A Novel Notch Pathway Inhibitor Blocks Osteoclast Activity and Synergistically Induces Apoptosis with the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cells.

Notch pathway inhibition in multiple myeloma (MM) cells is a promising new therapeutic approach since it controls myeloma cell growth as we previously demonstrated (Blood. 2004; 103:3511–3515). Notch signaling is involved in the tight interactions between myeloma cells and the bone marrow microenvironment and induces tumor cell growth in MM. We provided evidence that Notch receptors are expressed on MM cells and that Notch ligands on MM and bone marrow stromal cells activate Notch signaling through homotypic as well as heterotypic interactions in MM cells. In this study, we analyzed whether Notch signaling might be activated in osteoclasts, which express Notch receptors but not the ligands. To that end, we co-cultured MM cells and human osteoclasts, which were generated from mononuclear hematopoietic precursors of healthy donors using in vitro RANKL/M-CSF stimulation. Co-cultivation specifically activated Notch in osteoclasts and induced osteoclast activity as measured by mRNA expression of the tartrate-resistant acid phosphatase. The novel Notch pathway inhibitor, so called γ-secretase inhibitor 1 (GSI1), that we recently identified by structural comparison of known inhibitors with unknown compounds by data bank screening, specifically inhibited Notch signaling in osteoclasts and blocked their activity in this co-cultivation assay. In addition, GSI1 induced apoptosis in osteoclasts in higher concentrations. We suggest from our data that GSI treatment controls MM cell growth and concomitantly aberrant osteoclast activity in vitro and possibly in vivo, that is under current investigation. We further hypothesized that GSI1 can be combined with the proteasome inhibitor bortezomib, which has been known to have in vitro and in vivo activity against MM. We evaluated the activity of the combination of GSI1 and bortezomib against MM cell growth and survival. Proliferation of MM cell lines treated with GSI, bortezomib and their combination was determined by CellTiter-Glo® luminescent cell viability assay. AnnexinV-FITC/PI staining and cleaved poly (ADP-ribose) polymerase (PARP) staining were used to determine the degree of apoptosis. Although treatment of MM cell lines (OPM2, LP1) with either drug alone significantly inhibited proliferation and induced apoptosis with concentrations of GSI1 (30–60 μM) and bortezomib (1–4 nM), the combination resulted in synergistic inhibition of cell growth and survival. Our data suggest that combination of GSI1 and bortezomib is a rational novel treatment option in MM that simultaneously targets different proliferative and anti-apoptotic pathways. Whether this combination might also have synergistic activity against aberrant osteoclast activity in MM will be further investigated.