Abstract A27: Chronic exposure of malignant hematological cell lines to bortezomib induces de novo hot spot mutations in the PSMB5 gene
Authors: Franke Niels , Niewerth Denise , van Meerloo Johan , Vojtekova Katarina , Verbrugge Sue Ellen , Schimmer Aaron , Kaspers Gertjan , Jansen Gerrit , Cloos Jacqueline 
Published: January 2011

Abstract
The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome. 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. We have developed a BTZ-resistant cell line model by chronic exposure to stepwise increasing concentrations of BTZ, 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, Blood 2009 vol 114(22), abstr 940). 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 concentrations of BTZ up to 500 nM to provoke higher resistance levels. Abilities to resist a BTZ concentration of 100 nM could be achieved relatively fast in the CEM cell line (within 16 weeks), intermediate in the THP-1 cell line (within 22 weeks) and relatively slowly in the 8226 cell line (within 60 weeks). Subsequent sequencing of the PSMB5 gene, encoding the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones, all resulting in amino-acid changes residing within the highly conserved BTZ binding pocket.
The relatively fast induction of mutations provoked the question whether there might already exist a subclone within the cell line that harbours the mutation. To distinguish between the outgrowth of a pre-existing resistant subclone and the occurrence of de novo mutations, we generated new BTZ resistant CEM and THP-1 cells (CEM-BR2 and THP-1-BR2). Interestingly, the new CEM-BR2 cells had a different nucleotide change (G322A resulting in a Ala49Thr substitution) from the original BTZ resistant CEM-BR1 (C323T and G332T resulting in Ala49Val and Cys52Phe amino acid substitutions, respectively). Of note, this new CEM-BR2 mutation represented the same mutation as seen in the original BTZ resistant THP-1-BR1. In addition, the new THP-1-BR2 cells also showed a different mutation (A309G) in the PSMB5 gene, introducing a Met45Val amino-acid substitution) compared to the original BTZ resistant THP-1 cells that showed a Met45Iso substitution. These results indicate that the mutations are acquired during BTZ exposure and are mainly induced in specific hot-spots (dominantly Ala49) within the PSMB5 gene. To explore whether mutation-induced resistance could be bypassed, a new 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) was studied in our model system. 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.
To determine whether mutation induction also occurs in patients treated with BTZ, screening for their presence in clinical samples of BTZ refractory patients is warranted. The notion that 5AHQ can overcome BTZ resistance related to single and multiple mutations in the PSMB5 gene, supports further research in this drug and its analogues. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw).
Citation Information: Clin Cancer Res 2010;16(7 Suppl):A27