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Publication

NLRP3-independent inflammasome activation by proteasome inhibitors

Journal Contribution - Journal Abstract Conference Contribution

Introduction

Proteasome inhibitors (PIs) such as Bortezomib (BTZ) have revolutionized multiple myeloma (MM) treatment. However, their efficacy is limited by the emergence of drug resistance and treatment-limiting side effects, including peripheral neuropathy and lymphopenia. Treatment resistance in MM has been demonstrated to be promoted by an NLRP3 inflammasome-mediated innate immune response to MM-associated proteins. Thus, we set out to investigate the impact of PI treatment on innate immune cytokine production.

Materials and Methods

The PIs BTZ, marizomib, epoxomicin and onx-0914 were applied to a variety of inflammasome-competent human immortalized and primary cells, including the cell line THP-1 (monocytic), primary human peripheral blood mononuclear cells (PBMC), M-CSF or ascites-derived macrophages, keratinocytes, and epithelial cells. Inflammasome activation was characterized via Interleukin-1b (IL-1β) release in cellular supernatants (ELISA, all cell types) and ASC-speck formation using an ASC-GFP reporter cell line (THP-1). To characterize the contribution of Nod-like receptor pyrin domain-containing protein 3 (NLRP3) and caspase-1 (CASP-1) to inflammasome formation, NLRP3 and CASP-1-deficient THP1 cells were generated using CRISPR/Cas9-mediated genome engineering, and NLRP3 and caspase-1 were pharmacologically inhibited using MCC-950 (Invitrogen) and VX-765 (Invivogen).

Results

We report PI-mediated activation of the inflammasome in all cells tested. Treatment with PI induced secretion of IL-1b by THP1 cells and PBMC with an EC50 that was similar to the IC50 in BTZ-sensitive, but not BTZ-resistant, KMS-11 MM cells. We observed hallmarks of canonical inflammasome activation such as the formation of ASC specks in PI-exposed cells. Moreover, pharmacological inhibition of caspase-1 with VX-765 or genetic caspase-1 deficiency abrogated IL-1b. A variety of inflammatory stimuli, including soluble proteins secreted by MM cells, induce inflammasome formation via activation of NLRP3, the only inflammasome receptor currently targetable by a small molecule and investigated in clinical trials. Surprisingly, neither genetic ablation of NLRP3 in THP-1 cells nor MCC-950 treatment of PBMC inhibited IL-1b signaling following BTZ exposure.

Conclusion

Here, we identify PIs as activators of the inflammasome. In contrast to previously identified myeloma-intrinsic mechanisms leading to inflammasome assembly, PI-mediated IL-1b secretion was independent of NLRP3. Further investigation will include the identification of the receptor mediating BTZ-driven inflammasome assembly in order to identify mechanisms of action that may be amenable to therapeutic intervention.
Journal: Blood
ISSN: 0006-4971
Issue: Supplement 1
Volume: 140
Pages: 3125
Publication year:2022
Accessibility:Closed