Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines

Wei-Wen Chien, Céline Le Beux, Nicolas Rachinel, Michel Julien, Claire-Emmanuelle Lacroix, Soraya Allas, Pierre Sahakian, Aurélie Cornut-Thibaut, Loïc Lionnard, Jérôme Kucharczak, Abdel Aouacheria, Thierry Abribat, Gilles Salles, Wei-Wen Chien, Céline Le Beux, Nicolas Rachinel, Michel Julien, Claire-Emmanuelle Lacroix, Soraya Allas, Pierre Sahakian, Aurélie Cornut-Thibaut, Loïc Lionnard, Jérôme Kucharczak, Abdel Aouacheria, Thierry Abribat, Gilles Salles

Abstract

Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes (especially NFκB-related genes) are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility.

Conflict of interest statement

S.A. P.S., M.J. and T.A. are employees, and T.A. is a shareholder of Alizé Pharma II. Alizé Pharma II owns patent rights to pegylated r-crisantaspase. The remaining authors declare no competing financial interests.

Figures

Figure 1. Expression of phosphorylated elF2α, phosphorylated…
Figure 1. Expression of phosphorylated elF2α, phosphorylated Erk1/2, AsnS and GlnS following ASNase treatment.
B-ALL (RS(4, 11), Nalm-6) and malignant NK (MEC04, KHYG1) cell lines were treated with 5 U/ml of pegylated-r-crisantaspase for 6 h or 20 h. Non-treated cells served as controls. Protein expression was analyzed by Western Blot. For the proteins elF2α and elF2α-pS51, all samples were loaded in the same gel. For other proteins, samples were loaded in different gels (one gel for RS(4, 11) and Nalm-6 cell lines and one gel for MEC04 and KHYG1 cell lines). Two gels have been run under the same experimental conditions. The experiment was repeated twice with similar results.
Figure 2. Expression of phosphorylated 4E-BP1 following…
Figure 2. Expression of phosphorylated 4E-BP1 following ASNase treatment.
B-ALL (RS(4, 11), Nalm-6) and malignant NK (MEC04, KHYG1) cell lines were treated with 5 U/ml of pegylated-r-crisantaspase for 6 h or 20 h. Non-treated cells served as controls. Protein expression was analyzed by Western Blot. Samples were loaded in different gels (one gel for RS(4, 11) and Nalm-6 cell lines and one gel for MEC04 and KHYG1 cell lines). Two gels have been run under the same experimental conditions. The experiment was repeated twice with similar results.
Figure 3. Analysis of cell death modality…
Figure 3. Analysis of cell death modality and timing after exposure to ASNase.
B-ALL (RS(4, 11), Nalm-6) and malignant NK (MEC04, KHYG1) cell lines were treated with increasing doses of pegylated-r-crisantaspase (from 5 × 10−5 to 5 U/ml). Non-treated cells served as controls. The percentage of early apoptotic (Annexin V+/PI−) and late apoptotic (Annexin V+/PI+) was determined by flow cytometry after Annexin V/PI staining.
Figure 4. Analysis of BCL2A1 cleavage after…
Figure 4. Analysis of BCL2A1 cleavage after exposure to ASNase.
B-ALL (RS(4, 11), Nalm-6) and malignant NK (MEC04, KHYG1) cell lines were treated with 5 U/ml of pegylated-r-crisantaspase for 6 h. Non-treated cells served as controls. Protein expression was analyzed by Western Blot. All samples were loaded in the same gel.

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