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Trends in Endocrinology & Metabolism
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Amino Acid Depletion Therapies: Starving Cancer Cells to Death

Open AccessPublished:March 29, 2021DOI:https://doi.org/10.1016/j.tem.2021.03.003

      Highlights

      • Targeting amino acid metabolism is an attractive form of therapy, as it can mitigate long-term treatment-related toxicities by reducing the need for genotoxic agents.
      • The effect of amino acid depletion therapies can be further enhanced when combined with either conventional therapies or by targeting tumor cell-specific survival mechanisms.
      • A number of novel amino acid depletion strategies is currently under clinical evaluation.
      Targeting tumor cell metabolism is an attractive form of therapy, as it may enhance treatment response in therapy resistant cancers as well as mitigate treatment-related toxicities by reducing the need for genotoxic agents. To meet their increased demand for biomass accumulation and energy production and to maintain redox homeostasis, tumor cells undergo profound changes in their metabolism. In addition to the diversion of glucose metabolism, this is achieved by upregulation of amino acid metabolism. Interfering with amino acid availability can be selectively lethal to tumor cells and has proven to be a cancer specific Achilles’ heel. Here we review the biology behind such cancer specific amino acid dependencies and discuss how these vulnerabilities can be exploited to improve cancer therapies.

      Keywords

      Introduction

      Advances in chemotherapy and supportive care, but also the introduction of targeted- and immune-therapies, have led to increased survival of cancer patients over the past decades [World Health Organization, 2018 (https://www.who.int/news-room/fact-sheets/detail/cancer)]. However, a large number of cancer survivors, especially those treated at a young age, present with late effects, which severely impacts quality of life [
      • American Cancer Society
      Cancer Treatment & Survivorship: Facts & Figures 2019-2021.
      ]. Therefore, next to improving cure rates, alternative therapies that are not associated with long-term toxicities require attention. Tumor cell metabolism may prove to be a vulnerability that can be targeted with minimal collateral damage [
      • Vettore L.
      • et al.
      New aspects of amino acid metabolism in cancer.
      ,
      • Lieu E.L.
      • et al.
      Amino acids in cancer.
      ].
      To accommodate their enhanced proliferation, tumor cells increase their metabolic rate to provide sufficient cellular building blocks (proteins, DNA, RNA, and lipids), energy, and reducing agents [
      • Lieu E.L.
      • et al.
      Amino acids in cancer.
      ]. This often involves the activation of prominent oncogenes, including Ras and c-Myc or loss of tumor suppressors such as PTEN and P53, driving changes in cellular metabolism to meet the increased demand for metabolites [
      • Vettore L.
      • et al.
      New aspects of amino acid metabolism in cancer.
      ,
      • Kim J.
      • DeBerardinis R.J.
      Mechanisms and implications of metabolic heterogeneity in cancer.
      ] (Figure 1). Their elevated uptake of nutrients is already put to use in routine diagnostics, where intratumor accumulation of radiolabeled nutrients such as glucose but also specific amino acids, can be visualized using positron emission tomography (PET) scans [
      • Venneti S.
      • et al.
      Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo.
      ,
      • Sanderson S.M.
      • et al.
      Methionine metabolism in health and cancer: a nexus of diet and precision medicine.
      ]. While the cancer cell-specific diversion of carbohydrate intermediates from oxidative phosphorylation towards several anabolic pathways, known as the Warburg effect, has been long recognized, attention for amino acid dependencies in cancer cells came decades later. Tumor cells often rely on an exogenous supply of amino acids. Surprisingly, this not only holds true for essential amino acids (EAA), the type that the body cannot synthesize, but also several nonessential amino acids (NEAA) appear to be rate limiting for the growth of tumor cells [
      • Choi B.H.
      • Coloff J.L.
      The diverse functions of non-essential amino acids in cancer.
      ]. Here we discuss the biology behind the most prominent tumor-specific amino acid dependencies and their potential clinical applications.
      Figure 1
      Figure 1Commonly Deregulated Genes/Pathways Controlling Amino Acid Homeostasis and Metabolism in Cancer Cells.
      Import of amino acids from the bloodstream, amino acid biosynthesis, amino acids derived from autophagy, and micro/micropinocytosis contribute to the internal amino acid cell pool. These are used for energy production, macromolecular biosynthesis (proteins, nucleic acids, lipids, and glutathione), and methylation processes. The tight balance between amino acid uptake, biosynthesis, and catabolism is controlled by mammalian target of rapamycin complex 1 (mTORC1) and general control nonderepressible 2 (GCN2). mTORC1 facilitates amino acid metabolism in response to amino acid availability. In response to amino acid shortages, mTORC1 is inhibited while GCN2 is activated, resulting in the activation of the amino acid response pathway. This includes a global shutdown of translation, bringing cells into quiescence in order to be able to survive nutrient shortage. If these stress conditions persist, apoptosis is induced. To fulfill their enhanced demand for amino acids, cancer cells often upregulate amino acid homeostasis and metabolism (shown in red). This involves mutations in commonly mutated cancer genes, including c-Myc, Ras, PI3K, PTEN, and mTOR, but also genes directly implicated within pathways of amino acid homeostasis and metabolism (e.g., EIf4EBP and eIf4E). Abbreviation: ROS, reactive oxygen species.

      Amino Acid Availability as a Therapeutic Target for Cancer Therapy

      Next to forming the building blocks of proteins, amino acids provide many of the structural elements of a cell and are an important source of energy. It is therefore not surprising that cancer cells, although striving to maintain amino acid homeostasis (Box 1) by promoting amino acid synthesis or salvage (Figure 1), become more dependent on exogenous supply of amino acids. This increased demand for amino acids may even cause auxotrophy (i.e., the inability to sustain growth in the absence of a particular nutrient) for NEAAs [
      • Lomelino C.L.
      • et al.
      Asparagine synthetase: function, structure, and role in disease.
      ]. The latter can be either acquired during tumor progression, or related to the cell of origin, such as the insufficient expression of asparagine synthetase (ASNS) in leukemic cells and their origin, immature lymphocytes (Box 2) [
      • Lomelino C.L.
      • et al.
      Asparagine synthetase: function, structure, and role in disease.
      ]. Both the selective dependency and the potential to target a specific amino acid makes some more suitable as a therapeutic target than others [
      • Kim J.
      • DeBerardinis R.J.
      Mechanisms and implications of metabolic heterogeneity in cancer.
      ]. The importance of amino acid metabolism is underscored by the fact that limiting the availability of these nutrients can be selectively lethal to tumor cells.
      Sensors of Amino Acid Availability
      Amino acids, best known as the structural units that make up our proteins, also serve as a resource for various other key cellular processes, including the generation of other macromolecules, hormones and neurotransmitters, energy production, and methylation.
      To ensure sufficient supply of amino acids, both normal and transformed cells are able to sense and respond to conditions of limited nutrient availability, by coordinating amino acid uptake, biosynthesis, and catabolism (see Figure 1 in main text) [
      • Lieu E.L.
      • et al.
      Amino acids in cancer.
      ]. The mammalian target of rapamycin complex 1 (mTORC1) and general control nonderepressible 2 (GCN2) are the two most prominent nodes in the pathways controlling the cellular response to low amino acid availability. mTORC1 activation in response to amino acid availability is primarily mediated by RAG GTPases, which in turn are activated by a number of amino acid sensors, including solute carriers that transport amino acids over the cell membrane [
      • Shimobayashi M.
      • Hall M.N.
      Multiple amino acid sensing inputs to mTORC1.
      ]. Specific amino acids, including leucine, Arg, and Gln appear to be more relevant for mTORC1 signaling than others [
      • Shimobayashi M.
      • Hall M.N.
      Multiple amino acid sensing inputs to mTORC1.
      ].
      At the same time the cellular amino acid sensor GCN2 is activated by uncharged tRNAs or stalled ribosomes, suppressing global protein translation by phosphorylation of the eukaryotic initiation factor 2 (eIF2), effectively stalling CAP-dependent protein translation [
      • Donnelly N.
      • et al.
      The eIF2alpha kinases: their structures and functions.
      ]. Simultaneously, CAP-independent translation of activating transcription factor 4 (ATF4) is increased. This stress induced transcription factor controls expression of a wide range of adaptive genes within the amino acid response (AAR) pathway. These include amino acid transporters, enzymes that promote de novo synthesis of amino acids, as well as activators of autophagy that together act to restore homeostasis. However, under persistent stress conditions, ATF4 will trigger a transcriptional program that favors the induction of apoptosis [
      • Wortel I.M.N.
      • et al.
      Surviving stress: modulation of ATF4-mediated stress responses in normal and malignant cells.
      ].
      With nutrient sensing being crucial for tumor cell survival and proliferation, it is not surprising that essential sensors and effectors are frequently mutated or upregulated [
      • Lieu E.L.
      • et al.
      Amino acids in cancer.
      ]. Upstream regulators such as PI3K and RAS, but also mTORC1 itself are often subject to overactivation or mutations [
      • Kim J.
      • DeBerardinis R.J.
      Mechanisms and implications of metabolic heterogeneity in cancer.
      ,
      • Counihan J.L.
      • et al.
      Cancer metabolism: current understanding and therapies.
      ,
      • Tsai W.B.
      • et al.
      Activation of Ras/PI3K/ERK pathway induces c-Myc stabilization to upregulate argininosuccinate synthetase, leading to arginine deiminase resistance in melanoma cells.
      ]. Mutations in downstream effectors like 4E-BP1 and ELF4E have been found in certain tumors [
      • Bjornsti M.A.
      • Houghton P.J.
      Lost in translation: dysregulation of cap-dependent translation and cancer.
      ].
      Asparaginase, the Prime Example of Targeting Amino Acid Availability
      Although tumor cell metabolism has long been recognized as a potential therapeutic target and antimetabolites such as methotrexate and 5-fluoruracil (5-FU) were successfully introduced as anticancer therapy decades ago [
      • Lieu E.L.
      • et al.
      Amino acids in cancer.
      ,
      • Rosenzweig A.
      • et al.
      Beyond the Warburg effect: how do cancer cells regulate one-carbon metabolism?.
      ], most of these compounds fail to discriminate between the tumor and rapidly dividing normal tissues, such as skin, gut epithelium, and bone marrow [
      • Alexander S.
      • et al.
      Classification of treatment-related mortality in children with cancer: a systematic assessment.
      ]. However, some metabolic therapies, for instance those involving the selective depletion of a particular amino acid, can be quite tumor cell specific. Unlike other cell types in the body, lymphocytes, including leukemic blasts, are selectively dependent on Asn. As a consequence, the introduction of the Asn depleting enzyme asparaginase (ASNase) in the treatment of pediatric acute lymphoblastic leukemia, has profoundly improved cure rates [
      • Cools J.
      Improvements in the survival of children and adolescents with acute lymphoblastic leukemia.
      ]. Upon injection, the bacterially derived ASNase hydrolyses Asn to aspartic acid and ammonia, and Gln into glutamic acid, effectively depleting Asn from the blood. While most cells express asparagine synthetase (ASNS), the enzyme that converts aspartate into Asn, expression in immature lymphocytes and their malignant counterparts, leukemic blasts, is insufficient, making these cells auxotrophic for Asn. As a result, ASNase treatment kills the leukemic cells. ASNase treatment, is associated with several acute, but mostly manageable toxicities [
      • Aldoss I.
      • Douer D.
      How I treat the toxicities of pegasparaginase in adults with acute lymphoblastic leukemia.
      ], although in some cases severe pancreatitis, thrombosis, or allergic reactions develop. However, unlike genotoxic agents, late and long-term effects are mostly absent. Nowadays, ASNase is a key component of the multiagent therapy regimen that cures more than 90% of pediatric acute lymphoblastic leukemia (ALL) patients [
      • Cools J.
      Improvements in the survival of children and adolescents with acute lymphoblastic leukemia.
      ]. While ASNase is only one of the many drugs that are included in the treatment protocols, its added value becomes apparent from the fact that premature discontinuation immediately translates into an inferior disease-free survival [
      • Gupta S.
      • et al.
      Impact of asparaginase discontinuation on outcome in childhood acute lymphoblastic leukemia: a report from the children's oncology group.
      ]. Although initial trials with other tumor types showed disappointing results, more recently a better understanding of the intimate connection between ASNS expression and the response to therapy has rekindled the interest in this protein drug, which is now under clinical investigation for the treatment of lymphomas, acute myeloid leukemia (AML), natural killer (NK)/T cell lymphoma, pancreatic ductal adenocarcinoma, glioblastoma, ovarian carcinomas, breast cancer, gastric cancer, and liver cancer [
      • Knott S.R.V.
      • et al.
      Asparagine bioavailability governs metastasis in a model of breast cancer.
      ,
      • Herranz D.
      • et al.
      Metabolic reprogramming induces resistance to anti-NOTCH1 therapies in T cell acute lymphoblastic leukemia.
      ,
      • Chiu M.
      • et al.
      Asparagine synthetase in cancer: beyond acute lymphoblastic leukemia.
      ]. Moreover, upcoming amino acid targeting therapies will benefit greatly from the experience gained with the decades of using asparaginase therapy in clinical practice. These include the use of therapeutic drug monitoring, strategies to avoid immune response and related adverse effects, and management of acute toxicities.
      Targeting amino acid metabolism can be approached from different angles: inhibition of either amino acid transporters [
      • Lieu E.L.
      • et al.
      Amino acids in cancer.
      ], amino acid biosynthesis, or by depletion of amino acids. Although upregulated expression of amino acid transporters is used by cancer cells to meet an increased demand, the functional redundancies between amino acid transporters make these rather unattractive therapeutic targets. By contrast, inhibition of enzymes involved in de novo and/or salvage pathways of amino acid synthesis as, for example, phosphoglycerate dehydrogenase (PHGDH), part of the serine (Ser) biosynthesis pathway [
      • Counihan J.L.
      • et al.
      Cancer metabolism: current understanding and therapies.
      ] or glutaminase (GLS) [
      • Fung M.K.L.
      • Chan G.C.
      Drug-induced amino acid deprivation as strategy for cancer therapy.
      ], shows more promise. Amino acid depletion can also be achieved by the degradation of a specific amino acid in the bloodstream. When tumor cells are selectively dependent on exogenous supply of a specific amino acid, this will lead to amino acid starvation, cessation of growth, and ultimately, induction of apoptosis [
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ] (Figure 2). Here we will discuss different amino acids that are targets for amino acid depletion therapy.
      Figure 2
      Figure 2Schematic Overview of Different Strategies to Target Amino Acid Metabolism.
      Cancer cells that are dependent on endogenous amino acid biosynthesis can be targeted by amino acid pathway inhibitors (purple). Cancer cells that are dependent on the exogenous supply of a specific amino acid, can be targeted by depletion of this specific amino acid (blue). Normal cells show a much lower demand for amino acids and can survive either amino acid biosynthesis inhibition or depletion of amino acids (gray). Abbreviations: ADI, arginine deiminase; EAA, essential amino acid; GLS, glutaminase; NEAA, nonessential amino acid; PHGDH, phosphoglycerate dehydrogenase.

      Asparagine

      To date, asparagine (Asn) is the most successful and best documented target for amino acid depletion therapy in the treatment of cancer (Box 2). Particularly in pediatric acute lymphoblastic leukemia (ALL), the bacterially derived enzyme ASNase has become an essential component of ALL treatment [
      • Pieters R.
      • et al.
      L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase.
      ] and its therapeutic efficacy in some solid tumors is under clinical investigation. For instance, breast cancer cells were shown to be dependent on Asn, although these tumors show high ASNS expression. Two independent studies show that Asn can stimulate de novo glutamine (Gln) biogenesis and high levels even promote epithelial to mesenchymal transition (EMT), a crucial event in the cascade of events that drive metastasis. Accordingly, ASNase-mediated limitation of Asn repressed both primary tumor growth as well as the development of metastasis, not only by depriving the tumor of Asn, but by proxy depleting Gln [
      • Knott S.R.V.
      • et al.
      Asparagine bioavailability governs metastasis in a model of breast cancer.
      ,
      • Pavlova N.N.
      • et al.
      As extracellular glutamine levels decline, asparagine becomes an essential amino acid.
      ].

      Glutamine

      The finding that Gln availability can limit tumor cell proliferation may be surprising, given the fact that it is the most abundant amino acid in serum and, next to glucose, also the most consumed nutrient. In addition to its role as a proteogenic amino acid, Gln is the major source of α-ketoglutarate in the tricarboxylic acid (TCA) cycle. Furthermore, it is utilized in the biosynthesis of all NEAAs [
      • Choi B.H.
      • Coloff J.L.
      The diverse functions of non-essential amino acids in cancer.
      ] and its intermediate glutamate functions as an exchange factor for the import of EAAs [
      • Zhang J.
      • et al.
      Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine.
      ]. As a result of the high consumption, Gln is considered a conditionally EAA and, as discussed earlier, may be rate limiting to tumor growth. Gln metabolism is highly regulated. Both de novo biosynthesis of Gln and glutaminolysis are upregulated in several cancers, via common oncogenes/tumor suppressors including c-Myc and p53 [
      • Yang L.
      • et al.
      Glutaminolysis: a hallmark of cancer metabolism.
      ]. Increased Gln synthesis is often due to upregulation of Gln synthetase (GS) [
      • Zhang J.
      • et al.
      Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine.
      ], whereas enhanced glutaminolysis is caused by increased GLS activity and correlates with poor prognosis in glioblastoma, ovarian cancer, breast cancer, medulloblastoma, and lung cancer [
      • Yang L.
      • et al.
      Glutaminolysis: a hallmark of cancer metabolism.
      ,
      • Vanhove K.
      • et al.
      Glutamine addiction and therapeutic strategies in lung cancer.
      ].
      Tumors driven by c-Myc or KRAS are particularly dependent on exogenous Gln [
      • Altman B.J.
      • et al.
      From Krebs to clinic: glutamine metabolism to cancer therapy.
      ,
      • Najumudeen A.K.
      • et al.
      The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer.
      ]. This not only validates Gln as a therapeutic target, but can also be exploited for diagnostic imaging where tumors are detected in PET scans as a result of accumulation of radiolabeled Gln [
      • Venneti S.
      • et al.
      Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo.
      ].
      The identification of Gln metabolism as a therapeutic target has led to the development of Gln mimetics. Despite impressive efficacy in vitro, their high toxicity has precluded further development. Pharmacological inhibition of GLS shows more promise. Particularly CB-839 has shown encouraging results as it inhibits tumor cell proliferation in different cancer types both in vitro and in vivo including triple-negative breast cancer (TNBC) [
      • Luengo A.
      • et al.
      Targeting metabolism for cancer therapy.
      ], acute myeloid leukemia (AML) [
      • Jacque N.
      • et al.
      Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.
      ,
      • Gregory M.A.
      • et al.
      Targeting glutamine metabolism and redox state for leukemia therapy.
      ] and non-small cell lung cancer (NSCLC) [
      • Jacque N.
      • et al.
      Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.
      ]. CB-839, commercialized as Telaglenastat, has moved to Phase II clinical trials for hematological malignancies and solid tumors [
      • Fung M.K.L.
      • Chan G.C.
      Drug-induced amino acid deprivation as strategy for cancer therapy.
      ,
      • Altman B.J.
      • et al.
      From Krebs to clinic: glutamine metabolism to cancer therapy.
      ,
      • Jacque N.
      • et al.
      Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.
      ] (Figure 3). As mentioned before, while Asn is the prime target of ASNase, this enzyme also exhibits GLS activity. With ongoing efforts to study the efficacy of ASNase for the treatment of solid tumors, the GLS effect may become more valuable, and rational design of the enzyme could enhance the GLS activity and thereby the efficacy of ASNase in these distinct tumor contexts.
      Figure 3
      Figure 3Current Progress of Amino Acid Depletion Therapies in the Treatment of Cancer.
      Preclinical and clinical evidence for antitumor activity of the different amino acid therapies. Information on preclinical data was subtracted from Research Papers, whereas clinical information was subtracted from https://www.clinicaltrials.gov/. Abbreviations: ADI, arginine deiminase; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; BC, breast cancer; CC, colon cancer; CLL, chronic lymphocytic leukemia; CRC, colorectal cancer; GBM, glioblastoma multiforme; GLS, glutaminase; HCC, Hepatocellular carcinoma; HNSCC, head and neck squamous cell carcinoma; NHL, non-Hodgkin lymphoma; NK, natural killer; NSCLC, non-small cell lung carcinoma; RCC, renal cell carcinoma; TNBC, triple-negative breast cancer; WM, Waldenstrom macroglobulinemia.

      Arginine

      Arginine (Arg) is also a semi-essential amino acid: synthesized from Gln or proline, but with conditional dependence on dietary intake [
      • Choi B.H.
      • Coloff J.L.
      The diverse functions of non-essential amino acids in cancer.
      ]. As a charged amino acid, Arg is essential for the stabilization of protein structure. In addition, it is consumed by the TCA cycle and is a precursor for compounds such as creatine, polyamines, and nitric oxide (NO) [
      • Choi B.H.
      • Coloff J.L.
      The diverse functions of non-essential amino acids in cancer.
      ,
      • Albaugh V.L.
      • et al.
      Arginine-dual roles as an onconutrient and immunonutrient.
      ]. Polyamines and NO promote tumor cell proliferation and metastasis, the latter acting as a free radical causing DNA damage [
      • Albaugh V.L.
      • et al.
      Arginine-dual roles as an onconutrient and immunonutrient.
      ]. The enzymes involved in de novo synthesis of Arg, argininosuccinate synthetase (ASS1), and argininosuccinate lyase (ASL) are frequently deregulated in cancer cells. While elevated expression of these key enzymes is associated with poor survival in different cancer types including glioblastoma, ovarian cancer, and gastric cancer, in other tumors expression is suppressed, leading to Arg auxotrophy [
      • Vettore L.
      • et al.
      New aspects of amino acid metabolism in cancer.
      ,
      • Albaugh V.L.
      • et al.
      Arginine-dual roles as an onconutrient and immunonutrient.
      ,
      • Riess C.
      • et al.
      Arginine-depleting enzymes - an increasingly recognized treatment strategy for therapy-refractory malignancies.
      ]. The enzymes involved in the Arg salvage pathway, using ornithine as a source for Arg, are also subject to deregulation [
      • Albaugh V.L.
      • et al.
      Arginine-dual roles as an onconutrient and immunonutrient.
      ,
      • Riess C.
      • et al.
      Arginine-depleting enzymes - an increasingly recognized treatment strategy for therapy-refractory malignancies.
      ].
      In auxotrophic tumors, Arg depletion induces autophagy and apoptosis. By contrast, normal cells enter a state of quiescence and can survive long periods of starvation [
      • Scott L.
      • et al.
      Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells.
      ]. Enzymatic depletion of Arg by administration of the human arginase (ARGase) or the bacterial arginine deiminase (ADI) is being explored as anticancer therapy for pediatric patients with relapsed/refractory cancers [Register, E.U.C.T. 2020 (https://www.clinicaltrialsregister.eu/ctr-search/trial/2017-002762-44/NL)]. ARGase is part of the normal urea cycle and converts Arg to ornithine and urea. It is nonimmunogenic and shows no toxicity. However, the antitumor effects of ARGase have been rather disappointing, possibly as a result of a homeostatic feedback where the Arg salvage pathway is utilized to convert ornithine back into Arg, effectively relieving cells from starvation [
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ,
      • Riess C.
      • et al.
      Arginine-depleting enzymes - an increasingly recognized treatment strategy for therapy-refractory malignancies.
      ,
      • Mussai F.
      • et al.
      Arginine dependence of acute myeloid leukemia blast proliferation: a novel therapeutic target.
      ].
      ADI converts Arg into citrulline and ammonia and selectively kills cancer cells, while at the same time suppresses angiogenesis [
      • Fung M.K.L.
      • Chan G.C.
      Drug-induced amino acid deprivation as strategy for cancer therapy.
      ,
      • Riess C.
      • et al.
      Arginine-depleting enzymes - an increasingly recognized treatment strategy for therapy-refractory malignancies.
      ]. Although citrulline can be used for de novo synthesis of Arg, in auxotrophic cells this appears to be insufficient [
      • Albaugh V.L.
      • et al.
      Arginine-dual roles as an onconutrient and immunonutrient.
      ]. ADI is now under clinical evaluation for different types of cancers [
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ] with encouraging results: the treatment is well tolerated and shows a therapeutic benefit in Phase I/II clinical trials (Figure 3).

      Methionine

      Apart from its role in protein synthesis, and as a precursor for cysteine (Cys) and polyamine synthesis, the EAA methionine (Met) is indispensable for the generation of S-adenosylmethionine (SAM), the sole methyl donor for methylation of DNA, histones, and other proteins. Met is catabolized in a series of reactions known as the Met cycle, which is often hyperactivated in tumor cells as a result of upregulation of Met adenosyltransferase 2A (MAT2A) [
      • Wang Z.
      • et al.
      Methionine is a metabolic dependency of tumor-initiating cells.
      ]. In contrast to other amino acids, Met metabolism can play an active role in malignant transformation. Downstream of MAT2A, the methyltransferase nicotinamide N-methyltransferase (NNMT) catalyzes the conversion of SAM into S-adenosylhomocysteine (SAH). By effectively consuming all available SAM, this enzyme prevents DNA and histone methylation, affecting the epigenetic landscape of cancer cells [
      • Ulanovskaya O.A.
      • et al.
      NNMT promotes epigenetic remodeling in cancer by creating a metabolic methylation sink.
      ].
      The Met salvage pathway is the only source of Met apart from exogenous supply. This salvage route requires the activity of methylthioadenosine phosphorylase (MTAP) and Met synthase (MS) [
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ], enzymes that are often downregulated in malignant cells [
      • Chaturvedi S.
      • et al.
      Exploiting methionine restriction for cancer treatment.
      ]. Furthermore, MTAP is frequently codeleted with the cell cycle regulator CDKN2A [
      • Sanderson S.M.
      • et al.
      Methionine metabolism in health and cancer: a nexus of diet and precision medicine.
      ], rendering cells exclusively dependent on the import of Met from the extracellular environment [
      • Sanderson S.M.
      • et al.
      Methionine metabolism in health and cancer: a nexus of diet and precision medicine.
      ,
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ]. The increased uptake of Met is used for diagnostic purposes, for instance by visualizing intratumor accumulation of radiolabeled Met using a PET scan for high grade gliomas, but also to predict the therapeutic response in multiple myeloma and brain tumors [
      • Sanderson S.M.
      • et al.
      Methionine metabolism in health and cancer: a nexus of diet and precision medicine.
      ].
      With Met being an EAA and given its central role in methylation, it is a prime candidate for therapeutic targeting, particularly in tumors that are driven by mutations in epigenetic modifiers such as TET and IDH proteins and methyl transferases. Indeed, a Met-free diet in tumor bearing mice hampered tumor growth of TNBC, colorectal cancer, sarcoma, glioma, and mixed-lineage leukemia (MLL)-rearranged leukemia, and suppressed metastasis formation [
      • Sanderson S.M.
      • et al.
      Methionine metabolism in health and cancer: a nexus of diet and precision medicine.
      ,
      • Jeon H.
      • et al.
      Methionine deprivation suppresses triple-negative breast cancer metastasis in vitro and in vivo.
      ,
      • Barve A.
      • et al.
      Perturbation of methionine/S-adenosylmethionine metabolism as a novel vulnerability in MLL rearranged leukemia.
      ]. Exploring its potential in humans, a study showed that dietary Met restriction is relatively well tolerated over a period of 8–17 weeks, with limited side effects, apart from weight loss [
      • Epner D.E.
      • et al.
      Nutrient intake and nutritional indexes in adults with metastatic cancer on a phase I clinical trial of dietary methionine restriction.
      ].
      Alternatively, promising antitumor effects of enzymatic depletion of Met have been observed using the bacterially derived enzyme L-methionine-gamma-lyase (METase) in vitro as well as in vivo in neuroblastoma, colorectal cancer, melanoma, and brain tumors [
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ,
      • Hoffman R.M.
      • et al.
      Total methionine restriction treatment of cancer.
      ]. METase converts Met to α-ketobutyrate, ammonia, and methanethiol, and showed limited toxicity in Phase I trials [
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ]. By supplementing homocysteine, vitamin B12, and folate to promote Met synthesis using the salvage pathway in non-malignant cells, the toxicity of Met depletion can be mitigated in patients with tumors deficient in this route [
      • Epner D.E.
      • et al.
      Nutrient intake and nutritional indexes in adults with metastatic cancer on a phase I clinical trial of dietary methionine restriction.
      ].

      Serine and Cysteine

      Although selective dependencies on other amino acids have been reported, the development of these potential tumor vulnerabilities into therapeutic strategies lags behind the previously discussed amino acids. Ser is a NEAA and next to its role in biosynthesis of proteins, phospholipids and glycine, it feeds into the folate cycle for the production of nucleotides [
      • Shuvalov O.
      • et al.
      One-carbon metabolism and nucleotide biosynthesis as attractive targets for anticancer therapy.
      ]. Tumors, including those driven by the c-MYC oncogene [
      • Sun L.
      • et al.
      cMyc-mediated activation of serine biosynthesis pathway is critical for cancer progression under nutrient deprivation conditions.
      ], exploit the Ser biosynthesis route to become less dependent on exogenous supply by controlling expression of the enzymes involved [
      • Luengo A.
      • et al.
      Targeting metabolism for cancer therapy.
      ,
      • Locasale J.W.
      • et al.
      Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis.
      ]: PHGDH, phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). However, this dependency makes them vulnerable to inhibitors of this route. For example: PHGDH inhibitors suppress cancer cell proliferation in vitro and in patient-derived xenograft (PDX) models [
      • Luengo A.
      • et al.
      Targeting metabolism for cancer therapy.
      ,
      • Locasale J.W.
      • et al.
      Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis.
      ]. Conversely, other cancers become auxotrophic for Ser during tumor progression [
      • Luengo A.
      • et al.
      Targeting metabolism for cancer therapy.
      ], facilitating Ser limitation as a possible therapeutic approach. This is particularly the case in TP53 deficient tumors, as they lack the ability to mount an appropriate prosurvival response after Ser depletion [
      • Maddocks O.D.
      • et al.
      Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells.
      ]. Initial preclinical studies show that combinatorial dietary restriction of Ser and glycine suppresses tumor cell proliferation in mouse models of intestinal cancer and lymphoma [
      • Maddocks O.D.K.
      • et al.
      Modulating the therapeutic response of tumours to dietary serine and glycine starvation.
      ], as well as in a PDX colon cancer model [
      • Maddocks O.D.
      • et al.
      Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells.
      ], although information on toxicity profiling and efficacy is currently not available.
      As one of the few sulfur containing amino acids and one of the building blocks of glutathione, Cys is crucial for redox homeostasis [
      • Tabe Y.
      • et al.
      Amino acid metabolism in hematologic malignancies and the era of targeted therapy.
      ]. While Cys can be produced from Met via homocysteine, this is often insufficient to satisfy the demand, leading to Cys auxotrophy and susceptibility to Cys depletion therapy [
      • Cramer S.L.
      • et al.
      Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth.
      ]. In various tumor types, including gastrointestinal cancer, certain lymphomas, leukemias, breast cancers, and hepatocellular carcinoma, transcriptional silencing of enzymes involved in Cys synthesis lead to Cys auxotrophy [
      • Cramer S.L.
      • et al.
      Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth.
      ]. The first preclinical tests of enzymatic Cys depletion using Cyst(e)inase (CYSase) showed decreased tumor cell proliferation and increased survival of mice transplanted with PDX derived from prostate cancer [
      • Cramer S.L.
      • et al.
      Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth.
      ] or chronic lymphocytic leukemia (CLL) [
      • Jones C.L.
      • et al.
      Cysteine depletion targets leukemia stem cells through inhibition of electron transport complex II.
      ], potentially involving ferroptosis as an inducer of cell death [
      • Badgley M.A.
      • et al.
      Cysteine depletion induces pancreatic tumor ferroptosis in mice.
      ].

      The Effects of Amino Acid Interventions Are Highly Context Dependent

      Amino Acid Supplementation as an Antitumor Strategy

      In spite of the broad applicability of amino acid depletion therapies in the treatment of cancer, in some cases high abundance of a specific amino acid may have antitumor effects. This can be either intrinsic to the tumor cells or involve extrinsic mechanisms such as stimulation of an antitumor immune response. Dietary Gln supplementation for example, was shown to block melanoma tumor growth and prolong survival in a transgenic mouse model by affecting epigenetic reprogramming [
      • Ishak Gabra M.B.
      • et al.
      Dietary glutamine supplementation suppresses epigenetically-activated oncogenic pathways to inhibit melanoma tumour growth.
      ], whereas the supplementation of histidine (His) increased the sensitivity of leukemic xenografts to methotrexate (MTX) [
      • Kanarek N.
      • et al.
      Histidine catabolism is a major determinant of methotrexate sensitivity.
      ]. Hereby His flux drains the cellular pool of tetrahydrofolate, the enzymatic cofactor required for nucleotide biosynthesis, which is also targeted by MTX [
      • Kanarek N.
      • et al.
      Histidine catabolism is a major determinant of methotrexate sensitivity.
      ].

      Location of the Tumor

      The efficacy of therapeutic interventions using amino acid availability is determined not only by cancer cells themselves, but also by external factors, including the location of the tumor and their impact on immune surveillance (Figure 4) [
      • Kim J.
      • DeBerardinis R.J.
      Mechanisms and implications of metabolic heterogeneity in cancer.
      ]. Hence, it might not come as a surprise that the same tumor cells can show different metabolic and nutrient requirements at their primary or metastatic site. For instance, breast cancer cells were shown to upregulate Ser biosynthesis to support mammalian target of rapamycin complex 1 (mTORC1) growth signaling when metastasized to the lung, but not at their primary location [
      • Rinaldi G.
      • et al.
      In vivo evidence for serine biosynthesis-defined sensitivity of lung metastasis, but not of primary breast tumors, to mTORC1 inhibition.
      ], while Cys uptake via the transporter xCT appears to be more important for mammary metastases as compared with the primary tumor [
      • Conti L.
      • et al.
      Immunotargeting of the xCT cystine/glutamate antiporter potentiates the efficacy of HER2-targeted immunotherapies in breast cancer.
      ]. Moreover, the limited availability of Ser and Gly in the brain forces metastasized cells in this tissue to upregulate Ser biosynthesis making these, but not the extracranial growing tumors, sensitive to PHGDH inhibitors [
      • Ngo B.
      • et al.
      Limited environmental serine and glycine confer brain metastasis sensitivity to PHGDH inhibition.
      ].
      Figure 4
      Figure 4The Effects of Amino Acid Interventions are Highly Context Dependent.
      The efficacy of therapeutic interventions using amino acid availability is determined by different external factors including the type of tissue, the tumor microenvironment, and immune surveillance. Tumor cells originating from the same cancer have different metabolic and nutrient requirements at their primary or metastatic sites. Furthermore, cells from the tumor microenvironment can supply cancer cells with depleted amino acids. To effectively fight cancer cells, T cells need to get activated, proliferate, and differentiate, which consumes high amounts of amino acids. As a consequence, immune surveillance is strongly dependent on amino acid availability. Abbreviations: AA, amino acids; CAF, cancer associated fibroblast; MSC, mesenchymal stem cells.

      Immune Surveillance

      When it comes to antitumor immunity, amino acid depletion predominantly suppresses the antitumor activity of immune cells, including T cells. The clonal expansion and maturation process that is needed to generate effector T cells that target cancer cells, requires increased metabolic needs for glucose and amino acids. Cancer cells are able to suppress immune cell function by outcompeting immune cells for specific amino acids, and obviously, amino acid depletion strategies may antagonize an immune response [
      • Wang W.
      • Zou W.
      Amino acids and their transporters in T cell immunity and cancer therapy.
      ].
      The role of Arg, Cys, Gln, and Met in T cell function has been relatively well studied. Arg is one of the most essential amino acids for T cell proliferation, activation, and effector function and supplementation of Arg might thus be clinically beneficial for Arg non-auxotrophic tumors. Indeed, Arg supplementation promotes immune surveillance by supporting T cell mediated antitumor activity and even synergizes with (chemo-) immunotherapy in different cancer models [
      • Wang W.
      • Zou W.
      Amino acids and their transporters in T cell immunity and cancer therapy.
      ,
      • Satoh Y.
      • et al.
      Supplementation of l-arginine boosts the therapeutic efficacy of anticancer chemoimmunotherapy.
      ]. Furthermore, inhibition of the arginase Arg1 was shown to induce antitumor immunity by inhibition of myeloid cell mediated suppression of T cell proliferation [
      • Wang W.
      • Zou W.
      Amino acids and their transporters in T cell immunity and cancer therapy.
      ]. Also Met is critical for T cell survival and Met starvation causes alterations in histone methylation that impair T cell function [
      • Bian Y.
      • et al.
      Cancer SLC43A2 alters T cell methionine metabolism and histone methylation.
      ]. Dietary supplementation of Met can restore those epigenetic alterations and increase T cell immunity in tumor bearing mice and patients with colon cancer [
      • Bian Y.
      • et al.
      Cancer SLC43A2 alters T cell methionine metabolism and histone methylation.
      ]. Cys and Gln are essential for T cell expansion and activation, respectively [
      • Wang W.
      • Zou W.
      Amino acids and their transporters in T cell immunity and cancer therapy.
      ]. However, in other reports amino acid depletion was shown to have immunostimulatory effects. Single treatment with CYSase was shown to induce antitumor T cell responses and its cytotoxicity against tumor cells could be further increased by T cell mediated release of interferon γ induced by immunotherapy [programmed death-ligand 1 (PD-L1) blockade] [
      • Wang W.
      • Zou W.
      Amino acids and their transporters in T cell immunity and cancer therapy.
      ]. Gln blockade induced nutrient depletion in tumor cells and T cells and was shown to evoke different molecular responses in both cell types. T cells upregulate their oxidative metabolism resulting in long-lived and highly activated T cells, whereas tumor cells fail to do so and eventually die [
      • Leone R.D.
      • et al.
      Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion.
      ]. Given the diversity in effects of amino acid availability on immune surveillance, it will be important to investigate the efficacy of amino acid depletion therapies in immunocompetent models.
      In summary, before amino acid depletion can be clinically applied, not only do metabolic dependencies of a particular cancer type need to be investigated, but also extrinsic factors need to be considered, such as their location and metabolic crosstalk with other cell types including immune cells (Figure 4). Also, the effects of amino acid depletion on the efficacy of other treatments will have to be carefully investigated and scheduling and dosing will need to be optimized.

      Future Perspectives and Challenges for Amino Acid Depletion Therapies

      Synergy of Amino Acid Depletion with Other Therapies

      Amino acid starvation may enhance the efficacy of conventional chemotherapy: the induction of cell cycle arrest in normal cells [
      • Broer S.
      • Broer A.
      Amino acid homeostasis and signalling in mammalian cells and organisms.
      ] may protect these cells from the DNA damage inflicted by chemotherapeutics while synergizing with these drugs in killing tumor cells.
      Synergistic effects in combination with amino acid depletion have been reported with both chemotherapeutics and targeted therapies (Table 1). While the mechanism underlying these synergies often remains to be elucidated, for some combinations the mode of (inter)action is clearer: fluorouracil (5-FU), a pyrimidine analogue, and Met depletion converge on the folate cycle, both acting to inhibit thymidylate synthase (TS) function. Moreover, Met restriction leads to downregulation of O6-alkylguanine-DNA alkyltransferase (AGT), an enzyme that eliminates alkyl groups from DNA [
      • Kokkinakis D.M.
      • et al.
      Regulation of O6-methylguanine-DNA methyltransferase by methionine in human tumour cells.
      ], thereby enhancing the effect of alkylating agents. Interestingly, although amino acid depletion, unlike classical chemotherapeutic agents, does not act primarily by provoking DNA damage, amino acid depletion can lead to nucleotide imbalances that could affect mutational signatures. A positive effect from such changes can be the generation of neo-antigens on the tumor cells as targets for immunotherapy [
      • Lee J.S.
      • et al.
      Urea cycle dysregulation generates clinically relevant genomic and biochemical signatures.
      ].
      Table 1Drug Combinations with Amino Acid Depletion Therapies
      Depletion ofDrug combinationRefs
      GlutamineVenetoclax[
      • Jacque N.
      • et al.
      Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.
      ]
      FLT3 tyrosine kinase inhibitor[
      • Gregory M.A.
      • et al.
      Glutaminase inhibition improves FLT3 inhibitor therapy for acute myeloid leukemia.
      ]
      Notch inhibition[
      • Herranz D.
      • et al.
      Metabolic reprogramming induces resistance to anti-NOTCH1 therapies in T cell acute lymphoblastic leukemia.
      ]
      Glucose metabolism[
      • Lee Y.M.
      • et al.
      Inhibition of glutamine utilization sensitizes lung cancer cells to apigenin-induced apoptosis resulting from metabolic and oxidative stress.
      ,
      • Elgogary A.
      • et al.
      Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.
      ]
      Modulators of the integrated stress response[
      • Qing G.
      • et al.
      ATF4 regulates MYC-mediated neuroblastoma cell death upon glutamine deprivation.
      ]
      mTORC1[
      • Tsai W.B.
      • et al.
      Activation of Ras/PI3K/ERK pathway induces c-Myc stabilization to upregulate argininosuccinate synthetase, leading to arginine deiminase resistance in melanoma cells.
      ,
      • Tanaka K.
      • et al.
      Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment.
      ]
      Metformin[
      • Qie S.
      • et al.
      Targeting glutamine-addiction and overcoming CDK4/6 inhibitor resistance in human esophageal squamous cell carcinoma.
      ]
      AsparagineDoxorubicin[
      • Abakumova O.
      • et al.
      Antitumor activity of L-asparaginase from Erwinia carotovora against different leukemic and solid tumours cell lines.
      ]
      BH3 mimetics[
      • Kang M.H.
      • et al.
      Activity of vincristine, L-ASP, and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo.
      ]
      KRAS pathway inhibition[
      • Gwinn D.M.
      • et al.
      Oncogenic KRAS regulates amino acid homeostasis and asparagine biosynthesis via ATF4 and alters sensitivity to L-asparaginase.
      ]
      Chloroquine[
      • Takahashi H.
      • et al.
      Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells.
      ]
      Targeting Gln metabolism[
      • Chien W.W.
      • et al.
      Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines.
      ]
      GCN2 inhibition[
      • Nakamura A.
      • et al.
      Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response.
      ]
      GLS inhibition[
      • Chien W.W.
      • et al.
      Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines.
      ,
      • Rotoli B.M.
      • et al.
      Inhibition of glutamine synthetase triggers apoptosis in asparaginase-resistant cells.
      ]
      ZBTB1[
      • Williams R.T.
      • et al.
      ZBTB1 regulates asparagine synthesis and leukemia cell response to L-asparaginase.
      ]
      Wnt/STOP signaling[
      • Hinze L.
      • et al.
      Synthetic lethality of Wnt pathway activation and asparaginase in drug-resistant acute leukemias.
      ]
      Metformin[
      • Krall A.S.
      • et al.
      Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.
      ]
      ArginineCytarabine[
      • Mussai F.
      • et al.
      Arginine dependence of acute myeloid leukemia blast proliferation: a novel therapeutic target.
      ]
      Histone-deacetylase inhibitor (SAHA)[
      • Fiedler T.
      • et al.
      Arginine deprivation by arginine deiminase of Streptococcus pyogenes controls primary glioblastoma growth in vitro and in vivo.
      ]
      Chloroquine[
      • Bean G.R.
      • et al.
      A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas.
      ]
      Targeting Gln metabolism[
      • Long Y.
      • et al.
      Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.
      ,
      • Kremer J.C.
      • et al.
      Arginine deprivation inhibits the Warburg effect and upregulates glutamine anaplerosis and serine biosynthesis in ASS1-deficient cancers.
      ]
      MethionineFluorouracil[
      • Gao X.
      • et al.
      Dietary methionine influences therapy in mouse cancer models and alters human metabolism.
      ]
      Doxorubicin[
      • Stern P.H.
      • Hoffman R.M.
      Enhanced in vitro selective toxicity of chemotherapeutic agents for human cancer cells based on a metabolic defect.
      ]
      Vincristine[
      • Stern P.H.
      • Hoffman R.M.
      Enhanced in vitro selective toxicity of chemotherapeutic agents for human cancer cells based on a metabolic defect.
      ]
      BCNU[
      • Hoffman R.M.
      • et al.
      Total methionine restriction treatment of cancer.
      ]
      Temozolomide[
      • Hoffman R.M.
      • et al.
      Total methionine restriction treatment of cancer.
      ]
      Cisplatin[
      • Tan Y.
      • et al.
      Efficacy of recombinant methioninase in combination with cisplatin on human colon tumors in nude mice.
      ]
      TRAIL-R2 agonist[
      • Strekalova E.
      • et al.
      Methionine deprivation induces a targetable vulnerability in triple-negative breast cancer cells by enhancing TRAIL receptor-2 expression.
      ]
      LeucineEpidermal growth factor receptor (EGFR) inhibitors[
      • Wang Y.
      • et al.
      Branched-chain amino acid metabolic reprogramming orchestrates drug resistance to EGFR tyrosine kinase inhibitors.
      ]
      Tamoxifen[
      • Thewes V.
      • et al.
      The branched-chain amino acid transaminase 1 sustains growth of antiestrogen-resistant and ERalpha-negative breast cancer.
      ]
      CysteineBSO[
      • Cramer S.L.
      • et al.
      Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth.
      ]
      Curcumin[
      • Cramer S.L.
      • et al.
      Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth.
      ]
      Alternatively, strategies that enhance the effect of amino acid depletion may help to reduce the need for conventional chemotherapeutics, although the design and use of sensitizers is mostly in a preclinical phase. An obvious approach is to counteract cell-intrinsic mechanisms of therapy resistance. The most straightforward mechanism for auxotrophic tumor cells to acquire resistance, is by upregulating enzymes responsible for cellular production of the depleted amino acid. For example, tumors may induce ASS1 expression upon ADI treatment [
      • Tsai W.B.
      • et al.
      Activation of Ras/PI3K/ERK pathway induces c-Myc stabilization to upregulate argininosuccinate synthetase, leading to arginine deiminase resistance in melanoma cells.
      ] while ARGase treatment may promote ornithine recycling into Arg [
      • Fernandes H.S.
      • et al.
      Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.
      ]. ASNase sensitive NCSLC cell lines become resistant by inducing ASNS expression in a KRAS dependent manner. Combining ASNase treatment with KRAS pathway inhibition in vitro and in vivo, re-sensitizes cells to ASNase-induced cell death [
      • Gwinn D.M.
      • et al.
      Oncogenic KRAS regulates amino acid homeostasis and asparagine biosynthesis via ATF4 and alters sensitivity to L-asparaginase.
      ].
      Autophagy, the stress activated catabolism of macromolecules and even complete organelles in order to preserve and recycle energy and nutrients is a potent rescue mechanism for cells to overcome periods of limited availability of resources [
      • Takahashi H.
      • et al.
      Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells.
      ]. ASNase is known to induce cytoprotective autophagy in ovarian cancer, chronic myeloid leukemia (CML), and ALL [
      • Takahashi H.
      • et al.
      Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells.
      ] and also ADI therapy promotes autophagosome formation in vitro [
      • Bean G.R.
      • et al.
      A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas.
      ]. Autophagy inhibitors such as chloroquine (CG) can re-sensitize cells to those amino acid depletion therapies [
      • Takahashi H.
      • et al.
      Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells.
      ], although this may deprive normal cells from this cytoprotective process as well.
      Many mechanisms by which cells can acquire resistance are related to a switch in metabolic dependencies, frequently leading to the formation of another Achilles heel. For example, breast cancer cell lines resistant to the GLS inhibitor CD-839 show downregulated Gln consumption, but an increased dependence on exogenous Asn [
      • Krall A.S.
      • et al.
      Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor.
      ]. Conversely, increased activity of Gln transporters through post-translational modifications induces a Gln dependent resistance against ASNase [
      • Chien W.W.
      • et al.
      Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines.
      ] and similar mechanisms were found in ADI resistant cell lines [
      • Long Y.
      • et al.
      Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.
      ]. Both the ASNase [
      • Chien W.W.
      • et al.
      Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines.
      ,
      • Rotoli B.M.
      • et al.
      Inhibition of glutamine synthetase triggers apoptosis in asparaginase-resistant cells.
      ] and ADI [
      • Long Y.
      • et al.
      Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.
      ,
      • Kremer J.C.
      • et al.
      Arginine deprivation inhibits the Warburg effect and upregulates glutamine anaplerosis and serine biosynthesis in ASS1-deficient cancers.
      ] resistant tumor cells could be re-sensitized by targeting Gln metabolism.
      Our growing understanding of tumor cell metabolism also allows for rational design of combination therapies. Targeting two or more nutrients simultaneously could prevent cells from compensating one addiction with another. Furthermore, targeting modulators of the integrated stress response [
      • Qing G.
      • et al.
      ATF4 regulates MYC-mediated neuroblastoma cell death upon glutamine deprivation.
      ], mTORC1 [
      • Tsai W.B.
      • et al.
      Activation of Ras/PI3K/ERK pathway induces c-Myc stabilization to upregulate argininosuccinate synthetase, leading to arginine deiminase resistance in melanoma cells.
      ,
      • Tanaka K.
      • et al.
      Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment.
      ], redox homeostasis [
      • Cramer S.L.
      • et al.
      Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth.
      ], or oxidative phosphorylation [
      • Qie S.
      • et al.
      Targeting glutamine-addiction and overcoming CDK4/6 inhibitor resistance in human esophageal squamous cell carcinoma.
      ,
      • Krall A.S.
      • et al.
      Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.
      ] can enhance the antitumor response of amino acid depletion therapies.
      Other resistance mechanisms developing in response to nutrient depletion therapies have been observed, such as upregulation of eEF2 kinase by blocking translation elongation [
      • Leprivier G.
      • et al.
      The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.
      ] or drug-resistant mutations, such as GLS-K325A, which leads to resistance towards GLS inhibitors (BPTES, CD-839) [
      • Xiang Y.
      • et al.
      Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis.
      ]. Current efforts include drug screens and CRISPR/Cas9 based screens to identify actionable pathways that may enhance tumor cell killing in combination with amino acid depletion strategies [
      • He L.
      • et al.
      Methods for high-throughput drug combination screening and synergy scoring.
      ,
      • Fellmann C.
      • et al.
      Cornerstones of CRISPR-Cas in drug discovery and therapy.
      ]. These approaches recently led to the identification of ZBTB1 [
      • Williams R.T.
      • et al.
      ZBTB1 regulates asparagine synthesis and leukemia cell response to L-asparaginase.
      ] and Wnt/STOP signaling [
      • Hinze L.
      • et al.
      Synthetic lethality of Wnt pathway activation and asparaginase in drug-resistant acute leukemias.
      ] and BTK [
      • Miriam Butler M.
      • et al.
      A CRISPR/Cas9 based kinome screen identifies bruton tyrosine kinase (BTK) as an important determinant of asparaginase treatment response in acute lymphoblastic leukemia.
      ] as possible targets to enhance the efficiency of ASNase.

      Challenges Remaining

      As with all drugs, resistance, either intrinsic or acquired, remains a formidable challenge. Resistance can not only occur as a result of cell autonomous factors, as explained previously, but can also be induced by extrinsic factors such as the tumor environment. Suboptimal amino acid depletion may be sufficient to maintain tumor cells in a state of cellular quiescence rather than to induce apoptosis, increasing the chance of relapse once the treatment is discontinued. This can occur when amino acid depletion enzymes cannot reach the tumor cells because of poor penetrance of the drug at so-called sanctuary sites, such as the central nervous system or the bone marrow [
      • Rizzari C.
      • et al.
      Asparagine levels in the cerebrospinal fluid of children with acute lymphoblastic leukemia treated with pegylated-asparaginase in the induction phase of the AIEOP-BFM ALL 2009 study.
      ]. Furthermore, amino acids can be provided by cells present in the tumor microenvironment (Figure 4). For leukemia it was shown that mesenchymal stem cells (MSCs) in the bone marrow niche protect leukemic blasts from ASNase induced cytotoxicity [
      • Iwamoto S.
      • et al.
      Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase.
      ], whereas bone marrow stromal cells provide Cys for CLL cells [
      • Zhang W.
      • et al.
      Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia.
      ]. Moreover, obesity is known to impair the efficacy of therapy in ALL as adipocytes can release Gln causing leukemia cell resistance to ASNase [
      • Ehsanipour E.A.
      • et al.
      Adipocytes cause leukemia cell resistance to L-asparaginase via release of glutamine.
      ]. Similarly, cancer associated fibroblasts (CAFs) support solid tumor growth by providing Asp to carcinoma cells [
      • Bertero T.
      • et al.
      Tumor-stroma mechanics coordinate amino acid availability to sustain tumor growth and malignancy.
      ]. Interestingly, in this model, the tumor cells return the favor by providing glutamate that allows the CAFs to balance their redox state. Also, Ser-starved pancreatic ductal adenocarcinoma were shown to attract peripheral axons secreting Ser into the tumor [
      • Banh R.S.
      • et al.
      Neurons release serine to support mRNA translation in pancreatic cancer.
      ] while in neuroblastoma, tumor associated macrophages release interleukin 1β and tumor necrosis factor α in response to Arg depletion, leading to upregulation of Arg2 in the neuroblastoma cells, creating an immune suppressive environment, which correlates with a poor outcome [
      • Fultang L.
      • et al.
      Macrophage-derived IL1beta and TNFalpha regulate arginine metabolism in neuroblastoma.
      ] (Figure 4). Hence, there is a close interaction between tumor cells and cells within their tumor environment, altering the amino acid availability upon treatment.
      Another major challenge of therapies using therapeutic enzymes of non-human origin, is the immune response that is mounted following recognition of these therapeutic proteins as non-self. The development of inhibitory antibodies results in enhanced clearance of the enzyme, allowing amino acid concentrations to rapidly return to baseline levels [
      • Pieters R.
      • et al.
      L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase.
      ,
      • Schellekens H.
      Immunogenicity of therapeutic proteins: clinical implications and future prospects.
      ]. In addition, allergic reactions, ranging from mild hypersensitivity reactions up to anaphylactic shock, can prevent the continuation of treatment [
      • Tabe Y.
      • et al.
      Amino acid metabolism in hematologic malignancies and the era of targeted therapy.
      ,
      • Schellekens H.
      Immunogenicity of therapeutic proteins: clinical implications and future prospects.
      ]. To increase the half-life and reduce immunogenicity, therapeutic enzymes have been modified by conjugation to polyethylene glycol (PEG) [
      • Schellekens H.
      Immunogenicity of therapeutic proteins: clinical implications and future prospects.
      ], nanoparticle encapsulation [
      • Elgogary A.
      • et al.
      Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.
      ], or erythrocyte encapsulation [
      • Thomas X.
      • Le Jeune C.
      Erythrocyte encapsulated l-asparaginase (GRASPA) in acute leukemia.
      ], and the treatment can be combined with immune-suppressive agents such as prednisone and dexamethasone. Furthermore, in silico and in vitro immunogenicity prediction tools can be used to assess the immunogenicity potential of a protein drug beforehand [
      • Salazar-Fontana L.I.
      • et al.
      Approaches to mitigate the unwanted immunogenicity of therapeutic proteins during drug development.
      ].

      Concluding Remarks

      Amino acid depletion strategies show great promise in the treatment of cancer. A major advantage over other therapies is their limited toxicity and the absence of late effects as a result of DNA damage. However, before amino acid depletion can be applied more broadly in the clinic, the metabolic dependencies of particular cancer types and its tumor environment need to be investigated in detail, allowing selection of the right amino acid target. Also, therapeutic agents including metabolic inhibitors are unlikely to be effective as a single agent, as metabolic changes in tumor cells exposed to amino acid starvation may render cells resistant to the therapy (see Outstanding Questions). Therefore, future applications of amino acid depletion therapies will likely involve a combination with (targeted) agents in order to prevent resistance mechanisms to occur. In order to prevent tumor regrowth by the persistence of residual cancer cells, a deep initial response will have to be achieved. This implies a multidrug strategy that will simultaneously inhibit different (metabolic) pathways within cancer cells and takes into account reciprocal interactions with the tumor microenvironment. Therefore, amino acid depletion sensitizers will need to be developed that increase both the efficiency and durability of the response.
      How can we recognize specific amino acid vulnerabilities in particular cancer types?
      Can we identify sensitizers that increase the efficacy of amino acid depletion or prevent activation of salvage mechanisms?
      When using enzymatic depletion strategies, how can we prevent immune-related inactivation or toxicities
      How can we overcome rescue from amino acid depletion-induced cell death by the cellular microenvironment?

      Acknowledgments

      The authors apologize for not citing other relevant publications owing to space limitations. M.B. is supported by a PhD grant from the Radboud university medical center . L.T.v.d.M. and F.N.v.L. are supported by the Dutch Cancer Society (grant 10072 ) and Children Cancer-free Foundation (KiKa, grant 134 ).
      We thank Professor Rob Pieters for critically reviewing our manuscript.

      Declaration of Interests

      No interests are declared.

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