title: Anti-leukemic activity and mechanisms underlying resistance to the novel immunoproteasome inhibitor PR-924.
abstract: 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: 1.5-2.8 μM), 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 20 μM 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.
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: d.niewerth@vumc.nl.
authors.forename: Denise
authors.initials: D
authors.lastname: Niewerth
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands; Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: j.vanmeerloo@vumc.nl.
authors.forename: Johan
authors.initials: J
authors.lastname: van Meerloo
authors.affiliation: Department of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: g.jansen@vumc.nl.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.affiliation: The Fred Wyszkowski Cancer Research Lab, Technion-Israel Institute of Technology, Haifa, Israel. Electronic address: assaraf@technion.ac.il.
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: t_c_hendrickx@hotmail.com.
authors.forename: Tessa C
authors.initials: TC
authors.lastname: Hendrickx
authors.affiliation: Research Department, Onyx Pharmaceuticals Inc., South San Francisco, CA, USA. Electronic address: ckirk@onyx.com.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Kirk
authors.affiliation: Research Department, Onyx Pharmaceuticals Inc., South San Francisco, CA, USA. Electronic address: janderl@onyx.com.
authors.forename: Janet L
authors.initials: JL
authors.lastname: Anderl
authors.affiliation: Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: s.zweegman@vumc.nl.
authors.forename: Sonja
authors.initials: S
authors.lastname: Zweegman
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: gjl.kaspers@vumc.nl.
authors.forename: Gertjan J L
authors.initials: GJ
authors.lastname: Kaspers
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands; Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: j.cloos@vumc.nl.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cloos
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000473:pathology; D016133:Polymerase Chain Reaction; D061988:Proteasome Inhibitors / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D061988:Proteasome Inhibitors
keywords: Immunoproteasome; Leukemia; PR-924; Proteasome; Proteasome inhibitor