Acquisition of Mutations in the PSMB5 Gene during Chronic Exposure of Human Acute Lymphoblastic Leukemia CCRF-CEM Cells to the Proteasome Inhibitor Bortezomib

Abstract

Despite the good prognosis of children suffering from acute lymphoblastic leukemia (ALL), approximately 25% of the patients develop a relapse. The proteasome inhibitor bortezomib (BTZ) is a representative of a novel generation of therapeutic drugs that revealed promising activity in patients with multiple myeloma (MM) and certain types of lymphomas, and is being studied in the treatment of relapsed ALL. Whereas most research on the mechanism of action of BTZ has focused on MM or lymphoma, the present study was designed to examine the activity of BTZ in an ALL model and in particular to assess the propensity to develop resistance to BTZ after prolonged drug exposure. Previously, we have shown (Franke et al., ASH 2007) that BTZ-resistant subclones of the human T-ALL cell line CCRF-CEM could emerge relatively rapid (within 2–3 months) after chronic exposure to stepwise increasing concentrations (0.1–200 nM) of BTZ. These BTZ-resistant cells (CEM/BTZ) showed cross-resistance against other proteasome inhibitors while remaining completely sensitive to a broad spectrum of other anticancer drugs. Subsequent analysis of these BTZ-resistant subclones showed an upregulation of the catalytic proteasomal β-subunits at both mRNA and protein level. In the present study we further elucidated the molecular basis for BTZ-resistance in a panel of CEM/BTZ cells selected at 7 nM BTZ (CEM/BTZ7) up to 200 nM BTZ (CEM/BTZ200). Since the proteasome β5 subunit is the primary target of BTZ, we screened whether mutations in the PSMB5 gene could be involved in conferring resistance to BTZ. Indeed, sequencing of exon 2 of the PSMB5 gene for CEM/BTZ7 cells revealed a single G to T nucleotide shift at position 332 of this gene. In the mature and functional β5 proteasome subunit, this mutation introduces a Cys to Phe substitution at amino acid 52, residing in a highly conserved substrate/inhibitor binding domain of the β5 subunit. In this context, it was of interest to note that CEM/BTZ7 cells already displayed a marked level (28-fold) of cross-resistance to 4A6, a 6-mer peptide that specifically targets the proteasome β5 catalytic subunit, thus suggesting that this mutation is responsible for diminished inhibitor binding. Upon screening for mutations in CEM cells with higher BTZ resistance levels, an additional mutation in the PSMB5 gene was revealed in CEM/BTZ30 cells. Herein, an additional C to T nucleotide shift at position 323 of the PSMB5 gene was identified, introducing an Ala to Val amino acid substitution at amino acid 49 of the mature and functional β5 subunit protein. This amino acid also resides in a highly conserved substrate/inhibitor binding domain of the β5 subunit. Both Cys52Phe and Ala49Val mutations were retained, and no others revealed, in BTZ-resistant CEM cells up to CEM/BTZ200 cells. Of note, recently we have identified an Ala49Thr mutation in the BTZ binding domain of the proteasome β5 subunit in a BTZ-resistant subclone of the human acute myeloid leukemic cell line THP-1 (Oerlemans & Franke, Blood 2008). Moreover, an Ala49Thr mutation was also recently described in a BTZ-resistant Jurkat cell line (Lü et al., J Pharmacol Exp Ther 2008). Together, this study demonstrates that mutations in the PSMB5 gene are provoked relatively fast in ALL cells, already at low levels of BTZ resistance. Furthermore, given the notion that independent studies in 3 different leukemia cell line models demonstrated that BTZ-resistance is associated with a mutation introducing amino acid substitutions at position Ala49 in the highly conserved substrate/inhibitor binding domain of the β5 subunit, this position may be considered as a hot spot for mutations in the PSMB5 gene through which acquired resistance to BTZ may be facilitated. Analysis of mutational status of the PSMB5 gene in future clinical BTZ studies in ALL patients should further confirm the clinical relevance of our finding, and might be exploited in the development of next generation proteasome inhibitors, designed to overcome resistance.