation, survival, migration and homing to the bone marrow (BM). Materials and Methods Notch inhibition in MM cell lines was obtained by 6μM GSI-XII and 10-40μM SAHM1. Apoptosis was measured by Annexin V-FITC/PI staining and flow cytometry. CXCR4 and SDF1α expression were analyzed qRT-PCR and by flow cytometry. NOD/SCID mice were irradiated and MM cells were injected in the lateral tail vein. Ten days after, mice were divided in two groups and received 5 mg/g/day GSI-XII or an equal volume of DMSO i.p. Mice were sacrificed 30 days after MM cells injection and analyzed to detect tumor infiltration in the BM. Results Upon Notch withdrawal MM cells showed a decrease in cell proliferation and an increase in the apoptotic rate, confirming that dysregulated Notch signaling plays an oncogenic role in MM. Additionally, we observed reduced CXCR4 and SDF1α gene and protein expression levels along with a decreased MM cell ability to migrate and proliferate in response to SDF1α. These evidences and SDF1α ability to rescue GSI-XII-induced apoptosis suggest that CXCR4 signaling pathway is a downstream effector of the Notch oncogenic signaling in MM. These results were validated in vivo on NOD/SCID MM xenografts. GSI-XII treatment causes an ~80% decrease in CXCR4/active-Notch1 double positive cells in mice BM. This evidence further confirms in vitro results and indicates that Notch inhibition impairs MM cells homing to the BM reducing tumor engraftment. Conclusion We demonstrate for the first time that Notch signaling dysregulation in MM is crucial for the localization of MM cells in the BM and possibly for the recirculation of malignant plasma cells giving origin to multiple lesions. This study provides a rationale for using a Notch-tailored therapy to prevent the localization of MM cells in the BM along with the pathological interplay between MM cells and the supportive BM niche.