The Novel Proteasome Inhibitor 5-Amino-8-Hydroxyquinole (5AHQ) Overcomes Bortezomib Resistance in Malignant Hematological Cell Line Models Harboring Mutations in the PSMB5 Gene

• Published in: Blood Vol. 114; no. 22; p. 940
• Main Authors: Franke, Niels E, van Meerloo, Johan, Slot, Linda, Li, Xiaoming, Wood, Tabitha E, Vojtekova, Katarina, Verbrugge, Sue Ellen, Kaspers, Gertjan, Batey, Robert A., Schimmer, Aaron D., Jansen, Gerrit, Cloos, Jacqueline
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 20.11.2009
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V114.22.940.940

Abstract 940 The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome, thereby interfering in the degradation of proteins involved in cell-cycling, NF-kB activation, apoptosis induction and micro-environmental interactions. The introduction of BTZ has shown promising results in the treatment of Multiple Myeloma (MM), Non-Hodgkin lymphoma and leukemia. Despite these encouraging results, clinical trials in MM also revealed that a significant proportion of patients acquired resistance to BTZ mono-therapy. Previously we reported the development of three different BTZ-resistant cell line models, including the AML THP-1 cell-line (Oerlemans & Franke et al, Blood 2008), the T-ALL CCRF-CEM-C7 cell-line and the MM RPMI-8226 cell line (Franke et al, ASH 2008) after chronic exposure to stepwise increasing concentrations of BTZ. The aim of our current study was to further elucidate the molecular basis of BTZ resistance in these cell lines and investigate whether the resistant phenotype could be overcome by the second generation proteasome inhibitor 5-amino-8-hydroxyquinole (5AHQ), acting as a non-competitive inhibitor of the non-catalytic alpha-7 subunit of the proteasome (Li et al. ASH 2008). When added to intact cells, cell extracts, or isolated rabbit reticulocyte proteasomes (gift of A Navon, Weizmann Institute of Science, Israel), 5AHQ inhibited the chymotrypsin-like enzymatic activity of the proteasome with IC50's of 6.9 ± 0.05 uM, 4.2 ± 0.6 uM, and 2.1 ± 0.18 μM, respectively. Cells were initially selected for growth at 7 nM BTZ to acquire low levels of BTZ resistance (2-3 fold higher IC50 concentrations) and subsequently challenged to higher concentrations of BTZ (up to 500 nM) to provoke higher resistance levels. Sequencing of the PSMB5 gene, encoding for the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones. An overview of the mutation site in the PSMB5 gene, the amino acid position at which substitutions take place at the beta 5 subunit protein level, and the functional implication of each mutation are depicted in the table below. Cell lineMutationBTZ-resistant subcloneAmino acid changeFunctional effect on beta 5 subunit proteinTHP-1 (AML)NoneTHP1/BTZ7NoneG322ATHP1/BTZ30-200Ala49ThrBTZ binding pocketG322A G311TTHP1/BTZ500Ala49Thr Met45Ileenzyme-substrate interactions (hydrolysis of hydrophobic peptide bonds)CEM-C7 (ALL)G332TCEM/BTZ7Cys52Phesubstrate/inhibitor binding domainG332T C323TCEM/BTZ 30-200Cys52Phe Ala49ValBTZ binding pocket8226 (MM)None8226/BTZ7NoneA247G8226/BTZ14-30Thr21AlaBTZ binding pocketG322A8226/BTZ100Ala49ThrBTZ binding pocket Subsequently, we determined whether THP-1, CEM and 8226 cells with low and high levels of BTZ resistance would be sensitive towards the new proteasome inhibitor 5AHQ. Strikingly, all BTZ-resistant selectants retained full sensitivity towards 5AHQ (IC50: 4-7 μM, measured in a 4-day MTT cytotoxicity assay) as compared to parental cells. Together, these data indicate that in vitro selection of low and high levels of BTZ resistance in 3 hematological cell lines is accompanied by the introduction of multiple mutations of amino acids, all of which have key positions in BTZ binding within the active site of the proteasome beta 5 subunit. Notably, Ala49 mutations were independently observed in all 3 cell lines selected for BTZ concentrations > 100 nM. Knowledge of specific mutations in the PSMB5 gene that can confer BTZ resistance allows purposive screening for these types of mutations in clinical samples of BTZ refractory patients. In this respect, the notion that 5AHQ can bypass BTZ resistance related to single or multiple mutations in the PSMB5 gene, supports further preclinical development and clinical application of this drug. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw), The Leukemia and Lymphoma Society and the Ontario Institute for Cancer Research through funding from the Ministry of Research and Innovation, Province of Ontario. No relevant conflicts of interest to declare.