Ritonavir Sensitizes Multiple Myeloma Cells to Bortezomib-Induced Apoptosis

The ubiquitin-proteasome system plays a central role in the regulation of cell growth and cell proliferation by controlling the abundance of key cell cycle proteins. Increasing evidence indicates that unscheduled proteolysis of many cell cycle regulators contributes significantly to tumourigenesis and is indeed found in many types of human cancers, including colon and renal cell cancer, non hodgkin lymphomas, and multiple myelomas. Protein degradation pathways are also targets for cancer therapy, as shown by the successful introduction of bortezomib (B), an inhibitor of the 26S proteasome, for the therapy of multiple myeloma. Recently, protease inhibitors conventional in HIV therapy (e.g., ritonavir (R)) have been found to exert an antiproliferative effect on different tumor types, including multiple myeloma cells. More recently, ritonavir has been shown to induce endoplasmic reticulum stress and to sensitize sarcoma cells towards bortezomib induced apoptosis.

In this study, we show that the combination of therapeutic concentrations of bortezomib and ritonavir acts synergistically on cultured multiple myeloma cell lines (U266, RPMI 8226, ARH-77 (2 variants-One is more resistant)). Cell proliferation was significantly reduced in an overadditive fashion as compared to individual treatments (proliferation as % of DMSO control after 48hrs: CRL-1621 (ARH-77): B: 40%, R: 50%, R&B: 7%, CCL-155 (RPMI 8226): B: 37%, R: 50%, B+R: 6%; n=3). Moreover, we found proteasome inhibition by bortezomib to be associated with low levels of expression of Skp2 and consequent stabilization of its target p27^Kip1, a negative cell cycle regulator at the G1/S cell cycle transition. Accordingly, bortezomib induced cell cycle arrest at the G1/S transition followed by caspase dependent apoptosis. In contrast, only mild induction of apoptosis and no activation of Caspase 3 were observed in myeloma cells treated with ritonavir, although growth arrest was present. Interestingly, growth arrest in ritonavir-treated cells was associated with an accumulation in G2 phase.

In a limited clinical study, we treated two patients with stage III relapsed and refractory myeloma with a combination of bortezomib at standard doses and ritonavir at 600 mg twice a day. Both patients had MR or greater lasting for more than 2 months. Only grade 2 GI toxicity was seen.

Taken together, our findings suggest different mechanisms of action for bortezomib and ritonavir on myeloma cells and suggest that the combination of the two drugs may be a valid therapeutic strategy.