Interferon-γ-Induced Upregulation of Immunoproteasome Subunit Assembly Overcomes Bortezomib Resistance of Leukemia Cell Lines Harbouring Bortezomib-Induced Mutations in Constitutive PSMB5

Abstract 1346

Acquired resistance to the proteasome inhibitor (PI) bortezomib (BTZ) is an emerging factor limiting its efficacy in the treatment of hematologic malignancies. The clinical impact of acquired resistance has been shown in Multiple Myeloma (MM) patients who were re-treated with BTZ. Although BTZ-retreatment was found to be effective, the response rate as well as the duration of response were less as compared to initial treatment, indicating the development of BTZ-resistance in a subgroup of patients. In line with that, we previously found increased expression of constitutive proteasome (cP) subunit ß5 harbouring a mutation in the BTZ-binding pocket and a decreased expression of non-mutated immunoproteasome subunits in BTZ-resistant cell lines of hematologic malignancies (Franke and Niewerth et al, Leukemia 2012).

We here explore whether upregulation of immunoproteasome (iP) expression could restore sensitivity in BTZ-resistant leukemia cells towards BTZ and two epxoyketone-based irreversible PIs; carfilzomib (CFZ) and the ß5i-targeted ONX 0914. BTZ-resistant cell lines were of multiple myeloma (8226), T-cell (CEM) and myelomonocytic (THP1) origin and displayed resistance towards cell growth inhibition in the presence of 7–200 nM BTZ. Induction of iP in wild type (WT) and BTZ-resistant 8226, CCRF-CEM and THP1 cells was achieved by exposure to 100U/ml Interferon- γ (IFN-γ) for 6–72 h. IFN-γ transiently increased (maximum between 24–48 hours) mRNA levels of β5i, β1i, and β2i up to 8-fold, 30-fold and 4-fold, respectively. These findings were corroborated at the β5i, β1i and β2i protein expression level using Western blot analysis. Following IFN-γ exposure, chymotrypsin-like proteasome activity increased up to 2.5-fold compared to unstimulated controls, trypsin-like activity increased up to 1.5-fold, whereas caspase-like activity was slightly decreased. Consistent with increased proteasome activity, there was also an increased expression of cell surface HLA Class I molecules.

The impact of IFN-γ induced upregulation of iPs on the sensitivity to the PI BTZ, CFZ, and ONX 0914, defined by the decrease in IC₅₀, is summarized in Table 1. 8226/BTZ100 cells became 4-fold more sensitive towards BTZ after IFN-γ exposure, whereas THP1/BTZ200 and CEM/BTZ200 cells displayed nearly 2-fold increased sensitivity. For CFZ, a modest level of sensitization was observed in all cell lines with high level BTZ resistance. Interestingly, for the immunoproteasome inhibitor ONX 0914, IC₅₀ values were markedly decreased (7-fold for 8226/BTZ100 and 3-fold for THP1/BTZ200 and CEM/BTZ200 cells). Additionally, in 8226 cells with low levels of BTZ resistance (8226/BTZ7), IFN-γ restored parental cell sensitivity to ONX 0914. Restoration of PI sensitivity after IFN-γ exposure was further confirmed by activation of PARP cleavage and accumulation of ubiquitinated proteins, pointing to restoration of BTZ activity under proteasome inhibition and consequent induction of apoptosis. Finally, to provide evidence that upregulation of β5i and or β1i by IFN-γ was responsible for the observed sensitization, siRNA downregulation of β5i and β1i was applied prior to exposure to IFN-γ. Under these conditions, mRNA levels and proteasome activity of β5i remained suppressed, even after exposure to IFN-γ. Moreover, after β5i silencing, PI sensitization and apoptosis were attenuated. Silencing of β1i expression had no effect on PI-sensitization.

In conclusion, down-regulation of β5i subunit expression is a major determinant of BTZ-resistance and increasing its proteasomal assembly after IFN-γ exposure facilitates restoration of sensitivity in BTZ-resistant leukemia cells towards cP inhibitors and in particular iP inhibitors.

50% inhibitory concentration compared to untreated controls (IC₅₀ nM) as determined in a 4 days growth inhibition assay (MTT). Results depicted are means of at least 3 separate experiments. SF: sensitization factor: IC₅₀ control/IC₅₀ with IFN-g.