Combination of Proteasome Inhibitors Bortezomib and NPI-0052 Trigger In Vivo Synergistic Cytotoxicity in Multiple Myeloma.

Bortezomib (Velcade™), the first in class proteasome inhibitor is FDA approved drug for the treatment of relapsed and relapsed/refractory multiple myeloma (MM). However, as with other agents, dose-limiting toxicities and the development of resistance limits its long-term utility. Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in MM cells; and importantly, is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 + bortezomib-induced synergistic apoptosis is associated with:

1. activation of caspase-8, caspase-9, caspase-3, and PARP;

2. induction of ER-stress response and JNK;

3. inhibition of migration of MM cells and angiogenesis;

4. suppression of chymotrypsin-like (CT-L), caspase-like (C-L) and trypsin-like (T-L) proteolytic activities; and

5. blockade of NF-kappa B signaling.

Studies in a xenograft MM model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth. Importantly, analysis of resected xenografted tumors show that 30–40% proteasome inhibition of all three (CT-L, C-L and T-L) proteasomal activities is sufficient to trigger significant MM cell apoptosis, confirming both the sensitivity of MM cells to proteasome inhibition and the importance of inhibiting all three proteolytic activities to obtain maximum response. Immunohistochemical analysis of MM tumors excised from NPI-0052 + bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. The clinical observation that bortezomib therapy can be associated with toxicity and drug-resistance, coupled with our present preclinical findings demonstrating that low doses of bortezomib together with NPI-0052 trigger a potent anti-MM effect in vitro and in vivo, suggests the promise of combination treatment strategies to enhance anti-MM activity, reduce toxicity, overcome drug resistance, and improve outcome in MM patients. In addition to the above studies, data related to combination of NPI-0052 with lenalidomide (Revlimid™) and with histone deacetylase inhibitors such as MS-275, Tubacin or LBH589, will be presented.