1 Asparagine Synthetase: Regulation by Cell Stress and Involvement in Tumor Biology 2 3 Authors: Mukundh N. Balasubramanian, Elizabeth A. Butterworth, and Michael S. Kilberg

Affiliations: Department of Biochemistry and Molecular Biology, Shands Cancer Center and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida 32610 4 5 Running head: Regulation and Function of Asparagine Synthetase 6 7 Address Correspondence to: Michael S. Kilberg, PhD, Dept. of Biochemistry and Molecular 8 Biology, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, Tel: 9 352-392-2711, Fax: 352-392-6511, email: mkilberg@ufl.edu 10

Acronyms: AA, amino acids; AAR, amino acid response; AARE, amino acid response element; ALL, acute lymphoblastic leukemia; ASNase, asparaginase; ASNS, asparagine synthetase; ATF, activating transcription factor; CARE, C/EBP-ATF response elements; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; CHOP, C/EBP homology protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCN2, general control non-derepressible; HisOH, histidinol; HAT, histone acetyltransferase; JNK, JUN N-terminal kinase; MAPK, mitogen-activated protein kinases; mTOR, mammalian target of rapamycin; qPCR, real time quantitative PCR; SNAT2, sodium-dependent neutral amino acid transporter 2; Sp1 or Sp3, Specificity protein 1 or 3; TRB3, Tribbles homolog 3; UPR, unfolded protein response 11

Articles in PresS. Am J Physiol Endocrinol Metab (February 12, 2013). doi:10.1152/ajpendo.00015.2013

Copyright © 2013 by the American Physiological Society.

2 12 Abstract:

Asparagine synthetase (ASNS) catalyzes the conversion of aspartate and glutamine to

asparagine and glutamate in an ATP-dependent reaction. The enzyme is ubiquitous in its organ

distribution in mammals, but basal expression is relatively low in tissues other than the exocrine

pancreas. Human ASNS activity is highly regulated in response to cell stress, primarily by

increased transcription from a single gene located on chromosome 7. Among the genomic

elements that control ASNS transcription is the C/EBP-ATF response element (CARE) within the

promoter. Protein limitation or an imbalanced dietary amino acid composition activate the ASNS

gene through the amino acid response (AAR), a process that is replicated in cell culture through

limitation for any single essential amino acid. Endoplasmic reticulum stress also increases ASNS

transcription through the PERK-eIF2-ATF4 arm of the unfolded protein response (UPR). Both the

AAR and UPR lead to increased synthesis of ATF4, which binds to the CARE and induces ASNS

transcription. Elevated expression of ASNS protein is associated with resistance to asparaginase

therapy in childhood acute lymphoblastic leukemia and may be a predictive factor in drug

sensitivity for certain solid tumors as well. Activation of the GCN2-eIF2-ATF4 signaling pathway,

leading to increased ASNS expression appears to be a component of solid tumor adaptation to

nutrient deprivation and/or hypoxia. Identifying the roles of ASNS in fetal development, tissue

differentiation, and tumor growth may reveal that ASNS function extends beyond asparagine

biosynthesis.

Keywords: amino acid metabolism, asparaginase, ATF4, nutrient sensing, unfolded protein 13 response 14

3Asparagine Synthetase Enzyme Function 15

The mammalian asparagine synthetase (ASNS) enzyme catalyzes the ATP-dependent 16 conversion of L-aspartate and L-glutamine into L-asparagine and L-glutamate (reviewed in 73). 17 The biochemical characterization of ASNS activity has been studied following purification from 18 mammalian cells and recombinant systems of both bacterial and mammalian origin. Early 19 investigation revealed that the reaction requires magnesium ions and ATP, and involves formation 20 of a β-aspartyladenylate intermediate (68). The β-aspartyladenylate intermediate is converted to L-21 asparagine by transfer of ammonia from L-glutamine to yield L-glutamate and AMP as the 22 remaining products (74). There are two bacterial forms of ASNS, one that uses ammonia (AS-A) as 23 the nitrogen donor and a second that uses glutamine as the nitrogen donor (AS-B) (73). The latter 24 reaction is analogous to that performed by the mammalian enzyme and structural studies on the 25 Escherichia coli glutamine-dependent AS-B have revealed the presence of two distinct catalytic 26 domains, an N-terminal amido-transferase domain and a C-terminal ATP-pyrophosphatase domain 27 bridged by a intramolecular tunnel that allows for ammonia to shuttle between the two domains 28 (56). Whereas the protein’s name directs focus on its function in asparagine synthesis, the reaction 29 it catalyzes may impact the cellular levels of the other three reactants as well. Given the critical 30 function of glutamine as an oxidizable energy source, a key interorgan nitrogen carrier, and a 31 mammalian target of rapamycin (mTOR) regulator, the possible impact of ASNS activity should 32 also be considered when evaluating glutamine homeostasis. 33

The level of ASNS expression among tissues in adult animals varies considerably. Based 34 on a direct comparison of enzyme specific activity in many tissues, the pancreas was shown to 35 exhibit much greater expression than any other tissue analyzed (63, 64). This distribution is 36 consistent across many species including humans, rodents, birds, and ox (63), and the prevalence 37 of higher pancreatic ASNS expression has been confirmed at the protein level by immunoblotting 38 using both polyclonal (47) and monoclonal (Figure 1, Richard Hutson and Michael Kilberg, 39

4unpublished results) antibodies. As illustrated by immunohistochemistry of human pancreatic 40 tissue, pancreatic ASNS protein expression is largely associated with the exocrine cells (33). After 41 fasting mice for 54 h or feeding them an asparagine-free diet for 10 days, the pancreatic ASNS 42 activity was unaltered (64), in apparent contrast to the nutritional regulation of ASNS observed for 43 other tissues, as discussed below. The pancreas does not release significant amounts of 44 asparagine into the circulation and radioactive incorporation studies suggested that the bulk of 45 newly synthesized asparagine is used for protein synthesis (64). It is tempting to speculate that 46 serum ASNS activity may be a valuable marker for pancreatic exocrine cell lysis as Cooney et al. 47 observed release of ASNS protein from murine primary tumors into the serum at a rate proportional 48 to tumor growth (27). 49

50 The Mammalian Asparagine Synthetase Gene 51

The human ASNS gene, a schematic of which is illustrated in Figure 2, was first assigned to 52 chromosome 7 by analyses of somatic cell hybrids (9). Greco et al. established that the ASNS 53 locus is derived from the long arm of human chromosome 7 and in situ hybridization mapped the 54 locus more precisely to chromosome region 7q21.3 (38, 44). An ASNS cDNA was first cloned from 55 Chinese hamster ovary (CHO) cells (71) and subsequently, human clones containing the complete 56 ASNS coding sequence were obtained (6). Zhang et al. determined that the human genomic 57 sequence for ASNS was approximately 35 kb long and organized into 13 exons (100). The authors 58 assigned the transcriptional start site of ASNS to be 203 nucleotides (nt) upstream of the 59 translation start site. Greco et al. discovered that a temperature-sensitive hamster gene that 60 controlled cell cycle progression, called ts11, was ASNS and characterized the human ASNS 61 counterpart to show that the first two exons were non-coding, with translation starting in the third 62 exon (38, 39). Both the Andrulis and Basilico groups determined that the region surrounding the 63 transcription start site of the human ASNS gene has a high CG content, a circumstance that we 64

5now recognize as a promoter-associated CG Island (38, 100). Greco et al. (38) discovered that the 65 human ASNS gene exhibited multiple transcription start sites, with one major site at 117 nt 66 upstream of the translation start site. Chen et al. further characterized the multiple transcription 67 initiation sites for the human ASNS gene during AA deprivation of HepG2 human hepatoma cells 68 (24). Those investigators used both 5´ rapid amplification of cDNA ends and a ribonuclease 69 protection assay to establish that multiple transcription initiation sites were spread across a 70 nt 70 region for cells cultured in amino acid (AA) complete medium. However, following AA limitation, 71 which induces ASNS transcription, there was a shift towards a predominant utilization of the 72 transcription initiation site 117 nt upstream of the translation start site originally identified by Greco 73 et al. (38). This transcription start site will be used as nt +1 to designate all genomic sequences in 74 this review. 75

76 Regulation of ASNS by Nutrient Limitation and Other Forms of Cell Stress 77

Protein or Amino Acid Limitation: The Amino Acid Response 78 Mammals exhibit a wide spectrum of adaptive processes that serve to sense and respond to 79

fluctuations in the diet, including dietary protein or AA imbalance. To illustrate the dynamic nature of 80 this response, within 1 h after mice consumed a leucine-free diet there was increased expression of 81 AA-responsive genes; conversely, within 2 h after re-feeding a leucine-replete diet the changes were 82 reversed (21). Expression array analysis in rats revealed that a diet deficient in total protein or limited 83 in one or more of the essential AA, as many of the plant-derived proteins are, resulted in altered 84 gene expression well beyond that for AA metabolism. For example, Endo et al. discovered that 85 compared to casein, feeding rats a diet containing gluten, which is lysine- and threonine-deficient, 86 resulted in significant changes in enzymes involved in cholesterol metabolism (34). Likewise, 87 deprivation of cells in culture for a single essential AA (32) or feeding mice a leucine-free diet (41) 88 results in a suppression of fatty acid synthase expression and changes in lipid metabolism. 89

6Genomic analysis in vivo is complicated because most tissues contain multiple cell types, 90

which compromises the interpretation of mechanistic data with regard to cell-specific responses. 91 Furthermore, in vivo studies do not allow for a direct test of AA regulation because circulating AA 92 levels will modulate the release and action of hormones and growth factors (76). Consequently, 93 considerable work has been performed in cell culture model systems, which has allowed the 94 identification of multiple signaling pathways collectively referred to as the AA response (AAR) (Figure 95 3). The AAR controls gene expression through changes in chromatin structure, transcription start 96 site, transcription rates, mRNA splicing, RNA export, RNA turnover, and translation initiation 97 (reviewed in 52). Among the AAR pathways, GCN2-eIF2-ATF4 (general control non-derepressible 2 98 - eukaryotic initiation factor 2 - activating transcription factor 4) appears to be the predominant 99 signaling mechanism that activates transcription from the ASNS gene during the AAR as induction of 100 ASNS expression is blocked in ATF4-deficient cells (78, 81, 87). GCN2 is a well-characterized 101 cellular AA monitoring mechanism that is conserved from yeast to humans. The GCN2 protein has 102 an inherent kinase activity that is increased when the protein binds any one of the uncharged tRNA 103 molecules, thus GCN2 has the ability to sense the level of all 20 of the AA typically associated with 104 protein synthesis. Activated GCN2 phosphorylates eIF2 on its alpha subunit, which suppresses 105 general protein synthesis (reviewed in 45, 53, 95, 99), but promotes a paradoxical increase in 106 translation of selected mRNA species. As discussed in more detail below, in mammalian cells this 107 translational control mechanism includes the transcription factors ATF4 and ATF5 (62, 91, 102). 108

Long before molecular probes were available for ASNS analysis, Arfin et al. demonstrated 109 that CHO cells incubated in asparagine-free medium exhibit reduced aminoacylation of 110 asparaginyl-tRNA and an increased level of ASNS enzymatic activity (10). Likewise, Andrulis 111 showed that ASNS activity was elevated when cells containing a temperature-sensitive 112 asparaginyl-, leucyl-, methionyl- or lysyl-tRNA synthetase mutant were transferred to the non-113 permissive temperature (7). Those data are now understood to reflect the role of GCN2 as the 114 sensor of amino acid availability by monitoring the level of each of the uncharged tRNAs. After 115

7discovering that the cell cycle defect of cells harboring the ts11/ASNS mutation could be reversed 116 by asparagine supplementation, Basilico and colleagues were the first to show that ASNS mRNA 117 was induced by AA limitation of mammalian cells, not only for asparagine, but also for leucine, 118 isoleucine, or glutamine (37). Hutson and Kilberg extended those studies by reporting that ASNS 119 mRNA level increased following limitation for all AA or for a single essential AA (48). Consistent 120 with the earlier reports that asparagine levels regulated ASNS enzymatic activity (10), after AA 121 limitation there is increased association of ASNS mRNA with polysomes (50) and increased ASNS 122 protein production as revealed by pulse-chase labeling (49). 123 124 Endoplasmic Reticulum Stress: The Unfolded Protein Response 125

GCN2 is only one of four eIF2 kinases that mediate the response to a variety of cellular 126 stresses (reviewed in 95). A second eIF2 kinase that impacts ASNS expression is the double-127 stranded RNA-activated protein kinase-like endoplasmic reticulum (ER) kinase (PERK) (Figure 4). 128 The PERK signaling pathway, together with the ATF6 and IRE-1 pathways, make up what is referred 129 to as the Unfolded Protein Response (UPR), which is activated by cellular insults such as ER protein 130 synthesis overload, oxidative stress, or other circumstances that perturb ER function. Like GCN2, 131 activation of PERK leads to phosphorylation of eIF2, suppression of global translation, and increased 132 ATF4 synthesis (42). Consequently, the ATF4-dependent downstream transcriptional programs for 133 the AAR and UPR overlap. Thus, in many instances either AA limitation or ER stress will lead to 134 transcriptional activation of common ATF4-responsive target genes, including ASNS (14-16). 135 However, there are also differences in the gene profile activated by GCN2 and PERK signaling, 136 indicating that factors other than ATF4 add specificity to each pathway (29). 137 138

Genomic Sequences that Control ASNS Transcription During the AAR and UPR 139 140 C/EBP-ATF Response Element (CARE) 141

8After discovering that the ASNS gene was regulated by AA availability, the Basilico group 142

analyzed the promoter region to identify the essential cis-regulatory elements (40). A region 143 around nt -68 was indispensible for the response and was designated as an “amino acid response 144 element” (AARE). This was the first description of an AA-responsive genomic element in a 145 mammalian cell. To further dissect the ASNS promoter, Barbosa-Tessmann et al. used a 146 combination of in vivo footprinting, deletion analysis, and site-directed mutagenesis to identify the 147 specific nucleotide sequences that mediated the AAR-induced transcription (15). The authors 148 determined that two distinct regions within the human ASNS promoter were both necessary for 149 AAR-sensitivity, one from nt -68 to -60 with the sequence 5´-TGATGAAAC-3´ (initially called 150 “nutrient-sensing response element 1”, NSRE1) was the same as that identified by Basilico and 151 colleagues and a second previously undetected site from nt -48 to -43 with the sequence 5´-152 GTTACA-3´ (NSRE2). The NSRE1 sequence is now referred to as a C/EBP-ATF response 153 element (CARE). As is the hallmark of enhancer elements, the NSRE1/NSRE2 “unit” functioned 154 independently of location or orientation, and was also functional when linked to heterologous 155 promoters. However, Zhong et al. showed that the 5′ to 3′ orientation of NSRE1 relative to NSRE2 156 is not reversible and that the 11 nt spacer region between the NSRE1 and NSRE2 sequences is 157 required for enhanced transcription (101). The requirement for approximately 10 bp between the 158 NSRE1 and NSRE2 suggests that it is functionally necessary for these sites to face the same side 159 of the DNA helix. 160

While the trans-acting factors that bind to the NSRE2 site have not been identified, some of 161 those that bind to the NSRE1 sequence have been established and they include several members 162 of the activating transcription factor (ATF) and CCAAT/enhancer-binding protein (C/EBP) 163 subfamilies of the basic-leucine zipper (bZIP) superfamily of transcription factors (81, 82). The 164 ASNS NSRE1 sequence, composed of a half-site for the C/EBP family and a half-site for the ATF 165 family of transcription factors, is just one example of a family of related sequences with the 166

9consensus sequence of 5′-TGATGXAAX-3′ that are present in a wide spectrum of genes activated 167 by the AAR and/or the UPR (52). This type of “C/EBP-ATF composite site” element was first 168 identified by Wolfgang et al. within the C/EBP-homology protein (CHOP) gene (96) and the same 169 laboratory showed that following arsenite treatment, ATF4 binds to the element as an activator, but 170 is later displaced by ATF3 which represses transcription (35). To continue the use of the “C/EBP-171 ATF” nomenclature, we refer to this family of sequences, including the ASNS NSRE1 site, as 172 C/EBP-ATF response elements (CARE). Following the determination that ER stress, initiated by 173 either carbohydrate deprivation or disruption of protein folding, leads to induction of ASNS 174 transcription (14, 16), analysis of the promoter established that the same two elements that were 175 responsible for the response to AA limitation, NSRE1 and NSRE2, were also necessary for the 176 transcriptional response to the UPR (15). The sensitivity of the gene to limitation of either AA or 177 glucose is the reason that those investigators coined the term NSRE. Although these genomic 178 sequences exhibit “AARE activity”, their responsiveness to a range of other cellular stress signals 179 is also clear. 180

The in vivo footprinting studies of the human ASNS gene also revealed constitutive protein 181 binding to three GC-boxes located upstream of the promoter-localized CARE (15). These sites are 182 spread across the region covered by nt -153 to -93 and were discovered to be critical for 183 maintenance of basal transcription, but were also required for a maximal response to AA 184 deprivation. Upon further characterization of the three GC-boxes, it was established that 185 exogenous expression of Sp1 supported basal ASNS promoter activity only, whereas expression 186 of Sp3 enhanced the basal activity and permitted AAR-induced ASNS transcription (57). 187 188 The Network of Transcription Factors that Control the ASNS Gene 189

Chen et al. employed chromatin immunoprecipitation (ChIP) assays to show that within 30-190 45 min after initiating AA limitation in HepG2 hepatoma cells, newly synthesized ATF4 rapidly 191 translocated to the nucleus and was associated with the ASNS proximal promoter region (25). The 192

10amount of ATF4 binding continuously increased up to 4 h, coinciding with increased histone 193 acetylation, recruitment of the general transcription machinery, and the peak of ASNS 194 transcription. The authors observed that a subsequent decline in ASNS transcription between 4 195 and 24 h of continued AA limitation is mirrored by a gradual decline in ATF4 binding and a parallel 196 increase in ATF3, C/EBPβ, and CHOP recruitment to the CARE site (25, 85). This auto-regulatory 197 feedback cycle results from ATF4-dependent induction of the C/EBPβ, ATF3, and CHOP 198 transcription followed by a subsequent recruitment of the corresponding proteins to the ASNS 199 promoter (23, 66, 85). Subsequently, this self-limiting cycle for ATF4-dependent transcriptional 200 activation was also observed for numerous CARE-containing genes (58, 66). While the 201 transcriptional induction of other AA-responsive genes, such as CHOP, requires the histone 202 acetyltransferase ATF2, ASNS exhibits less dependence on this ATF family member (18, 36). 203 Therefore, although histone acetylation is an important component of the transcriptional activation 204 of the ASNS gene following AA limitation, additional acetyltransferases appear to be involved (13, 205 25). 206

The ATF5 protein is homologous to ATF4 (3) and its synthesis is also subject to AA-207 dependent control through the GCN2-eIF2 translation mechanism (93, 102). Over-expression of 208 ATF5 activates transcription driven by the ASNS promoter through the CARE site and, like CHOP’s 209 antagonism of ASNS induction by ATF4 (85), CHOP also counteracts ATF5 with regard to ASNS 210 regulation (3). Interestingly, Al Sarraj et al. showed that ATF4 was required to induce ATF5 211 expression and to support the ATF5-dependent transcription of ASNS. Although the ATF4-212 dependence for induction of ATF5 expression was confirmed by Su et al. (87), they did not 213 observe a decrease in ASNS induction by AA limitation following ATF5 knockdown. Thus, the 214 exact role of ATF5 in regulating the ASNS gene remains unresolved. However, an additional link 215 between ASNS expression and ATF5 arises from the observation that ATF5 polymorphisms have 216 been linked to an altered response to childhood acute lymphoblastic leukemia (ALL) therapy. 217 During treatment for childhood ALL, patients are given Asparaginase (ASNase), a component of 218

11combination chemotherapy (see below). ASNase therapy causes depletion of plasma asparagine 219 followed by the loss of intracellular asparagine (26). Due to the lack of a rapid up-regulation of 220 ASNS protein content in ALL cells, they are preferentially sensitive to ASNase (11, 86). Rousseau 221 et al. observed that ALL patients with a T1562C polymorphism within the ATF5 gene have 222 decreased event-free survival times after ASNase therapy (77). The T1562C polymorphism results 223 in higher ATF5 promoter activity and the investigators proposed that elevated ATF5-driven 224 expression of ASNS within the leukemia cells causes decreased sensitivity to ASNase therapy. 225

226 Clinical Relevance of ASNS 227

ASNS and ASNase Treatment of Childhood Acute Lymphoblastic Leukemia 228 Asparagine metabolism in transformed cells has received considerable attention after it was 229

determined that certain types of tumors are susceptible to ASNase treatment (17). Primary ALL 230 cells and many ALL cell lines exhibit a particularly low level of ASNS expression (12, 75) and 231 therefore, are unusually sensitive to asparagine depletion. Conversely, drug-selected ASNase-232 resistant ALL cell lines exhibit elevated expression of ASNS (11, 30) and over-expression of 233 exogenous ASNS protein alone results in an ASNase-resistant phenotype in the absence of drug 234 selection (11). Clinically, the relationship is less clear. In a survey of 173 patients, Holleman et al. 235 observed a correlation between in vitro ASNase sensitivity and ASNS mRNA abundance 236 measured by oligonucleotide arrays (46). Although ASNS was not one of the top genes associated 237 with ASNase resistance, ASNS mRNA levels were about 3-fold higher in the resistant patients. 238 However, several clinical studies have shown a lack of correlation between ASNS mRNA levels 239 and ASNase sensitivity in ALL patients (8, 46, 55, 83, 84). One feature of all of these investigations 240 was that ASNS mRNA was analyzed, rather than ASNS protein content or enzymatic activity. Su et 241 al. addressed this apparent conflict by demonstrating that ASNS protein level, not mRNA content, 242 serves as a prognostic indicator of ASNase sensitivity in ALL cells (86). The molecular basis for 243

12this observation appears to be a significant time delay between up-regulation of ASNS mRNA 244 following ASNase treatment and the translation of that mRNA into ASNS protein. Interestingly, the 245 correlation of ASNase sensitivity to ASNS protein, rather than mRNA, was later observed for 246 several human ovarian tumor lines as well (59). The molecular mechanism for the delay between 247 mRNA synthesis and protein expression has not been established, nor is there any published 248 evidence for post-translational modification of the ASNS protein. 249

One aspect of ASNase treatment that has not been fully explored is the effect of the drug on 250 normal tissues, despite the fact that ASNase therapy is associated with toxicity. Bunpo et al. 251 demonstrated that ASNase treatment leads to the induction of the AAR in the liver, as illustrated by 252 induction of hepatic ASNS and down regulation of mTOR (20). Using a Gcn2 knockout mouse 253 model, the authors showed that the ASNase action was mediated through a Gcn2-dependent 254 mechanism. In a subsequent study, they also documented that the Gcn2 dependence extended to 255 the immunosuppressive action of ASNase on the thymus and spleen (19). The studies in Gcn2 256 knockout mice suggest that if there are humans with a deficiency in GCN2 activity they may be 257 even more susceptible to ASNase toxicity. Collectively, these results provide mechanistic insight 258 into the consequences of ASNase anti-tumor therapy on normal tissues and suggest that co-259 treatment with ASNase and GCN2-specific inhibitors may be even more effective than ASNase 260 alone. 261 262 Methylation of the ASNS Gene 263

A number of investigations have focused on a possible correlation between ASNase sensitivity 264 and the DNA methylation status of the ASNS gene. Sugiyama et al. proposed that the ASNS gene 265 in asparagine-auxotrophic Jensen rat sarcoma cells was silenced by DNA hypermethylation 266 because ASNS expression was detectable after 5-azacytidine (5-Aza-C) treatment, which leads to 267 hypomethylation of the genome (88). Subsequently, several studies in asparagine-dependent and 268 asparagine-independent cell lines have revealed a correlation between the DNA methylation within 269

13the ASNS gene locus and ASNS expression (5, 70, 97). In the context of human leukemic cell 270 lines, Ren et al. also observed that a high degree of ASNS promoter methylation correlated with 271 lack of ASNS expression and that 5-Aza-dC treatment enhanced ASNS expression (72). 272 Furthermore, by electromobility shift analysis with nuclear extracts from the leukemia cells, the 273 authors detected the presence of a “methyl binding protein” that was bound to an oligonucleotide 274 that contained a methylated CpG site near the ASNS CARE sequence. Interestingly, those authors 275 observed that binding of ATF4 and several C/EBP members was significantly diminished when the 276 methylated oligonucleotide was compared to the same sequence in the unmethylated state (72). 277 They postulated that in vivo the methyl binding protein might compete with transcriptional 278 activators of ASNS expression, including ATF4. Ding et al. co-cultivated asparagine-dependent 279 human leukemia cells with mouse peritoneal macrophages and observed subsequent 280 demethylation of the ASNS promoter, enhanced ASNS expression, and acquisition of asparagine-281 independence (31). In ALL bone marrow samples, 74% of B-cells and 83% of T-cells displayed 282 methylation of the ASNS promoter, in contrast with a lack of methylation observed in brain and 283 breast tumors (2). Based on these studies, Akagi et al. hypothesized that ASNS methylation may 284 underlie the susceptibility of ALL cells to ASNase chemotherapy (2). 285 286 ASNS and Solid Tumor Growth 287

In contrast to ALL, the potential relationship between ASNS expression and solid tumor 288 initiation/promotion has not been as extensively studied. Koumenis and colleagues investigated 289 the role of ATF4 in tumor cell survival and proliferation using an ATF4 shRNA knockdown strategy 290 (98). They observed that after ATF4 knockdown in HT1080 fibrosarcoma and DLD1 colorectal 291 adenocarcinoma cells, survival was reduced in the absence of non-essential AA. The authors 292 showed that reduced proliferative capacity and increased apoptosis was correlated with decreased 293 ASNS expression in the ATF4 deficient cells. Supplementation of shATF4-expressing tumor cells 294

14with asparagine, but no other individual amino acid, reversed the increased apoptosis and 295 autophagy leading to increased cell survival. Ye et al. sought to further define the mechanism of 296 ASNS activation in transformed cells and discovered that GCN2 was activated in response to 297 shATF4 knockdown, presumably due to lower ASNS expression and therefore, asparagine 298 deficiency (98). Once again, supplementation of the cells with asparagine repressed the activation 299 of GCN2. When the authors probed for the ability of transformed GCN2+/+ and GCN2-/- cells to form 300 tumors in a xenograft model, they observed that tumor growth was impeded in the GCN2-/- cells. 301 As eIF2 is the sole known substrate for GCN2, the researchers concluded that activation of the 302 GCN2-eIF2-ATF4 pathway: 1) increases the survival and proliferative capabilities of tumor cells 303 undergoing nutrient limitation, 2) is required for starvation-activated autophagy in transformed 304 cells, and 3) induces ASNS as a key factor in tumor initiation and growth under AA limiting 305 conditions (98). The exact role of ASNS and its product asparagine in modulating tumor growth is 306 unknown. The most obvious explanation, protein synthesis, seems too simplified as other AA 307 synthesis pathways are not as highly regulated and other AA do not appear to play as critical a 308 role. 309

Dufour et al. used immunohistochemistry to screen 98 human pancreatic ductal carcinomas 310 and determined that ASNS expression was low or below detection in about 70% of the patients 311 (33). These results suggest that some pancreatic tumors may be susceptible to ASNase therapy, 312 although clinical trials using ASNase were disappointing because of toxicity. Cui et al. used 313 expression array analysis to identify genes that were induced in pancreatic tumor cells exposed to 314 low glucose and one of those identified was ASNS (28). As mentioned above, ASNS transcription 315 is induced via the UPR pathway in response to glucose deprivation (14-16). Cui et al. proposed 316 that the function of ASNS up-regulation in response to low glucose is to protect the pancreatic 317 cancer cells from apoptosis based on the observation that ASNS over-expression suppressed JUN 318 N-terminal kinase (JNK) activation and reduced apoptosis, whereas ATF4 knockdown increased 319 the susceptibility of the cells to apoptosis. In addition to greater tolerance for glucose limitation, 320

15pancreatic cancer cells over-expressing ASNS exhibited increased resistance to apoptosis induced 321 by cis-diamine-dichloroplatinum (CDDP), a result also linked to the suppression of JNK activation 322 by ASNS (28). 323 In addition to JNK, extracellular-regulated kinase (ERK) is activated by AA limitation and 324 influences downstream signaling in the AAR (1, 22, 36, 65, 69, 90). Among the AAR-associated 325 targets of JNK and ERK are ATF2 (22) and cJUN (36). The AP-1 family of transcription factors can 326 form complexes composed of homo- or hetero-dimers of cJUN, JUN-B, JUN-D, cFOS, FOS-B, 327 Fra1, and Fra2 (80) and of these, the cJUN, cFOS, JUN-B, and FOS-B genes are activated by the 328 AAR in HepG2 human hepatoma cells, whereas those for JUN-D, FRA-1, and FRA-2 are not (36, 329 79). Fu et al. showed that for some, but not all cell lines from several human tissues, the relative 330 induction of cJUN expression was greater in transformed cells compared to non-transformed cells, 331 independent of cell growth rate (36). Those authors also showed that over-expression of cJUN 332 exhibited a concentration-dependent activation of both the basal and AAR- or ATF4-induced 333 ASNS-driven transcription, whereas a dominant negative cJUN form suppressed the increased 334 ASNS transcription. The results of Fu et al. also revealed that existing cJUN protein is 335 phosphorylated through a cascade that involves both ERK and JNK and subsequently, cJUN-ATF2 336 dimers induce transcription from the cJUN gene itself. Presumably, homo- or hetero-dimers 337 containing cJUN then activate additional downstream genes. Given that cJUN promotes cell 338 growth by increasing cyclin D expression (92), induction of cJUN by the AAR may contribute to 339 tumor cell survival in the presence of a limited AA supply. 340

Lorenzi et al. screened the NCI-60 human cancer cell panel using microarray assays and 341 noted a negative correlation between ASNS mRNA expression and susceptibility to ASNase 342 treatment in several ovarian cancer cells (60). They also observed increased ASNase sensitivity 343 after siRNA knockdown of ASNS expression. In a second study with a larger number of ovarian 344 cell lines, the same group determined that whereas the correlation between ASNase efficacy and 345 ASNS mRNA expression was weak, there was a stronger correlation between ASNase efficacy 346

16and ASNS protein levels (59). As mentioned above, this result confirmed previous observations in 347 human MOLT4 leukemia cells (86). These ovarian cell culture studies may lead to the use of ASNS 348 as a biomarker in ovarian cancer screening (61). 349 350 ASNS and Tumor Metastasis 351

Metastasis is a complicated and incompletely understood process, but some evidence 352 suggests that ASNS may be important in certain metastatic mechanisms. For cancer to spread 353 from the primary tumor to distant sites, a cell or group of cells must detach from the primary tumor 354 and enter the bloodstream where they exist in suspension until they reach the metastatic site. 355 Patrikainen et al. mimicked this transition by adapting PC-3 prostate cancer cells from adherent to 356 suspension culture and then examined changes in gene expression concomitant with suspension-357 adaption (67). They discovered that the ASNS expression was 6-fold greater in the suspension-358 adapted PC-3 cells compared to the adherent cells. Subsequently, Ameri et al. created orthotopic 359 xenografts using human MDA-MB-231 breast cancer cells in an established metastatic mouse 360 model (4). The authors observed that the protein abundance for ATF4 and ATF3, as well as their 361 target gene, ASNS, were elevated in circulating tumor cells isolated from mouse blood compared 362 with the parental MDA-MB-231 cell line. When returned to in vitro culture and exposed to hypoxia, 363 the circulating tumor cells showed higher basal expression and greater induction of ATF4 and 364 ASNS than the parental MDA-MB-231 cell line. Furthermore, the circulating tumor cells had an 365 increased capacity for colony formation in a soft agar assay under hypoxic conditions and grew 366 faster when re-implanted as xenografts. The authors speculated that circulating tumor cells may 367 represent a subpopulation that are selected for their ability to survive insults such as hypoxia and 368 nutrient deprivation as the result of up-regulation of factors such as ATF4, ATF3, and ASNS. With 369 specific regard to ASNS, its increased abundance in metastasizing cells suggests that ASNS 370 activity is beneficial for cancer cell survival once they detach from the primary tumor and enter the 371 bloodstream. 372

17 These studies documenting the up-regulation of the ATF4 and its target genes ATF3 373

and ASNS in metastasis reinforce studies revealing the importance of the GCN2-ATF4 (98) and 374 PERK-ATF4 pathway, as well as their downstream target ASNS, in transformed cells. As 375 illustrated in Fig. 3, activation of either eIF2 kinase GCN2 or PERK will lead to ATF4 production 376 and the subsequent downstream transcriptional programs. However, two points are noteworthy 377 and indicate that further investigation is required to fully understand the roles of these kinases in 378 tumor biology. First, while some of the cell stress circumstances that trigger each of these kinases 379 are known, such as AA limitation for GCN2 and ER stress/hypoxia for PERK, others may yet to be 380 discovered (54, 94). Second, while both PERK and GCN2 catalyze the phosphorylation of eIF2, 381 which leads to ATF4 synthesis, the translational and transcriptional output for the two kinases 382 differs suggesting that additional modulating signals have yet to be discovered. (29). 383

384 Examples of the Many Remaining Questions 385

Though many facets of ASNS regulation and function have been characterized over the 386 past several decades, there remain major gaps in our knowledge. The exact roles and 387 physiological impact that this enzyme activity plays in maintaining homeostasis of two substrates 388 and two products remains largely unexplored. For example, the name implies a focus on 389 asparagine biosynthesis, but the activity could have significant effects on cellular glutamine 390 content, especially during periods of long-term up-regulation. Knockout animals have not been 391 investigated to address the in vivo impact during embryonic development or physiological stress 392 states during adulthood. The striking level of ASNS expression in the exocrine pancreas is an 393 interesting opportunity for further exploration. The high ASNS content may simply be related to the 394 need for asparagine availability for glycoprotein synthesis and secretion, but the liver also has high 395 rates of glycoprotein secretion with what appears to be much less ASNS activity. The importance 396 of increased ASNS expression in tumor proliferation in solid tumors and development of resistance 397 to ASNase chemotherapy in leukemia illustrates that a better comprehension of ASNS regulation, 398

18as well as the AAR in general, is needed in transformed cells. Additional investigation of the 399 impact of tumor-associated changes in ASNS regulation, such as the polymorphisms in the ATF5 400 gene, are likely to contribute to our understanding of the interesting links between aberrant ASNS 401 expression and transformed cell growth that have been observed for many tumors. 402

19Acknowledgements: This research was supported by grants to MSK from the Institute of

Diabetes, Digestive and Kidney Diseases, the National Institutes of Health (DK92062 and

DK94729). The authors thank Tracy Anthony, Rutgers University, for helpful discussion about

asparaginase toxicity. The authors acknowledge the contribution of Dr. Richard Hutson who

generated the data shown in Figure 1 while a member of the Kilberg laboratory. The authors wish

to thank other members of the laboratory for helpful discussion.

Author Contributions: All three authors contributed to the writing, editing, and final preparation of

the text.

403

20REFERENCES 404

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26FIGURE LEGENDS 691

Figure 1. Expression of asparagine synthetase (ASNS) protein in rat tissues. The indicated 692 tissues were harvested from rats fed a control chow and immunoblotting of the resulting 693 protein extracts was used to illustrate the basal expression of ASNS. These data are 694 consistent with those of Milman et al. who measured the tissue distribution of ASNS enzyme 695 activity across many species, including humans (63). 696 697 Figure 2. The gene structure and proximal promoter sequence for human asparagine 698 synthetase (ASNS). Panel A shows the exon-intron structure and size of the human ASNS 699 gene. The translation start site is within exon 3 and the stop site within exon 13, so the tan 700 exon boxes represent the 3′ and 5′ untranslated regions of the mRNA and the protein 701 coding exons are shown in green. Panel B shows the sequence of the proximal 173 bp for 702 the human ASNS promoter. Designated are a number of transcription factor binding sites 703 that have been identified by in vivo footprinting and single nucleotide mutagenesis to 704 contribute to either basal or stress-induced transcription (15, 57, 101). Details of the role for 705 each of these sequences are discussed in the text. 706

707 Figure 3. The multiple signal pathways that make up the amino acid response (AAR) in 708 mammals. The AAR is a collection of signaling pathways that result in an integrated 709 transcriptional program. Whereas uncharged tRNA-activation of the GCN2 kinase has been 710 documented to be the AA sensor for the pathways leading to increased NF-κB activity (51) 711 and ATF4 synthesis (42), the sensor that leads to MEK (90) and GPCR12 (22) activation 712 has not been identified (indicated by the dashed lines). Amino acid transporters have been 713 proposed as possible sensor molecules (89). 714

715

27Figure 4. The eIF2 kinase sensors for the AAR versus the UPR. Limitation for one or more 716 amino acids causes an increased abundance of the corresponding uncharged tRNA that in 717 turn binds to and activates the GCN2 kinase. On the other hand, other cellular stress 718 events including glucose starvation or tunicamycin treatment that leads to glycoprotein 719 accumulation, thapsigargin-induced inhibition of calcium homeostasis, viral overload, 720 oxidative stress, and a wide range of disease states or drug treatments trigger endoplasmic 721 reticulum (ER) stress (43). The resulting ER stress causes stimulation of a collection of 722 signaling pathways collectively referred to as the unfolded protein response (UPR). One of 723 these pathways involves activation of the ER-bound kinase PERK. Both GCN2 and PERK 724 are members of a family of kinases that phosphorylate the eukaryotic translation initiation 725 factor eIF2 (95). Phospho-eIF2 leads to a suppression of global protein synthesis, but 726 enhanced translation of select mRNA species, such as the transcription factors ATF4 and 727 ATF5, that contain short upstream opening reading frames that serve as regulatory 728 sequences. Among the hundreds of ATF4 target genes is GADD34, which directs protein 729 phosphatase 1 (PP1) to p-eIF2 and thus, returns the translation factor to it’s 730 dephosphorylated state and the promotion of global translation. As an ATF4-responsive 731 enhancer element, the CARE sequence in ASNS, and many other CARE-containing genes, 732 results in activation of transcription during either the AAR or the UPR. 733

734

Asparagine Synthetase Protein Expression in Rat Tissues

B. HUMAN ASPARAGINE SYNTHETASE PROMOTER SEQUENCE

-173 CAAAAGAGCT CCTCCTTGCG CCCTTCCGCC GCCCCACTTA GTCCTGCTCC GCCCC!GTTTTCTCGA GGAGGAACGC GGGAAGGCGG CGGGGTGAAT CAGGACGAGG CGGG!

GC-1 GC-2 GGACA !

GCCTGT!

TACTAC TT!TGAAGGGC!ATGATG AA!ACTTCCCG!

GC-3 NSRE1/CARE -113 CCCCGCGGCC CCGCCCCTGT GCGCGCTGGT TGGTCCTCGC AGGC!

GGGGCGCCGG GGCGGGGACA CGCGCGACCA ACCAGGAGCG TCCG!

TT!A!C!NSRE-2

-53 CACGCG! AGGAGCCAGG TCGGTATAAG CGCCAGCGGC CTCGCCGCCC GTC!aagctgt!AA!G!T!GTGCGC! TCCTCGGTCC AGCCATATTC GCGGTCGCCG GAGCGGCGGG CAG!ttcgaca!

TATA

A. HUMAN ASPARAGINE SYNTHETASE GENE STRUCTURE

1 2 3 4 5 6 7 8 9 10 11 12 13

~ 20 kb