Identification of Chemotherapy-Resistant Mechanisms in Childhood T-ALL Cell Lines: Correlation with Genetic Profiles in Patients Having Early Treatment Failure.

Abstract

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.