Anti-leukemic activity and mechanisms underlying resistance to the novel immunoproteasome inhibitor PR-924

• Published in: Biochemical pharmacology 2014-05, Vol.89 (1), p.43-51
• Main Authors: Niewerth, Denise, van Meerloo, Johan, Jansen, Gerrit, Assaraf, Yehuda G., Hendrickx, Tessa C., Kirk, Christopher J., Anderl, Janet L., Zweegman, Sonja, Kaspers, Gertjan J.L., Cloos, Jacqueline
• Format: Article
• Language: English
• Subjects: Base Sequence; DNA Primers; Drug Resistance, Neoplasm; Humans; Immunoproteasome; Leukemia; Leukemia - drug therapy; Leukemia - pathology; Polymerase Chain Reaction; PR-924; Proteasome; Proteasome inhibitor; Proteasome Inhibitors - pharmacology
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2014.02.005

Despite potent immunoproteasome β5i-subunit inhibition, PR924-sensitive cells require concomitant constitutive β5-subunit inhibtion for apoptosis induction. PR924-resistant cells upregulate mutated (Met45Ile*) β5-subunit with less effective catalytic activity inhibition and attenuated apoptosis induction. PR-924 is a novel prototypic immunoproteasome inhibitor bearing markedly enhanced specificity for the β5i immunoproteasome subunit, compared to the classical proteasome inhibitor bortezomib. Here, we assessed the growth inhibitory potential of PR-924 in three human hematologic malignancy cell lines (CCRF-CEM, THP1, and 8226) and their bortezomib-resistant sublines. Parental cells displayed equal sensitivity to PR-924 (IC50: 1.5–2.8μM), whereas their bortezomib-resistant tumor lines displayed a 10–12 fold cross-resistance to PR-924. However, PR-924 cross-resistance factors for bortezomib-resistant sublines were markedly lower compared to the resistance factors to bortezomib. Proteasome inhibition experiments confirmed that PR-924 specifically inhibited β5i activity, even far below concentrations that exerted anti-proliferative activity. We further determined whether PR-924 activity might be compromised by acquisition of drug resistance phenomena. Indeed, CEM cells rendered stepwise resistant to 20μM PR-924 (CEM/PR20) displayed 13-fold PR-924-resistance and 10-fold cross-resistance to bortezomib. CEM/PR20 cells were devoid of mutations in the PSMB8 gene (encoding β5i), but acquired Met45Ile mutation in the PSMB5 gene (encoding constitutive β5), consistent with β5 mutations observed in bortezomib-resistant cells. Furthermore, compared to parental CEM cells, CEM/PR20 cells exhibited 2.5-fold upregulation of constitutive proteasome subunit expression, whereas immunoproteasome subunit expression was 2-fold decreased. In conclusion, PR-924 displayed potent anti-leukemic activity including toward bortezomib-resistant leukemia cells. Despite the specificity of PR-924 to the β5i immunoproteasome subunit, its anti-leukemic effect required concentrations that blocked both β5 and β5i subunits. This is underscored by the emergence of mutations in PSMB5 rather than in PSMB8.