Ex Vivo Activity of Bortezomib and Dexamethasone Combinations Against Childhood Acute Leukemia Cells.

Response to glucocorticoid (GC) treatment is of prognostic value for children with acute lymphoblastic leukemia (ALL). In this context, sensitization of leukemic cells for GC may further improve the prognosis. In recent studies (Oerlemans et al, 2007) we showed that chronic inhibition of the nuclear transcription factor NFkB sensitized leukemia cells for GC treatment, including GC-resistant acute myeloid leukemia (AML) cells. The proteasome inhibitor bortezomib (BTZ) exerts part of its mechanism of action via inhibition of NFkB activation. The aim of our study was to determine the cytotoxic effects of BTZ, the GC dexamethasone (DEX), and their combination, against childhood leukemia cells and normal bone marrow (BM) cells as controls. Drug (combination) responses in samples of childhood leukemia and normal BM cells were evaluated after a 4 day drug exposure using the MTT-cytotoxicity assay. The drug concentration that was lethal to 50% of cells compared to control cells without drugs was defined as LC50. Synergism between the 2 drugs was established when the percentage of leukemic cell survival (LCS) of the combination was smaller than the % LCS BTZ x % LCS DEX/100. Inclusion criteria for the MTT analysis were a percentage of blasts present in the leukemic samples > 80%, and a MTT-OD read-out of control cells at day 4 minus the blank control > 0.060. Overall analyses included 5 normal BM samples, 18 ALL and 9 AML pediatric patient samples. All normal BM cells were insensitive to DEX (LC50 values > 15.3 uM). AML cells were, as expected, significantly (p<0.01) more resistant to DEX (median LC50 >15.3 uM) as compared to ALL cells (LC50 = 0.41 uM). Within the group of AML samples 8 out of 9 (89%) had an LC50 > 15.3 uM, while only 6 of 18 (34%) of the ALL samples had such a high LC50 value. For BTZ cytotoxicity similar differences were found. With a median LC50 of 14.6 nM (range: 10–236 nM), normal BM samples were significantly (p<0.01) less sensitive for BTZ than ALL cells (median LC50 5.7 nM, range: 4–10 nM). BTZ-sensitivity of AML cells (median LC50: 14.6 nM, range: 10–26 nM) was not significantly different from normal BM cells. Based on the dose-response analysis of BTZ, 4 different concentrations of BTZ were used (ranging from 0.3 to 34 nM) in combination with 6 concentrations of DEX (ranging from 0.0046 to 15.3 uM). The drug combination predominantly revealed an additive effect, although a low level of synergism was observed at the highest concentration of BTZ and those patient samples that were resistant to DEX. This particularly referred to 6 of 9 (67%) AML samples but was also noted in 2 of 5 (40%) normal BM samples. 3 of 18 (17%) ALL samples showed synergism for the drug combination. In conclusion, BTZ displayed potent cytotoxic effects against pediatric ALL cells. This antileukemic effect may be further enhanced in combination with DEX, based on frequent additive and synergistic drug interactions we observed. The combination of BTZ and DEX is therefore a candidate drug combination for further clinical evaluation in the treatment of especially childhood ALL.