Bortezomib Resistant Constitutive NF-κB Activation in Mantle Cell Lymphoma.

Bortezomib is a proteasome inhibitor whose antineoplastic effects include inhibition of NF-κB, a transcription factor whose deregulation may play a central role in mantle cell lymphoma (MCL) pathogenesis. Bortezomib has shown clinical efficacy in relapsed and refractory cases of MCL with response rates of 40%. NF-κB can be activated through several pathways, including a proteasome inhibitor resistant pathway. It remains unknown whether MCL harbors bortezomib resistant constitutive NF-κB activity, but characterization of this may have important implications in elucidating bortezomib resistance and also in establishing rational therapeutic combinations. We investigated the effect of bortezomib on constitutive NF-κB activity in 3 EBV-negative MCL cell lines (Jeko, Rec-1 and Z138) and 20 MCL patient samples. Electrophoretic mobility supershift assay demonstrated that each of the cell lines had distinct NF-κB complexes with the Jeko and Rec-1 containing mainly p50/p65 and p50/cRel heterodimers, and Z138 comprised almost entirely of p52/RelB heterodimers. At a physiologically achievable dose of bortezomib (20nM), a live cell-based proteasome inhibition assay demonstrated greater than 80% proteasome inhibition in all three cell lines. Treatment of Jeko cells with such a dose resulted in a 50% decrease of NF-κB DNA binding, in contrast to a 10 to 30% increase of DNA binding in Rec-1 and Z138 cells by electrophoretic mobililty shift assay. Of 10 MCL patient samples from which results could be obtained, only 2 demonstrated a greater than 50% decrease in NF-κB DNA binding after treatment with 20nM and 100nM of bortezomib, whereas the remainder showed either no inhibition or even increased binding. Thus, bortezomib resistant constitutive NF-κB activity appears to be present in Rec-1, Z138, and a majority of MCL cases. Cytotoxicity assessed by flow cytometry following staining with propidium iodide showed Rec-1 and Z138 cells had greater resistance to bortezomib induced apoptosis (82 ± 5% and 69 ± 5% viability) than Jeko cells (47 ± 6% viability) after 20nM bortezomib treatment for 24 hours. Combining bortezomib with perillyl alcohol, a known suppressor of proteasome inhibitor resistant NF-κB activation, resulted in a synergistic cytotoxic effect in all 3 cell lines as assessed by the combination index (CI) method with CIs of 0.31, 0.32, and 0.60 for Jeko, Rec-1, and Z138 cells respectively, where CI of 0.1–0.3 is strong synergism, 0.3–0.7 is synergism, 0.7–0.85 is moderate synergism and 0.85–0.9 is slight synergism. In conclusion, our findings suggest that bortezomib resistant NF-κB activity is present in a significant subset of MCL cases, and the combination of bortezomib with a suppressor of proteasome inhibitor resistant NF-κB activity may elicit a synergistic cytotoxic effect in MCL.