Pediatrics and Neonatology (2015) 56, 287e293

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REVIEW ARTICLE Asparaginase Therapy in Pediatric Acute Lymphoblastic Leukemia: A Focus on the Mode of Drug Resistance Shih-Hsiang Chen

Division of Hematology/Oncology, Department of Pediatrics, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, 5 Fu-Shin Street, Kwei-Shan 333, Taoyuan, Taiwan

Received Apr 24, 2014; received in revised form Aug 28, 2014; accepted Oct 6, 2014 Available online 23 December 2014

Key Words Asparaginase is one of the most important chemotherapeutic agents against pediatric acute acute lymphoblastic lymphoblastic leukemia (ALL), the most common form of childhood cancer. The therapeutic leukemia; efficacy (e.g., chemoresistance) and adverse effects of asparaginase (e.g., hypersensivity asparaginase; and pancreatitis) have been investigated over the past four decades. It was suggested early chemotherapy; on that leukemic cells are resistant to asparaginase because of their increased asparagine syn- children; thetase activity. Afterward, other mechanisms associated with asparaginase resistance were resistance reported. Not only leukemic cells but also patients themselves may play a role in causing as- paraginase resistance, which has been associated with unfavorable outcome in children with ALL. This article will briefly review asparaginase therapy in children with ALL and comprehen- sively analyze recent reports on the potential mechanisms of asparaginase resistance. Copyright ª 2015, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved.

1. Introduction Taiwan Childhood Cancer Foundation, there are about 130e140 newly diagnosed ALL children (younger than Acute lymphoblastic leukemia (ALL) is the most common 18 years), accounting for 25% of all childhood cancers in cancer in children. An estimated 6000 new cases of ALL are Taiwan. The treatment of childhood ALL in Taiwan has diagnosed yearly in the United States; roughly 60% of cases improved during the past decade. Overall survival improved e occur in people aged younger than 20 years.1 Recent ad- significantly from 75.5% in 1997 2001 to 83.5% in e 10 vances in intensive chemotherapy, as well as adequate 2002 2007. The high cure rate for ALL is attributable to supportive care, have made significant improvements in the improved supportive care, more precise risk stratifica- tion, and personalized chemotherapy based on the char- childhood ALL, so that approximately 80% of the children 12 are cured (Table 1).2e11 According to the registry of the acteristics of leukemic cells and hosts. Asparaginase is an enzyme isolated from various natural sources. However, only Escherichia coli and Erwinia chrys- E-mail address: [email protected]. anthemi asparaginase are currently available for medical http://dx.doi.org/10.1016/j.pedneo.2014.10.006 1875-9572/Copyright ª 2015, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved. 288 S.-H. Chen

Table 1 Results of selected clinical trials for pediatric acute lymphoblastic leukemia. Study Years of study No. of Age Event-free survival Overall survival Reference patients range (y) at 5 y (%) at 5 y (%) BFM-95 1995e1999 2169 0e18 79.6 0.9 87.0 0.7 Mo¨ricke et al3 COG 2000e2005 7153 0e21 NA 90.4 0.5 Hunger et al2 DCOG-9 1997e2004 859 1e18 80.6 1.4 86.4 1.2 Veerman et al4 DFCI 00-01 2000e2004 492 0e18 80.0 2.0 91.0 1.0 Vrooman et al5 JCCLSG ALL 2000 2000e2004 305 1e15 79.7 2.4 89.2 1.8 Yamaji et al6 Ma-Spore ALL 2003 2002e2011 556 0e18 80.6 3.5 89.2 2.7 Yeoh et al7 MRC UKALL 2003 2003e2011 3126 1e25 87.2 14 91.5 1.2 Vora et al8 NOPHO-2000 2002e2007 1023 1e15 79.4 1.5 89.1 11 Schmiegelow et al9 TPOG-2002 2002e2007 788 0e18 77.4 1.7 83.5 1.6 Liang et al10 SJCH XV 2000e2007 498 1e18 85.6 2.9 93.5 1.9 Pui et al11 Note. From “Biology, risk stratification, and therapy of pediatric acute leukemias: an update,” by C.H. Pui, W.L. Carroll, S. Meshinchi, R.J. Arceci, 2011, J Clin Oncol, 29, p. 551e65. ª 2011 American Society of Clinical Oncology. Adapted with permission.52. BFM Z BerlineFrankfurteMu¨nster; COG Z Children’s Oncology Group; DCOG Z Dutch Children’s Oncology Group; DFCI Z DanaeFarber Cancer Institute Consortium; JCCLSG Z Japanese Children’s Cancer and Leukemia Study Group; Ma-Spore Z MalaysiaeSingapore; MRC UKALL Z Medical Research Council United Kingdom Acute Lymphoblastic Leukemia; NA Z not available; NOPHO Z Nordic Society of Pediatric Hematology and Oncology; TPOG Z Taiwan Pediatric Oncology Group; SJCRH Z St. Jude Children’s Research Hospital. use. Polyethylene glycol (PEG)easparaginase is a conjugate regimens in pediatric ALL.21e23 Resistance to asparaginase of the native E. coli asparaginase covalently linked to PEG has been demonstrated to be an unfavorable prognostic at sites not affecting enzymatic activity. The characteris- factor.24e27 One may reasonably postulate that resistant tics of various asparaginase preparations are summarized in leukemic cells would have accesses to increase their aspara- Table 2. Asparaginase has been highly successful for gine in sufficient quantity to meet their needs. In recent years, inducing remissions in acute leukemia since 1967.13 Aspar- considerable effort has been made to address fundamental aginase exerts its antileukemic activity by converting aspar- questions about the role of ASNS in asparaginase resistance. agine to aspartic acid in the extracellular fluid.14 Most normal However, data from some recent studies on the importance of cells can synthesize asparagine, but leukemic lymphoblasts ASNS are conflicting (Table 3). In addition to the leukemic are sensitive to the depletion of extracellular asparagine, cells, there is increasing evidence of host factors that can putatively because they express low levels of asparagine affect asparaginase resistance. In this article, the mechanisms synthetase (ASNS)15e17 or they lack the ability to upregulate contributing to asparaginase resistance will be reviewed. the expression of ASNS when exposed to asparaginase.18,19 The relative inability of leukemic lymphoblasts to produce their own asparagine to compensate for the shortage of 1.1. Baseline high ASNS expression level in extracellular asparagine reduces asparagine-dependent leukemic cells synthesis, resulting in leukemic cell death.20 Extensive clinical data support the benefit of intensive Horowitz et al15 reported experiments on the ASNS activity asparaginase treatment compared with less intensive of several transplanted mouse leukemias. Seven different

Table 2 Main characteristics of various asparaginase preparations. Native Escherichia coli Erwinia chrysanthemi Pegylated form of native asparaginase asparaginase E. coli asparaginase Trade name Elspar Erwinase Oncospar Leunase Kidrolase Crasnitin Asparaginase medac FDA approval 1978 2011 1994 Half-life (d)47,53,54 1.08 e 1.35 (IM) 0.27 (IV) 5.5 e 5.7 (IM) 0.65 (IM) Route IV, IM IV, IM IV, IM Estimated equivalent doses 6000e10,000 U/m2 20,000e25,000 U/m2 2500e3500 U/m2 every 2 wk for complete asparagine every 2e3d every 2e3d depletion for 2 wk Frequency of antiasparaginase 20e42% 8e33% 2e11% antibody formation55e59 FDA Z Food and Drug Administration; IM Z intramuscular; IV Z intravenous. saaiaei ct ypolsi leukemia lymphoblastic acute in Asparaginase

Table 3 Summary of recent studies with conflicting conclusions on the impact of asparagine synthesis on asparaginase sensitivity in leukemic cells. Sample origin Specific cell lines/ALL subtypes Sample Methods to determine Relationship between The comparison of ASNS level Reference No. ASNS activity ASNS and ASP sensitivity in one subtype to other subtypes 28 NCI60 cancer cell lines * 6 Microarray ASNS[,GI50 [ Scherf et al Primary ALL TEL/AML1 and non-TEL/AML1 ALL 47 qRT-PCR No correlation TEL ALL > non-TEL ALL Stams et al32 Primary ALL TEL/AML1 ALL 30 qRT-PCR No correlation Stams et al32 Primary ALL Non-TEL/AML1 ALL 17 qRT-PCR No correlation Stams et al32 34 Primary ALL Non-TEL/AML1 ALL 117 qRT-PCR and microarray ASNS[,LC50 [ Stams et al y 30 Leukemic cell lines 16 Microarray ASNS[,LC50 [ Fine et al Primary ALL 38 Microarray No correlation Fine et al30 29 Primary ALL 173 Microarray ASNS[,LC50 [ Holleman et al Primary ALL TEL ALL 20 qRT-PCR Not mentioned TEL ALL > non-TEL ALL Krejci et al33 Non-TEL/AML1 ALL 23 Primary ALL TEL/AML1 ALL 57 Microarray Not mentioned TEL ALL < non-TEL ALL Iwamoto et al35 Non-TEL/AML1 ALL 231 Primary ALL Hyperdiploid ALL 51 Microarray Not mentioned Hyperdiploid ALL < Iwamoto et al35 Nonhyperdiploid ALL 237 nonhyperdiploid ALL Primary ALL T-ALL 47 Microarray Not mentioned T-ALL > B-lineage ALL Iwamoto et al35 B-lineage ALL 241 60 Leukemic cell lines Jurkat, SUP T1 2 qRT-PCR ASNS[,EC50 [ Estes et al Leukemic cell lines MOLT-4, Nalm6, REH 3 qRT-PCR No consistent correlation Su et al36 36 Leukemic cell lines MOLT-4, Nalm6, REH 3 Immunoblotting ASNS[,IC50 [ Su et al Note. From “A genome-wide approach identifies that the aspartate metabolism pathway contributes to asparaginase sensitivity,” by S.H. Chen, W Yang, Y Fan, G Stocco, K.R. Crews, J.J. Yang, et al., 2011, Leukemia, 25, p. 66e74.44 ª2011 Macmillan Publishers Ltd. ALL Z acute lymphoblastic leukemia; ASNS Z asparagine synthetase; ASP Z asparaginase; EC50 Z effective concentration leading to 50% cell death; GI50 Z concentration with 50% growth inhibition of cells; IC50 Z concentration with 50% inhibition to cell growth; LC50 Z concentration lethal to 50% of cells. * MOLT4, CEM, HL-60 (TB), SR, RPMI-8226, K562. y Nalm20, Nalm21, Nalm29, Nalm30, SUP-B15, HB, RS411, BV173, HAL, UOC, MV, Nalm27, Nalm6, REH, 697, RCH-ACV. 289 290 S.-H. Chen asparaginase-sensitive mouse leukemias proved to have no Abbatiello et al37 developed a highly sensitive and selective or only slight ASNS activity. In contrast, asparaginase- nanoflow reversed-phase liquid chromatography-tandem resistant leukemias showed appreciable ASNS activity. mass spectrometry (RPLC-MS/MS) technique to quantify Haskell and Canellos16 studied the ASNS levels in 18 leu- ASNS protein levels. They successfully measured the ASNS kemia patients who were subsequently treated with protein concentration directly in four patient blast sam- asparaginase. They found that the ASNS in leukemic cells ples. This new technique may help to reassess the corre- was nearly undetectable prior to therapy regardless of lation between ASNS expression and asparaginase subsequent response to asparaginase treatment, although resistance in the future. the ASNS level in the asparaginase-resistant group was In conclusion, a different sample size, determination of slightly higher than in the asparaginase-sensitive group. expressions, and origin of sample may account for the Kiriyama et al17 later showed that asparaginase-sensitive discrepancy in the results. However, these studies indicate human leukemic cell lines exhibited a lower baseline that the baseline ASNS level plays a role but is not the only ASNS level than asparaginase-nonsensitive ones. ASNS was factor for leukemic cells’ resistance to asparaginase. also reported as one of the top with at least 2-fold expression level in two resistant T-ALL cell lines at 17 1.2. Upregulation of ASNS expression in leukemic different maturation stages. cells in asparagine-depleted environment profiling studies by microarray analysis revealed conflicting results. Scherf et al28 showed that six In nine leukemia patients with paired ASNS activity deter- leukemic cell lines had very high negative correlation be- mination before and during/after asparaginase treatment, tween the ASNS expression and asparaginase sensitivity. the asparaginase-sensitive group showed no change in ASNS Holleman et al29 found that ASNS was one of the genes with therapy; however, there was a 7-fold increase in the discriminating asparaginase-sensitive and asparaginase- mean level of ASNS levels in the asparaginase-resistant resistant leukemic cells from newly diagnosed pediatric group.16 The difference between the ASNS levels in the ALL patients. Fine et al30 demonstrated that ASNS was one of sensitive and resistant groups, determined during/after the genes that had different asparaginase-sensitive and asparaginase treatment, was highly significant. asparaginase-resistant cell lines in accordance with previous Hutson et al19 also demonstrated that resistance to reports. However, baseline ASNS expression was not associ- asparaginase in leukemic cell lines was mediated in vitro by ated with asparaginase sensitivity in clinical ALL samples. an upregulation of ASNS expression in response to aspara- The t(12;21)þ ALL, which harbors the TEL/AML1 gene gine depletion of the culture medium. Aslanian et al18 fusion and accounts for about 25% of pediatric ALL, was further examined the reversibility of the upregulation of found to be significantly related to in vitro drug sensitivity ASNS caused by asparaginase treatment in asparaginase- for asparaginase.31 The simple paradigm would be sensitivity resistant leukemic cells. They found that the elevated to asparaginase in t(12;21)þ ALL emerged via a low baseline ASNS expression in the resistant cells was not fully reversed expression of ASNS. Surprisingly, Stams et al32 showed that even 6 weeks after asparaginase had been removed from the expression of ASNS in t(12;21)þ ALL was significantly the culture medium. higher than in t(12;21) ALL. The ASNS expression did not However, asparaginase-sensitive leukemic cells were differ in sensitive and resistant t(12;21)þ ALL, either. The later revealed also to upregulate ASNS after asparaginase same trend was also demonstrated by Krejci et al.33 Both treatment. Stams et al32 exposed samples from t(12;21)þ studies had only a small number of patient samples and used and t(12;21) ALL patients in vitro to asparaginase. Almost real-time quantitative polymerase chain reaction (RTQ-PCR) all samples showed upregulation of ASNS, which was inde- to determine ASNS mRNA expression. Although ASNS pendent from t(12;21) status and cellular sensitivity to expression was not linked to asparaginase resistance in asparaginase. Appel et al38 also demonstrated that the t(12;21)þ ALL, Stams et al34 later reported that the ASNS upregulation of ASNS occurred in both sensitive and resis- expression determined by either RTQ-PCR or microarray tant cases within 24 hours of in vivo exposure to aspar- significantly differed between sensitive and resistant aginase. These data imply that mechanisms other than t(12;21)þ ALL. With a larger sample size and microarray upregulated expression levels of ASNS contribute to cellular analysis, a different result was shown by Iwamoto et al.35 The asparaginase resistance. ASNS expression levels were found to be significantly lower in t(12;21)þ ALL compared to t(12;21) ALL. Notably, the quantification of ASNS expression in 1.3. Increased substrates for ASNS reaction in leukemic cells by mRNA is limited by the fact that the leukemic cells relationship between the ASNS mRNA and intracellular protein concentrations in primary ALL has not been estab- Aslanian and Kilberg39 found that multiple adaptive changes lished. Su et al36 tested the correspondence between ASNS affect the ASNS substrate availability in asparaginase- mRNA expression by RTQ-PCR and ASNS protein content by resistant leukemic cell line. These changes appear to sup- immunoblotting. Asparaginase resistance was estimated by port increased asparagine biosynthesis by increasing the half-maximal inhibitory concentration (IC50) after exposure intracellular levels of aspartate and glutamine. Asparaginase- to asparaginase. Their observations demonstrated that the resistant cells produced more glutamine by an increase in correlation between ASNS protein and IC50 was much better glutamine synthetase (GS) activity. These cells also enhanced than that between the IC50 and the ASNS mRNA content. the uptake of extracellular glutamine via secondary active This may imply that measuring the ASNS protein rather than Naþ-dependent transporters. However, there was little or mRNA can predict asparaginase resistance. More recently, no aspartate uptake by these cells. Recently, Appel et al25 Asparaginase in acute lymphoblastic leukemia 291 demonstrated that the levels of intracellular amino acids did genetic variation is a key determinant for the interindi- not significantly differ among different in vitro sensitivity vidual differences in treatment resistance. Luo et al43 cases or in vivo clinical responders. Therefore, the contri- identified that the single nucleotide polymorphism in the bution of the ASNS substrate, aspartate, and glutamine’s promotor region of the ASNS gene, e92G>A, is associated resistance to asparaginase remains unclear. with increased ASNS expression and is more common in pediatric leukemic patients than among normal control in- 1.4. Influence on protein synthesis and in dividuals. We used an agnostic genome-wide approach to leukemic cells identify inherited genomic variants that contribute to asparaginase resistance using normal lymphoblastoid cell lines from 87 trio members of European ancestry and 54 Protein synthesis is fundamental in all living cells, both primary ALL leukemic blast samples at diagnosis.44 We benign and malignant. Amino acid deficiency impairs pro- found that the top-ranked pathway was that of aspartate tein synthesis and leads to apoptosis and cell death. Many metabolism, which may be linked directly to the mecha- tRNA syntheses and amino acid transporters were shown to nism of action of asparaginase. The two most highly ranked increase expression in human cells with amino acid star- genes (ADSL and DARS ) in this pathway encompassed seven vation.30 In asparaginase-resistant ALL cells, several ribo- single nucleotide polymorphisms. Moreover, we found that somal protein genes were overexpressed, indicating the more sensitive ALL subtypes (hyperdiploid and TEL-AML1) possible role of protein synthesis in asparaginase resis- had lower ADSL expression than the more resistant sub- tance.29 Holleman et al40 further analyzed the expression types (T-ALL), which is consistent with higher ADSL patterns of 70 apoptosis genes and tested the association expression associated with resistance. between the expression of these genes and in vitro drug Because asparaginase is a foreign protein, hypersensi- resistance in B-lineage ALL cells. Of the 70 apoptosis genes, tivity reactions due to antiasparaginase antibody produc- three were associated with asparaginase resistance. Over- tion are common, occurring in up to 45% of patients.45 The expression of BCL2L13, one of the genes associated with development of these antibodies appears to be more asparaginase resistance, was also associated with an unfa- commonly observed in native E. coli asparaginase than in vorable clinical outcome. the pegylated enzyme.46 The development of anti- asparaginase antibodies may shorten the half-life of 2. Host factors asparaginase, prevent or delay absorption after intramus- cular injection of asparaginase, or interfere with aspar- There is increasing interest in the host factors resulting in aginase activity.47e49 Therefore, it confers resistance to asparaginase resistance of leukemic cells. Iwamoto et al35 asparaginase therapy and reduced therapeutic efficacy in described the protective effect of mesenchymal cells in some clinical studies.50,51 leukemic cells against asparaginase treatment in bone marrow microenvironment. They showed that bone marrow 3. Conclusion mesenchymal cells express significantly higher amounts of ASNS than leukemic cells. They then showed that the resistance of leukemic cell lines and some specimens of The intensive administration of asparaginase improved the primary leukemia to asparaginase correlated with the treatment outcomes in children with ALL. It is crucial to expression of ASNS by mesenchymal cells. Forced increased recognize the mechanisms for the development of both expression of ASNS by the mesenchymal cell leads to in vitro and in vivo resistance by leukemic cells. The role of increased resistance of the ALL cells; conversely, reduced the host factor is increasing. Adequate asparaginase expo- expression of ASNS by the mesenchymal cell leads to sure may result in a lower clearance of steroids, which may enhanced sensitivity of the ALL cells. potentiate the cytotoxicity of vincristine. Therefore, it is The childhood obesity problem has become epidemic in important for clinicians to choose the appropriate man- the United States and other industrialized countries. In vivo agement in patients with asparaginase resistance. Switch- and in vitro models have shown that obesity impairs the ing to another preparation may be a difficult choice. efficacy of chemotherapeutics against ALL cells, likely Measurement of asparaginase level during asparaginase mediated by adipocytes.41 Ehsanipour et al42 examined treatment may help to guide the decision of switching bone marrow biopsy specimens from four obese and four preparations or dose escalation. Further studies should lean adolescent leukemia patients for expression of ASNS continue investigating and promoting effective aspar- and GS. They found that the GS expression was markedly aginase therapy individually. increased in bone marrow adipocytes after induction chemotherapy, whereas ASNS expression appeared to be Conflicts of interest unaltered after treatment. They further cultured ALL cells over fibroblasts or adipocytes in media with asparaginase. The author has no conflicts of interest relevant to this They observed that adipocytes protected ALLs from aspar- article. aginase treatment via release of glutamine, which com- plemented the previous finding from Iwamoto et al.35 The protective effect from such microenvironment is still open Acknowledgments to debate. In recent years, pharmacogenetics/pharmacogenomics The author thanks Dr Mary Relling of St. Jude Children’s in childhood ALL has become a major field of research. The Research Hospital in Memphis, Tennessee, for advice. 292 S.-H. Chen

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