Summary Acquired drug resistance eventually leads to treatment failure in T‐cell acute lymphoblastic leukaemia (T‐ALL). Immunophenotypic and cytogenetic heterogeneities within T‐ALL influence susceptibility to cytotoxic therapy, and little is known about the mechanisms of drug resistance at specific stages of T‐cell ontogeny. We developed tolerance to therapeutic concentrations of daunorubicin (DNR) and l‐asparaginase (l‐asp) in Jurkat (CD1a−, sCD3+) and Sup T1 (CD1a+, sCD3−) cell lines, having respective ‘mature’ and ‘cortical’ stages of developmental arrest. DNR resistant cells acquired multidrug resistance: 310‐fold increased resistance to vincristine (VCR) and a 120‐fold increased resistance to prednisolone (PRED). Microarray analysis identified upregulation of asparagine synthetase (ASNS) and argininosuccinate synthase 1 (ASS1) to cell lines with acquired resistance to l‐asp, and in the case of DNR, upregulation of ATP‐binding cassette B1 (ABCB1). Suppression of ABCB1, ASNS and ASS1 by RNA interference revealed their functional relevance to acquired drug resistance. Expression profiling of these genes in 80 T‐ALL patients showed correlation with treatment response. This study expands the pool of available drug resistant cell lines having cortical and mature stages of developmental arrest, introduces three new drug resistant T‐ALL cell lines, and identifies gene interactions leading to l‐asp and DNR resistance.

Acquisition of drug resistance in childhood T-cell acute lymphoblastic leukemia (T-ALL) is a major cause of treatment failure. Drug resistant cell lines are effective tools for elucidating mechanisms of acquired drug resistance. In this study, we developed novel drug resistant cell lines that could be employed to identify mechanisms of drug resistance. We established three new drug resistant T-ALL cell lines: 2 resistant to L-asparaginase (L-asp), and one resistant to daunorubicin (DNR). The DNR-resistant line (Jurkat) acquired a 117-fold increase in EC50 to DNR, from 20.6 nM to 2.4 μM, while the L-asp resistant cells (Jurkat and Sup T1) showed respective increases in resistance of 320-fold (0.003 IU/mL to 0.962 IU/mL) and 29-fold (0.042 IU/mL to 1.22 IU/mL). The DNR resistant cell line acquired a multidrug resistant phenotype, showing 310 and 120-fold increase in resistance to vincristine and prednisolone, respectively. Resistance to L-asp was unchanged. Microarray analysis showed that ABCB1 (MDR1, P-glycoprotein) was significantly upregulated (567-fold) in DNR resistant cells. siRNA experiments that reduced ABCB1 mRNA levels by 74% restored DNR sensitivity. In L-asp resistant T-ALL cells (Jurkat and Sup T1), two notable genes were upregulated, asparagine synthetase (ASNS) which catalyzes synthesis of asparagine (41-fold and 1.5-fold) and argininosuccinate synthase (ASS)(32-fold and 6.5-fold), respectively. Reduction of ASNS with siRNA restored drug sensitivity in both cell lines. Interestingly, siRNA suppression of ASS in conjunction with ASNS achieved an exaggerated restoration of drug sensitivity compared to ASNS alone. We next examined the microarray profiles of drug resistant cells with those of 86 T-ALL patients; of which 8 failed induction (IF). Interestingly, these 3 key genes are upregulated in 25–62% of IF cases. Although ABCB1 overexpression has been shown to be a mechanism of DNR resistance in many cancers, there is a paucity of resistant T-ALL cell lines to adequately model the effect of stage of differentiation and genetic heterogeneity underlying drug resistance in T-ALL. With the establishment of 3 new T-ALL cell lines in this report, there exist 5 T-ALL drug resistant cell lines, representing the spectrum of T-cell differentiation (pre-T, cortical T, and mature T cells). Finally, this is the first report of the potential contribution of ASS in addition to ASNS to L-asp resistance in leukemia cells.

Acquisition of drug resistance in tumor cells in children with T-cell acute lymphoblastic leukemia (T-ALL) during chemotherapy results in relapse and poor outcome. T-ALL cell lines that have acquired resistance to chemotherapeutics are therefore critical tools for the study of acquired resistance, yet there is a paucity of cell lines available for study. In this study, we hypothesize that drug resistant T-ALL cells can be produced by prolonged exposure to chemotherapeutics and that microarray analysis can be employed to identify the gene products responsible for acquired drug resistance. By incrementally increasing the drug concentration in growth media, we have produced T-ALL cell lines (Jurkat and Sup T1) that grow well in the presence of therapeutic concentrations of L-asparaginase (ASNase) and daunorubicin (DNR). The genetic profiles of the drug-resistant cell lines were compared to their parental progenitors using the Affymetrix HG-U133Plus2 GeneChip platform, capable of hybridizing ~54,000 genes and ESTs/chip. Signal intensity was normalized using the robust multi-array average (RMA) technique in GeneSpring 7.2. The Sup T1 and Jurkat ASNase-resistant cell lines increased their IC50s 26-fold (0.044 IU/mL to 1.14 IU/mL) and 320-fold (0.003 IU/mL to 0.96 IU/mL), respectively. The IC50 of the Jurkat DNR resistant cell line increased 77-fold (30 nM to 2300 nM), and 4.0-fold, (0.46 nM to 1.85 nM), respectively. Notably, DNR resistant Jurkat cells were also resistant to therapeutic concentrations of vincristine and prednisolone, but not ASNase. In contrast, the ASNase resistant cell lines remained sensitive to DNR, vincristine, and prednisolone. Microarray data comparing DNR-resistant and parental cell lines showed 288 genes upregulated >1.5-fold in the resistant line. Two sets of genes were the most upregulated in the drug resistant cells in comparison to parental cells. ABCB1 (ABC transporter P-glycoprotein) was upregulated ~940-fold and genes coding for 6 different killer-cell immunoglobulin-like receptors (KIRs) were upregulated >6-fold. In the case of the ASNase-resistant cell lines, 96 genes were found to be upregulated >1.5-fold in both Jurkat and Sup T1 lines. The most highly upregulated gene in both cell lines was argininosuccinate synthase (ASS), 32-fold upregulated in Jurkat and 6.5-fold in Sup T1. All expression results were confirmed by qRT-PCR. These genes have previously been implicated in the acquisition of drug resistance: ASS is critical for responding to asparagine depletion caused by ASNase. ABCB1 acts as a molecular pump capable of lowering intracellular concentrations of substrate chemotherapeutics such as DNR, vincristine, and prednisolone, consistent with our observation of multi-drug resistance in that cell line. To our knowledge, this is the first description of DNR and ASNase resistant Jurkat and Sup T1 T-ALL cell lines. In addition, our results suggest that microarray technology is a valid method for elucidating the genetic nature of drug resistance in T-ALL cell lines, making it a productive approach to identify mechanisms of chemotherapy resistance. Finally, these cell lines will serve as useful tools for studying mechanisms of chemotherapeutic resistance in T-ALL.