Background/Purpose:

The proteasome inhibitor (PI) PR-957/ONX 0914 blocks cytokine production in vitro and attenuates disease progression in experimental models of rheumatoid arthritis (RA) (Nature Medicine 2009 15;781-788). While these anti-inflammatory effects were demonstrated to be due to inhibition of the immunoproteasome versus the constitutive proteasome, there remained a question as to which immunoproteasome subunits, LMP7, LMP2, or MECL-1, were necessary and sufficient for these effects. Here we utilize subunit-selective PIs to address this question.

Methods:

Proteasome subunit inhibition was measured in mice following administration and/or in MOLT-4 cells (human leukemia) and human PBMCs following exposure to the immunoproteasome subunit-selective inhibitors, ONX 0914 (LMP7, LMP2, MECL-1), KZR-329 (LMP7), KZR-504 (LMP2), and KZR-082 (MECL-1), via a subunit active site occupancy assay (ProCISE). Cytokine release (TNF-α, IL-6, IL-12/23p40 [p40], and IFN-γ) was measured in human PBMCs stimulated with either endotoxin (LPS) or antibodies to CD3 and CD28 (anti-CD3/CD28) via Meso Scale Discovery electrochemiluminescent detection (MSD). Cell viability was measured by CellTiter-Glo. The therapeutic effect of selective inhibitors alone or in combination was evaluated in the Collagen Antibody-Induced Arthritis (CAIA) mouse model of RA.

Results:

Consistent with previous studies, at a concentration that inhibited multiple immunoproteasome subunits, ONX 0914 blocked secretion of all cytokines tested in stimulated PBMCs. KZR-329 partially blocked secretion of p40 following LPS stimulation and IFN-γ following anti-CD3/CD28 stimulation, but had minimal effect on TNF-α and IL-6. KZR-504 and KZR-082 had no effect on cytokine production. A combination of KZR-329 and either KZR-504 or KZR-082 resulted in a blockade of cytokine release similar to that of ONX 0914. Blockade of all 3 immunoproteasome subunits resulted in enhanced cytokine inhibition compared to the combination of any 2 selective inhibitors, although the triple combination also resulted in a slight decrease in cell viability. In the CAIA model, KZR-329 resulted in a slight attenuation of disease progression, while KZR-504 treatment had no effect. However, in combination, these two inhibitors prevented disease progression at a level similar to ONX 0914.

Conclusion:

Selective inhibition of individual immunoproteasome subunits resulted in minimal decreases in cytokine production in vitro, and was not sufficient to prevent disease progression in a mouse model of RA. Combined inhibition of LMP7 and LMP2, comparable to that achieved with ONX 0914, blocked multiple inflammatory cytokines in vitro and attenuated disease progression in mice. These data suggest that development of selective inhibitors of the immunoproteasome that target multiple subunits will be necessary in order to achieve a therapeutic benefit in rheumatic diseases such as RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/selective-inhibition-of-the-immunoproteasome-subunit-lmp7-is-not-sufficient-for-blocking-cytokine-production-or-attenuating-progression-of-experimental-arthritis/