Introduction: Mantle cell lymphoma (MCL) is not curable and novel therapeutic agents and their rational combinations are needed. The ubiquitin-proteasome pathway is an attractive target for antitumor therapy, as its persistent activity is associated with tumor formation, growth, metastasis, and drug resistance in many cancer types, including B-cell non-Hodgkin lymphoma. 20S proteasome activity is regulated by the catalytic subunits β1, β2, and β5 or in the case of the immunoproteasome (i20S) by the inducible catalytic subunits LMP2 (β1i), MECL-1(β2i), and LMP7 (β5i). Carfilzomib (CFZ), a proteasome inhibitor, inhibits the proteasome by binding specifically and irreversibly to the catalytic subunit β5/β5i.

Materials and Methods: CFZ was supplied by Onyx Pharmaceuticals (South San Francisco, CA). The Btk inhibitor ibrutinib (PCI-32765) was provided by Pharmacyclics (Sunnyvale, CA). Human MCL cell lines (Mino, Jeko-1, Z138, and Rec-1) were either created by our laboratory or obtained from American Type Culture Collection. Primary cells were obtained from newly diagnosed MCL patients with informed consent. Cell viability was assessed by an MTT assay, apoptosis by Annexin V binding assays, and proteasome expression by Western blotting.

Results: We found that MCL cells had different responses to CFZ. For the CFZ-sensitive MCL cell lines Mino and Z138 and freshly isolated MCL cells from 6 patients, CFZ significantly induced the growth inhibition and apoptosis at a low dose (IC50 2-6 nM). In contrast, for the CFZ-resistant MCL cell line (Rec-1) and fresh primary MCL cells from patients with clinical resistance, CFZ did not induce cell death (IC50 not reached at 100 nM). Next, Western blot analysis showed that the CFZ-resistant cells lacked expression of the i20S subunits LMP2, LMP7, and MECL-1. By contrast, the CFZ-sensitive cells showed high levels of LMP2, LMP7, and MECL-1. These results suggest that an intact immunoproteasome is necessary for CFZ-induced anti-MCL activity. In an attempt to overcome the CFZ resistance, we studied the effect of ibrutinib in CFZ-resistant MCL cells. Ibrutinib alone was toxic to both CFZ-sensitive and CFZ-resistant MCL cells with IC50 values ranging from 3 to 12.5 μM. Furthermore, we found that ibrutinib synergized with CFZ in CFZ-sensitive cell lines, and primary MCL cells. The Chou-Talay combination index was < 1, indicating synergism between the two drugs. Most importantly, the CFZ-resistant MCL cells Rec-1 were sensitive to ibrutinib, with an IC50 of 6 uM.

Conclusion: Our data suggest that LMP2, LMP7, and MECL-1 may serve as potential biomarkers for patients who are likely to be sensitive to CFZ. Our data also suggest that ibrutinib may be useful in patients with CFZ-resistant MCL. Our preclinical data provide a strong rationale for testing the ibrutinib-CFZ combination clinically.

Citation Format: Zhishuo Ou, Liang Zhang, Kate Newberry, Luhong Sun, Kirk J. Christopher, Alex Rollo, Archito Tamayo, John Lee, Richard J. Ford, Michael Wang, Lan V. Pham. The bruton's tyrosine kinase inhibitor ibrutinib synergized with the proteasome inhibitor carfilzomib and overcame immunoproteasome-mediated carfilzomib resistance in mantle cell lymphoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2432. doi:10.1158/1538-7445.AM2013-2432