Abstract
BackgroundDespite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the β5 subunit. Here we investigated whether up-regulation of immunoproteasomes by exposure to interferon-γ restores sensitivity to bortezomib in myeloma and leukemia cell lines with acquired resistance to bortezomib.MethodsRPMI-8226 myeloma, THP1 monocytic/macrophage and CCRF-CEM (T) parental cells and sub lines with acquired resistance to bortezomib were exposed to Interferon-γ for 24-48 h where after the effects on proteasome subunit expression and activity were measured, next to sensitivity measurements to proteasome inhibitors bortezomib, carfilzomib, and the immunoproteasome selective inhibitor ONX 0914. At last, siRNA knockdown experiments of β5i and β1i were performed to identify the contribution of these subunits to sensitivity to proteasome inhibition. Statistical significance of the differences were determined using the Mann-Whitney U test.ResultsInterferon-γ exposure markedly increased immunoproteasome subunit mRNA to a significantly higher level in bortezomib-resistant cells (up to 30-fold, 10-fold, and 6-fold, in β1i, β5i, and β2i, respectively) than in parental cells. These increases were paralleled by elevated immunoproteasome protein levels and catalytic activity, as well as HLA class-I. Moreover, interferon-γ exposure reinforced sensitization of bortezomib-resistant tumor cells to bortezomib and carfilzomib, but most prominently to ONX 0914, as confirmed by cell growth inhibition studies, proteasome inhibitor-induced apoptosis, activation of PARP cleavage and accumulation of polyubiquitinated proteins. This sensitization was abrogated by siRNA silencing of β5i but not by β1i silencing, prior to pulse exposure to interferon-γ.ConclusionDownregulation of β5i subunit expression is a major determinant in acquisition of bortezomib-resistance and enhancement of its proteasomal assembly after induction by interferon-γ facilitates restoration of sensitivity in bortezomib-resistant leukemia cells towards bortezomib and next generation (immuno) proteasome inhibitors.
Highlights
Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, calling for novel strategies to overcome bortezomib-resistance
Characterization of bortezomib-sensitive and bortezomib-resistant hematologic tumor cell lines Cell lines were of multiple myeloma (8226), T-cell leukemia (CCRF-CEM) and myelomonocytic leukemia (THP1) origin and their bortezomib-resistant sublines displayed 40-150 fold bortezomib resistance upon cell growth inhibition [8,9]
When studying the absolute protein expression in ng subunit/μg total protein of the catalytically active subunits in wild type (WT) and bortezomibresistant sublines, all WT tumor cell lines harbored a lower amount of immunoproteasomes (β1i + β2i + β5i) than constitutive proteasomes (β1 + β2 + β5) (8226/WT: 24% vs. 76%, THP1/WT: 43% vs. 57%, and CEM/WT: 37% vs. 63%, respectively)
Summary
Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, calling for novel strategies to overcome bortezomib-resistance. To investigate possible mechanisms of bortezomib resistance, we previously developed in vitro cell line models of hematologic malignancies in which acquired resistance to bortezomib was provoked by chronic exposure to gradually increasing bortezomib concentrations [8,9]. These bortezomib-resistant cell lines were characterized by an increased expression of the constitutive proteasome subunit β5 harboring mutations in the bortezomibbinding pocket, along with a decreased expression of non-mutated immunoproteasome subunits. There is evidence for pre-clinical activity of the β5i-specific proteasome inhibitor ONX 0914 [21] in experimental autoimmune disease models, data justifying its use for the treatment of hematologic malignancies is limited [2]
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