Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer.

Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.

Suppression of asparagine synthetase enhances the antitumor potency of ART and artemalogue SOMCL-14-221 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality. Artemisinin (ART) and SOMCL-14-221 (221), a spirobicyclic analogue of ART, have been reported to inhibit the proliferation of A549 cells with unclear underlying mechanism. In the present study, we validated that both ART and 221 inhibited the proliferation and migration of NSCLC cells and the growth of A549 xenograft tumors without appreciable toxicity. The proteomic data revealed proteins upregulated in ART and 221 groups were involved in "response to endoplasmic reticulum stress" and "amino acid metabolism". Asparagine synthetase (ASNS) was identified as a key node protein in these processes. Interestingly, knockdown of ASNS improved the antitumor potency of ART and 221 in vitro and in vivo, and treatments with ART and 221 disordered the amino acid metabolism of A549 cells. Moreover, ART and 221 activated ER stress, and inhibition of ER stress abolished the anti-proliferative effects of ART and 221. In conclusion, this study demonstrates that ART and 221 suppress tumor growth by triggering ER stress, and the inhibition of ASNS enhances the antitumor activity of ART and 221, which provides new strategy for drug combination therapy.

An Antitumor Bis(N-Heterocyclic Carbene)Platinum(II) Complex That Engages Asparagine Synthetase as an Anticancer Target.

New anticancer platinum(II) compounds with distinctive modes of action are appealing alternatives to combat the drug resistance and improve the efficacy of clinically used platinum chemotherapy. Herein, we describe a rare example of an antitumor PtII complex targeting a tumor-associated protein, rather than DNA, under cellular conditions. Complex [(bis-NHC)Pt(bt)]PF6 (1 a; Hbt=1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone) overcomes cisplatin resistance in cancer cells and displays significant tumor growth inhibition in mice with higher tolerable doses compared to cisplatin. The cellular Pt species shows little association with DNA, and localizes in the cytoplasm as revealed by nanoscale secondary ion mass spectrometry. An unbiased thermal proteome profiling experiment identified asparagine synthetase (ASNS) as a molecular target of 1 a. Accordingly, 1 a treatment reduced the cellular asparagine levels and inhibited cancer cell proliferation, which could be reversed by asparagine supplementation. A bis-NHC-ligated Pt species generated from the hydrolysis of 1 a forms adducts with thiols and appears to target an active-site cysteine of ASNS.

Glutaminase Activity of L-Asparaginase Contributes to Durable Preclinical Activity against Acute Lymphoblastic Leukemia.

We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNaseWT) and its glutaminase-deficient Q59L mutant (ASNaseQ59L) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo, even against ASNS-negative cancer types.

Asparagine protects tumor cells from glutamine depletion-induced cell death.

Asparagine promotes the survival of cancer cells in response to glutamine withdrawal.

Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.

Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.

Knockdown of asparagine synthetase A renders Trypanosoma brucei auxotrophic to asparagine.

Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg(2+). TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.

A sulfoximine-based inhibitor of human asparagine synthetase kills L-asparaginase-resistant leukemia cells.

An adenylated sulfoximine transition-state analogue 1, which inhibits human asparagine synthetase (hASNS) with nanomolar potency, has been reported to suppress the proliferation of an l-asparagine amidohydrolase (ASNase)-resistant MOLT-4 leukemia cell line (MOLT-4R) when l-asparagine is depleted in the medium. We now report the synthesis and biological activity of two new sulfoximine analogues of 1 that have been studied as part of systematic efforts to identify compounds with improved cell permeability and/or metabolic stability. One of these new analogues, an amino sulfoximine 5 having no net charge at cellular pH, is a better hASNS inhibitor (K(I)(∗)=8 nM) than 1 and suppresses proliferation of MOLT-4R cells at 10-fold lower concentration (IC(50)=0.1mM). More importantly, and in contrast to the lead compound 1, the presence of sulfoximine 5 at concentrations above 0.25 mM causes the death of MOLT-4R cells even when ASNase is absent in the culture medium. The amino sulfoximine 5 exhibits different dose-response behavior when incubated with an ASNase-sensitive MOLT-4 cell line (MOLT-4S), supporting the hypothesis that sulfoximine 5 exerts its effect by inhibiting hASNS in the cell. Our work provides further evidence for the idea that hASNS represents a chemotherapeutic target for the treatment of leukemia, and perhaps other cancers, including those of the prostate.

Proteomic analysis of proteins responsible for the development of doxorubicin resistance in human uterine cancer cells.

Drug resistance is a common cause of failure in cancer chemotherapy treatments. In this study, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin-resistant MES-SA/Dx5 as a model system to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes induced by doxorubicin treatment and doxorubicin resistance. A proteomic study revealed that doxorubicin-exposure altered the expression of 87 proteins in MES-SA cells, while no significant response occurred in similarly treated MES-SA/Dx5 cells, associating these proteins with drug specific resistance. By contrast, 37 proteins showed differential expression between MES-SA and MES-SA/Dx5, indicating baseline resistance. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against asparagine synthetase (ASNS) and membrane-associated progesterone receptor component 1 (mPR) proteins, to monitor and evaluate their potency on the formation of doxorubicin resistance. The proteomic approach allowed us to identify numerous proteins, including ASNS and mPR, involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.

[Development and application of screening method for asparagine synthetase B inhibitors by high performance liquid chromatography].

A screening method for asparagine synthetase B (AS-B) inhibitors by reversed-phase high performance liquid chromatography (RP-HPLC) has been established. The contents of asparagines produced in the reaction system can be analyzed by HPLC after the derivatization with 1-fluoro-2,4-dinitrobenzene (DNFB) and used to calculate the total activity of AS-B. The sample was separated on an Agilent C18 column (250 mm x 4.6 mm, 5 microm) at the temperature of 30 degrees C with the elution of 50 mmol/L sodium acetate buffer (pH 6.2)-acetonitrile (15:85, v/v) as mobile phase at a flow rate of 1.0 mL/min. The detection wavelength was set at 365 nm. The enzyme reaction system consisted of 100 mmol/L Tris (tris(hydroxymethyl)aminomehane)-HCl buffer (pH 8.0), 100 mmol/L NaCl, 10 mmol/L MgCl2, 5 mmol/L adenosine triphosphate (ATP), 10 mmol/L L-aspartate, 10 mmol/L L-glutamine and 2 microg recombinant soybean AS-B (1 mL of the total volume), then mixed for 1 min and incubated for 15 min at 37 degrees C. After quenching with ethanol and centrifugation, the supernatant was derivatized by DNFB and then separated by HPLC. The amino acids in the reaction system were baseline separated within 6 min. The quantitative analysis of AS-B inhibition was performed by determining its dynamic parameters. The inhibitor L-glutamic acid gamma-methyl ester was used in the enzyme reaction system to test this method and its inhibition constant obtained was close to the literature value. The established method is fast, accurate, sensitive and suitable for high throughput screening AS-B inhibitors.

A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase.

The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability.

Declined asparagine synthetase mRNA expression and enhanced sensitivity to asparaginase in HL-60 cells committed to monocytic differentiation.

During 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human promyelocytic leukemia HL-60 cells toward maturing monocytes/macrophages, asparagine synthetase (ASNS) mRNA expression declined time and dose-dependently. The effect of TPA was inhibited by inhibitors for PKC and MEK 1/2, but not by those for JNK and p38 MAPK. Combination treatment with TPA and asparaginase synergistically enhanced the growth retardation accompanied by apoptotic cell death characterized by internucleosomal DNA fragmentation. These data suggest the possible involvement of MEK1/2 MAPK in the inhibitory effect of TPA on ASNS mRNA expression and that the induction of the down-regulation of ASNS (via MEK1/2 activation) may be a new strategy for the treatment of leukemia blast cells.

Correlation between asparaginase sensitivity and asparagine synthetase protein content, but not mRNA, in acute lymphoblastic leukemia cell lines.

BACKGROUND: Asparaginase (ASNase) is an essential component of most treatment protocols for childhood acute lymphoblastic leukemia (ALL). Although increased asparagine synthetase (ASNS) expression may contribute to ASNase resistance, there is conflicting data from patient samples with regard to correlation between ASNS mRNA content and ASNase sensitivity. PROCEDURE: Both T-cell and B-cell derived ALL cell lines were treated with ASNase and then monitored for cell proliferation, cell death, and ASNS mRNA and protein expression. RESULTS: Despite elevated ASNS mRNA following ASNase treatment, different ALL cell lines varied widely in translation to ASNS protein. Although ASNS mRNA levels did not consistently reflect ASNase sensitivity, there was an inverse correlation between ASNS protein and ASNase-induced cell death. Expression of ASNS in an ASNase-sensitive cell line resulted in enhanced ASNase resistance, and conversely, siRNA-mediated inhibition of ASNS expression promoted increased drug sensitivity. CONCLUSIONS: These observations provide an explanation for the ASNase sensitivity of ALL cells and demonstrate the importance of measuring ASNS protein rather than mRNA in predicting ASNase responsiveness.

Asparagine synthetase chemotherapy.

Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzyme-based methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. Significant side effects can arise in these protocols and, in many cases, patients develop drug-resistant forms of the disease that may be correlated with up-regulation of the enzyme glutamine-dependent asparagine synthetase (ASNS). Though the precise molecular mechanisms that result in the appearance of drug resistance are the subject of active study, potent ASNS inhibitors may have clinical utility in treating asparaginase-resistant forms of childhood ALL. This review provides an overview of recent developments in our understanding of (a) the structure and catalytic mechanism of ASNS, and (b) the role that ASNS may play in the onset of drug-resistant childhood ALL. In addition, the first successful, mechanism-based efforts to prepare and characterize nanomolar ASNS inhibitors are discussed, together with the implications of these studies for future efforts to develop useful drugs.

Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase.

Several lines of evidence suggest that up-regulation of asparagine synthetase (AS) in human T-cells results in metabolic changes that underpin the appearance of asparaginase-resistant forms of acute lymphoblastic leukemia (ALL). Inhibitors of human AS therefore have potential as agents for treating leukemia and tools for investigating the cellular basis of AS expression and drug-resistance. A critical problem in developing and characterizing potent inhibitors has been a lack of routine access to sufficient quantities of purified, reproducibly active human AS. We now report an efficient protocol for preparing multi-milligram quantities of C-terminally tagged, wild type human AS in a baculovirus-based expression system. The recombinant enzyme is correctly processed and exhibits high catalytic activity. Not only do these studies offer the possibility for investigating the kinetic behavior of biochemically interesting mammalian AS mutants, but such ready access to large amounts of enzyme also represents a major step in the development and characterization of inhibitors that might have clinical utility in treating asparaginase-resistant ALL.

Kinetic mechanism of asparagine synthetase from Vibrio cholerae.

Asparagine synthetase B (AsnB) catalyzes the formation of asparagine in an ATP-dependent reaction using glutamine or ammonia as a nitrogen source. To obtain a better understanding of the catalytic mechanism of this enzyme, we report the cloning, expression, and kinetic analysis of the glutamine- and ammonia-dependent activities of AsnB from Vibrio cholerae. Initial velocity, product inhibition, and dead-end inhibition studies were utilized in the construction of a model for the kinetic mechanism of the ammonia- and glutamine-dependent activities. The reaction sequence begins with the ordered addition of ATP and aspartate. Pyrophosphate is released, followed by the addition of ammonia and the release of asparagine and AMP. Glutamine is simultaneously hydrolyzed at a second site and the ammonia intermediate diffuses through an interdomain protein tunnel from the site of production to the site of utilization. The data were also consistent with the dead-end binding of asparagine to the glutamine binding site and PP(i) with free enzyme. The rate of hydrolysis of glutamine is largely independent of the activation of aspartate and thus the reaction rates at the two active sites are essentially uncoupled from one another.

Synthesis and characterization of an N-acylsulfonamide inhibitor of human asparagine synthetase.

[structure: see text] The synthesis of N-acylsulfonamide 6, which is an analogue of beta-aspartyl-AMP, is described. This compound appears to be the first and only potent inhibitor of human asparagine synthetase that has been described to date. The N-acylsulfonamide 6 exhibits slow-onset inhibition kinetics, with a K(i) of 728 nM. Preparation and characterization of two additional N-acylsulfonamide analogues has also demonstrated the importance of hydrogen-bonding interactions in the recognition of the AS inhibitor with the enzyme. These observations provide the basis for the discovery of new compounds with application in the treatment of drug-resistant leukemia.

Inhibition of plant asparagine synthetase by monoterpene cineoles.

Asparagine (Asn) synthetase (AS) is the key enzyme in Asn biosynthesis and plays an important role in nitrogen mobilization. Despite its important physiological function, little research has been done documenting inhibitors of plant AS. Plant growth inhibition caused by the natural monoterpene 1,4-cineole and its structurally related herbicide cinmethylin was reversed 65% and 55%, respectively, by providing 100 microM Asn exogenously. Reversion of the phytotoxic effect was dependent on the concentration of Asn. The presence of either 1,4-cineole or cinmethylin stimulated root uptake of [(14)C]Asn by lettuce (Lactuca sativa) seedlings. Although the physiological responses suggested that both compounds affected Asn biosynthesis, biochemical analysis of AS activity showed that the natural monoterpene was a potent inhibitor (I(50) = approximately 0. 5 microM) of the enzyme, whereas the commercial product was not inhibitory up to levels of 10 mM. Analysis of the putative metabolite, 2-hydroxy-1,4-cineole, showed that the cis-enantiomer was much more active than the trans-enantiomer, suggesting that the hydroxyl group was involved in the specific ligand/active site interaction. This is the first report that AS is a suitable herbicide target site, and that cinmethylin is apparently a proherbicide that requires metabolic bioactivation via cleavage of the benzyl-ether side chain.

Kinetic mechanism of Escherichia coli asparagine synthetase B.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.