Acquired Resistance to Bortezomib in Human RPMI-8226 Multiple Myeloma Cells: Molecular Characterization, Cross-Resistance with Other Proteasome Inhibitors but Marked Sensitization to Glucocorticoids

The proteasome inhibitor bortezomib (BTZ, PS-341, Velcade^®) has proven to be an effective drug in the treatment of multiple myeloma (MM) patients, both in first line as in refractory disease. However, emergence of drug resistance, for which the molecular mechanism(s) remain elusive, can hamper its clinical efficacy. Recently, we showed (Oerlemans & Franke, Blood 2008) that resistance to BTZ in human THP1 acute myeloid leukemia cells was conferred by an acquired mutation in the PSMB5 gene. This mutation introduced an amino acid substitution in the highly conserved substrate binding pocket of the β5 subunit of the proteasome, which is the primary target of BTZ. In the present study we investigated whether chronic exposure to BTZ could provoke acquired resistance to BTZ in the human RPMI-8226 MM cell line model and whether a PSMB5 gene mutation would be responsible. To this end, 8226 cells were exposed in vitro to stepwise increasing concentrations of BTZ from 0.1 nM BTZ up to 7nM (8226/BTZ7) over a period of 6 months and up to 30nM (8226/BTZ30) over a period of 12 months. In parallel to these selective concentrations, RPMI-8226 cells were cultured without BTZ (8226/WT). Characterization of 8226/BTZ7 vs 8226/WT cells revealed:

• 2.6-fold resistance to BTZ along with cross-resistance to other peptide-based proteasome inhibitors; MG132 (2.2-fold), MG262 (1.9-fold) and 4A6 (4.9 fold),

• unchanged sensitivity to other anti-MM drugs, including doxorubicin, melphalan and thalidomide,

• a marked gain in sensitivity for the glucocorticoids dexamethasone (IC50: 25 nM vs >10 uM, respectively) and prednisolone (IC50: 2 uM vs > 1000 uM, respectively),

• other than 8226/WT cells, no G2M cell cycle arrest after BTZ exposure in 8226/BTZ7 cells,

• no significant changes in mRNA and functional activity of the three dominant catalytic subunits of the proteasome; β1, β2 and β5, although at a protein level β5 expression was markedly increased (5–10 fold), and

• sequencing of the PSMB5 gene in 8226/BTZ7 and 8226/BTZ30 cells, and one clinical sample of plasma cells of a BTZ-resistant plasma cell leukemia patient, showed no evidence for PSMB5 mutations.

Microarray experiments were initiated to further elicit possible molecular mechanism(s) of BTZ resistance in 8226/BTZ7 cells. Initial analysis showed upregulation of several genes involved in microenvironment interaction and plasma cell differentiation. In this context, Gene Set Enrichment Analysis (GSEA) showed enrichment in the 8226/BTZ7 for the gene sets related to CD138 upregulation and MYC upregulation, indicative for a more differentiated phenotype of 8226/BTZ7 cells. Consistently, immune phenotypic characterization showed a shift to a dominant CD138^dim/CD45⁺/CD20^dim/CD27^dim cell population for 8226/WT cells grown in parallel with 8226/BTZ7, which retained a CD138⁺/CD45⁻/CD20⁻/CD27⁻ marker profile. Collectively, these data indicate that, other than in acute leukemia, the onset of acquired resistance to BTZ in 8226 MM cells proceeds very slowly, is associated with the upregulation of a (non-mutated) β5 catalytic proteasome subunit, and goes along with a shift towards a more mature immune phenotype in combination with an enrichment of genes that support cell growth. Notwithstanding this fact, the marked sensitization of BTZ-resistant 8226 MM cells for glucocorticoids may further corroborate the use of these drugs in combination with BTZ.