Ibrutinib Overcomes Carfilzomib Resistance In Immunoproteasome-Deficient Mantle Cell Lymphoma

The biological significance of the ubiquitin-proteasome system in the control of cellular processes has been well-recognized; however, the pathophysiological importance of the immunoproteasome (i-proteasome), the inducible form of the proteasome, has not been well-appreciated in cancer cells, particularly in mantle cell lymphoma (MCL), a clinically challenging, aggressive B-cell non-Hodgkin’s lymphoma (NHL-B). The proteasome activity is regulated by standard catalytic subunits β1, β2 and β5, or inducible catalytic subunits LMP2 (β1i), MECL-1 (β2i), and LPM7 (β5i). The inducible catalytic subunits have been reported to be highly expressed in hematological malignancies. Our results indicated that inducible catalytic subunits of i-proteasome are highly expressed in both established and primary MCL cells. Interestingly, we found that the expression of i-proteasome subunits were required for the anti-MCL activity of carfilzomib, a novel second-generation proteasome inhibitor, in MCL cells. Both primary and established MCL cells with high expression of i-proteasome subunits were more responsive to carfilzomib, even at low dose treatment (IC50, between 1.2nM to 8.6nM). In contrast, MCL cells with low/no expression of i-proteasome subunits are highly resistant to carfilzomib (IC50 >200 nM). These results suggested that the i-proteasome could play an important role in determining the sensitivity of carfilzomib.

To elucidate the mechanism responsible for carfilzomib sensitivity in MCL cells, we used IFN-γ to stimulate carfilzomib-resistant MCL cell line Rec-1, which lacked the expression of i-proteasome subunits especially LMP2. We found that pretreatment of Rec-1 cells with IFN-γ for 48 hours increased i-proteasome subunit LMP2 but not c-proteasome subunit b1, and led to sensitization of Rec-1 cells to carfilzomib. To provide direct evidence that upregulation of i-proteasome subunit LMP2 by IFN-γ was responsible for the observed carfilzomib sensitization, siRNA downregulation of LMP2 was applied prior to exposure to IFN-γ. After LMP2 knockdown, carfilzomib sensitization was attenuated. The data show that an intact i-proteasome, especially LMP2, appears to be necessary for its anti-MCL activity, suggesting that i-proteasome could serve as a biomarker for identifying patients who will benefit from carfilzomib.

Since bruton’s tyrosine kinase (Btk) is pivotal for B-cell lymphoma growth and its downstream signals are associated with NF-κB and other important survival pathways, we combined ibrutinib, a novel Btk inhibitor, with carfilzomib for treatment of MCL cells. We found that ibrutinib sensitized carfilzomib-resistant MCL cells, and overcame carfilzomib resistance in these cells. Taken together, i-proteasome could be indispensable for the action of carfilzomib in MCL cells. However, ibrutinib overcame carfilzomib resistance in MCL cells indicating ibrutinib has different therapeutic targets that are not associated with i-proteasome. Our data could demonstrate the profound potential of using an ibrutinib and carfilzomib combination in clinical trials for patients with relapsed or refractory MCL.