Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells

Abstract

Donor-specific antibodies (DSAs) have a deleterious effect on allografts and remain a major immunologic barrier in transplantation. Current therapies to eliminate DSAs are ineffective in highly HLA-sensitized patients. Proteasome inhibitors have been employed as a strategy to target bone marrow plasma cells (BMPCs), the source of long-term antibody production; however, their efficacy has been limited by poorly defined drug-resistance mechanisms. Here, we performed transcriptomic profiling of CD138+ BMPCs that survived in vivo desensitization therapy with the proteasome inhibitor carfilzomib to identify mechanisms of drug resistance. The results revealed a genomic signature that included increased expression of the immunoproteasome, a highly specialized proteasomal variant. Western blotting and functional studies demonstrated that catalytically active immunoproteasomes and the immunoproteasome activator PA28 were upregulated in carfilzomib-resistant BMPCs. Carfilzomib-resistant BMPCs displayed reduced sensitivity to the proteasome inhibitors carfilzomib, bortezomib, and ixazomib, but enhanced sensitivity to an immunoproteasome-specific inhibitor ONX-0914. Finally, in vitro carfilzomib treatment of BMPCs from HLA-sensitized patients increased levels of the immunoproteasome β5i (PSMB8) catalytic subunit suggesting that carfilzomib therapy directly induces an adaptive immunoproteasome response. Taken together, our results indicate that carfilzomib induces structural changes in proteasomes and immunoproteasome formation. Donor-specific antibodies (DSAs) have a deleterious effect on allografts and remain a major immunologic barrier in transplantation. Current therapies to eliminate DSAs are ineffective in highly HLA-sensitized patients. Proteasome inhibitors have been employed as a strategy to target bone marrow plasma cells (BMPCs), the source of long-term antibody production; however, their efficacy has been limited by poorly defined drug-resistance mechanisms. Here, we performed transcriptomic profiling of CD138+ BMPCs that survived in vivo desensitization therapy with the proteasome inhibitor carfilzomib to identify mechanisms of drug resistance. The results revealed a genomic signature that included increased expression of the immunoproteasome, a highly specialized proteasomal variant. Western blotting and functional studies demonstrated that catalytically active immunoproteasomes and the immunoproteasome activator PA28 were upregulated in carfilzomib-resistant BMPCs. Carfilzomib-resistant BMPCs displayed reduced sensitivity to the proteasome inhibitors carfilzomib, bortezomib, and ixazomib, but enhanced sensitivity to an immunoproteasome-specific inhibitor ONX-0914. Finally, in vitro carfilzomib treatment of BMPCs from HLA-sensitized patients increased levels of the immunoproteasome β5i (PSMB8) catalytic subunit suggesting that carfilzomib therapy directly induces an adaptive immunoproteasome response. Taken together, our results indicate that carfilzomib induces structural changes in proteasomes and immunoproteasome formation.