Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines

Abstract

Background: Despite 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.

Methods: RPMI-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.

Results: Interferon-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.

Conclusion: Downregulation 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.

Figure 1

Proteasome subunit expression in bortezomib-resistant and bortezomib-sensitive 8226 (MM), THP1 (AML) and CEM (ALL) cells. Protein expression levels of constitutive proteasome subunits (β5, β1, and β2) and immunoproteasome subunits (β5i, β1i, and β2i) were determined by ProCISE and expressed in ng/μg total protein for 8226/WT, CEM/WT, THP1/WT, 8226/BTZ100, THP1/BTZ200 and CEM/BTZ200 cells. Percentages of individual subunits are given. Results depicted represent the mean (± SD) of 3 separate experiments.

Figure 2

Effect of IFN-γ exposure on constitutive and immunoproteasome subunits mRNA and protein levels in bortezomib-resistant and bortezomib-sensitive 8226 (MM), THP1 (AML) and CEM (ALL) cells. (A) Expression levels of β5, β1, β2, β5i, β1i and β2i mRNA were monitored after 6-72 hrs exposure of 8226/BTZ, THP1/BTZ, CEM/BTZ and (B) their parental counter parts to 100 U/ml IFN-γ. Results are presented relative to untreated controls conditions and depicted as mean (± SD) of 3 individual experiments for bortezomib-resistant cells and mean of two experiments performed in duplicate for parental cells. (C) Western blot analysis of β1i, β1, β5i and β5 protein expression after 6-72 hrs exposure of 8226/BTZ, THP1/BTZ, CEM/BTZ and (D) their parental counter parts to 100 U/ml IFN-γ. One representative example of 3 experiments is shown.

Figure 3

Impact of IFN-γ exposure on proteasome catalytic activity in intact cells and cell extracts of bortezomib-resistant and bortezomib-sensitive 8226 (MM), THP1 (AML) and CEM (ALL) cells. (A) Chymotrypsin-like, caspase-like and trypsin-like proteasome activity in intact bortezomib-resistant and (B) bortezomib-sensitive 8226, THP1 and CEM cells before and after 6-72 hrs exposure 100 U/ml IFN-γ. Results are presented relative to untreated controls and represent the mean (± SD) of 3 individual experiments. (C) β5, β5i and β1i-associated catalytic activity in cell extracts of 8226/WT, 8226/BTZ100, THP1/WT, THP1/BTZ, CEM/WT, and CEM/BTZ200 cells after 24 hr and 48 hr exposure to 100 U/ml IFN-γ. Activity assays in cell extracts employed subunit-specific substrates. Results represent the mean (± SD) of 3 experiments.

Figure 4

Sensitivity of bortezomib-resistant cell lines to proteasome inhibitors after IFN-y pre-exposure. Sensitivity of 8226/BTZ100, THP1/BTZ200 and CEM BTZ/200 cells to (A) bortezomib (BTZ) (with and without IFN-γ), (B) Carfilzomib (CFZ) (with and without IFN-γ), and (C) ONX 0914 (with and without IFN-γ), compared to parental cell sensitivity, and (D) and the sensitivity of 8226/BTZ7 cells to BTZ, CFZ, and ONX 0914, as determined by MTT cytotoxicity assays after 4 days drug exposure. Pre-exposure with 100 U/ml IFN-y was for 24h prior to 4-day BTZ, CFZ and ONX 0914 addition. Results represent the mean (± SD) of 3 individual experiments. P-values indicate differences between BTZ-resistant cells exposed or unexposed to IFN-γ.

Figure 5

Apoptosis induction and accumulation of ubiquitinated proteins in bortezomib-resistant 8226 (MM), THP1 (AML) and CEM (ALL) cells after sensitizing cells for bortezomib with IFN-γ. Western blot analysis of PARP cleavage and expression of NOXA as indicators of apoptosis and accumulation of polyubiquitinated proteins in untreated cells, after 24 h exposure to bortezomib (100 nM for 8226/BTZ100 and 200 nM for both CEM/BTZ200 and THP1/BTZ200), single IFN-γ (100 U/ml), or the combination of IFN-γ and bortezomib.

Figure 6

Effect of IFN-γ after siRNA knockdown of immunosubunits PSMB8 (β5i) and PSMB9 (β1i). (A)PSMB8 mRNA expression after PSMB8 siRNA with and without 100 U/ml IFN-γ for 24h compared to non-target siRNA with and without 100 U/ml IFN-y, (B)PSMB9 mRNA expression after PSMB9 siRNA with and without 100 U/ml IFN-γ for 24 h compared to non-target siRNA with and without 100 U/ml IFN-γ. Results are presented as percentage relative to controls (mean (± SD) of 3 individual experiments). (C) β5i protein expression after PSMB8 silencing for 48 h compared to non-target control siRNA (58% downregulation) and protein expression of the mature protein form of β1i after PSMB9 silencing for 48 h compared to non-target control siRNA (78% downregulation). (D) Chymotrypsin-like activity of non-target siRNA as compared to PSMB8 or PSMB9 siRNA with and without 100 U/ml IFN-y for 24 h. Results are the means of 4 separate experiments. *P < 0.05

Figure 7

PSMB8 (β5i) siRNA attenuates sensitizing effect of IFN-γ to bortezomib and ONX 0914. Cell growth inhibitory effects of (A) bortezomib and (B) ONX 0914 after PSMB8 or PSMB9 silencing in THP1/BTZ200 cells. Effects of PSMB8 and PSMB9 silencing on IFN-y sensitization of growth inhibition of THP1/BTZ200 cells to (C) bortezomib and (D) ONX 0914. Results depicted are the means (± SD) of 3 independent experiments.