Targeting Notch and Hedgehog Embryonic Signaling Pathways Has Potent Anti-Tumor Activity in Myeloma and Is Effective in Myeloma Bone Disease.

Abstract 2938

Notch and Hedgehog are embryonic signaling pathways which regulate normal stem cells and cancer stem cells in a variety of solid and hematopoietic malignancies. It has been demonstrated that Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma (MM). We previously demonstrated that aberrant activity of Notch is a hallmark of MM cells and is indispensable for their survival. MM cells interact with their microenvironment and activate osteoclasts via Notch in vitro, possibly contributing to characteristic osteolytic lesions in MM. Hence, we speculated that Notch pathway inhibition has anti-tumor activity and controls bone disease in a murine MM model. Recently, we used the gamma-secretase inhibitor (GSI) XII, which blocks ligand-induced cleavage of Notch receptors and hinders release of the activated intracellular part of Notch and transcriptional Notch target gene activation. In this study, we treated the newly established BALB/c-derived plasmacytoma cell line MOPC315.BM.Luc with GSI XII in vitro and assessed its anti-tumor activity. In vivo-selected MOPC315.BM.Luc cells have a tropism for bone marrow and cause neoplastic lesions which closely resemble human disease with particular respect to (1) secretion of high monoclonal dinitrophenyl(DNP)-specific immunoglobulin (Ig)A serum levels and (2) bone lesions. DNP-IgA levels correlate with tumor burden in this MM model. MOPC315.BM.Luc cells show activated Notch signaling, as evidenced by high nuclear levels of intracellular Notch1. GSI XII treatment of MOPC315.BM.Luc cells resulted in decreased Notch activity and subsequently induced apoptosis in vitro. For in vivo experiments, MOPC315.BM.Luc cells were injected into tail veins of recipient BALB/c mice. Bone lesions were detected and quantified employing micro-CT analysis. High-resolution CT scans allowed for quantitative assessment of bone density of cortical and trabecular bone, trabecular numbers and bone mineral content. Large lesions appeared as punctured bone in CT images or even as macroscopically visible holes in bone cortex. 10 mg/kg GSI XII was administered intraperitoneally for two weeks as soon as DNP-specific IgA levels reached 20 μg/ml. As a result GSI XII-treated mice exhibited significantly slower increases of DNP-IgA levels as compared to vehicle treated controls. In addition, in a small number of mice micro-CT scans of tibiae and femora revealed higher relative bone volume and bone mineral content in GSI XII treated versus control animals. Therefore, our data indicated anti-tumor activity of GSI XII treatment in the murine MOPC315.BM.Luc MM model. We further studied simultaneous targeting of Notch and the aberrantly activated embryonic signaling pathway Hedgehog in MM. It has been shown that cross-talk interaction between Notch and Hedgehog regulates self-renewal of cancer cell lines. Hedgehog inhibition through the Smoothened antagonists cyclopamine and GDC-0449 drastically sensitized MOPC315.BM.Luc cells to GSI XII treatment in vitro. Further in vivo studies with Hedgehog inhibitors and analysis of the molecular basis for potential cross-talk of both pathways in MM are under current investigation.