Targeting Therapy for Resistant Multiple Myeloma with a Novel Inhibitor of NF-KB, NPI-1387.

Transcription factor NF-KB is linked to growth and survival of multiple myeloma (MM)cells; blockade of NF-KB activity is therefore an attractive therapeutic strategy. Here we describe NPI-1387, a potent inhibitor of NF-KB activation and its effects on MM cells, including those resistant to conventional agents dexamethasone or doxorubicin. Cell-based assays were used to screen a library of 200 semi-synthetic analogs derived from the pimarane diterpene, Acanthoic acid. Among these analogs, NPI-1387 inhibited LPS-induced TNF-A synthesis in the murine macrophage-like RAW 264.7 cells most potently. Importantly, NPI-1387 reduced TNF-A-induced NF-KB activation in a HEK293 NF-KB/luciferase reporter cell line. Therefore additional studies were initiated to define the biological activities in MM. Treatment of MM cells lines (MM.1S, MM.1R, OCI-My5, OPM1, Dox-40) with NPI-1347 for 48h induces a dose-dependent significant (P < 0.004) decrease in cell viability in all cell lines at pharmacologically achievable concentrations (IC₅₀ range 25–40 micromolar). To determine whether NPI-1387-decreased cell viability is due to apoptosis, various MM cell lines were treated at their respective IC₅₀ for 48h; harvested; and analyzed for apoptosis. NPI-1387 triggered significant apoptosis in these cells, as measured by a marked increase in nuclear condensation reflected by dense pattern of DAPI stain under phase contrast microscopy. In contrast, untreated control cells exhibited homogeneous and intact nuclei. Besides nuclear condensation, NPI-1387 triggered proteolytic cleavage of poly (ADP ribose) polymerase (PARP), a hallmark of apoptosis. Examination of purified patient MM cells demonstrated similar results. Notably, NPI-1387 decreases the viability of cells obtained from Bortezomib-refractory MM patient. In contrast, no significant toxicity of NPI-1387 was observed against peripheral blood mononuclear cells from normal healthy donors or CD138⁻ MM patient cells. Moreover, NPI-1387 does not affect the viability of MM patient-derived bone marrow stromal cells (BMSCs). Genetic and biochemical evidence indicates that apoptosis proceeds by two major cell death pathways: an intrinsic pathway that involves mitochondrial membrane permeabilization and release of several apoptogenic factors, followed by caspase-9 activation; and an extrinsic apoptotic signaling pathway that occurs via caspase-8 activation. Both caspase-8 and caspase-9 activate downstream caspase-3. We therefore next examined whether NPI-1387 triggers extrinsic or intrinsic apoptotic signaling pathways. Our results show that NPI-1387 (25 micromolar) induces activation of caspase-8, and caspase-9, followed by caspase-3 cleavage. These data suggest that NPI-1387-triggered MM cell apoptosis predominantly proceeds via caspase-8/caspase-9>>>>caspase-3 signaling pathway. Together, these findings provide the rationale for clinical evaluation of NPI-1387 to induce MM cell killing, overcome drug-resistance, and improve patient outcome in MM.