WO2016207867A1

WO2016207867A1 - Nat8l and n-acetylaspartate in cancer

Info

Publication number: WO2016207867A1
Application number: PCT/IB2016/053810
Authority: WO
Inventors: Karsten HILLER; Daniel WEINDL
Original assignee: Université Du Luxembourg
Priority date: 2015-02-25
Filing date: 2016-06-27
Publication date: 2016-12-29

Abstract

The present invention relates to an Inhibitor of NAT8L and/or an inhibitor of the function and/or production of N-Acetylaspartic acid for use in the treatment of cancer. The present invention further provides for a method for detecting mitochondrial Acetyl CoA, a method for screening for an inhibitor or activator of NAT8L and/or an inhibitor of the function and/or production of N-acetylaspartic acid, method for detecting a cancer cell and a method for determining whether or not a cancer cell is susceptible to the treatment with an inhibitor of the present invention.

Description

NAT8L and N -acetyl aspartate in cancer

INVENTION

[001] The present invention relates to an Inhibitor of NAT8L and/or an inhibitor of the function and/or production of N-Acetylaspartic acid for use in the treatment of cancer. The present invention further provides for a method for detecting mitochondrial Acetyl CoA, a method for screening for an inhibitor or activator of NAT8L and/or an inhibitor of the function and/or production of N-acetylaspartic acid, method for detecting a cancer cell and a method for determining whether or not a cancer cell is susceptible to the treatment with an inhibitor of the present invention.

DESCRIPTION

[002] NAcAsp is well-known to occur in very high abundance in neuronal tissue where it is synthesized from aspartic acid and acetyl-CoA by the long unknown enzyme NAT8L. Its biosynthesis in non-neuronal mammalian cells has so far not been shown. In the mammalian brain NAcAsp is known as the precursor of the neuropeptide N-acetylaspartylglutamic acid (Becker et al. (2010) Molecular characterization of N-acetylaspartylglutamate synthetase. J Biol Chem 285: 29156-29164). Additionally, NAcAsp was shown to be an essential carrier of acetyl units for their transport from neurons to oligodendrocytes where it is cleaved by aspartic acid acylase (ASPA) to be used for myelin biosynthesis (Chakraborty et al. (2001 ) Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase. J Neurochem 78: 736-745; Madhavarao et al. (2005) Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease. Proc Natl Acad Sci U S A 102: 5221-5226). Perturbation of this transport is known to lead to pathological conditions known as Canavan's disease (Matalon et al. (1988) Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with canavan disease. American Journal of Medical Genetics 29: 463-471 ). In addition, recently NAT8L was found to be expressed in brown adipose tissue where it increases lipid turnover (Pessentheiner et al, 2013 as cited herein). [003] Non-targeted isotope labeling used by the present invention revealed the production of NAcAsp by lung cancer cells. This was unexpected because NAcAsp was only known to be produced in neuronal cells where it acts as a acetyl carrier between different cells of the nervous system.

[004] The technical problem can be seen in an alternative/improved treatment of cancer. The technical problem is solved by the embodiments reflected in the claims, described in the description, and illustrated in the Examples and Figures.

[005] Thus, the present invention relates to an Inhibitor of NAT8L and/or an inhibitor of the function and/or production of N-Acetylaspartic acid for use in the treatment of cancer.

[006] In addition, the present invention relates to an inhibitor of aspartoacylase (ASPA) for use in the treatment of cancer.

[007] The present invention also relates to the use of an inhibitor of NAT8L and an inhibitor of ATP-dependent citrate lyase (ACLY) for use in the treatment of cancer.

[008] The present invention further relates to a method for detecting mitochondrial Acetyl CoA, the method comprising

(a) using N-acetylaspartic acid.

[009] Furthermore, the present invention relates to a use of N-acetylaspartic acid for the detection of mitochondrial Acetyl CoA.

[010] In addition, the present invention relates to a method for screening for an inhibitor or activator of NAT8L and/or an inhibitor or activator of the function and/or production of N-acetylaspartic acid, the method comprising

(a) contacting cancer cells with a nucleic acid molecule such as a siRNA or miRNA, binding protein, small molecule or compound of interest;

(b) measuring proliferation of cancer cells, wherein a decrease in the proliferation of said cancer cells compared to said cancer cells before contacting indicates that the nucleic acid molecule such as the siRNA or miRNA, binding protein, small molecule or compound of interest serves as an inhibitor of NAT8L and/or as an inhibitor of the function and/or production of N- acetylaspartic acid, or wherein an increase in the proliferation of said cancer cells compared to said cancer cells before contacting indicates that the nucleic acid molecule such as the siRNA or miRNA, binding protein, small molecule or compound of interest serves as an activator of NAT8L and/or as an activator of the function and/or production of N- acetylaspartic acid.

[011] The present invention also relates to a method for determining whether or not a cancer cell is susceptible to the treatment with an inhibitor as described herein, comprising determining whether or not said cancer cell expresses NAT8L or said cancer cell comprises N-acetylaspartic acid.

[012] Additionally the present invention relates to a method for detecting a cancer cell, wherein the method comprises

(a) using primers/oligonucleotides specific for exon2 of NAT8L.

[013] Moreover, the present invention relates to a kit comprising mass spectrometric fragments of N-acetylaspartic acid.

[014] The figures show:

[015] Figure 1 : Neuronal metabolism of N-acetylaspartic acid. N-acetylaspartic acid is produced from acetyl-CoA and aspartic acid by NAT8L. It can be hydrolyzed to aspartic acid and acetic acid by ASPA or elongated to N-acetylaspartylglutamic acid by N-acetylaspartylglutamic acid synthetase (NAAGS).

[016] Figure 2: Significantly changed metabolite levels 48 h after transfection with NAT8L-targeting siRNA. Heatmap of raw signal intensities normalized to row mean. Metabolites with Welch's t-test p < 0.05 are shown. Unidentified compounds are labeled with their Rl. Rl 2718.62 has been identified as N-acetylaspartylglutamic acid [-H2O] 2TMS. Identified compounds from (A). Signal intensity after knockdown normalized to siCtrl.

[017] Figure 3: Occurrence of N-acetylaspartic acid in cancer cells. Tumorigenic RWPE-2 cells show higher levels of N-acetylaspartic acid than non-tumorigenic RWPE-1 cells (Welch's t-test, n = 3, p = 0.053). N-acetylaspartic acid levels in lung tissue of cancer patients. Abundance is higher in most tumor samples. Points are means of three technical replicates. One outlier not plotted (24 χ 105 signal intensity in tumor sample). Paired t-test, n = 19, p = 0.1 1.

[018] Figure 4: NAT8L mRNA splicing and protein domains. nat8l codes for three exons. The protein has a predicted transmembrane domain. TM: predicted transmembrane domain; NAT: N-acetyltransferase domain. (Source: NCBI protein/gene/nuccore databases). [019] Figure 5: Glycerol-3-phosphate (G3P) metabolism. G3P is a precursor for glycerolipids and part of the G3P shuttle. Cytosolic dihydroxyacetone phosphate (DHAP) can be reduced to G3P by NAD-dependent cytosolic GPD1 or GPD1 L and reoxidized by mitochondrial membrane-bound GPD2 with concomitant reduction of FAD, effectively transferring two electrons to the mitochondrium. GPD gene expression analysis in A549 cells. GPD1 L expression is slightly reduced in siNAT8L transfected cells. GPD2 is induced in siNAT8L transfected cells under normoxia. Bars show mean±SD of two replicates (Welsh's t-test, n = 2, p > 0.05).

[020] Figure 6: Changes in MIDs due to NAT8L knockdown after labeling with [U- 13C]glucose and [U-13C]glutamine under (A) normoxia and (B) hypoxia. Welch's t- test, n = 3, ^*: p < 0.05, ^**: p < 0.01 , ^***: p < 0.001 .

[021] Figure 7: Model of cytosolic-mitochondrial acetyl-CoA transport under hypoxia where acetyl-CoA-carbon is mainly derived from glutamine. Classical model for providing cytosolic acetyl-CoA via oxidative (red) or reductive (blue) glutamine metabolism, not considering a role of NAT8L. Both pathways rely on slow citrate formation via citrate synthase, and reductive IDH flux and citrate synthase reaction possibly suffer from product inhibition by citrate. Proposed alternative model based on the N-acetylaspartic acid shuttle for acetyl transport. N-acetylaspartic acid is formed by NAT8L in the mitochondrium, transported to the cytosol where it is cleaved and the resulting acetate is used to provide acetyl-CoA. Here, no competing citrate- forming reactions are necessary.

[022] Figure 8: Gene expression levels in response to NAT8L silencing. NAT8L silencing is stronger under normoxia. It induces gene expression of ACSS1 and ACSS2 and represses citrate synthase expression. ACLY gene expression is not affected. ^*: Welch's t-test p < 0.05 (n = 3), ^**: p < 0.01 , ^***: p < 0.001 . n = 3, except for ACLY expression and "1 % 0₂ siNAT8L" levels, where n = 2.

[023] Figure 9: Model of the role of N-acetylaspartic acid and NAT8L in normoxic cancer cells where cytosolic acetyl-CoA is mainly derived from glucose. Classical model for providing cytosolic acetyl-CoA via glycolysis, PDH, citrate synthase, and ACLY. Citrate synthesis is slow and a potential bottleneck for NADH and FADH2 production when it is exported to the cytosol. The alternative model based on the N- acetylaspartic acid shuttle can export acetyl-CoA from the mitochondrium without interfering with the TCA cycle, assuming that there is enough mitochondrial acetyl- CoA available. The slow (Yudko et al. (1994) Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle. J Biol Chem, 1994;269(44):27414{27420) citrate synthase reaction is circumvented.

[024] Figure 10: A549 cell growth during 72 h of NAT8L, ACLY, and combined NAT8L+ACLY silencing under normoxic and hypoxic conditions. Mean±standard deviation of the mean (SD) of cell numbers of three replicates are shown. Welch's t- test, n = 3, ^*: p < 0.05.

[025] Figure 1 1 : Proxies for acetyl-CoA labeling. From the mass spectra of the acetylated compounds Rl 1823 and N-acetylaspartic acid, the labeling in their acetyl- moiety can be determined (Example 10). Graph shows the ¹³C enrichment after [U- ¹³C]glutamine labeling. The different enrichments point to two distinct subcellular acetyl-CoA pools.

[026] Figure 12: Workflow for non-targeted mass isotopolome analysis. Non- targeted approaches are valuable scouting experiments for hypothesis generation. These hypotheses are tested by more targeted techniques leading to refined hypotheses and biological insights. Data-driven analysis of non-targeted stable isotope labeling experiments. After stable isotope labeling experiments, active pathways and changed fluxes can be detected in a non-targeted manner from MIDs and changes therein. Compound identity and additional biochemical knowledge is only needed for further interpretation. Analysis of MID similarity between compounds can aid their identification or help to determine their biosynthetic pathway. Yellow boxes highlight novel analysis techniques presented in this work.

[027] Figure 13: Changes in fluxes are necessary, but not sufficient for MID changes. MIDs after stable isotope labeling with [U-¹³C]glutamine in glutamine catabolism and a simplified section of the TCA cycle. Only these isolated reactions are considered and treated as irreversible and in isotopic and metabolic steady state (metabolite concentrations and MIDs are constant over time). Isotopic labeling is fully defined by the tracer and metabolic fluxes. MIDs alone can only provide relative flux information: If both IDH and GLS fluxes change proportionally, i.e. their ratio is constant, this change cannot be recognized from the MIDs (A,C). However, if their flux ratio changes, the MIDs of downstream metabolites will change accordingly (A,B,D, blue MIDs). Grey MIDs do not depend on either IDH or GLS flux, and thus, are not informative. [028] Figure 14: MID similarity analysis for pathway contextualization and detection of metabolically related compounds. A The pairwise similarities of all MIDs is determined. A similarity threshold is applied, and compounds with highly similar MIDs are visualized as network. Networks derived from different experimental conditions can be overlaid for more information. B Before the distance calculation the MID vectors are aligned to account for gains or losses of labeled fragments, which would otherwise conceal the metabolic proximity of these compounds.

[029] Figure 15: Effects of different oxygen concentrations on MIDs and metabolic fluxes in lung cancer cells fed with [1 ,2-¹³C]glucose and [U-¹³C]glucose. A Analysis of mass isotopomer abundance variation revealed that the MIDs of citric acid cycle associated metabolites and some unidentified compounds were the most affected ones. Arrows indicate the mass isotopomers with the highest abundance variation. Unidentified compounds are named by their chromatographic retention index (Rl). B Simplified model of how these strong changes in MIDs at low oxygen can be explained by the relative reduction of PDH flux and inversion of IDH flux directionality. C MID-similarity aided compound identification. The network shows the compounds with the closest MIDs to the unidentified compound 'Rl 1651 ' after glucose or glutamine labeling. Edge color represents the condition at which the high MID similarity was Unidentified acetylated hexosamine Rl 1823 Another initially un-known compound was found to exhibit strong alterations in MIDs with changing oxygen concentrations. Abbreviations: ASAT aspartate aminotransferase; IDH/MDH/FUM/SDH/OGDH/PDH/GLUD isocitrate/malate/fumarate/succinate/2- oxoglutarate/pyruvate/glutamate dehydrogenase; ACL ATP-dependent citrate lyase; AT aminotransferases; ACO aconitase; Glc glucose; Pyr pyruvic acid; AcCoA acetyl- coenzyme A; Cit citrate; OAA oxaloacetic acid; G3P glycerol-3-phosphate.

[030] Figure 16: List of sequences.

[031] Cancer cells are known to have a high requirement of fatty acids for lipid biosynthesis to sustain cell proliferation and, therefore, increased fatty acid uptake and de novo biosynthesis. De novo fatty acid biosynthesis requires cytosolic acetyl- CoA, which was traditionally thought to derived from citrate by ATP-dependent citrate lyase (ACLY).

[032] The present invention shows that - in addition to citrate-derived acetyl-CoA - there is a significant contribution from N-acetylaspartate. In the brain, N- acetylaspartate (NAA) is known as part of an inter-cellular acetyl-CoA shuttle between neurons and oligodendrocytes. Starting from neuronal NAA biosynthesis from aspartate and acetyl-CoA by NAT8L, transport to oligodendrocytes, hydrolysis by ASPA, the resulting acetate is activated to acetyl-CoA by cytosolic ACSS2 and used for lipid biosynthesis. The present invention surprisingly shows that the same reaction sequence is used in cancer cells for intra- not inter-cellular transport across the mitochondrial membrane.

[033] The discovery of this additional transport mechanism is highly relevant for cancer therapy. Inhibition of NAT8L and/or the function and/or production of N- acetylaspartic acid therefore provides for a new therapy for treating cancer.

[034] In addition, the ACLY-inhibitor hydroxycitrate has been shown to slow down cancer growth, but these cells most likely still have an operating NAA-based pathway in place. Combined targeting of ACLY and NAT8L or ASPA leads to a synergistic inhibitory effect on cancer cell proliferation. The present invention relates to an inhibitor of NAT8L and/or an inhibitor of the function and/or production of N- Acetylaspartic acid for use in the treatment of cancer. The present invention also relates to the use of an Inhibitor of NAT8L and an inhibitor of ATP-dependent citrate lyase (ACLY) for use in the treatment of cancer.

[035] In general a "therapy" or "treatment" seeks remediation of a health problem such as cancer, usually following a diagnosis. In the medical field, this term is synonymous with treatment of a disease or disorder. Therefore, in this context, a therapy also includes the administration of an inhibitor of NAT8L and/or an inhibitor of the function and/or production of N-Acetylaspartic acid. Likewise, a "therapeutic effect" relieves to some extent one or more of the symptoms of the abnormal condition, such as cancer.

[036] The present invention contemplates any inhibitors that can serve as an inhibitor of NAT8L. The determination of whether or not a compound is an inhibitor of NAT8L and/or an inhibitor of the function and/or production of N-Acetylaspartic acid is within the skill of one of ordinary skill in the art. Examples of assays useful to identify inhibitor of NAT8L and/or an inhibitor of the function and/or production of N- Acetylaspartic acid include those as described in the Examples.

[037] An example of how one could determine if a compound is an inhibitor of NAT8L would be to isolate the NAT8L protein. For example, the amino acid sequence of the protein that encodes human NAT8L is Uniprot number Q8N9F0 (SEQ ID NO: 1 ). The amino acid sequence of the protein that encodes mouse NAT8L is Uniprot number Q3UGX3, that encodes nat8L in zebrafish is Uniprot number F1 QNT7, that encodes Nat8L in rat is uniprot number D3ZVU9. The protein can be isolated from cells where the NATSL is naturally expressed or where it has been overexpressed by means of transfection of a genetic construct or infection with a virus that directs the expression of the NATSL. The nucleic acid sequence of the mRNA that encodes NATSL is NCBI Reference Sequence: NM_178557.3 (SEQ ID NO: 2) or N __001001985.3 (SEQ ID NO. 3). Also the mRNA can be isolated from a ceil and e.g. be expressed in a host ceil. NAT8L can for example be expressed recombinantly.

[038] An inhibitor to NATSL may be effective in any possible way. For example, the expression of NATSL (e.g. of NATSL protein, mRNA or DNA) may be inhibited/reduced. Another possibility can be that the function of NATSL may be inhibited/reduced/decreased.

[039] In general any reduction in expression as described herein can be measured by any technique, which is known to the skilled person. For example, such measurement can be performed by "real-time PCR" or "Real-time Polymerase Chain Reaction (RT~PCR)" or qPCR. This technique has the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected throughout the PCR process, rather than at the end of the PCR. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. There are two main methods used to perform quantitative PCR: dye-based and probe-based detection. Both methods rely on calculating the initial (zero cycle) DNA concentration by extrapolating back from a reliable fluorescent signal. The basic principle of this method is known in the art (Arya M, Shergiii I S, Williamson M, Gommersall L, Arya N, Patel HRH "Basic principles of quantitative real time PCR" Expert Rev. ol. Diagn. 5(2):209-219). An inhibitor of NATSL can thus descrease the expression of an amino acid sequence or nucleic acid molecule comprising SEQ ID NO. 1 , SEQ ID NO. 2 and/or SEQ ID NO. 3 or an amino acid sequence or nucleic acid molecule having at least 80 %, 70 %, 80 % 90 % 95 % 99 % sequence identity to any of SEQ ID No. 1 , 2 and/or 3 e.g. in a cell,

[040] An inhibitor may additionally or alternatively inhibit/reduce/decrease NAT8L (function) by 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more when compared to the activity of NAT8L without the addition of the inhibitor or compared to the acitivty of NAT8L before the addition of the inhinitor. A block of NAT8L (function) to be inhibited is present when the enzymatic activity of NAT8L is inhibited by 100 % when compared to the enzymatic activity of NAT8L without the addition of the inhibitor or compared to the acitivty of NAT8L before the addition of the inhinitor. It is also contemplated by the present invention that the inhibitor inhibits a protein that catalyzes the synthesis of Acetyl-CoA and/or L-aspartate.

[041] Upon isolating the NAT8L protein a person of ordinary skill in the art can measure its activity in the presence or absence of a potential NAT8L inhibitor, preferably using positive and/or negative controls. Notably, NAT8L catalyzes the following reaction: Acetyi-CoA + L-aspartate = CoA + N-acetyl-L-aspartate. If the activity of NAT8L is less in the presence of the inhibitor than in the absence of an alleged inhibitor, then this inhibitor truly is an NAT8L inhibitor. For example, an assay comprising an inhibitor, NAT8L, Acetyl-CoA and L-aspartate may generate less CoA and/or N-acetyl-L-aspartate (N-Acetylaspartic acid) than the same assay without the inhibitor. Then the inhibitor decreases AT8L function.

[042] The levels of the compounds as described herein, such as e.g. N-acetyl-L- aspartate, Acetyl-CoA etc. can be measured by any technique known by the skilled artesian. For exampe, measurement can be performed by immunihisto- or cytology using antibodies directed against the compound to be measured or by spectometry. The method of spectrometry is described elswhere herein.

[043] To confirm that a compound is a NAT8L inhibitor useful to treat cancer, the inhibitor may be tested in a routine (cancer cell) apoptosis assay or proliferation assay to confirm and assess its activity to induce apoptosis or reduce proliferation. As used herein ceil growth (proliferation) of cancer ceils can also be called tumor growth.

[044] The present invention further contemplates that the NAT8L inhibitor and/or the ACLY inhibitor can inhibit the growth/proliferation of cancer/tumor ceils of around 5 %, 10 %, 15 %, 20 % 25 %, 30 %, 35 %, 40 %, 45 %, 50 % or more compared to the proliferation/growth measured before addition of the inhibitor(s). The inhibition of cell proliferation of cancer/tumor ceils can take place under normoxic or hypoxic conditions. Growth inhibitory effect of the inhibitor(s) may be lower under hypoxia than under normoxia.

[045] An example of how one could determine if a compound is an inhibitor the function and/or production of N-Acetyiaspartic acid can be an assay as described for NAT8L. This is because an inhibitor of NAT8L can result in reduced production of N- Acetylaspartic acid. To confirm that such an inhibitor or any other inhibitor as described herein is useful to treat cancer, the found inhibitor may be tested in a routine (cancer eel!) apoptosis or proliferation assay to confirm and assess its activity to induce apoptosis or reduce proliferation (of cancer cells). The inhibitors for use of the present invention can for example be a siRNA, miRNA, binding protein, small molecule or compound. The inhibitors for use of the present invention can also be a nucleic acid molecule such as siRNA or miRNA.

[046] The term "nucleic acid molecule" when used herein encompasses any nucleic acid molecule having a nucleotide sequence of bases comprising purine- and pyrimidine bases which are comprised by said nucleic acid molecule, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences can include DNA, cDNA, genomic DNA, RNA, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. A polynucleotide can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

[047] A variety of modifications can be made to DNA and RNA; thus, the term "nucleic acid molecules" can embrace chemically, enzymatically, or metabolically modified forms. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine.

[048] The nucleic acid molecule can, for example, be a siRNA or a miRNA. Further, the nucleic acid molecule can, for example, be designed with regard to a target sequence. The target sequence can, for example, be a nucleic acid molecule of any of SEQ ID NO. 16, 17, 18 and/or 19. The nucleic acid molecule that can be used in the present invention can therefore comprise a sequence that is complementary to a sequence that comprises any of SEQ ID NO: 16, 17, 18 and/or 19. The present invention also encompasses nucleic acid sequences (in particular siRNA sequences) which are 50 %, 60 %, 70 %, 80 %, 85 %, 90 %, 95 %, 97 %, 99 % or 100 % complementary to a nucleic acid molecule that comprises a sequence of SEQ ID NO: 16, 17, 18 and/or 19.

[049] In accordance with the present invention, the term "identical" or "percent identity" in the context of two or more nucleic acid molecules or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 95 %, 96 %, 97 %, 98 % or 99 % identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 80 % to 95 % or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

[050] Also available to those having skill in this art are the BLAST and BLAST 2.4 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses as defaults a word size (W) of 28, an expectation (E) of 10, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 6, and an expectation (E) of 10. Furthermore, the BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) can be used.

[051] For example, BLAST2.4, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), can be used to search for local sequence alignments.

[052] The inhibitors for use of the present invention can for example be a binding protein. Exemplary binding proteins include an antibody such as a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule with antibody-like binding properties.

[053] Such an "antibody" can be a full length antibody, a recombinant antibody molecule, or a fully human antibody molecule. A full length antibody is any naturally occurring antibody. The term "antibody" also includes immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as lgG1 , lgG2 etc.). Such full length antibodies can be isolated from different animals such as e.g. different mammalian species. The "recombinant antibody molecule" refers to an antibody molecule, the genes of which have been cloned, and is produced recombinantly in a host cell or organism, using well-known methodologies of genetic engineering. Typically, a recombinant antibody molecule has been genetically altered to comprise an amino acid sequence, which is not found in nature. Thus, a recombinant antibody molecule can be a chimeric antibody molecule or a humanized antibody molecule.

[054] The antibody/inhibitor can also be an "antibody fragment". Such antibody fragments comprise any part of an antibody, which comprises a binding site. Illustrative examples of such an antibody fragment are single chain variable fragments (scFv), Fv fragments, single domain antibodies, such as e.g. VHH (camelid) antibodies, di-scFvs, fragment antigen binding regions (Fab), F(ab')₂ fragments, Fab' fragments, diabodies or domain antibodies, to name only a few (Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM. Domain antibodies: proteins for therapy. Trends Biotech nol. 2003 Nov; 21 (1 1 ):484-90).

[055] For example, an inhibitor/antibody used in the present invention can be a divalent antibody fragment such as an (Fab)₂' -fragment or a divalent single-chain Fv fragment. Therefore, an antibody/inhibitor used in the present invention can be an antibody or antibody fragment, which has an antibody format as described in International patent application WO2013/092001 . Alternatively, the inhibitor/antibody might also be a bivalent proteinaceous artificial binding molecule such as a lipocalin mutein that is also known as "duocalin".

[056] An inhibitor or an antibody used in the present invention may only have a single binding site, i.e., may be monovalent. Examples of monovalent inhibitors include, but are not limited to, a monovalent antibody or antibody fragment, a monovalent proteinaceous binding molecule with antibody-like binding properties. Examples of monovalent antibody fragments include, but are not limited to a Fab fragment, a Fv fragment, and a single-chain Fv fragment (scFv).

[057] In some embodiments, antibody derived inhibitors that are used in the present invention may comprise an attenuated Fc-part. An Fc-part is, for example, attenuated, when such an antibody molecule is not able to bind via the CH2 or the CH3 domain to Fc receptors anymore, or binds less efficiently to them than a parent antibody. Examples of mutations that can be introduced into the CH2 or CH3 domain to achieve such Fc attenuation are described in International patent application WO2013/092001 (cf. for example, Figures 1 N, O of WO 2013/092001 ). In other embodiments, antibody derived inhibitors used in the present invention may comprise no Fc part at all.

[058] The binding protein as used in the present invention can thus be selected from the group consisting of an (full length, recombinant, chimeric) antibody, a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule with antibody-like binding properties.

[059] The present invention further envisiones that the divalent antibody fragment can be an (Fab)2'-fragment, a divalent single-chain Fv fragment, a bsFc-1/2-dimer or a bsFc-CH3-1/2 dimer.

[060] It is also contemplated by the present invention that the monovalent antibody fragment is selected from the group consisting of a Fab fragment, a Fv fragment, a single-chain Fv fragment (scFv) or an scFv-Fc fragment.

[061] An inhibitor used in the present invention can also be a proteinaceous binding molecule with antibody-like binding properties. Non-limiting examples of a proteinaceous binding molecule with antibody-like binding properties inlcude an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, an avimer or a (recombinant) receptor protein.

[062] Illustrative examples of proteinaceous binding molecules with antibody-like binding properties that can be used as inhibitor include, but are not limited to, an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, an avimer, a EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a G1 a domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an immunoglobulin domain or a an immunoglobulin-like domain (for example, domain antibodies or camel heavy chain antibodies), a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP-type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin- type EGF-like domain, a C2 domain, "Kappabodies" (III CR1 , Gonzales JN, Houtz EK, Ludwig JR, Melcher ED, Hale JE, Pourmand R, Keivens VM, Myers L, Beidler K, Stuart P, Cheng S, Radhakrishnan R. Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions. Protein Eng. 1997 Aug; 10(8):949-57) "Minibodies" (Martin F, Toniatti C, Salvati AL, Venturini S, Ciliberto G, Cortese R, Sollazzo M. The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6. EMBO J. 1994 Nov 15;13(22):5303-9), "Janusins" (Traunecker A, Lanzavecchia A, Karjalainen K. Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells. EMBO J. 1991 Dec; 10(12):3655-9 and Traunecker A, Lanzavecchia A, Karjalainen K. Janusin: new molecular design for bispecific reagents. Int J Cancer Suppl. 1992;7:51 -2), a nanobody, a tetranectin, a microbody, an affilin, an affibody or an ankyrin, a crystallin, a knottin, ubiquitin, a zinc-finger protein, an autofluorescent protein, an ankyrin or ankyrin repeat protein or a leucine-rich repeat protein, an avimer (Silverman J, Liu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H, Whitehorn E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Stemmer WP. Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol. 2005 Dec;23(12): 1556-61 ); as well as multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains as also described in Silverman et al. (2005) cited herein). In some embodiments, the inhibitor used in the present invention is a proteinaceous binding molecule with antibody-like binding properties, which is selected from the group of an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, and an avimer.

[063] Alternatively, an inhibitor used in the present invention can also be a non- proteinaceous aptamer. Such an aptamer is an oligonucleic acid that binds to a specific target molecule. These aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist. More specifically, aptamers can be classified as: DNA or RNA aptamers. They consist of (usually short) strands of oligonucleotides. Therefore, a proteinaceous aptamer as described above may also include an oligonucleotide portion in addition to a protein portion.

[064] The inhibitors for use of the present invention can also be a small molecule. Such a small molecule can have a low molecular weight of less than 900 daltons (da), less than 800 da, less than 700 da, less than 600 da or less than 500 da. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry.

[065] The inhibitors for use of the present invention can also be a compound. The term "compound" embraces any compound that may serve as an inhibitor for the enzymes as described herein and/or as an inhibitor of the function and/or production of N-Acetylaspartic acid.

[066] The inhibitor of the function and/or production of N-Acetylaspartic acid for use of the present invention can for example be selected from the group consisting of roteneone, myxothiazol, cyanide or oligomycin. An exemplary inhibitor for ACLY is hydroxycitrate.

[067] The inhibitors for use in the present invention can for example reduce the availability of Acetyl-CoA in the cytosol of cancer cells compared to availability of Acetyl CoA before addition of the inhibitor.

[068] Additionally or alternatively, the inhibitors for use in the present invention can reduce proliferation of cancer cells. Cell proliferation can produce two cells from one, and it can require cell growth followed by cell division. Uncontrolled cell proliferation can, for example, be a hallmark of cancer. Methods to detect proliferation of cells are well known to the skilled artesian. E.g. cell proliferation can be measured by immunohistological staining for Ki-67. For example, proliferation can be measured before administration/addition of the inhibitor(s) and the obtained results can then be compared to the measured proliferation after administration/addition of the inhibitor(s). If the measured proliferation after administration/addition of the inhibitor is lower than the masured proliferation before administration/addition, then the inhibitor reduces cell proliferation.

[069] Additionally or alternatively, the inhibitors for use in the present invention can reduce proliferation of cancer cells under normoxic and hypoxic conditions. A "normoxic condition" as used herein refers to a normal oxygen concentration. Such a normal oxygen concentration typically is about 20-21 % in the atmosphere, or 2-3% in physiological contexts. A "hypoxic condition" is a condition, which has a lower oxygen concentration than what is the normal oxygen condition. For example, a hypoxic condition can be a condition in which the oxygen concentration is below 20% in the atmosphere, or below 2% in physiological contexts.

[070] Additionally or alternatively, the inhibitors for use in the present invention can increase the de novo synthesis of glycerol-3-phosphate.

[071] An inhibitor of NAT8L may for example decrease the abundance of N-aspartic acid and/or the abundance glycerol-3-phospbate levels. In this regard, for example, glycerol-3-phosphate levels can be decreased to 60 %, 50 %, 40 %, 30 %. 20 % 10 % in the presence of the inhibitor compared to the glyceroi-3-phosphate level measured in the absence of the inhibitor or before addition of the inhibitor. Giyceroi- 3-phosphate levels can also be decreased to 13 % in the presence of the inhibitor compared to the glyceroi-3-phosphate level measured in the absence of the inhibitor or before addition of the inhibitor. Additionally or alternatively, the inhibitors for use in the present invention can reduce the glycerol-3-phosphate from lipid turnover.

[072] Additionally or alternatively the inhibitor of NAT8L may increase the expression of Glycerol-3-phosphate dehydrogenase 2 (GPD2). The GPD2 may have a protein sequence comprising SEQ ID NO. 20 (Uniprot Number: P43304). The increase in expression of GPD2 may take place under normoxic conditions as described herein. Thus, additionally or alternatively, the inhibitors for use in the present invention can increase expression of GPD2 under normoxia.

[073] It is further envisioned that the inhibitors used in the present invention can increase GPD2 expression by 10 %, 20 %, 30 % 40 % 50 % or more compared to GPD2 expression measured before the addition of the inhibitor or in the absence of the inhibitor. For example, the NAT8L inhibitor may increase GPD2 expression by 40 % at normoxia compared to GPD2 expression before additon of the NAT8L inhibitor.

[074] Additionally or alternatively, the inhibitor for use in the present invention does, for example, virtually not affect expression of GPDL1 under normoxia and hypoxia.

[075] The inhibitors disclosed are used to treat cancer. Non-limiting examples of cancers include adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non- Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, rectum cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, basal and squamous cell cancer, melanoma, merkel cell cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor. For example, the cancer can be a liver cancer, prostate cancer or lung cancer, preferably a lung cancer.

[076] Additionally or alternatively, the inhibitors for use in the present invention can reduce the invasiveness of the cancer. The invasiveness of cancer or cancer cells as used herein relates to the ability of these cancers/cells to spread beyond the layer of tissue in which it/they developed and its ability to grow into surrounding, healthy tissues. These motile cells may then pass through the basement membrane and extracellular matrix, may progress to intravasation as they penetrate the lymphatic or vascular circulation. The metastatic cells may then journey through the circulatory system and may invade the vascular basement membrane and extracellular matrix in the process of extravasation. Ultimately, these cells may attach at a new location and proliferate to produce a secondary tumor (or metastasis).

[077] Notably, several members of the Ras superfamilyfamilies of small GTP-binding proteins whose functions of proteins have been implicated in these (invasive) processes as described in Hernandez-Alcoceba et al. (2000) "The Ras family of GTPases in cancer cell invasion" Cell. Mol. Life Sci. 57; 65-76.

[078] An effect of the inhibitors concerning a reduction of the invasiveness of a cancer/cancer cell can be measured as e.g. described in Example 2. For example, firstly some cells are transformed with v-Ki-ras (e.g. RWPE-2 cells). Then the inhibitor(s) are added to these cells and e.g. abundance of N-acetylaspartic acid or any other compound suitable for detection of the enzymatic acitvity of the enzyme (as also described herein) to be inhibited can be measured. If, the abundance of N- acetylaspartic acid or any other suitable compound is lower than the abundance of N- acetylaspartic acid or any other suitable compound before addition of the inhibitor, the inhibitor also reduces the invasiveness of a cancer/cancer cells. [079] It is also envisioned by the present invention that the inhibitor(s) further comprise an inhibitor of ATP-dependent citrate lyase (ACLY). The present invention contemplates any inhibitors that can serve as an inhibitor of ACLY. There are many examples of ACLY inhibitors in the art. Some are for example mentioned in US 5,447,954 or in Barrow, et al. (1997) "Antimycins, Inhibitors of ATP-Citrate Lyase, from a Streptomyces sp.", Journal of Antibiotics, vol. 50, No. 9, pp. 729. Other known inhibitors can for example include (-)hydroxycitrate, (R,S)-S-(3,4-dicarboxy-3- hydroxy-3-methyl-butyl)-CoA. radicicol, tartrate and S-carboxymethyl-CoA, cell- penetrant gamma-lactone or the chemical inhibitor SB-2G4990, a prodrug of SB- 201076. ACLY can also be inhibited by other known strategies known to the skilled artesian such as by RNAi or anti-ACLY antibodies. Multiple agents, such as a-lipoic acid, statins, capsaicin, a Met kinase inhibitor (SU 1 1274), etc., have been found to enhance the effects of ACLY inhibitors in small studies of tumors. Thus, these agents may be combined with the inhibitor of ACLY. The determination of whether or not a compound is an ACLY inhibitor is within the skill of one of ordinary skill in the art.

[080] An inhibitor to ACLY may work in any way. For example, the inhibitor may inhibit/decrease/reduce the expression of ACLY (e.g. of ACLY protein, mRNA or DNA) as described herein. Therefore, the inhibitor of ACLY may reduce the expression of an amino acid sequence comprising SEQ ID NO. 4 (Uniprot number: P53396). An inhibitor of ACLY can thus descrease/reduce the expression of an amino acid sequence comprising SEQ ID NO. 4 or an amino acid sequence having at least 60 %, 70 %, 80 % 90 % 95 % 99 % sequence identity to any of SEQ ID No. 4 e.g. in a cell. Additionally or alternatively, the inhibitor may reduce the expression of mouse acly (Uniprot Number: Q91V92-1 ).

[081] An inhibitor of ACLY may additionally or alternatively reduce/inhibit/decrease ACLY (function). For example, the inhibitor may reduce the (enzymatic) activity of ACLY by 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more when compared to the measured activity of ACLY without the addition of the inhibitor or before addition of the inhibitor. A block of ACLY (function) to be inhibited is present when the enzymatic activity of ACLY is inhibited by 100 % when compared to the enzymatic activity of ACLY without the addition of the inhibitor or before the addition of the inhibitor.

[082] Notably, ACLY catalyzes the following reaction: ADP + phosphate + acetyl- CoA + oxaloacetate = ATP + citrate + CoA. If the activity of ACLY is less in the presence of the inhibitor than in the absence of an alleged inhibitor then this inhibitor truly is an ACLY inhibitor. For example an assay comprising an inhibitor, ACLY, ADP, phosphate, acetyi-CoA, oxaloacetate may generate less ATP, citrate and/or CoA than the same assay without the inhibitor or before the addition of the inhibitor. Then the inhibitor decreases ACLY function.

[083] Additionally or alternatively, the inhibitor can further comprise an inhibitor of aspartoacylase (ASPA). The present invention contemplates any inhibitors that can serve as an inhibitor of ASPA. An exemplary ASPA inhibitor is diisopropyl fluorophosphate.

[084] An inhibitor to ASPA may work in any way. For example, the inhibitor may reduce/inhibit the expression of ASPA (e.g. ASPA protein, mRNA and/or DNA). An inhibitor of ASPA can thus descrease the expression of an amino acid sequence comprising SEQ ID NO. 5 (Uniprot number; P45381 ) or an amino acid sequence having at least 60 %, 70 %, 80 % 90 % 95 % 99 % sequence identity to SEQ ID No. 5 e.g. in a cell. Additionally or alternatively, the inhibitor may reduce the expression of mouse aspa (Uniprot Number: Q8R3P0).

[085] An inhibitor of ASPA may additionally or alternatively reduce or decrease the (enzymatic) activity of ASPA (ASPA function) by 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more when compared to the activity of ASPA without the addition of the inhibitor or compared to the activity of ASPA before the addition of the inhibitor. A block of ASPA (function) to be inhibited is present when the enzymatic activity of ASPA is inhibited by 100 % when compared to the enzymatic activity of ASPA without the addition of the inhibitor or to the enzymatic acitivty measured before the addition of the inhibitor.

[086] Notably, ASPA catalyzes the following reaction: N-acyi-L-aspartate + H₂0 = a carboxylate + L-aspartate. If the activity of ASPA is less in the presence of the inhibitor than in the absence of an alleged inhibitor, then this inhibitor truly is an ASPA inhibitor. For example, an assay comprising an inhibitor, ASPA, N-acyl~L- aspartate, H₂0 may generate less a carboxylate and/or L-aspartate than the same assay without the inhibitor. Then this inhibitor is an ASPA inhibitor.

[087] Additionally or alternatively, the inhibitor can further comprise an inhibitor of acyl-CoA synthetase (ACSS). The present invention contemplates any inhibitors that can serve as an inhibitor of ACSS. Exemplary inhibitors of ACSS include triacsins, long-chain acyl-coenzyme A (CoA) compounds (palmityl, stearyl, and oleyl) of Saccharomyces cerevisiae strain LK2G12 from aerobic cells, allicin, rosiglitazone, Triacsin C, VPA and the like.

[088] An inhibitor to ACSS may work in any way. For example, the inhinitor may reduce/decrease/inhibit the expression of ACSS (e.g. the expression of ACSS protein, mRNA and/or DNA). Therefore, the inhibitor of ACSS may reduce the expression of an amino acid sequence comprising SEQ ID NO. 6 (Uniprot number: 014975; Very long-chain acyl-CoA synthetase), SEQ ID NO. 7 (Uniprot number: Q53FZ2; Acyl-coenzyme A synthetase ACSIV13, mitochondrial), SEQ ID NO: 8 (Uniprot Number: Q6NUN0; Acyl-coenzyme A synthetase ACSfvIS, mitochondrial), SEQ ID No. 9 (Uniprot Number: Q08AH1 ; Acyl-coenzyme A synthetase ACSM1 , mitochondrial), SEQ ID No. 10 (Uniprot Number: Q08AH1 ; Acyl-coenzyme A synthetase ACSIV12A, mitochondrial), SEQ ID No. 1 1 (Uniprot Number: Q68CK6; Acyl-coenzyme A synthetase AC81V12B, mitochondrial), SEQ ID No. 12 (Uniprot Number: Q6P461 ; Acyl-coenzyme A synthetase ACSM8, mitochondrial), and/or SEQ ID No. 13 (Uniprot Number: P0C7M7; Acyl-coenzyme A synthetase ACSM4, mitochondrial).

[089] An inhibitor of ACSS may additionally or alternatively reduce or decrease enzymatic activity of ACSS (ACSS function) by 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more when compared to the activity of ACSS measured without the addition of the inhibitor or measured before the addition of the inhibitor. A block of ACSS (function) to be inhibited is present when the enzymatic activity of ACSS is inhibited by 100 % when compared to the enzymatic activity of ACSS without the addition of the inhibitor or measured before the addition of the inhibitor.

[090] Notably, ACSS catalyzes the following reaction: ATP + a carboxyiate + CoA = AMP + diphosphate + an acyi-CoA. If the activity of ACSS is less in the presence of the inhibitor than in the absence of an alleged inhibitor, then this inhibitor truly is an ACSS inhibitor. For example an assay comprising an inhibitor, ACSS, ATP, a carboxyiate and CoA may generate less AMP, diphosphate and/or an acyl-CoA than the same assay without the inhibitor. Then this inhibitor is an ACSS inhibitor.

[091] In general, all the inhibitors mentioned herein can also be such that they hybridize to the mRNA that can be translated to the protein sequences as described herein. For example such mRNA may be translated into a protein comprising any of amino acid sequences of SEQ ID NO. 1 , 3-13. Furthermore, the inhibitor may hybridize to a sequence comprising a nucleic acid sequence of SEQ ID NO. 2 and/or SEQ ID NO. 3.

[092] The term "hybridizes" as used in accordance with the present invention may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001 ); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well known within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as O. lxSSC, 0.1 % SDS at 65°C. Non- stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6xSSC, 1 % SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Notably variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[093] Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).

[094] In addition, the inhibitors can be a nucleic acid sequence that is 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % complementary to a mRNA that will be translated into an amino acid sequence comprising any of SEQ ID Nos. 1 , 3-13 or comprising an amino acid sequence having a sequence identity of 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % to any of SEQ ID NO. 1 , 3-13 or to the nucleic acid sequence compising SEQ ID NO. 2 and/or SEQ ID NO. 3 or a nucleic acid seuquence having a sequence identity of 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % to SEQ ID NO. 2 and/or 3.

[095] The terms "complementary" or "complementarity" refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.

[096] Inhibitors used in the present invention can also be used in co-treatment with other therapies such as other anti-cancer therapies. This co-treatment can include administration of an inhibitor used in the present invention, preferably in the form of a medicament, to a subject suffering from a condition comprising cancer. Similarly included is the administration of an inhibitor used in the present invention, preferably in the form of a drug/medicament, to a subject.

[097] The inhibitor(s) used in the present invention can be administered by any suitable route. Exemplary routes of administration include oral, intravenous, intrapleural, intramuscular, topical or via inhalation.

[098] The inhibitors used in the present invention may also be comprised in a pharmaceutical composition. The pharmaceutical composition can be administered to a subject. Such pharmaceutical compositions can be administered in any suitable unit dosage form. Suitable oral formulations can be in the form of tablets, capsules, suspension, syrup, chewing gum, wafer, elixir, and the like. Pharmaceutically acceptable carriers such as binders, excipients, lubricants, and sweetening or flavoring agents can be included in the pharmaceutical compositions. If desired, conventional agents for modifying tastes, colors, and shapes of the special forms can also be included.

[099] For injectable formulations, the pharmaceutical compositions can be in lyophilized powder in admixture with suitable excipients in a suitable vial or tube. Before use in the clinic, the inhibitors may be reconstituted by dissolving the lyophilized powder in a suitable solvent system to form a composition suitable for intravenous or intramuscular injection.

[100] The present invention also relates to a method for the prophylaxis and/or treatment of cancer in a subject, comprising administering a therapeutically effective amount of one or more inhibitors as described herein or a pharmaceutical composition as described herein to the subject.

[101] Also the present invention provides for a use of one or more inhibitors as described herein or a pharmaceutical composition as described herein for the preparation of a medicament.

[102] In addition the present invention relates to a use of one or more inhibitors as described herein or a pharmaceutical composition as described herein for the prophylaxis and/or treatment of cancer.

[103] Similarly, the present invention also provides for a method for the prophylaxis and/or treatment of cancer in a subject, comprising administering a therapeutically effective amount of one or more inhibitor(s) as described herein or a pharmaceutical composition as described herein to the subject.

[104] The "subject", which may be treated with one or more inhibitors or pharmaceutical compositions as described herein, can be a vertebrate. The vertebrate can further be a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. Preferably, a mammal is a human, dog, cat, cow, pig, mouse, rat etc., particularly preferred, it is a human. In the context of the present invention the term "subject" can mean an individual in need of a treatment and/or prophylaxis of cancer. The subject can also be a patient suffering from cancer or being at a risk thereof.

[105] The present invention also relates to a method for detecting mitochondrial Acetyl CoA, the method comprising (a) using N-acetylaspartic acid. The detection can be for example performed via mass spectrometry.

[106] Mass spectrometry (MS) as used herein encompasses all techniques which allow for the determination of the molecular weight (i.e. the mass) or a mass variable corresponding to a compound/metabolite/molecule such as e.g. N-Aspartic acid, to be determined/analyzed. Mass spectrometry can be coupled to different chromatographic techniques. Such chromatographic separation techniques can, for example, be selected from the group consisting of liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography, size exclusion or affinity chromatography, ion exchange chromatography, expanded bed adsorption (EBA) chromatographic separation, reversed-phase chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography or countercurrent chromatography. The chromatographic separation technique can furthermore be coupled to mass spectrometry.

[107] Also these methods are all known to the person skilled in the art and, for example, described in Gowda and Djukovic (2014) Overview of Mass Spectrometry- Based Metabolomics: Opportunities and Challenges" Methods Mol Biol. 1 198: 3-12. For example, mass spectrometry may be used in combination of gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), direct infusion mass spectrometry or Fourier transform ion-cyclotrone-resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis mass spectrometry (OEMS), high-performance liquid chromatography coupled mass spectrometry (HPLC- MS), quadrupole mass spectrometry, any sequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICP- MS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry or time of flight mass spectrometry (TOF). Thus, mass spectrometry as used herein can relate to GC-MS, LC-MS, direct infusion mass spectrometry, FT-ICR-MS, CE-MS, HPLC- MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry such as MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF or any combined approaches using the techniques described herein. These techniques are disclosed in, e.g., Nissen 1995, Journal of Chromatography A, 703: 37-57, US 4,540,884 or US 5,397,894.

[108] How to apply these techniques is well known to the person skilled in the art. Moreover, suitable devices are commercially available. Mass spectrometry as used herein can relate to LC-MS and/or GC-MS, i.e. to mass spectrometry being operatively linked to a prior chromatographic separation step. Mass spectrometry as used herein can also encompass quadrupole MS. [109] Liquid chromatography as described herein refers to all techniques which allow for separation of compounds (i.e. metabolites) in liquid. Liquid chromatography is characterized in that compounds in a mobile phase are passed through the stationary phase. When compounds pass through the stationary phase at different rates they become separated in time since each individual compound has its specific retention time (i.e. the time which is required by the compound to pass through the system). Liquid chromatography as used herein also includes HPLC. Devices for liquid chromatography are commercially available, e.g. from Agilent Technologies, USA.

[110] Gas chromatography as applied in accordance with the present invention, in principle, operates comparable to liquid chromatography. However, rather than having the compounds (i.e. metabolites) in a liquid mobile phase, which is passed through the stationary phase, the compounds/metabolites will be present in a gaseous volume. The compounds/metabolites pass the column which may contain solid support materials as stationary phase or the walls of which may serve as or are coated with the stationary phase. Again, each compound/metabolite has a specific time which is required for passing through the column.

[111] As an alternative or in addition to mass spectrometry techniques, the following techniques may be used for compound/metabolite determination: nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier transform infrared analysis (FT-IR), ultraviolet (UV) spectroscopy, refraction index, fluorescent detection, radiochemical detection, electrochemical detection, light scattering (LS), dispersive Raman spectroscopy or flame ionization detection (FID). These techniques are well known to the person skilled in the art.

[112] The techniques described herein can be assisted by automation, for example, sample processing or pre-treatment can be automated by robotics. Data processing and comparison can be assisted by suitable computer programs and databases. Automation as described herein allows using the method/uses of the present invention in high-throughput approaches.

[113] The present invention also relates to a use of N-acetylaspartic acid for the detection of mitochondrial Acetyl CoA.

[114] In addition, the present invention relates to a method for screening for an inhibitor or activator of NAT8L and/or an inhibitor or activator of the function and/or production of N-acetylaspartic acid, the method comprising (a) contacting cancer cells with a nucleic acid such as siRNA, miRNA, binding protein, small molecule or compound of interest;

(b) measuring proliferation of cancer cells, wherein a decrease in the proliferation of said cancer cells compared to said cancer cells before contacting indicates that the nucleic acid such as siRNA, miRNA, binding protein, small molecule or compound of interest serves as an inhibitor of NAT8L and/or an inhibitor of the function and/or production of N-acetylaspartic acid, or wherein an increase in the proliferation of said cancer cells compared to said cancer cells before contacting indicates that the nucleic acid such as siRNA, miRNA, binding protein, small molecule or compound of interest serves as an activator of NAT8L and/or as an activator of the function and/or production of N-acetylaspartic acid.

[115] The present invention also relates to a method to identify an anti-cancer compound, nucleic acid molecule, small molecule and/or binding protein, the method comprising measuring the ability of the compound, nucleic acid molecule, small molecule binding protein to inhibit one or more of the enzymes (NAT8L, ASPA, ACLY; ACSS) as described herein, wherein the ability to inhibit one or more of the enzymes as described herein indicates that the compound, nucleic acid molecule, small molecule and/or binding protein is an anti-cancer compound, nucleic acid molecule, small molecule and/or binding protein.

[116] Also, the present invention relates to a method for determining whether or not a cancer cell is susceptible to the treatment with an inhibitor as disclosed herein, comprising determining whether or not said cancer cell expresses NAT8L or said cancer cell comprises N-acetylaspartic acid.

[117] In addition, the present invention relates to a method for detecting a cancer cell, wherein the method comprises

(a) using primers specific for exon2 of NAT8L.

[118] The term "primer" as used herein refers to a oligonucleotide capable of acting as a point of initiation of template-directed nucleic acid synthesis when placed under conditions in which polynucleotide extension is initiated (e.g., under conditions comprising the presence of requisite nucleoside triphosphates (as dictated by the template that is copied) and a polymerase in an appropriate buffer and at a suitable temperature or cycle(s) of temperatures (e.g., as in a polymerase chain reaction). Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as initiators of de novo RNA synthesis and in vitro transcription- related processes (e.g., nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), etc.). A primer is typically a single- stranded oligonucleotide (e.g., oligodeoxyribonucleotide). The appropriate length of a primer depends on the intended use of the primer but typically ranges from 6 to 40 nucleotides, more typically from 15 to 35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the target sequence on a template.

[119] A primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in ELISA assays), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available. The primer can be a Lux primer, a scorpion primer, or a radiolabeled a primer.

[120] Also envisaged by the present invention is a primer/oligonucleotide having 70 %, 80 % 90 %, 95%, 99 %, 100 % sequence identity to SEQ ID NO. 14 and/or SEQ ID NO. 15 for the detection of exon 2 of NAT8L. It is also contemplated by the present invention that for the detection of exon 2 of NAT8L that one primer/oligonucleotide has 70 %, 80 % 90 %, 95%, 99 %, 100 % sequence identity to SEQ ID NO. 14 and another primer/oligonucleotide has 70 %, 80 % 90 %, 95%, 99 %, 100 % sequence identity to SEQ ID NO. 15, wherein both primers are used in combination.

[121] The term "oligonucleotide" refers to a nucleic acid that includes at least two nucleic acid monomer units (e.g., nucleotides). An oligonucleotide typically includes from 5 to 175 nucleic acid monomer units, more typically from eight to 100 nucleic acid monomer units, and still more typically from 10 to 50 nucleic acid monomer units (e.g., about 15, about 20, about 25, about 30, about 35, or more nucleic acid monomer units). The exact size of an oligonucleotide will depend on many factors, including the ultimate function or use of the oligonucleotide. Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99, 1979); the phosphodiester method of Brown et al. (Meth. Enzymol. 68: 109-151 , 1979); the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22: 1859-1862, 1981 ); the triester method of Matteucci et al. Am. Chem. Soc. 103:3185-3191 , 1981 ); automated synthesis methods; or the solid support method of U.S. Pat. No. 4,458,066, or other methods known to those skilled in the art.

[122] Further, the present invention relates to a kit comprising mass spectrometric fragments of N-acetylaspartic acid. The kit may further comprise (i) acetyl- hexosamine, and/or (ii) 2-acetamidoglucal.

[123] In addition, the present invention relates to an inhibitor of aspartoacylase (ASPA) for use in the treatment of cancer.

EXAMPLES

[124] EXAMPLE 1 NAT8L and N-acetylaspartic acid-based acetyl shuttling in lung cancer cells. N-acetylaspartic acid was one of the metabolites popping up in a mass isotopomer abundance variation analysis (Example 10). N-acetylaspartic acid is well-known to occur in very high abundance in neuronal tissue (Birken & Oldendorf (1989) N-acetyi-l-aspartic acid: a literature review of a compound prominent in 1 h-nmr spectroscopic studies of brain. Neurosci Biobehav Rev; 13(1 ):23-31 ), where it is synthesized from aspartic acid and acetyl-CoA by the long unknown enzyme NAT8L, which is also known as Shati (Wiame et al. Molecular identification of aspartate n- acetyltransferase and its mutation in hypoacetylaspartia. Biochem J, 425(1 ): 127-136). A function of N-acetylaspartic acid in non-neuronal mammalian cells has not been described so far. However, NAT8L expression was recently shown to play an important role in lipid turnover in brown adipocytes by an unknown mechanism (Pessentheiner et al cited herein). It was therefore surprising at first to find /V-acetylaspartic acid biosynthesis in cancer cells. However, the measurement of an authentic standard confirmed its identity and stable isotope labeling clearly excluded an exogenous origin.

[125] In the mammalian brain, /V-acetylaspartic acid is known as the precursor of the neuropeptide /V-acetylaspartylglutamic acid (Figure 1 ) (Becker et al. (2010) Molecular characterization of n-acetylaspartylglutamate synthetase. J Biol Chem; 285(38):29156-29164). Potential functions of neuronal /V-acetylaspartic acid have been proposed (Moffett et al. (2007) N-acetylaspartate in the ens: from neurodiagnostics to neurobiology. Prog Neurobiol; 81 (2):89-131 ) but its role in cancer cells has not been elucidated yet. Its acetyl-transport function in the brain, together with the known increase in lipid biosynthesis of cancer cells, make it interesting to speculate about a role of /V-acetylaspartic acid in tumor cells.

[126] To find out whether /V-acetylaspartic acid is produced by NAT8L in A549 cells, an siRNA-mediated knockdown of NAT8L was performed. Due to the lack of adequate primers for NAT8L, silencing efficiency could not be assessed. With the primers used by Pessentheiner et al. (Pessentheiner et al. (2013) Nat8l (n- acetyltransferase 8-like) accelerates lipid turnover and increases energy expenditure in brown adipocytes. J Biol Chem; 288(50):36040-36051 ). NAT8L in human neuroepithelial stem cells could be amplified, but did not yield any product in A549 cells. However, siNAT8L-transfection had a significant effect on the levels of several metabolites (Figure 2). /V-acetylaspartic acid levels dropped significantly to 7.5% of the levels measured in the siCtrl-transfected cells, thus providing strong evidence that NAT8L is responsible for /V-acetylaspartic acid production in A549 cells. Besides /V-acetylaspartic acid, glycerol-3-phosphate levels were significantly decreased to 13% of the levels in siCtrl-transfected cells. Another compound, glsRI 2718.62 was later identified as N-acetylaspartylglutamic acid 3TMS.

[127] EXAMPLE 2: Occurrence of N-acetylaspartic acid and NAT8L across other non-neuronal cell types. After having confirmed the identity and endogenous origin of /V-acetylaspartic acid, as well as its production by NAT8L, the question was if this metabolite and enzyme also occurs in other cell types and tissues. Therefore other GC-MS measurements were analyzed and it was found that /V-acetylaspartic acid is present also in human hepatocellular carcinoma cells (HepG2) and human prostate epithelial cells (RWPE-1 and RWPE-2). Interestingly, /V-acetylaspartic acid levels in RWPE-2 cells were higher than in RWPE-1 cells (Figure 3A). The two cell lines are identical, except that RWPE-2 was further transformed with v-Ki-ras, rendering the cells tumorigenic.

[128] Furthermore, it was found that /V-acetylaspartic acid is present in primary lung tissue. For a previous study, lung biopsies from cancer patients were taken from healthy and tumor tissue of the same patient. /V-acetylaspartic acid levels were increased in most tumor samples, pointing towards a role in cancer metabolism ((paired t-test, n = 19, p = 0.1 1 , Figure 3B). For a subset of these lung tissue samples, NAT8L mRNA levels were determined and found to be higher in tumor tissue with a median difference of 8% (paired t-test, n = 1 1 , p = 0.19; data not shown). Moreover, using the GENEVESTIGATOR (Hruz et al. (2008) Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinformatics,;2008:420747; Huang et al. (2012) Phospho- np63 /srebfl protein interactions: Bridging cell metabolism and cisplatin chemoresistance. Cell Cycle; 1 1 (20):3810-3827) gene expression search engine, high NAT8L expression in a large number of cancer cell lines was found (data not shown).

[129] EXAMPLE 3: Hints towards alternative splicing of NAT8L in A549 cells.

When trying to verify the NAT8L silencing efficiency by qPCR, initially no NAT8L cDNA could be amplified. Multiple primer pairs were tested. One pair of primers that failed to amplify NAT8L in A549 was working well for human neuroepithelial stem cells and was used by Pessentheiner et a/.(cited herein) in adipocytes.

[130] nat8l codes for three exons (Figure 4). The amplicon of aforementioned primer pair spanned the exon 1 - exon 2 junction. To account for potential alternative mRNA splicing, primers for amplicons within either exon 1 or exon 2 were designed. Using the exon 2-specific primers, NAT8L cDNA could be amplified in A549 cell extracts, but not with the exon 1 -specific primers, suggesting that exon 1 is not expressed in A549 cells. There are no alternative splicing variants of human NAT8L reported and the biological relevance is unclear.

[131] Absence of exon 1 would leave a single translation initiation site with the same reading frame at 505 bp that would give rise to a truncated 134 aa long, 15.5 kDa heavy protein, still containing the N-acetyltransferase domain. This protein would lack the predicted transmembrane domain at aa 121 to aa 141 . The N- terminal region may furthermore contain subcellular localization signals and alternative splicing could explain the different protein localization reported by different groups (Birken & Oldendorf (1989) N-acetyl-l-aspartic acid: a literature review of a compound prominent in 1 h-nmr spectroscopic studies of brain. Neurosci Biobehav Rev, 13(1 ):23-31 ; Arun et al. (2009) Evidence for mitochondrial and cytoplasmic n-acetylaspartate synthesis in sh-sy5y neuroblastoma cells. Neurochem Int; 55(4):219-225; Ariyannur et al. (2008) N-acetylaspartate synthesis in the brain: mitochondria vs. microsomes. Brain Res; 1227:34-41 ; Lu et al. (2004) N- acetylaspartate synthase is bimodally expressed in microsomes and mitochondria of brain. Brain Res Mol Brain Res, 2004; 122(1 ):71 -78). However, no localization signals were reported within the N-terminal region.

[132] EXAMPLE 4: NAT8L and glycerol-3-phosphate. NAT8L knockdown significantly changed the levels of several metabolites (Figure 3). The metabolite, the levels of which were most affected, was glycerol-3-phosphate. Glycerol-3- phosphate is a lipid building block as well as a key metabolite in electron shuttling to the mitochondrium via the glycerol-3-phosphate shuttle (Figure 5A). In humans, there exist multiple enzymes that are involved in glycerol-3-phosphate metabolism. Several O-acyltransferases are able to transfer fatty acids from and onto glycerol-3-phosphate, and glycerol-3-phosphate dehydrogenases catalyze the reversible NAD⁺/NADH dependent oxidation to the glycolytic intermediate dihydroxyacetone phosphate. Glycerol-3-phosphate dehydrogenase (GPD)2 together with GPD1 or GPD1 L constitute the glycerol-3-phosphate shuttle that transfers electrons from cytosolic NADH to mitochondrial FAD (Mracek T, Drahota Z, & Houstek J. (2013) The function and the role of the mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissues. Biochim Biophys Acta; 1827(3):401 -410). The resulting FADH2 can transfer the electrons to the respiratory chain. GPD1 L and GPD2, but not GPD1 were found to be expressed in A549. GPD1 L ex-pression was only little affected by NAT8L silencing, under both normoxia and hypoxia (Figure 5B). GPD2 expression, however, increased by over 40% at normoxia. GPD2 is the mitochondrial membrane-bound, usually glycerol-3-phosphate consuming enzyme. Its induction could explain the observed reduction of glycerol-3-phosphate levels after NAT8L silencing (Figure 2).

[133] EXAMPLE 5: NAT8L, glycerol, and reductive glutamine metabolism. To obtain hints on the functional role of /V-acetylaspartic acid and NAT8L in A549 cells and to find the link to glycerol-3-phosphate metabolism, a stable stable isotope labeling experiment was performed using [U-¹³C]glucose and [U-¹³C]glutamine, both under normoxia and hypoxia. NAT8L silencing was confirmed by qPCR and found to be more efficient under hypoxia (Figure 8). [134] Under normoxia, the most significantly changed MIDs were those of glycerol and glycerol-3-phosphate (Figure 6A). In siNAT8L-transfected cells, the relative abundance of fully labeled glycerol-3-phosphate increased significantly by about 5 percentage points, whereas the overall levels dropped dramatically by almost 90% (Figure 2). Fully labeled glycerol-3-phosphate is derived from the glycolytic intermediate dihydroxyacetone phosphate via GPD and the unlabeled isotopologue is derived from breakdown of unlabeled lipids from the growth medium (Figure 5). Therefore, the labeling experiment shows a relative increase in its de novo synthesis and a relative decrease in the contribution of lipid turnover.

[135] The labeling of glycerol is puzzling. Although not statistically significant, the labeling of glycerol changed inversely to glycerol-3-phosphate. This difference points to the irreversibility of glycerol-3-phosphate dephosphorylation, potentially due to the lack of glycerol kinase. However, isotopic enrichment in glycerol was higher than in glycerol-3-phosphate, which is surprising because it is derived from the latter and is therefore expected to be rather less enriched. Furthermore, the M₂ abundance in glycerol but not in glycerol-3-phosphate cannot be explained from its glycolytic origin, raising the question of other biosynthetic pathways leading to glycerol (Figure 6A).

[136] Under hypoxia, glycerol-3-phosphate MIDs showed a similar change as under normoxia (Figure 6B). However, its overall isotopic enrichment was about 25% lower, indicating a higher contribution from lipid turnover than under normoxia. An increased turnover of exogenous lipids under hypoxia has also been observed by others. For glycerol, no significant difference in isotopic enrichment, but again a M₂ fraction of unknown origin was observed.

[137] Whereas under normoxia NAT8L knockdown barely affected MIDs of TCA cycle-associated metabolites, neither from [U-¹³C]glucose nor [U-¹³C]glutamine labeling, the effect under hypoxia was quite drastic (Figure 6). There was a massive shift from M₃ to M₄ abundance of malic acid and aspartic acid after [U-¹³C]glutamine labeling in siNAT8L cells. [¹³C3]malic acid is produced from reductive glutamine metabolism via carboxylation of [¹³Cs]2-oxoglutarate followed by ACLY-action;

[¹³C₄]malic acid is produced from glutamine oxidation in the TCA cycle (Example 10). Therefore, the changes in MIDs indicate that NAT8L knockdown inhibits reductive glutamine metabolism or induces its oxidative metabolism. Reductive glutamine metabolism provides a means to sustain lipid biosynthesis fully on glutamine, independent of glycolysis and PDH activity (Metallo et al. (2012) Reductive glutamine metabolism by idhl mediates lipogenesis under hypoxia. Nature; 481 (7381 ):380-384).

[138] EXAMPLE 6: Model of N-acetylaspartic acid function in cancer cells.

Several potential functions of /V-acetylaspartic acid, including a role as osmoregulator (Mcintosh & Cooper (1965) Studies on the function of n-acetyl aspartic acid in brain. Journal of Neurochemistry; 12(9-10):825-835), have been proposed (Moffett et al. (2007) N-acetylaspartate in the ens: from neurodiagnostics to neurobiology. Prog Neurobiol, 2007; 81 (2):89-131 ) but its role in cancer cells has not been elucidated yet. The change in the ratio of oxidative and reductive glutamine metabolism upon NAT8L knockdown may be due to implications of NAT8L with mitochondrial-cytosolic-acetyl-transport (Figure 6B). Since cancer cells require high amounts of cytosolic acetyl-CoA for de novo lipid biosynthesis, such an acetyl-shuttling mechanism could be beneficial. Traditionally, cytosolic acetyl-CoA is thought to be derived from citrate by ACLY. In the following, it was postulated that an /V-acetylaspartic acid-based acetyl-shuttle exists and the aforementioned data will be discussed in that light, starting with metabolic fluxes under hypoxia, where NAT8L knockdown led to larger MID changes.

[139] Reductive glutamine metabolism under hypoxia. With decreasing oxygen concentrations, the cytosolic acetyl-CoA pool is increasingly supplied from glutamine catabolism (Metallo et al. (2012) Reductive glutamine metabolism by idhl mediates lipogenesis under hypoxia. Nature; 481 (7381 ):380-384). At the stage of 2- oxoglutaric acid, glutamine carbon can be oxidized by OGDH or reduced by IDH. Oxidative glutamine metabolism by OGDH, succinate dehydrogenase (SDH), malate dehydrogenase (MDH), malic enzyme, and PDH can produce mitochondrial acetyl- CoA (Figure 7A). This acetyl-CoA can be exported to the cytosol as citrate. The oxaloacetate resulting from ACLY action is transported back as malate. Reductive glutamine metabolism generates cytosolic citrate which can be cleaved by ACLY to provide acetyl-CoA. The resulting oxaloacetic acid can be transported to the mitochondrium, to form another molecule of acetyl-CoA via malic enzyme and PDH, which can be exported again as citrate to provide a second cytosolic acetyl-CoA molecule. However, in this scenario, citrate synthesis or transport may pose a bottleneck, because there are two competing reactions, via citrate synthase and IDH, producing citrate which could lead to product inhibition (Fendt et al. (2013) Reductive glutamine metabolism is a function of the -ketoglutarate to citrate ratio in cells. Nat Commun; 4:2236), limiting cytosolic acetyl-CoA.

[140] An N-acetylaspartic acid shuttle avoids product inhibition in reductive carboxylation of 2-oxoglutarate. N-acetylaspartic acid is likely produced in the mitochondrium (Pessentheiner et al cited herein). Assuming its mitochondrial biosynthesis and a cytosolic hydrolyzing enzyme, which may or may not be identical with neuronal ASPA, an acetyl-shuttling from the mitochondrium would be possible (Figure 7B). Mitochondrial synthesis of N-acetylaspartic acid, export to the cytosol and deacetylation could provide cytosolic acetate which can be activated to acetyl-CoA by acyl-CoA synthetase (ACSS). Acetate was recently shown by multiple groups to be a lipogenic substrate in hypoxic cancer cells (Mashimo et al. (2014) Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell; 159(7): 1603-1614; Kamphorst et al. (2014) Quantitative analysis of acetyl-coa production in hypoxic cancer cells reveals substantial contribution from acetate. Cancer & Metabolism; 2(1 ):23; Schug et al. (2015) Acetyl-coa synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress. Cancer Cell; 27(1 ):57-71 ). Although the net carbon transport in this scenario does not differ from the ACLY-based one (Figure 7B), the N- acetylaspartic acid model (Figure 7B) does not suffer from product inhibition by citrate and circumvents the citrate synthase reaction, which was shown to proceed rather slow in comparison to the transamination of oxaloacetate to aspartate.

[141] The proposed model explains the observed relative decrease in reductive carboxylation of 2-oxoglutarate upon NAT8L silencing: Without the possibility of N- acetylaspartic acid-based acetyl-transport, reductive glutamine metabolism is inhibited by citrate production from citrate synthase and its export to the cytosol. However, as a consequence of NAT8L silencing ACLY should become more important for cytosolic acetyl-CoA. A respective induction of ACLY gene expression, was not observed (Figure 8) unlike in brown adipocytes as reported by Pessentheiner et al. cited herein. The observed induction of both cytosolic and mitochondrial ACSS and the repression of citrate synthase in response to NAT8L silencing may also point towards a lack of acetyl-CoA (Figure 8): ACSS induction can replenish acetyl-CoA and reduced citrate synthesis via citrate synthase helps to conserve the available acetyl-CoA.

[142] Electron flow is affected under normoxia. Under normoxia, changes in MIDs were smaller than under hypoxia and there was no significant induction in ACLY expression. However, there was an induction in GPD2 gene expression. An N- acetylaspartic acid shuttle explains these observations. Under normoxia, most cytosolic acetyl-CoA is derived from glucose via glycolysis, PDH, citrate synthase, and ACLY (Figure 7A). PDH flux, mitochondrial acetyl-CoA availability and citrate synthesis are high at normoxia; citrate and ACLY can satisfy the demand in cytosolic acetyl-CoA, detectable flux ratios and MIDs of TCA cycle intermediates do not change. However, the citrate detracted from the TCA cycle is not available for oxidation and concomitant reduction of NAD⁺ and FAD to fuel oxidative phosphorylation. Therefore, GPD2 expression is induced to balance mitochondrial FADH₂ levels via the glycerol-3-phosphate shuttle and to regenerate cytosolic NAD⁺ to sustain a high glycolytic rate (Figure 5).

[143] The N-acetylaspartic acid shuttle mostly decouples acetyl-CoA export from NADH and FADH₂ production in the TCA cycle (Figure 7B). Provided there is enough mitochondrial acetyl-CoA, the N-acetylaspartic acid shuttle can deliver cytosolic acetyl-CoA and mitochon-drial NADH, independently of the TCA cycle. The TCA cycle can produce more reducing equivalents for oxidative phosphorylation as no citrate is detracted.

[144] EXAMPLE 7: NAT8L silencing lowers cell proliferation, even more than ACLY silencing. To further assess if the proposed model is adequate, a NAT8L, ACLY, and combined NAT8L+ACLY silencing was performed and cell proliferation was monitored. It was expected that the individual silencing of NAT8L and ACLY can be balanced by the respective other enzyme, but that their combined silencing, in which case the cells fully depend on acetate or fatty acid from the growth medium, would significantly reduce their proliferation rate.

[145] Under normoxic conditions, the individual or combined gene silencing led to a growth inhibition of around 30% (Figure 10). The effect of the combined silencing was not significantly different from the individual ones.

[146] Under hypoxia, the growth inhibitory effect of NAT8L silencing was 20% after 72 h and therefore lower than under normoxia (Figure 10). More surprisingly, ACLY silencing seemed to not affect cell growth under hypoxia. The lower effect of NAT8L silencing under hypoxia could be explained by the increased reductive glutamine metabolism leading to citrate that can provide cytosolic acetyl-CoA by ACLY. However, then the combined silencing should result in increased growth inhibition which was not observed.

[147] EXAMPLE 8: Validation of the model. To validate the postulated function of NAT8L and N-acetylaspartic acid, additional experiments were required. A stable isotope labeling experiment using N-([ ³C2]acetyl)-aspartic acid as tracer will show the fate of the acetyl-moiety of N-acetylaspartic acid in cancer cells, which is expected to mostly fuel fatty acid production and cytosolic or nuclear acetylation reactions (e.g. histones). The effect of NAT8L overexpression in cancer cells should be analyzed and an increased NAT8L activity to confer a proliferative advantage over normal cells is expected to take place. Furthermore, if the proposed model is correct, NAT8L over-expression should rescue the impaired proliferation in response to ACLY-silencing. Downstream of NAT8L action, a knockdown of ACSS2 should be performed in an acetate free growth medium, where it has a similar effect on metabolic fluxes as the NAT8L knockdown.

[148] For the suggested acetyl-shuttling mechanism, an N-acetylaspartic acid- hydrolyzing enzyme is required Benuck & already reported that N-acetylaspartic acid can be hydrolyzed in several peripheral tissues D'Adamo (Benuck M & D'Adamo A Jr. (1968) Acetyl transport mechanisms, metabolism of n-acetylaspartic acid in the non-nervous tissues of the rat. Biochim Biophys Acta; 152(3):61 1 -618). The obvious enzyme candidate is ASPA, which is responsible for oligodendrocytic N-acetylaspartic acid hydrolysis. However, its expression was not detected in A549 cells by qPCR. Furthermore, the subcellular localization of NAT8L in A549 cells needed to be confirmed.

[149] Summary. The presented experiments provide evidence for an N- acetylaspartic acid-based mitochondrial-cytosolic acetyl-shuttle in cancer cells. Such a mechanism would explain the recently reported importance of ACSS for the activation of acetate (Mashimo et al. (2014) Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell; 159(7): 1603-1614; Kamphorst et al. (2014) Quantitative analysis of acetyl-coa production in hypoxic cancer cells reveals substantial contribution from acetate. Cancer & Metabolism; 2(1 ):23; Schug et al. (2015) Acetyl-coa synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress. Cancer Cell; 27(1 ):57-71 ). However, not exogenous but N-acetylaspartic acid-derived, endogenous acetate is the main substrate of ACSS. NAT8L expression may play a role in the invasiveness of cancer cells as suggested by its elevated levels in tumorigenic RWPE-2 cells as compared to non-tumorigenic RWPE-1 cells, and by the correlation of N-acetylaspartic acid levels with prostate cancer progression as observed by others (Sreekumar et al. (2009) Metabolomic pro les delineate potential role for sarcosine in prostate cancer progression. Nature; 457(7231 ):910-914). Furthermore, NAT8L or the not yet identified hydrolase may provide a potential drug target for peripheral cancer. Most non-cancer cells, besides adipocytes and oligodendrocytes, have a lower acetyl- CoA demand and might therefore not suffer from inhibition of the N-acetylaspartic acid-shuttle.

[150] EXAMPLE 9: Acetylated compounds as proxies for acetyl-CoA labeling.

Acetyl-CoA is at the interface of glycolysis, the TCA cycle, fatty acid biosynthesis, terpene biosynthesis, β-oxidation and several other pathways, therefore it represents an important metabolic hub. Isotopic enrichment of acetyl-CoA after stable isotope labeling experiments can provide important information on fractional contribution of the given tracer to the aforementioned pathways. Unfortunately, acetyl-CoA cannot be analyzed with GC-MS due to its low volatility. Other sophisticated methods like isotopomer spectral analysis (ISA) and mass isotopomer distribution analysis (Hellerstein et al. (1999) Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations. Am J Physiol; 276(6 Pt1 ):E1 146-E1 170) have been used to deduce acetyl-CoA labeling from the labeling of fatty acids.

[151] In Example 10 it is demonstrated how the labeling of mass spectrometric fragments of N-acetylaspartic acid can be used to infer acetyl-CoA labeling (Figure 1 1 ). This approach has the advantage, that it does not require the separate work-up and measurement of fatty acids. Besides N-acetylaspartic acid, any other purely endogenous acetylated compound holds information on acetyl-CoA labeling. To determine the labeling of the acetyl-moiety either suitable mass spectrometric fragments need to be found, or it must be ensured that the non-acetyl-moiety cannot be labeled from the applied tracer. A candidate for the latter case is the putative 2- acetamidoglucal. For example in the case of glutamine labeling in non- gluconeogenetic cells under isotopic steady state the 2-acetamidoglucal MID represents the acetyl-CoA MID, since the glucal-carbon cannot be labeled from the glutamine tracer.

[152] Moreover, the two compounds N-acetylaspartic acid and 2-acetamidoglucal may be suitable to determine compartment-specific acetyl-CoA MIDs. If the above model (Figure 7) is correct, then N-acetylaspartic acid is synthesized in the mitochondrium and, thus, provides labeling information on the mitochondrial acetyl- CoA pool, whereas hexosamine acetylation occurs in the cytosol (Boehmelt et al. (2000) Cloning and characterization of the murine glucosamine-6-phosphate acetyltransferase emeg32. differential expression and intracellular membrane association. J Biol Chem; 275(17): 12821 -12832) and 2-acetamidoglucal or other acetyl-hexosamines provide information on the cytosolic acetyl-CoA pool. The mitochondrial acetyl-CoA labeling provides information on acetyl-CoA derived from acetate via ACSS1 , β-oxidation of fatty acids, and most importantly PDH. The cytosolic acetyl-CoA is a mixture of exported mitochondrial acetyl-CoA, and acetyl- CoA produced from reductive carboxylation of 2-oxoglutarate and ACLY action. The observed differences in acetyl-labeling of 2-acetamidoglucal and N-acetylaspartic acid support this hypothesis: The enrichment from [U-¹³C]glutamine is higher in the assumed cytosolic pool, which is composed of acetyl-CoA exported from the mitochondrium, but additionally enriched by labeled acetyl-CoA derived from reductive glutamine metabolism (Figure 1 1 ).

[153] Summary. This analysis of hypoxic cancer cells shows how non-targeted data acquisition and data analysis approaches are valuable tools to generate initial hypotheses leading to new biological insights. After performing stable isotope labeling experiments, mass spectrometric analysis, and the non-targeted detection of isotopically enriched compounds, qualitative analysis of isotopic enrichment provided information on active fluxes (e.g. production of /V-acetylaspartic acid) and the general fate of the metabolic tracer (Figure 12B). MIDs from different experimental conditions have been systematically analyzed to detect changes in metabolic fluxes (Example 10; e.g. decreased PDH flux and decreased IDH flux). Because MID similarity may indicate metabolic proximity, MIDs of compounds of interest have been compared to all other MIDs for pathway contextualization and to facilitate identification of unidentified compounds (/V-carboxyglutamic acid, 2-acetamidoglucal). Overall, this non-targeted approach (Figure 12B) provides information on: 1 ) active pathways, 2) changed fluxes and 3) compound identities. This information holds biological insights itself and will furthermore generate hypotheses for subsequent analyses (Figure 12A).

EXAMPLE 10 Systematic non-target mass isotope analysis to reveal metabolic flux changes

[154] Mass isotopolome analysis— Theoretical back-ground

The starting point of our workflow is a stable isotope labeling experiment. After mass spectrometric measurements of metabolite extracts, MIDs, corrected for natural isotope abundance, can be obtained in a non-targeted manner (Hiller et al. (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal Chem 81 : 3429- 3439; Creek et al. (2012) Stable isotope-assisted metabolomics for network-wide metabolic pathway elucidation. Anal Chem 84: 8442-8447; Huang Xet al. (2014) X(13)CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics. Anal Chem). We analyze these metabolome- wide MIDs, the mass isotopolome, to detect changes in metabolic fluxes and exploit MID similarity between compounds for their pathway contextualization.

[155] MID changes are linked to flux changes

When a tracer is taken up and metabolized by a cell, the MIDs of downstream metabolites will be identical to those of the tracer, until there is a loss or gain of labeled atoms, or a dilution by a converging flux carrying a different MID. We will illustrate that for the case of ¹³C-labeling in a simple metabolic model in metabolic and isotopic steady state (Fig 13). When glutamine is used as a tracer, glutamic acid will always have the same MID as glutamine, no matter how the GLS flux changes. On the other hand, the MID of 2OG is strongly dependent on the flux ratio of GLS or IDH. These fluxes lead to differently labeled 2OG. The MID of 2OG is the average of the MID of 2OG derived from either flux, weighted by the flux ratios (Fig 13). Thus, if all producing fluxes change proportionally, they will leave the MID of the product unchanged (Fig.13 A,C). Therefore, MIDs can only provide relative metabolic flux information (Wiechert W, de Graaf AA (1997) Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments. Biotechnol Bioeng 55: 101-1 17). Furthermore, that means that MIDs are fully determined by fluxes. If no fluxes are changing, neither will MIDs. In summary, this leads to the following consequences for flux information contained in MIDs: MIDs alone can only provide relative flux information.

Changes in MIDs must be caused by changes in metabolic fluxes.

Not all changes in metabolic fluxes manifest in MID changes.

[156] Locating flux changes by non-targeted mass isotopomer abundance variation analysis. Since changes in MIDs can only be a consequence of altered metabolic fluxes, we can detect metabolic flux changes by looking for changes in the mass isotopolome. Therefore, MIDs of identical compounds need to be matched across different experimental conditions to detect differences in relative mass isotopomer abundances. As a measure of variation, for each isotopically enriched compound we calculate the maximal standard deviation of relative mass isotopomer abundance across the different experimental conditions. We assume that large flux changes will lead to large changes in mass isotopomer abundance. Thus, to find the most significant flux changes, we rank metabolites by their variation score. Like any MID analysis, this approach is subject to the limitations described above. Apart from that, this systematic analysis of mass isotopomer abundance variation detects flux changes without the requirement of any biochemical a priori knowledge on the system of interest. It is only biased by analytical restrictions and the choice of the isotopic tracer, and will consider any unanticipated reactions or metabolites which cannot be accounted for in current flux analysis techniques.

[157] MID similarity indicates metabolic proximity

For subsequent interpretation of the detected changes in mass isotopomer abundances, the respective compounds need to be identified. This is usually done by matching their mass spectra against reference libraries (Wegner A, Weindl D, Jager C, Sapca u SC, Dong X, Stephanopoulos G, Hiller K (2014) Fragment Formula Calculator (FFC): Determination of Chemical Formulas for Fragment Ions in Mass Spectrometric Data. Anal Chem 86: 2221-2228), but the available libraries are far from comprehensive. Although thousands of chromatographic/mass spectrometric features, and among them at least several hundreds of metabolites can be analytically detected (Zhou et al. (2014) IsoMS: automated processing of LC-MS data generated by a chemical isotope labeling metabolomics platform. Anal Chem 86: 4675-4679; Bueschl et al. (2013) A novel stable isotope labelling assisted workflow for improved untargeted LC-HRMS based metabolomics research. Metabolomics: 1- 16), only a fraction thereof can be identified, rendering compound identification a major bottleneck in current metabolomics research (Sevin et al. (2015) Biological insights through nontargeted metabolomics. Current Opinion in Biotechnology 34: 1 - 8, systems Biology · Nanobiotechnology). Without identification of at least their pathways or compound classes, these features provide, apart from a potential application as biomarkers, only limited insights. Hence, compound identification is, however cumbersome, still highly important.

[158] To use MID similarity for pathway contextualization of unidentified compounds, we pairwisely compare MIDs of all isotopically enriched compounds (Fig. 14A). To account for potential losses or additions of isotopically enriched fragments to the molecules which would shift the MIDs we perform a Needleman-Wunsch alignment (Needleman SB, Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48: 443-453) on the MID vectors prior to the similarity calculation (Fig. 14B). This step is for example necessary to reveal the high similarity and metabolic proximity of isocitric acid and 2- oxoglutaric acid (20G), or 20G and succinic acid as shown in Figure 13. As a similarity measure, we compare the Canberra distances (see Materials and methods) of all pairwisely aligned MIDs. This pairwise comparison results in a distance or similarity matrix. After applying an empirically determined distance cutoff, we create a network of compounds with higher MID similarity. The resulting graph is likely to show metabolically connected compounds. However, the MID similarity can, dependent on tracer and pathways, be ambiguous. The specificity can be increased by using distinct tracers and multiple experimental conditions (Fig 14A). Edges in the graph occurring in multiple conditions are more likely to be biologically meaningful.

[159] In summary, MID similarity between compounds can indicate proximity within the metabolic network. This can be used to associate unidentified compounds to identified ones, and to map them to specific pathways. This itself is valuable information and can furthermore be a strong hint for subsequent compound identification. For both identified and unidentified compounds such an MID similarity analysis can reveal new biosynthetic pathways or help to distinguish between different known ones.

[160] Method summary

Non-targeted data acquisition and data analysis approaches are valuable tools to generate initial hypotheses, especially when little a priori information is available on the organism or subject of interest. We show how stable isotope labeling experiments and subsequent non-targeted mass iso-topolome analysis can be used for metabolic flux profiling and hypothesis generation (Fig. 13)

The proposed workflow starts with stable isotope labeling experiments, mass spectrometric analysis, and the non-targeted detection of isotopically enriched compounds (Fig 14B). In addition to MIDs, such an analysis yields, for each compound, the labeled and unlabeled mass spectra, as well as the chromatographic retention time, often normalized as retention index (Rl). Qualitative analysis of isotopic enrichment provides information on active fluxes and the general fate of the metabolic tracer. MIDs from different experimental conditions are systematically analyzed to detect changes in metabolic fluxes. MID similarity may indicate metabolic proximity, hence, MIDs of compounds of interest are compared to all other MIDs for path-way contextualization, discovery of potential precursors, or to facilitate identification of unidentified compounds. So overall, this non-targeted approach provides information on: 1 ) active pathways, 2) changed fluxes, and 3) compound identities. This information holds biological insights itself and will furthermore generate hypotheses for subsequent analyses (Fig. 13).

[161] Non-targeted mass isotopolome analysis of hypoxic cancer cells

We illustrate the described approach in the analysis of human lung cancer cell metabolism at different oxygen levels. Therefore, we performed stable isotope labeling experiments with [1 ,2-¹³C]glucose and [U-¹³C]glutamine at oxygen concentrations ranging from severe hypoxia at 1 % O2 to atmospheric 21 % O2. We chose glucose and glutamine as isotopic tracers because they are the major carbon sources of most mammalian cells and therefore lead to a good metabolome coverage of isotopic enrichment. After GC-MS analysis of the metabolite extracts, we determined all isotopically enriched compounds along with their MIDs in an automated and non-targeted manner. This way we detected 61 compounds which were labeled from [U-¹³C]glutamine and 83 labeled from [1 ,2-¹³C]glucose.

[162] Non-targeted flux profiling reveals changes in intermedi-ary metabolism

To detect hypoxia-induced metabolic flux changes we analyzed the variation in relative mass isotopomer abundances (see herein). With the chosen cut-off value of 0.05 for the variation score we ended up with six compounds from the glutamine labeling experiments and four from glucose labeling with one of these compounds common to both sets (Fig. 15A). With decreasing O2 concentration, the compounds with high MID varia-tion after glucose labeling showed an increase in the unlabeled (M₀) fraction and a concomitant decrease in the abundances of heavier mass isotopomers, indicating decreased glucose contribution to their biosynthesis (Fig. 15A). The compounds with changed MIDs after glutamine labeling had an either relatively constant or decreasing M₀ abundance, with the latter being indicative of increasing glutamine contribution to their biosynthesis. Additionally, these compounds showed a switch of the most abundant mass isotopomer indicating a switch of the biosynthesis route.

[163] Whereas the detection of these changes in labeling patterns was fully non- targeted and did not require compound identification, the further interpretation requires knowledge on the identity of the compounds, as well as detailed knowledge on the metabolic network, including carbon atom transitions. Hence, to interpret the observed changes in isotopic labeling, we tried to identify the corresponding compounds by matching their mass spectra against an in-house reference library. This way we identified three of the highest-ranking metabolites from glutamine labeling as the trimethylsilyl (TMS) derivatives of aspartic acid, glutamic acid and citric acid. Three other compounds remained unidentified (Fig. 15A). From glucose labeling we identified citric acid and again three compounds, which were distinct from the ones before, remained unidentified.

[164] Citric acid, aspartic acid, and glutamic acid are all associated with the tricarboxylic acid (TCA) cycle. With decreasing O2 concentration, the isotopic enrichment of citric acid from [1 ,2-¹³C]glucose was significantly reduced, indicating a relative decrease in glucose-carbon entering the TCA cycle (Fig. 15B). Glucose carbon enters the TCA cycle via pyruvate de-hydrogenase (PDH) or pyruvate carboxylase, hence the reduced glucose contribution is indicative of a relative flux decrease through these reactions.

[165] With [U-¹³C]glutamine labeling the relative amount of labeled glutamic acid was relatively constant, but with decreasing oxygen availability the M₃ fraction disappeared whereas the M₅ fraction increased. M₅ glutamic acid is derived from deamidation of the glutamine tracer without further changes in its carbon backbone, whereas the M₃ is formed from M₅ after deamination to 2-oxoglutaric acid (20G), one "round" in the TCA cycle back to 20G, and reamination to glutamic acid (Fig. 15B). The MIDs of glutamic acid show, that the relative glutamine contribution to its biosynthesis is unaffected by changes in O2 levels, but that its oxidative metabolisation via the TCA cycle decreases with lower O2 availability.

[166] A similar trend was observed in citric acid MIDs after [U-¹³C]glutamine labeling (Fig. 15AB): The unlabeled fraction is relatively constant, whereas M₂ and M₄ are replaced by M₅ at lower O2 levels. The M₄ fraction is derived from fully labeled glutamine entering the TCA cycle as 20G and subsequent decarboxylation, oxidation, and condensation with unlabeled acetyl-CoA. Further oxidative metabolism in the TCA cycle converts the M₄ citric acid to M₂. The M₅ fraction is generated from fully labeled 20G which is carboxylated by isocitrate dehydrogenase (IDH). So at low O2 availability there is a switch from oxidative to reductive glutamine metabolism and the relative reductive IDH flux becomes many times higher than at normoxia. These changes observed in citric acid propagate via oxaloacetic acid to aspartic acid (Fig. 12AB). At high oxygen levels aspartic shows mostly M₂ and M₄ labeling as explained for citric acid. At lower oxygen levels, when IDH flux is increasingly reversed, citric acid is cleaved by ATP-dependent citrate lyase (ACL) which generates cytoplasmic acetyl-CoA for lipid biosynthesis and M₃ oxaloacetic acid. Transamination to M₃ oxaloacetic acids leads to the observed M₃ aspartic acid at hypoxia. This switch from M₂ and M4 enrichment to almost exclusive M₃ enrichment also becomes apparent in MIDs of the other C₄ TCA cycle intermediates.

[167] The relative increase in glutamine contribution to citrate and subsequent ACL activity at low oxygen concentrations was also observed in slight changes in the MIDs of free palmitic acid from [U-¹³C]glutamine labeling. With decreasing oxygen concentration there was a shift in enrichment towards heavier mass isotopomers showing a higher contribution of glutamine carbon to de novo fatty acid synthesis.

[168] Acetylated compounds as proxy for compartment-specific acetyl-CoA labeling Besides any potential biological role NAcAsp is also of analytical interest. As mentioned before, the MID of the fragment m/z 245 of NAcAsp 3TMS was found to be almost identical to the MID of aspartic acid. Combinatorial analysis of possible fragment formulas using FFC (Wegner et al, 2014 cited herein) confirmed that this fragment most probably arises from loss of the acetyl-moiety of NAcAsp. This is of interest because it allows for the deconvolution of the MIDs of the aspartyl- and acetyl-moiety of NAcAsp and hence can be used as a proxy to assess acetyl-CoA labeling at isotopic steady state. Acetyl-CoA is a hub of many anabolic and catabolic reactions and its labeling, thus, very informative. Its labeling pattern is important for ¹³C-MFA applications for which often GC-MS measurements are used which cannot cover acetyl-CoA directly due to its large size after chemical derivatization.

[169] PDH flux is only sensitive to O2 at physiological concentrations

We used the previously detected acetylated compounds to analyze isotopic enrichment in the acetyl-CoA pools at different oxygen concentrations. The observed ACL flux (Fig. 13B) produces cytosolic acetyl-CoA which is mostly used for lipid biosynthesis. Using NAcAsp as a proxy, we analyzed changes in acetyl-CoA labeling which provides a clearer measure of glucose or glutamine utilization in the TCA cycle than the other intermediates. After [U-¹³C]glutamine labeling the fully labeled acetyl (M₂) fraction represents the glutamine contribution to acetyl-CoA via ACL (Fig. 13B). Plotting this glutamine contribution to the acetyl-CoA pool over the oxygen concentrations reveals a switching point in oxygen response at an O2 concentration of around 10% (Fig. 15). Below that point, the relative contribution of reductive glutamine metabolism shows high sensitivity towards changes in oxygen concentrations, whereas above these 10% O2 there is only a slight response. It is noteworthy, that this sensitive range matches the range of physiological oxygen concentrations which in most tissues lie below 10% O2 (Carreau et al. (201 1 ) Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med 15: 1239-1253). A similar trend was previously observed for the HIF-1 protein in HeLa cells, the amount of which was dramatically increasing at oxygen levels below 6% (Jiang BH, Semenza GL, Bauer C, Marti HH (1996) Hypoxia- inducible fac-tor 1 levels vary exponentially over a physiologically relevant range of 02 tension. Am J Physiol 271 : C1 172-C1 180).

[170] Discussion

To analyse metabolic changes in human adenocarcinoma cells in response to varying oxygen availability we performed stable isotope labeling experiments with ¹³C labeled glucose and glutamine at various oxygen concen-trations and analyzed the resulting mass isotopolome in a non-targeted manner. Unlike previous studies, we were not interested in the mere statistical differences in mass isotopomer abundances (Huang et al, 2014 cited herein), but our declared goal was their biological interpretation. Using a novel comparative stable isotope labeling analysis, we showed that lower oxygen availability leads to a relative reduction of glucose oxidation in the TCA cycle, a reversal of isocitrate dehydrogenase flux directionality, and a relative increase in glutamine utilization. Additionally, we found A549 cells to produce N-acetylaspartic acid, a compound which is well known to have an important function in neuronal tissue, but not known to be produced in other tissues. We demonstrated how NAcAsp and an unidentified compound can be used to determine compartment-specific information on isotopic enrichment.

[171] Locating flux changes by non-targeted mass iso-topomer abundance variation analysis. We globally analyzed mass isotopomer abundance variation across different experimental conditions to detect metabolic flux changes in a non-targeted manner. Because MIDs are determined by metabolic fluxes, changed MIDs are indicative of changed metabolic fluxes. In this differential analysis MID variation and, thus, flux changes are detected without considering the identity of the compounds. Therefore, even if such flux changes occurred in yet unknown reactions, the results would pop up in this analysis. Only later, for the interpretation of the observed flux changes, the analytes need to be identified. Since for many organisms or cell types the metabolic network is not fully known, especially in respect to compartmentalization of eukaryotic cells, such a non-targeted and data-driven approach is highly desirable. It can be used as a valuable scouting experiment to not miss unanticipated reactions or metabolites and to validate the assumptions required for example for ¹³C-MFA.

[172] The observed changes in cancer cell metabolism under hypoxia have been observed before and are mostly mediated by HIF-1 and PHDs (Henze et al. (2014) Loss of PHD3 allows tumours to over-come hypoxic growth inhibition and sustain proliferation through EGFR. Nat Commun 5: 5582). HIF-1 -mediated expression of pyruvate dehydrogenase kinase 1 (PDK1 ) leads to PDH in-hibition which, together with increased LDH expression (Le et al. (2010) Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci U S A 107: 2037-2042), redirects pyruvic acid away from the TCA cycle (Kim et al. (2006) HIF-1 -mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 3: 177-185). Enhanced reductive glutamine metabolism produces acetyl-CoA which flows into increased fatty acid biosynthesis (Wise et al, 201 1 cited herein; Metallo et al, 2012 cited herein).

To maintain high energy levels when oxidative phosphorylation is inhib-ited, increased substrate uptake is required and mediated by increased glu-cose transporter expression. These previous studies confirm the changed fluxes we found in a non-targeted manner and validate our approach.

[173] MID similarity as a measure for metabolic proximity Because MID similarity often correlates with metabolic proximity, comparison of MIDs of different compounds can reveal metabolic similarity. Addressing a current bottleneck in metabolomics studies, we demonstrated how the similarity in MIDs after stable isotope labeling can be of great help to identify unknown compounds. Although MID similarity analysis will not always allow for compound identification, it can still be used to put unknown compounds into the context of certain biochemical pathways, or to provide hypotheses on chemical substructures. Furthermore, this analysis can help to elucidate unknown biosynthetic pathways by revealing potential precursors. However, MID similarity can, dependent on tracer and pathways, sometimes be ambiguous. This ambiguity can be reduced by the use of distinct tracers and multiple experimental conditions as done in this study.

[174] Production of NAcAsp by lung cancer cells

Non-targeted isotope labeling revealed the production of NAcAsp by lung cancer cells. This was unexpected because NAcAsp was only known to be produced in neuronal cells where it acts as a acetyl carrier between different cells of the nervous system. Together with the known increased fatty acid biosynthesis in tumor cells this led us to the hypothesis that NAcAsp might have a similar role there to shuttle substrates between organelles or tumor cells.

[175] Acetyl-CoA proxies

Non-targeted mass isotopolome analysis. An advantage of non-targeted isotope labeling analysis is that, depending on the proper tracer choice, it clearly shows whether a given compound is formed by the organism or was externally introduced as ingredient of an undefined growth medium or as contamination and thus provides an additional quality control. An analytical benefit of non-targeted MID analysis is that they are more robust to technical variation than metabolite levels.

[176] Conclusion

We applied stable isotope labeling and illustrated a novel non-targeted mass isotopolome analysis approach to systematically analyze metabolic hypoxia response of human lung cancer cells. We employed non-targeted mass isotopomer abundance variation analysis for non-targeted metabolic flux change profiling. This approach can also account for unknown or unanticipated reactions, which is not possible using current flux analysis techniques. With this data-driven approach we detected most known hypoxia-induced metabolic effects, validating our approach. Furthermore, we detected several unanticipated metabolites and showed how MID similarity can assist compound identification, addressing a major bottleneck of current metabolomics research. We illustrated our workflow on GC-MS data, however, it can also directly be used in LC-MS experiments which may provide higher metabolome coverage. In summary, this non-targeted approach provided biological insights and proved to be a fruitful methodology for hypothesis generation and providing a start, not an alternative, for subsequent more targeted analyses.

[177] Materials and methods

All chemicals were bought from Sigma-Aldrich, unless indicated differently. All solvents were of grade Chromasolv or higher.

[178] Cell culture & stable isotope labeling

Human lung adenocarcinoma A549 cells (ATCC CCL-185, Giard et al. (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51 : 1417-1423) were grown in RPMI 5030 without glutamine and glucose, supplemented with either unlabeled 25 mM glucose and 5 mM glutamine or ¹³C labeled analogues thereof. Cells were grown at 37 °C and 5% O2. Stable isotope labeling were performed by adding 50% or 100% [U-¹³C]-D-glucose, [U-¹³C]-L-glutamine or [1 ,2-¹³C]-D-glucose instead of the unlabeled counterpart. For branched chain amino acid labeling [U-¹³C]-L-valine, [U-¹³C]-L-leucine or [U-¹³C,¹⁵N]- L-isoleucine were added to the medium, resulting in 50% isotopic enrichment.

[179] Metabolite extraction

Intracellular metabolites as in (Sapcariu et al. (2014) Simultaneous extraction of proteins and metabolites from cells in culture. MethodsX 1 : 74 - 80). be. isotope ratios robust to analytical variance.

FAMEs:

[180] GC-MS data processing and determination of isotopic enrichment

Deconvolution of mass spectra, label-free metabolomics data analysis as well as targeted MID determination were performed using the Metabolit-eDetector software (Hiller et al, 2009 cited herein). Detection of non-targeted stable isotope labeling and mass isotopolome analysis were performed using an in-house software based on the NTFD algorithm (Hiller et al. (2010) Nontargeted elucidation of metabolic pathways using stable-isotope tracers and mass spectrometry. Anal Chem 82: 6621-6628 Hiller et al. (2013) NTFD-a stand-alone application for the non-targeted detection of stable isotope-labeled compounds in GC/MS data. Bioinformatics 29: 1226-1228). Known contaminants like siloxanes were excluded from the data analysis. [181] Mass isotopomer abundance variation

To detect compounds with most varying labeling patterns, we used the maximal standard deviation in relative mass isotopomer abundance for every compound which was detected in at least three out of five conditions:

where p._j is the relative abundance of the M_j isotopologue of the given compound in the i-th dataset. The MIDs of the heaviest common fragments across all conditions were used and the MIDs of unlabeled compounds were not considered. The threshold was set to 0.05.

[182] MID Distance calculation

Compounds of the unlabeled samples where matched by their mass spectra and retention index. The mass isotopomer of the largest common frag-ment with R² < .95 was used. The pairwise distances of all aligned MID vectors were calculated. The Canberra distance of two MID vectors A and B was calculated as

and normalized b the sum of the dimensions of

MID vectors

Distances below an empirically determined threshold were considered as potentially metabolically connected. From the thresholded distance matrix a compound graph was plotted to visualize the network. Used this score instead of statistical test, so that no replicates are required

[183] MID deconvolution of aspartyl and acetyl moiety of NAcAsp

The mass spectrometric fragment ions m/z 304 and m/z 245 of N-acetylaspartic acid 3TMS represent the [M-CH₃]+ and [M-AcTMS]+ fragments respec-tively. The MID of N-acetylaspartic acid (given by [M-CH₃]+) is the con-volution or Cauchy product (Antoniewicz et al. (2006) Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab Eng 8: 324-337) of the MIDs of the aspartyl MIDAsp (given by [M-AcTMS]+) and the acetyl moiety MIDAc of the molecule: MI D,_.., * MID_AF;

This equation system was solved for MIDAc using a least squares approach using the Isfit routine of the R statistics environment (R Core Team (2013) R: A Language and Environment for Statistical Com-puting. R Foundation for Statistical Computing, Vienna, Austria). Determined acetyl MIDs with∑, | M, |> 1.01 were discarded.

[184] FAME analysis

For the relative contribution of glucose and glutamine to fatty acid biosyn-tesis we determinened the fractional ¹³C content of palmitic acid after [U-¹³C]glucose and [U- ¹³C]glutamine labeling. The ¹³C content is calculated as

where n is the carbon number of a given FAME and M, is the relative abundance of the M+i mass isotopomer after correction for natural isotope abundance. As the contribution of fatty acids from the medium we used the unlabeled fraction after simultaneous [U-¹³C]glucose and [U-¹³C]glutamine labeling. There the probability for the de novo synthesis of a completely unlabeled palmitic acid molecule is close to zero and thus all M₀ has to be derived from the medium. The fraction of "others" comprises carbon from breakdown of amino acids or other unlabeled medium components and is calculated as the ¹²C content after simultaneous [U-¹³C]glucose and [U-¹³C]glutamine labeling excluding the M₀ fraction which is completely derived from the medium:

It should be noted that the glucose and glutamine fraction also include the contribution of turnover of amino acids synthesized from these substrates and that the "other" fraction may also contain a probably small fraction of medium fatty acids turnover.

[185] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[186] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[187] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[188] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

[189] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[190] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[191] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[192] When used herein, the term "about" is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value. For example, if a formulation comprises about 5 mg/ml of a compound, this is understood to mean that a formulation can have between 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml. As used herein, an interval which is defined as "(from) X to Y" equates with an interval which is defined as "between X and Y". Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of "5 mg/ml to 10 mg/ml" or "between 5 mg/ml and 10 mg/ml" includes a concentration of 5, 6, 7, 8, 9, and 10 mg/ml as well as any given intermediate value.

REFERENCES

Arya M, Shergill I S, Williamson M, Gommersall L, Arya N, Patel HRH "Basic principles of quantitative real time PCR" Expert Rev. Mol. Diagn. 5(2):209-219

Mcintosh JC & Cooper JR. Studies on the function of n-acetyl aspartic acid in brain. Journal of Neurochemistry, 1965; 12(9-10):825-835

FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245)

Fendt SM, Bell EL, Keibler MA, Olenchock BA, Mayers JR, Wasylenko TM, et al. Reductive glutamine metabolism is a function of the -ketoglutarate to citrate ratio in cells. Nat Commun, 2013;4:2236

Hernandez-Alcoceba et al. (2000) "The Ras family of GTPases in cancer cell invasion" Cell. Mol. Life Sci. 57; 65-76

Mashimo T, Pichumani K, Vemireddy V, Hatanpaa KJ, Singh DK, Sirasanagandla S, et al. Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell, 2014; 159(7): 1603-1614

Martin F, Toniatti C, Salvati AL, Venturini S, Ciliberto G, Cortese R, Sollazzo M. The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6. EMBO J. 1994 Nov 15;13(22):5303-9

Nissen 1995, Journal of Chromatography A, 703: 37-57

Kamphorst J, Chung M, Fan J, & Rabinowitz J. Quantitative analysis of acetyl-coa production in hypoxic cancer cells reveals substantial contribution from acetate. Cancer & Metabolism, 2014;2(1 ):23

Schug ZT, Peck B, Jones DT, Zhang Q, Grosskurth S, Alam IS, et al. Acetyl-coa synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress. Cancer Cell, 2015;27(1 ):57-71

Silverman J, Liu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H, Whitehorn E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Stemmer WP. Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotech not. 2005 Dec;23(12): 1556-61

Thompson Nucl. Acids Res. 2 (1994), 4673-4680)

Traunecker A, Lanzavecchia A, Karjalainen K. Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells. EMBO J. 1991 Dec; 10(12):3655-9

Traunecker A, Lanzavecchia A, Karjalainen K. Janusin: new molecular design for bispecific reagents. Int J Cancer Suppl. 1992;7:51 -2

Benuck M & D'Adamo A Jr. Acetyl transport mechanisms, metabolism of n-acetyll- aspartic acid in the non-nervous tissues of the rat. Biochim Biophys Acta, 1968;

152(3):61 1 -618

Boehmelt G, Fialka I, Brothers G, McGinley MD, Patterson SD, Mo R, et al. Cloning and characterization of the murine glucosamine-6-phosphate acetyltransferase emeg32. differential expression and intracellular membrane association. J Biol Chem, 2000; 275(17): 12821 -12832.

Sreekumar A, Poisson LM, Rajendiran TM, Khan AP, Cao Q, Yu J, et al. Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature, 2009;457(7231 ):910-914.

Hellerstein MK & Neese RA. Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations. Am J Physiol, 1999;276(6 Pt 1 ):E1 146-E1 170.

Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM. Domain antibodies: proteins for therapy. Trends Biotechnol. 2003 Nov; 21 (1 1 ):484-90

Birken DL & Oldendorf WH. N-acetyl-l-aspartic acid: a literature review of a compound prominent in 1 h-nmr spectroscopic studies of brain. Neurosci Biobehav Rev, 1989; 13(1 ):23-31

Hiller et al. (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal Chem 81 : 3429- 3439

Creek et al. (2012) Stable isotope-assisted metabolomics for network-wide metabolic pathway elucidation. Anal Chem 84: 8442-8447

Huang Xet al. (2014) X(13)CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics. Anal Chem

III CR, Gonzales JN, Houtz EK, Ludwig JR, Melcher ED, Hale JE, Pourmand R, Keivens VM, Myers L, Beidler K, Stuart P, Cheng S, Radhakrishnan R. Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions. Protein Eng. 1997 Aug; 10(8):949-57

Wiechert W, de Graaf AA (1997) Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experi-ments. Biotechnol Bioeng 55: 101-1 17

Zhou R, Tseng CL, Huan T, Li L (2014) IsoMS: automated processing of LC-MS data generated by a chemical isotope labeling metabolomics platform. Anal Chem 86: 4675-4679

Bueschl C, Kluger B, Lemmens M, Adam G, Wiesenberger G, Maschietto V, Marocco A, Strauss J, Bodi S, Thallinger G, Krska R, Schuhmacher R (2013) A novel stable isotope labelling assisted workflow for improved untargeted LC-HRMS based metabolomics research. Metabolomics : 7- 16

Sevin DC, Kuehne A, Zamboni N, Sauer U (2015) Biological insights through nontargeted metabolomics. Current Opinion in Biotechnology 34: 1 - 8, systems Biology · Nanobiotechnology

Wegner A, Weindl D, Jager C, Sapcariu SC, Dong X, Stephanopoulos G, Hiller K (2014) Fragment Formula Calculator (FFC): Determination of Chemical Formulas for Fragment Ions in Mass Spectrometric Data. Anal Chem 86: 2221-2228

Narang et al. Meth. Enzymol. 68:90-99, 1979

Brown et al. Meth. Enzymol. 68: 109-151 , 1979

Beaucage et al. Tetrahedron Lett. 22: 1859-1862, 1981

Matteucci et al. Am. Chem. Soc. 103:3185-3191 , 1981

Needleman SB, Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48: 443-453

Carreau A, El Hafny-Rahbi B, Matejuk A, Grillon C, Kieda C (201 1 ) Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med 15: 1239-1253

Jiang BH, Semenza GL, Bauer C, Marti HH (1996) Hypoxia-inducible fac-tor 1 levels vary exponentially over a physiologically relevant range of 02 tension. Am J Physiol 271 : C1 172-C1 180

Henze AT, Garvalov BK, Seidel S, Cuesta AM, Ritter M, Filatova A, Foss F, Dopeso H, Essmann CL, Maxwell PH, Reifenberger G, Carmeliet P, Acker-Palmer A, Acker T (2014) Loss of PHD3 allows tumours to over-come hypoxic growth inhibition and sustain proliferation through EGFR. Nat Commun 5: 5582

Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, Royer RE, Vander Jagt DL, Semenza GL, Dang CV (2010) Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci U S A 107: 2037- 2042

Kim Jw, Tchernyshyov I, Semenza GL, Dang CV (2006) HIF-1 -mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 3: 177-185

Becker I, Lodder J, Gieselmann V, Eckhardt M (2010) Molecular charac-terization of N-acetylaspartylglutamate synthetase. J Biol Chem 285: 29156-29164

Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51 : 1417-1423

Gowda and Djukovic (2014) Overview of Mass Spectrometry-Based Metabolomics: Opportunities and Challenges" Methods Mol Biol. 1 198: 3-12

Sapcariu SC, Kanashova T, Weindl D, Ghelfi J, Dittmar G, Hiller K (2014) Simultaneous extraction of proteins and metabolites from cells in culture. MethodsX 1 : 74 - 80

Hiller K, Metallo CM, Kelleher JK, Stephanopoulos G (2010) Nontargeted elucidation of metabolic pathways using stable-isotope tracers and mass spectrometry. Anal Chem 82: 6621-6628

Hiller K, Wegner A, Weindl D, Cordes T, Metallo CM, Kelleher JK, Stephanopoulos G (2013) NTFD-a stand-alone application for the non-targeted detection of stable isotope-labeled compounds in GC/MS data. Bioinformatics 29: 1226-1228

Antoniewicz MR, Kelleher JK, Stephanopoulos G (2006) Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab Eng 8: 324-337

R Core Team (2013) R: A Language and Environment for Statistical Com-puting. R Foundation for Statistical Computing, Vienna, Austria

Matalon R, Michals K, Sebesta D, Deanching M, Gashkoff P, Casanova J, Optiz JM, Reynolds JF (1988) Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with canavan disease. American Journal of Medical Genetics 29: 463-471

Madhavarao CN, Arun P, Moffett JR, Szucs S, Surendran S, Matalon R, Garbern J, Hristova D, Johnson A, Jiang W, Namboodiri MAA (2005) Defective N- acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease. Proc Natl Acad Sci U S A 102: 5221-5226

Chakraborty G, Mekala P, Yahya D, Wu G, Ledeen RW (2001 ) Intraneu-ronal N- acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin- associated aspartoacylase. J Neurochem 78: 736-745

Claims

1. Inhibitor of NAT8L and/or an inhibitor of the function and/or production of N- Acetylaspartic acid for use in the treatment and/or prophylaxis of cancer.

2. Inhibitor for the use of claim 1 , wherein the inhibitor is a nucleic acid molecule, a binding protein, a small molecule or a compound.

3. Inhibitor for the use of claim 2, wherein the binding protein is selected from the group consisting of an antibody, or a proteinaceous binding molecule with antibody-like binding properties.

4. Inhibitor for the use of claim 2, wherein the nucleic acid molecule is a siRNA or a miRNA.

5. Inhibitor for the use of claim 3, wherein the proteinaceous binding molecule with antibody-like binding properties is selected from the group of an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, an avimer or a (recombinant) receptor protein.

6. Inhibitor for the use of claim 1 or 2, wherein the inhibitor of N-Acetylaspartic acid is selected from the group consisting of roteneone, myxothiazol, cyanide or oligomycin.

7. The inhibitor for the use of any one of claims 1-6, wherein the inhibitor reduces the availability of Acetyl-CoA in the cytosol of cancer cells compared to availability of Acetyl CoA before addition of the inhibitor.

8. Inhibitor for the use of any one of claims 1-7, wherein the inhibitor reduces proliferation of cancer cells.

9. Inhibitor for the use of claim 8, wherein the inhibitor reduces proliferation of cancer cells under normoxic and hypoxic conditions.

10. Inhibitor for the use of any one of claims 1-9, wherein the inhibitor increases the de novo synthesis of glycerol-3-phosphate.

11. Inhibitor for the use of any one of claims 1-10, wherein the inhibitor reduces the glycerol-3-phosphate from lipid turnover.

12. Inhibitor for the use of any one of claims 1-11 , wherein the inhibitor increases expression of GPD2 under normoxia.

13. Inhibitor for the use of any one of claims 1-12, wherein the inhibitor does virtually not affect expression of GPDL1 under normoxia and hypoxia.

14. Inhibitor for the use of any one of claims 1-13, wherein the cancer is selected from the group consisting of adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, rectum cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, basal and squamous cell cancer, melanoma, merkel cell cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor.

15. Inhibitor for the use of claim 14, wherein the cancer is a liver cancer, prostate cancer or lung cancer, preferably a lung cancer.

16. Inhibitor for the use of any one of claims 1-15, wherein the inhibitor reduces the invasiveness of the cancer.

17. Inhibitor for the use of any one of claims 1-16, wherein the inhibitor further comprises an inhibitor of ATP-dependent citrate lyase (ACLY).

18. Inhibitor for the use of any one of claims 1-17, wherein the inhibitor further comprises an inhibitor of aspartoacylase (ASPA).

19. Inhibitor for the use of any one of claims 1-18, wherein the inhibitor further comprises an inhibitor of acyl-CoA synthetase (ACSS).

20. Method for detecting mitochondrial Acetyl CoA, the method comprising

(a) using N-acetylaspartic acid.

21. Method of claim 20, wherein the detection is performed via mass spectrometry.

22. Use of N-acetylaspartic acid for the detection of mitochondrial Acetyl CoA.

23. Method for screening for an inhibitor or activator of NAT8L and/or an inhibitor of the function and/or production of N-acetylaspartic acid, the method comprising

(a) contacting cancer cells with a nucleic acid molecule, binding protein, small molecule or compound of interest;

(b) measuring proliferation of cancer cells,

wherein a decrease in the proliferation of said cancer cells compared to said cancer cells before contacting indicates that the nucleic acid molecule, binding protein, small molecule or compound of interest serves as an inhibitor of NAT8L and/or as an inhibitor of the function and/or production of N- acetylaspartic acid, or

wherein an increase in the proliferation of said cancer cells compared to said cancer cells before contacting indicates that the nucleic acid molecule, binding protein, small molecule or compound of interest serves as an activator of NAT8L and/or as an activator of the function and/or production of N- acetylaspartic acid.

24. Method for determining whether or not a cancer cell is susceptible to the treatment with an inhibitor as defined in any one of claims 1-19, comprising determining whether or not said cancer cell expresses NAT8L or said cancer cell comprises N-acetylaspartic acid.

25. Method for detecting a cancer cell, wherein the method comprises

(a) using primers/oligonucleotides specific for exon2 of NAT8L.

26. Kit comprising mass spectrometric fragments of N-acetylaspartic acid.

27. Kit of claim 26, further comprising one or more of

(i) acetyl-hexosamine,

(ii) 2-acetamidoglucal.