Patient-derived mutations impair PI response to Carfilzomib and Ixazomib.
Cytotoxicity assay and IC50 values of RPMI-8226 MM cell lines harboring two different stably expressed patient-derived mutations in proteasome subunits. The mutants were more resistant to the proteasome inhibitors Carfilzomib and Ixazomib compared to the controls (*) harboring lentivirally-encoded WT sequence of PSMC6/PSMD1. Shown are the means and standard deviations of three independent experiments (biological replicates) and three technical replicates in each experiment. The PSMC6 and PSMD1 expression levels were equivalent for the mutants and the controls.
Funding
Stiftung zur Forderung der Krebsforschung an der Universität Würzburg
Stifterverband
CDW Stiftung
Bayerisch-Tschechische Hochschulagentur (BTHA)
Deutsche Krebshilfe (German Cancer Aid)
Mildred Scheel Early Career Center (MSNZ)
Deutsche Forschungsgemeinschaft (DFG)
Instituto de Salud Carlos III (ISCIII)
ARTICLE ABSTRACT
Proteasome inhibitors (PI) are the backbone of various treatment regimens in multiple myeloma. We recently described the first in-patient point mutations affecting the 20S subunit PSMB5 underlying PI resistance. Notably, in vivo, the incidence of mutations in PSMB5 and other proteasome encoding genes is too low to explain the development of resistance in most of the affected patients. Thus, additional genetic and epigenetic alterations need to be explored.
We performed DNA methylation profiling by Deep Bisulfite Sequencing in PSMB5, PSMC2, PSMC5, PSMC6, PSMD1, and PSMD5, a subset of proteasome subunits that have hitherto been associated with PI resistance, recruited from our own previous research, the literature, or a meta-analysis on the frequency of somatic mutations. Methylation was followed up on gene expression level and by dual-luciferase reporter assay. The KMS11 cell line served as a model to functionally test the impact of demethylating agents.
We identified PSMD5 promoter hypermethylation and subsequent epigenetic gene silencing in 24% of PI refractory patients. Hypermethylation correlated with decreased expression and the regulatory impact of this region was functionally confirmed. In contrast, patients with newly diagnosed multiple myeloma, along with peripheral blood mononuclear cells and CD138+ plasma cells from healthy donors, generally show unmethylated profiles.
Under the selective pressure of PI treatment, multiple myeloma cells acquire methylation of the PSMD5 promoter silencing the PSMD5 gene expression. PSMD5 acts as a key orchestrator of proteasome assembly and its downregulation was described to increase the cell's proteolytic capacity. PSMD5 hypermethylation, therefore, represents a novel mechanism of PI tolerance in multiple myeloma.
