Molecular Mechanisms of Bortezomib Resistance in Acute Lymphoblastic Leukemia Cells in Comparison with Multiple Myeloma Cells.

Abstract

Children suffering from acute lymphoblastic leukemia (ALL) have a relatively good prognosis (overall survival 80%). Twenty percent of the children with ALL, however, display inherent resistance to current therapies or acquire resistance during treatment, thus requiring implementation of novel therapeutic drugs. The proteasome inhibitor bortezomib (BTZ) is a representative of a novel generation of therapeutic drugs that revealed promising activity in patients with therapy- refractory multiple myeloma (MM). The aim of our study was to determine whether ALL cells and MM cells would retain sensitivity to BTZ upon long-term treatment or face the onset of resistance to BTZ after dose escalations. To address this issue, human CEM-C7 cells, representing a model cell line for T-ALL, were exposed in vitro to stepwise increasing concentrations of BTZ over a period of 6 months. In parallel, the same protocol was applied for human MM cell line 8226. CEM-C7 and 8226 cells had approximately similar baseline sensitivities for growth inhibition by BTZ (IC50, 4 days drug exposure: 2 nM and 5.5 nM, respectively). Following chronic drug exposure, a high level of resistance to BTZ (up to 170-fold) could be provoked in CEM-C7 cells, while over the same time period resistance levels to BTZ in 8226 cells were less than 2-fold compared to baseline sensitivities. Further characterization of BTZ-resistant CEM-C7 cells revealed a marked cross-resistance to other proteasome inhibitors, MG132 (110-fold) and MG262 (25-fold), with preservation of full sensitivity to a broad series of anti-leukemic drugs, including daunorubicin, vincristine, L-asparaginase, Ara-C, methotrexate, and even a 2 to 3 fold increased sensitivity to dexamethasone. When CEM-C7 cells with the highest (170-fold) level of BTZ resistance were cultured for 2 months in the absence of BTZ, resistance levels for BTZ slowly declined to 30-fold, but high levels of resistance were rapidly re-induced upon re-exposure to BTZ. To unravel the molecular basis for BTZ resistance in CEM-C7 cells, mRNA and protein levels were analyzed of those proteasome subunits that harbor the proteasome's proteolytic activity, i.e. β1, β2, and β5, the latter being the primary target of BTZ. In CEM-C7 cells with the highest level of BTZ resistance, a markedly induced expression of all three subunits was observed: β5- (12.5-fold), β2- (9-fold) and β1-subunits (7-fold), part of which was accompanied by increased mRNA levels for β5- (3.5-fold), β2- (2-fold) and β1-subunits (2-fold). Together, these results demonstrate that BTZ exhibits potent activity against CEM-ALL and 8226 MM cells. Upon chronic exposure to BTZ, CEM-C7-ALL cells have a greater propensity to develop resistance to this drug due to an intrinsic property to upregulate the expression of the catalytic subunits of the proteasome. These data suggest that MM cells largely retain BTZ sensitivity upon prolonged exposure to BTZ, while in ALL drug resistance phenomena may be more likely to occur.