SCREENING METHOD FOR THE IDENTIFICATION OF NOVEL THERAPEUTIC COMPOUNDS

Abstract

The present invention pertains to a method for identifying and/or characterizing a compound suitable for the prevention and/or treatment of a disease. The invention is based on the finding that the glycolytic enzyme Enolase 1 (ENO1) binds RNA, and its enzymatic activity is thereby regulated. The invention is further based on the finding that riboregulation of ENO1 affects cell differentiation, which plays a pivotal role in cancer. Accordingly, the invention provides a screening method for novel therapeutic compounds based on the binding of RNA to ENO1. Compounds screened according to the present invention can affect the binding of RNA to ENO1, which harbors the therapeutic potential for the treatment of diseases, in particular proliferative diseases, such as cancer. Methods of treatment using these compounds, as well as pharmaceutical compositions thereof, are also provided.

Claims

1. A method for identifying and/or characterizing a compound suitable for the prevention and/or treatment of a disease, the method comprising the steps of: (a) Providing at least one enzyme of the glycolytic pathway, at least one nucleic acid, and a candidate compound; (b) Bringing into contact the at least one enzyme of the glycolytic pathway, the at least one nucleic acid and the candidate compound; (c) Detecting and/or quantifying a binding between the at least one enzyme of the glycolytic pathway and the at least one nucleic acid; wherein a differential level of the binding between the at least one enzyme of the glycolytic pathway and the at least one nucleic acid contacted with the candidate compound compared to the binding between the at least one enzyme of the glycolytic pathway and the at least one nucleic acid not contacted with the candidate compound indicates the candidate compound as suitable for the prevention and/or treatment of the disease.

2. The method according to claim 1, wherein steps (b) and (c) are performed in a cell-free system, or in a cell, such as in a biological assay cell or in a cell derived from a biological sample, such as a tissue sample or a body liquid sample of a subject, for example a blood sample.

3. The method according to claim 1 or 2, wherein the at least one nucleic acid is a functional or non-functional RNA polynucleotide molecule or a functional or non-functional DNA polynucleotide molecule, such as a single-stranded or doubled-stranded RNA polynucleotide molecule or DNA polynucleotide molecule, or a fragment or derivative thereof, for example an mRNA molecule, an RNA mimic, an RNA precursor, an RNA analogue, an RNA antisense molecule, an inhibitory RNA molecule, a ribozyme, an RNA antisense expression molecule, an RNA interference (RNAi) molecule, an siRNA molecule, an esiRNA molecule, an shRNA molecule, a miRNA molecule, a DNA mimic, a DNA precursor, a DNA analogue, an antisense DNA, a DNA aptamer, a decoy molecule, a GapmeR, a PNA (peptide nucleic acid) molecule, an LNA molecule (locked nucleic acid), a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct, a guide nucleic acid (gRNA or gDNA), and/or a tracrRNA, optionally wherein the at least one nucleic acid comprises at least one modification, for example a chemical modification selected from a modified internucleoside linkage, a modified nucleobase, or a modified sugar moiety, such as a 2′-O-alkyl modification, for example a 2′-O-methoxy-ethyl (MOE) or 2′-O-Methyl (OMe) modification, an ethylene-bridged nucleic acid (ENA), a 2′-fluoro (2′-F) nucleic acid, such as 2′-fluoro N3-P5′-phosphoramidite, a 1′,5′-anhydrohexitol nucleic acid (HNA), or a locked nucleic acid (LNA).

4. The method according to any one of claims 1 to 3, wherein the candidate compound is selected from a small molecular compound (“small molecule”), a polypeptide, a peptide, a glycoprotein, a peptidomimetic, an antigen binding construct (for example, an antibody, antibody-like molecule or other antigen binding derivative, or an antigen binding fragment thereof), a nucleic acid, such as a DNA or RNA, for example an antisense or inhibitory DNA or RNA, a ribozyme, an RNA or DNA aptamer, RNAi, siRNA, shRNA and the like, including variants or derivatives thereof, such as a peptide nucleic acid (PNA), a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct, a guide nucleic acid (gRNA or gDNA), and/or a tracrRNA.

5. The method according to any one of claims 1 to 4, wherein the detecting and/or quantifying in step (c) involves at least one of: (i) UV cross-linking, immunoprecipitation and radioactive labelling of co-purified RNA (PNK assay); (ii) Enhanced Crosslinking and Immunoprecipitation (eCLIP); (iii) Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immuno-precipitation (PAR-CLIP); (iv) RNA immunopurification followed by microarray hybridization (RIP-chip); (v) RNA immunopurification followed by high throughput sequencing (RIP-seq); (vi) RNA-protein crosslink; (vii) RNA pulldown; (viii) Mass-spectrometry; (ix) Proximity Extension Assay; (x) Immunofluorescent based assays; (xi) Proximity Ligation Assay; (xii) Förster resonance energy transfer (FRET), and/or (xiii) any other method for reporting at least one bi-molecular interaction.

6. The method according to any one of claims 1 to 5, wherein the at least one enzyme of the glycolytic pathway is Enolase 1 (ENO1), or a derivative, a precursor, a mutant, or a functional fragment thereof, comprising the amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% sequence identity to SEQ ID NO: 1.

7. A mutated Enolase 1 (ENO1) enzyme, or a functional fragment thereof, wherein the mutated ENO1 enzyme amino acid sequence when aligned to the amino acid sequence of SEQ ID NO: 1 comprises not more than 50 amino acid substitutions, deletions, and/or additions, preferably not more than 40, more preferably not more than 30, even more preferably not more than 10, even more preferably not more than 5, even more preferably not more than 4, even more preferably not more than 2, and most preferably not more than 1 amino acid substitution, deletion, and/or addition of an amino acid sequence according to SEQ ID NO: 1.

8. The mutated ENO1 enzyme, or the functional fragment thereof, according to claim 7, wherein the amino acid sequence of the mutated ENO1 enzyme, or of the functional fragment thereof, when aligned to the amino acid sequence of SEQ ID NO: 1, comprises at least one amino acid substitution, deletion, and/or addition in an amino acid at positions 57-132 of SEQ ID NO: 1, or at position 343 of SEQ ID NO: 1, preferably wherein the amino acid sequence of the mutated ENO1 enzyme, or of the functional fragment thereof, comprises at least one amino acid substitution, deletion, and/or addition at position 89, 92, 105, and/or 343 of SEQ ID NO: 1, and more preferably comprises the amino acid sequence of any of SEQ ID NOs: 7 to 14, or comprises not more than 50 amino acid substitutions, deletions, and/or additions, preferably not more than 40, more preferably not more than 3o, even more preferably not more than 10, even more preferably not more than 5, even more preferably not more than 4, even more preferably not more than 2, and most preferably not more than 1 amino acid substitution, deletion, and/or addition of an amino acid sequence according to the sequence of any of SEQ ID NOs: 7 to 14.

9. The mutated ENO1 enzyme, or the functional fragment thereof, according to claim 7 or 8, comprising at least one amino acid substitution selected from K89A, K92A, and Kio5A at positions 89, 92, and 105 in SEQ ID NO: 1, or comprising the amino acid substitutions K89A and K92A at positions 89, and 92 in SEQ ID NO: 1, or comprising the amino acid substitutions K89A and K105A at positions 89, and 105 in SEQ ID NO: 1, or comprising the amino acid substitutions K92A and K105A at positions 92, and 105 in SEQ ID NO: 1, or comprising the amino acid substitutions K89A, K92A, and K105A at positions 89, 92, and 105 in SEQ ID NO: 1, wherein said mutated ENO1 enzyme is characterized by an enhanced binding of at least one nucleic acid to the mutated ENO1 enzyme compared to the binding of the at least one nucleic acid to the wild type ENO1 enzyme comprising the amino acid sequence of SEQ ID NO: 1, preferably wherein the mutated ENO1 enzyme comprises the amino acid sequence of any of SEQ ID NOs: 7 to 13, or comprises not more than 50 amino acid substitutions, deletions, and/or additions, preferably not more than 40, more preferably not more than 30, even more preferably not more than 10, even more preferably not more than 5, even more preferably not more than 4, even more preferably not more than 2, and most preferably not more than 1 amino acid substitution, deletion, and/or addition of an amino acid sequence according to the sequence of any of SEQ ID NOs: 7 to 13.

10. The mutated ENO1 enzyme, or the functional fragment thereof, according to any one of claims 7 to 9, comprising at least one amino acid substitution at position 343 in SEQ ID NO: 1, such as a K343A amino acid substitution at position 343 in SEQ ID NO: 1, wherein said mutated ENO1 enzyme is characterized by a reduced binding of at least one nucleic acid to the mutated ENO1 enzyme compared to the binding of the at least one nucleic acid to the wild type ENO1 enzyme comprising the amino acid sequence of SEQ ID NO: 1, preferably wherein the mutated ENO1 enzyme comprises the amino acid sequence of SEQ ID NO: 14, or comprises not more than 50 amino acid substitutions, deletions, and/or additions, preferably not more than 40, more preferably not more than 30, even more preferably not more than 10, even more preferably not more than 5, even more preferably not more than 4, even more preferably not more than 2, and most preferably not more than 1 amino acid substitution, deletion, and/or addition of an amino acid sequence according to SEQ ID NO: 14.

11. An isolated nucleic acid, comprising a sequence coding for the mutated ENO1 enzyme, or the functional fragment thereof, according to any one of claims 7 to 10, or a vector, comprising the nucleic acid, optionally wherein the vector is an expression vector, comprising a promoter sequence operably linked to the nucleic acid.

12. A recombinant cell comprising a mutated ENO1 enzyme, or the functional fragment thereof, according to any one of claims 7 to 10, or a nucleic acid or a vector according to claim 11.

13. A pharmaceutical composition comprising the mutated ENO1 enzyme, or the functional fragment thereof, according to any one of claims 7 to 10, a nucleic acid or a vector according to claim 11, or a recombinant cell according to claim 12, together with a pharmaceutically acceptable carrier, stabilizer and/or excipient.

14. A compound for use in the treatment of a disease, the compound being selected from a mutated ENO1 enzyme, or the functional fragment thereof, according to any one of claims 7 to 10, a nucleic acid or a vector according to claim 11, a recombinant cell according to claim 12, and a pharmaceutical composition according to claim 13, wherein the disease is preferably a proliferative disease, such as cancer, diabetes, an infectious disease, a metabolic disease, an immune-related disease, a degenerative disease, such as a neurodegenerative disease, for example Alzheimer's disease, and/or aging.

15. A method for identifying and/or characterizing a compound suitable for the prevention and/or treatment of a disease, the method comprising the steps of: (a) Providing at least one enzyme of the glycolytic pathway, and a candidate compound; (b) Bringing into contact the at least one enzyme of the glycolytic pathway, and the candidate compound; (c) Detecting and/or quantifying at least one modification in the at least one enzyme of the glycolytic pathway; wherein a differential level of the at least one modification in the at least one enzyme of the glycolytic pathway contacted with the candidate compound compared to the at least one modification in the at least one enzyme of the glycolytic pathway not contacted with the candidate compound indicates the candidate compound as suitable for the prevention and/or treatment of the disease, and wherein the differential level of the at least one modification is indicative for a differential level of a binding of the at least one enzyme of the glycolytic pathway to at least one nucleic acid, preferably wherein the modification is selected from ubiquitination, acetylation, phosphorylation, methylation, glycosylation, lipid-conjugation, functionalization, heterodimerization, homodimerization, oxidation, hydroxylation, or any other natural or artificial post-translational modification, or combinations thereof.

16. A method for diagnosing, prognosing, stratifying and/or monitoring of a therapy, of a disease in a subject, comprising the steps of: (a) Providing a sample comprising at least one enzyme of the glycolytic pathway from the subject, at least one nucleic acid, and optionally at least one agent for detection of the at least one enzyme of the glycolytic pathway, the at least one nucleic acid, and/or a binding between the at least one enzyme of the glycolytic pathway and the at least one nucleic acid, such as an antigen binding construct (for example, an antibody, an antibody-like molecule or other antigen binding derivative, or an antigen binding fragment thereof), a nucleic acid, including an RNA or DNA aptamer, and the like; (b) Optionally, isolating the at least one enzyme of the glycolytic pathway from the sample; (c) Bringing into contact the at least one enzyme of the glycolytic pathway, and the at least one nucleic acid, and (d) Detecting and/or quantifying the binding between the at least one enzyme of the glycolytic pathway and the at least one nucleic acid; wherein a differential level of the binding between the at least one enzyme of the glycolytic pathway and the at least one nucleic acid in the sample from the subject as detected and/or quantified in step (d) compared to a control or reference value is indicative for the diagnosis, prognosis, stratification and/or monitoring of a therapy, of the disease in the subject.

Description

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

[0147] The figures show:

[0148] FIG. 1 shows that the glycolytic enzyme Enolase 1 (ENO1) binds RNA in vivo. a. ENO1 immunoprecipitation (IP) after crosslinking RNA-RBP complexes with UV light. Polynucleotide kinase labels RNA with radioactive .sup.32P-γATP (PNK assay). Western blot staining for ENO1 and LARP1 (RNA-binding protein) of the same experiment. Three biological replicates are shown for ENO1 and an IgG control of the same species. b. Volcano plot of differential crosslinking site occurrences as determined by DEWseq; each dot corresponds to a window of genomic region (50 nts), grey colouring indicates significant enrichment in ENO1 IPs (IHW-adjusted p-value <0.1, loge FC>0.5). The data are based on five biological experiments and were normalized to the background. Only the positive enrichment is displayed. c. Top: HeLa cell lysates were treated with RNase I/A/T1 or left untreated, subjected to sucrose density gradient (5-25%), centrifugation and fractionation. A protein's RNase-sensitive fraction moves from the higher fraction number (heavy) to the lower fraction number (light). Bottom: Quantification of biological replicates for the experimental setup on the top for ENO1 immunoblots (n=3). The standard deviation is given.

[0149] FIG. 2 shows ENO1's specific RNA binding in HeLa cells. ENO1's binding profile as identified by eCLIP relative to the input control is used for the identification of target and control sites. Top: Enriched ENO1-binding sites are normalised by the length of the feature. Bottom: CL motif analysis based on DEWseq results of ENO1 eCLIP (n=5). Logo representation of the top scoring motifs using DREME. The top scoring two sequence motifs (FIG. 2: TTTTTTBTTTTTT, and CCCAGRC) jointly account for ˜22% of all ENO1 binding sites.

[0150] FIG. 3 shows ENO1's specific RNA binding in vitro. a. Schematic of ENO1's binding profile as identified by eCLIP relative to the input control used for the identification of target and control sites. Every line represents an accumulation of crosslink sites at an individual nucleotide. b. Overlay of1H,15N-HSQC spectra of free ENO1 and ENO1 incubated with twofold excess of RNA (FTH1 18-mer start, black). Full titration points (in 1:0.1/0.2/0.4/0.8/1.2/2 ratios) for some residues are shown in insets. c. Comparative electromobility shift assay (EMSA) for target versus control RNA using radioactively labelled PABPC1 target 35-mer as a probe and unlabelled competitor RNA from either the target or control region as indicated in a. d. Inhibition constants (Ki) for target and control RNAs for the binding to ENO1wt (n=3). The Ki was calculated using a non-linear fitting with least squares regression.

[0151] FIG. 4 shows in vitro analyses of ENO1 RNA binding specificity by competitive EMSAs. a. Quantification of competition EMSA experiments using FTH1 target RNA as a probe and different unlabelled 18-mer FTH1 RNA competitors (n=3). b. Quantification of competition EMSA experiments using PABPC1 target RNA as a probe and unlabelled PABPC1 control or target RNA competing for the binding to ENO1 (n=3). c. Quantification of competition EMSA experiments using FTH1 target RNA as a probe and unlabelled control or target FTH1 RNA as competitors (n=3). d. Quantification of competition EMSA experiments using PTP4A1 target RNA as a probe and unlabelled control or target (n=3).

[0152] FIG. 5 shows riboregulation of ENO1's enzymatic activity in vitro. a. Enzymatic activity assay of recombinant human ENO1 with increasing enzyme concentrations exposed to different control and target RNAs (constant conc. 100 nM). b. Measurement of recombinant ENO1wt activity with increasing concentrations of control and target PABPC1 RNA (n=3). The standard deviation is given and the statistically significant differences were detected using two-way ANOVA and Sidak-correction for multiple comparison testing. c. Inhibition constants (Ki) for target and control RNAs for the binding to ENO1down determined by competitive EMSA (n=3). The Ki was calculated using a non-linear fitting with least squares regression. The respective curves are shown in FIGS. 6a-c. d. Measurement of recombinant ENO1down activity with increasing concentrations of control and target PABPC1 RNA (n=3). e. EMSA with recombinant human ENO1 and a labelled PABPC1 target RNA probe in competition with substrate of the forward (2-phosphoglycerate) and reverse reaction (phosphoenol pyruvate) or a control glycolytic metabolite (3-phosphoglycerate). f. Quantification of replicates for the experimental setup in e, including the inhibitor constants (n=3). The Ki was calculated using a non-linear fitting with least squares regression.

[0153] FIG. 6 shows the effect of RNA on the enzymatic activity of ENO1down in vitro. a. Quantification of competition EMSA experiments using PABPC1 target RNA as a probe and unlabelled PABPC1 control or target RNA competing for the binding to ENO1down (n=3). b. Quantification of competition EMSA experiments of ENO1down using FTH1 target RNA as a probe and unlabelled control or target FTH1 RNA as competitors (n=3). c. Quantification of competition EMSA experiments of ENO1down using PTP4A1 target RNA as a probe and unlabelled control or target (n=3). d. Measurement of recombinant ENO1down enzymatic activity with increasing concentrations of control and target FTH1 RNA (n=3). The standard deviation is given and the statistically significant differences were detected using two-way ANOVA and Sidak-correction for multiple comparison testing. e. Experimental setup as in f for control and target PTP4A1 RNA.

[0154] FIG. 7 shows Riboregulation of ENO1 in HeLa cells. A. Immunoprecipitation (anti-FLAG) and PNK assay of transiently expressed ENO1-Flag-HA proteins. RNA binding of tagged wild-type ENO1 (ENO1wt), a mutant with increased RNA binding (ENO1up) and reduced RNA binding (ENO1down) are being compared. B. Representative image of a proximity ligation assay (RNA-PLA) for ENO1wt with its mRNA target FTH1. ENO1 is being detected using an anti-FLAG antibody and the endogenous ENO1 is knocked down using siRNAs. The PLA signal is presented as a maximal projection of a Z-stack (10 pictures for 10 μm stack). Pictures for the DAPI (nuclear staining) and cellular outline (grey) are taken in one plane. The scale bar is equivalent to 20 μm. C. Representative image of an RNA-PLA for ENO1down with its mRNA target FTH1. D. Representative image of an RNA-PLA for ENO1up with its mRNA target FTH1. E. Quantification of RNA-PLA signals as dots per cell from three biological replicates with at least 30 cells in total. Statistically significant differences were detected using one-way ANOVA and Tukey-correction for multiple comparison testing. F. Immunofluorescent staining for ENO1wt, ENO1down and ENO1up in transfected HeLa cells using an anti-FLAG antibody, an anti-ENO1 antibody and DAPI. The scale bar is equivalent to 20 μm. G. Representative image of a control RNA-PLA for ENO1wt without the addition of a biotinylated probe. ENO1 is being detected using an anti-FLAG antibody and the endogenous ENO1 is knocked down using siRNAs. The RNA-PLA signal is presented as a maximal projection of a Z-stack (10 pictures for 10 μm stack). Pictures for the DAPI (nuclear staining) and cellular outline (grey) are taken in one plane. The scale bar is equivalent to 20 μm. H. Quantification of RNA-PLA signal of endogenous ENO1 with FTH1 mRNA as dots per cell from three replicates with at least 30 cells in total, comparing samples without probe, cells treated with control siRNA and ENO1 siRNA. Statistically significant differences were detected using one-way ANOVA and Tukey-correction for multiple comparison testing.

[0155] FIG. 8 shows the behaviour of ENO1 mutants in HeLa cells and riboregulation in mESCs. a. Comparison of ENO1's RNA-binding (PNK), and ENO1's enzymatic activity in HeLa cells, indirectly measured by the accumulation of lactate in the medium (n=3). b. Michaelis-Menten saturation curve of the basal enzymatic activity of recombinant ENO1wt, ENO1down and ENO1up in vitro in the absence of RNA using a non-linear curve fitting with least squares regression (n=3). c. Vmax and Km measurements for ENO1wt, ENO1down and ENO1up as determined from the Michaelis-Menten saturation curve in c. The asymmetrical confidence interval (CI) is given (n=3). d. Three distinct sets of target and control RNAs (5 μM) were nucleofected into mESCs. Upon nucleofection of the control or target RNAs, the lactate accumulation in the medium was measured after 30, 60 and 90 minutes and used to estimate the accumulation rate by calculating the slope. The R2 value was used as a quality control. The standard deviation is given and the two-tailed Student's t-test is used to detect statistically significant differences (n=3).

[0156] FIG. 9 shows riboregulation of ENO1 in HeLa cells. a. Relative enzymatic activity of ENO1wt, ENO1up, ENO1down and ENO1 as determined with recombinant proteins in vitro (n=3). b. Increasing concentrations of target and control PABPC1 were nucleofected into HeLa cells. Upon nucleofection of the control or target RNAs, the lactate accumulation in the medium was measured after 30, 60 and 90 minutes and used to estimate the accumulation rate (n=3). The standard deviation is given and the statistically significant differences were detected using two-way ANOVA and Sidak-corrected for multiple comparison testing. c. Same experimental setup as in b for FTH1 control and target RNA.

[0157] FIG. 10 shows analysis of ENO1 and mutant variants in mESCs. a. Lactate accumulation rate of pluripotent mESCs after LIF withdrawal for a period of seven days (n=3). The standard deviation is given and the two-tailed Student's t-test is used to detect statistically significant differences. b. Oxygen consumption rate of cells was measured with the Oxytherm System (n=3). The standard deviation is given and the two-tailed Student's t-test is used to detect statistically significant differences. c. Quantification of replicates of ENO1 PNK assays after UV-crosslinking for pluripotent mESCs and mouse cells after 7 days of LIF withdrawal (n=3). The standard deviation is given and the two-tailed Student's t-test is used to detect statistically significant differences. d. Immunoblot of ENO1, FLAG and HuR (loading control) for three clones for ENO1 knockout and constitutive transgenic expression of Flag-HA-tagged ENO1wt, ENO1up and ENO1down from the Rosa26 locus. e. The schematic shows that RNA riboregulates the enzymatic activity of ENO1, thereby inhibiting glycolysis and affecting mESC differentiation.

[0158] FIG. 11 shows that riboregulation of ENO1 affects mESC differentiation. a. Cells expressing Eomes-mCherry or Brachyury-BFP after seven days of LIF withdrawal were sorted for the respective fluorescent marker proteins, cultured for an additional five days and lactate accumulation in the culture medium was quantified (n=3). The standard deviation is given and the statistically significant differences were detected using the two-tailed Student's t-test. b. Cellular RNA-protein interactions were cross-linked with UV and a PNK assay was performed for cell populations described in a. A representative experiment is shown and was performed for a total of three times. Non-crosslinked (No CL), unsorted (us.) mESC were used as specificity controls. Immunoblots of ENO1 and actin are shown as controls. c. Cell lines were established for ENO1 knockout and constitutive transgenic expression of Flag-HA-tagged ENO1wt, ENO1up and ENO1down from the Rosa26 locus. RNA binding was measured for the three ENO1 variants by Flag immunoprecipitation and PNK assay after UV cross-linking in pluripotent mESCs. Three independent clones were used. The standard deviation is given and the statistically significant differences were detected using the two-tailed Student's t-test when comparing to ENO1wt. d. Measurement of the lactate accumulation in the culture medium for three independent clones of ENO1wt, ENO1up and ENO1down in pluripotent mESCs. The standard deviation is given and the statistically significant differences were detected using the two-tailed Student's t-test when comparing to ENO1wt. e.-g. Differentiation of mESCs and qPCRs of lineage marker for ENO1wt (e.), ENO1up (f.) and ENO1down (g.) after seven days of LIF withdrawal for three independent clones (from c.) in three experiments. The standard deviation is given and the statistically significant differences were detected using two-way ANOVA with Tukey-corrected multiple comparison testing when comparing to ENO1wt.

[0159] FIG. 12. Riboregulation of ENO1 affects mESC differentiation. A.-B. Cell lines were established for a transient ENO1 depletion using the auxin-inducible degron system. Four days after withdrawing LIF from the mESC medium, DMSO (A) or auxin (B) was added for 48 hours. The cells were differentiated for a total of seven days and RT-qPCRs were performed for differentiation and lineage marker. This experiment was performed with two independent clones in three biological replicates. The standard deviation is given, and the statistically significant differences were detected using two-way ANOVA with Sidak-corrected multiple comparison testing when comparing to the DMSO-treated samples. C. Immunoblot for auxin-induced degradation of ENO1 in two independent clones after 48 hours of treatment with auxin. Actin and Tubulin were stained as loading controls. D. Expression level differences of differentiation and lineage-relevant markers after treating wild-type mESCs with auxin or DMSO for seven days after withdrawing LIF. Statistically significant differences were detected using two-way ANOVA and Sidak-correction for multiple comparison testing (n=3).

[0160] FIG. 13 shows an analysis of ENO1 ubiquitination. a. TRIM21 (E3 ligase) co-immunoprecipitates (co-IP) with ENO1. b. Immunoprecipitation (IP) of ubiquitin shows enhanced ubiquitination of ENO1wt compared to ENO1up. The data supports that riboregulation of ENO1 is (at least partially) controlled by ubiquitination of ENO1.

[0161] FIG. 14 shows that acetylation activates ENO1's RNA binding. A. PNK assay of ENO1 in HeLa cells after a 16-hour treatment with the histone deacetylation (HDAC) inhibitor, sodium butyrate, with concentrations ranging from 2.5-10 mM. Western blotting was performed for ENO1 and using an antibody for acetylated lysine (acetyl-K). B. Western blot for SIRT2 and ENO1 after siRNA-mediated knockdown of SIRT2 in HeLa cells. C. Immunoprecipitation of ENO1 from siControl or siSIRT2-treated HeLa cells. Western blotting was performed for ENO1 and acetylated lysine (acetyl-K). D. Representative PNK assays for FLAG-tagged ENO1wt, ENO1up and ENO1down in HeLa cells after siRNA-mediated knockdown of SIRT2. Western blotting was performed for the FLAG-tagged ENO1 variants, SIRT2 to assess the knock-down efficiency, and Nucleolin as a loading control. E. Quantification of three biological replicates of the experimental setup in D. RNA binding as detected by the autoradiograph was normalized to the FLAG immunoprecipitation efficiency. Statistically significant differences were detected using two-way ANOVA and Sidak-correction for multiple comparison testing (SD, n=3). F. In vitro deacetylation assay coupled with mass spectrometry using recombinant SIRT2 wildtype or the enzymatically dead mutant SIRT2 H187Y and in vitro acetylated ENO1 wildtype. Two amino acids on the same peptide were found to respond to SIRT2 deacetylation. G. PNK assay of ENO1 in HeLa cells after a 24-hour treatment with 0.1% DMSO or the SIRT2 inhibitor, SirReal2 at a 12.5 μM. Western blotting was performed for ENO1 and nucleolin. H. PNK assay of ENO1 in HeLa cells after a 24-hour treatment with 0.1% DMSO or the SIRT2 inhibitor, Thiomyristoyl at a 10 μM. Western blotting was performed for ENO1 and nucleolin. I. Representative PNK assay for FLAG-tagged ENO1wt, ENO1up, ENO1down and an acetylation-mimicking version of the ENO1up mutant (ENO1KtoQ) in HeLa cells. Western blotting was performed for the FLAG-tagged ENO1 variants and Nucleolin as a loading control. J. Quantification of three biological replicates of the experimental setup in H. RNA binding as detected by the autoradiograph was normalized to the FLAG immunoprecipitation efficiency. Statistically significant differences were detected using two-way ANOVA and Sidak-correction for multiple comparison testing (SD, n=3).

[0162] FIG. 15 shows time- and germ layer-dependent changes in ENO1's RNA binding and acetylation level during mESC differentiation. A. Formaldehyde (0.1%)-crosslinked RNA-immunoprecipitation of ENO1 or an isotype-matched IgG from pluripotent mESCs, cells differentiated for three, five and seven days (-LIF). RT-qPCR for five specific and two control mRNAs. The RNA enrichment is calculated relative to the mean of the IgG samples and normalized to the input. Statistically significant differences were determined using two-way ANOVA and Sidak-correction for multiple comparison testing. B. Input for ENO1 immunoprecipitation in FIG. 15C. C. ENO1 immunoprecipitation from pluripotent mESCs, cells differentiated for three, five and seven days (-LIF), followed by immunoblotting using an antibody detecting acetylated lysine (acetyl-K). The ratio of acetyl-K signal in comparison to ENO1 staining was calculated for three biological replicates. The ratio of acetyl-K over ENO1 steadily increases and is significantly elevated at day seven (unpaired Student's T-test, p-value=0.030), highlighted in blue, relative to the ratio in pluripotent cells. D. Input for ENO1 immunoprecipitation in cells expressing Eomes-mCherry or Brachyury-BFP after seven days of LIF withdrawal were sorted for the respective fluorescent marker proteins, cultured for an additional five days and lysates were prepared. E. ENO1 immunoprecipitation in cells from FIG. 15D and staining with an antibody detecting acetylated lysine (acetyl-K). The ratio of acetyl-K signal in comparison to ENO1 staining was calculated for three biological replicates.

[0163] The sequences according to SEQ ID NOs. 1 to 14 show:

TABLE-US-00001 Amino acid sequence of human ENO1 SEQ ID NO: 1 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEKIDKLMIEMDGTENKSKFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK sequence motif 1 SEQ ID NO: 2 TTTTTTBTTTTTT sequence motif 2 SEQ ID NO: 3 CCCAGRC Amino acid sequence of human Polyadenylate- binding protein cytoplasmic 1 (PABPC1) SEQ ID NO: 4 MNPSAPSYPMASLYVGDLHPDVTEAMLYEKFSPAGPILSI RVCRDMITRRSLGYAYVNFQQPADAERALDTMNFDVIKGK PVRIMWSQRDPSLRKSGVGNIFIKNLDKSIDNKALYDTFS AFGNILSCKVVCDENGSKGYGFVHFETQEAAERAIEKMNG MLLNDRKVFVGRFKSRKEREAELGARAKEFTNVYIKNFGE DMDDERLKDLFGKFGPALSVKVMTDESGKSKGFGFVSFER HEDAQKAVDEMNGKELNGKQIYVGRAQKKVERQTELKRKF EQMKQDRITRYQGVNLYVKNLDDGIDDERLRKEFSPFGTI TSAKVMMEGGRSKGFGFVCFSSPEEATKAVTEMNGRIVAT KPLYVALAQRKEERQAHLTNQYMQRMASVRAVPNPVINPY QPAPPSGYFMAAIPQTQNRAAYYPPSQIAQLRPSPRWTAQ GARPHPFQNMPGAIRPAAPRPPFSTMRPASSQVPRVMSTQ RVANTSTQTMGPRPAAAAAAATPAVRTVPQYKYAAGVRNP QQHLNAQPQVTMQQPAVHVQGQEPLTASMLASAPPQEQKQ MLGERLFPLIQAMHPTLAGKITGMLLEIDNSELLHMLESP ESLRSKVDEAVAVLQAHQAKEAAQKAVNSATGVPTV Amino acid sequence of human Ferritin heavy chain 1 (FTH1) protein SEQ ID NO: 5 MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSY YFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGR IFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHK LATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMG APESGLAEYLFDKHTLGDSDNES Amino acid sequence of human Protein tyrosine phosphatase type IVA (PTP4A1) SEQ ID NO: 6 MARMNRPAPVEVTYKNMRFLITHNPTNATLNKFIEELKKY GVTTIVRVCEATYDTTLVEKEGIHVLDWPFDDGAPPSNQI VDDWLSLVKIKFREEPGCCIAVHCVAGLGRAPVLVALALI EGGMKYEDAVQFIRQKRRGAFNSKQLLYLEKYRPKMRLRF KDSNGHRNNCCIQ Amino acid sequence of ENO1-K89A mutant SEQ ID NO: 7 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEAIDKLMIEMDGTENKSKFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK Amino acid sequence of ENO1-K92A mutant SEQ ID NO: 8 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEKIDALMIEMDGTENKSKFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK Amino acid sequence of ENO1-K105A mutant SEQ ID NO: 9 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEKIDKLMIEMDGTENKSAFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK Amino acid sequence of ENO1-K89A/K92A mutant SEQ ID NO: 10 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEAIDALMIEMDGTENKSKFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK Amino acid sequence of ENO1-K89A/K105A mutant SEQ ID NO: 11 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEAIDKLMIEMDGTENKSAFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK Amino acid sequence of ENO1-K92A/K105A mutant SEQ ID NO: 12 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAST GIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSKK LNVTEQEKIDALMIEMDGTENKSAFGANAILGVSLAVCKA GAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAGN KLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKYG KDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKVV IGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLYK SFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDLT VTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLAQ ANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCRS ERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK Amino acid sequence of ENO1-K89A/K92A/ K105A mutant SEQ ID NO: 13 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEAIDALMIEMDGTENKSAFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK Amino acid sequence of ENO1- K343A mutant SEQ ID NO: 14 MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGAS TGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSK KLNVTEQEKIDKLMIEMDGTENKSKFGANAILGVSLAVCK AGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAG NKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKY GKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKV VIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLY KSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDL TVTNPKRIAKAVNEKSCNCLLLAVNQIGSVTESLQACKLA QANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCR SERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAK

[0164] Mutated amino acids in SEQ ID NOs: 7 to 14 are highlighted. The mutated amino acids in SEQ ID NOs: 7 to 14 are Lysine to Alanine mutations, when compared to the sequence of SEQ ID NO: 1.

EXAMPLES

[0165] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examplesrefe of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0166] The examples show:

Example 1: Enolase 1 Binds to Specific Transcriptomic Sites

[0167] The inventors used a PNK assay [1] to confirm that human ENO1 binds RNA in HeLa cells (FIG. 1a). The inventors estimated that under basal conditions around 10% of HeLa cell ENO1 is sensitive to RNase treatment when exposing RNase-treated or untreated lysates to sucrose density gradient centrifugation (FIG. 1c). The inventors also determined the RNA-binding sites of ENO1 in the transcriptome by applying an enhanced crosslinking and immunoprecipitation (eCLIP) protocol [2] CLIP of ENO1, which enabled to identify RNA-binding sites on a transcriptome-wide scale, and revealed RNA-binding sites at nucleotide resolution. The inventors attained that ENO1 interacts with a wide range of RNAs in HeLa cells with a preference towards the 5′untranslated region (5′UTR) of mRNAs (FIG. 2). Based on the exact crosslinking sites, the inventors identified approximately two thousand direct ENO1-binding sites across the transcriptome (FIG. 1b) that do not display striking linear sequence motif recognition. The top scoring two sequence motifs (FIG. 2: TTTTTTBTTTTTT, and CCCAGRC) jointly account for only ˜22% of all ENO1 binding sites.

[0168] For validation experiments, the inventors synthesized RNAs of 35 nucleotides in length that either correspond to ENO1's binding site or a GC-matched control, derived from a region of the same mRNA downstream from its binding site (schematic in FIG. 3a). The inventors tested an exemplary target and control RNA, derived from the PABPC1 5′UTR in a competition electromobility shift assay (EMSA) using recombinant human ENO1 (FIGS. 3c and d; Ki.sub.target:27±19 nM; Kicontrol: 2587±9 nM, FIG. 6). Similar results were obtained for two additional target and control pairs derived from the PTP4A1 and FTH1 mRNAs, respectively (FIG. 3d, FIG. 6a and b). Using NMR, the inventors observed RNA-induced chemical shift perturbations and line broadening of ENO1 resonances in .sup.1H, .sup.15N-HSQC spectra, confirming direct RNA binding in vitro (shortened FTH1 target RNA (18-mer), FIG. 3b). Taken together, the inventors showed that ENO1 specifically binds RNA targets, in particular, ENO1 binds RNA at numerous transcriptomic sites in human cells with two orders of magnitude difference between specific and non-specific interactions.

Example 2: Development of ENO1 Mutants Characterized by Enhanced and/or Decreased RNA Binding to ENO1

[0169] Instructed by RBDmap data, the inventors further generated an ENO1 mutant (K343A; ENO1down) with ˜5-10-fold decreased RNA binding (FIGS. 5c and d; Kitarget: 224±12 nM; Kicontrol: 3261±12 nM, FIG. 6) compared to ENO1wt as measured by competitive EMSA (FIGS. 5c and d, FIG. 5). Importantly, ENO1down displays no discernible alteration in its enzymatic activity with any of the RNAs tested (FIG. 5d, FIG. 6). Thus, both control RNAs and the ENO1 down mutant corroborate that RNA specifically riboregulates ENO1's activity in vitro.

[0170] Next, the inventors tested whether ENO1's enzymatic substrate binding and RNA binding are competitive. For this reason, the inventors performed EMSA experiments utilizing 2-PG and PEP as competitors. While both substrates compete with RNA for ENO1's binding, the specificity control 3-phosphoglycerate (3-PG), the immediate precursor of 2-PG with an identical molecular mass fails to compete (FIGS. 5e and f).Thus, the inventors observe specific competition between substrates and RNA targets for binding to ENO1. The inventors' data demonstrate that ENO1's enzymatic activity is reduced when incubated with specific RNAs, supporting a role of RNA as a regulator of ENO1.

[0171] In addition to ENO1down, the inventors generated an ENO1up mutant. The design of ENO1up was also guided by RBDmap data and entails the change of lysine residues (K89A/K92A/K105A) along the inferred interaction region of RNA with the enzyme. After knocking down endogenous HeLa cell ENO1 and rescue with the respective Flag-tagged ENO1 variant, ENO1up displays increased RNA binding compared to ENO1wt, as measured by PNK assays (FIG. 7a). In line with the in vitro data, ENO1down displays substantially decreased RNA binding in HeLa cells relative to ENO1wt (FIG. 7a).

[0172] The inventors independently confirmed the differential RNA binding of the ENO1up and ENO1down mutants using an immunofluorescence-based, UV crosslinking-independent RNA proximity ligation assay (PLA), as disclosed in [3] (Zhang et al., 2016) that enables the in situ detection of endogenous or tagged proteins with their RNA targets. ENO1's association with the FTH1 mRNA ligand was validated by the combination of an antisense probe hybridizing close to ENO1's FTH1 mRNA-interaction site and an antibody specifically recognizing the Flag-tagged ENO1 variants. Using this orthogonal assay, the inventors validated the differential RNA binding of ENO1wt (FIG. 7B, E), ENO1down (FIG. 7C, E) and ENO1up (FIG. 7D, E) in HeLa cells, ensuring that the expression levels and localization of the ENO1 variants were comparable (FIG. 7F) and that the PLA signal is specific (FIG. 7G and H).

[0173] When the inventors tested the ENO1down and ENO1up mutants for their ability to rescue glycolysis (lactate accumulation in the medium) in HeLa cells after knock-down of the endogenous ENO1, ENO1wt rescued lactate production (FIG. 8a). Of note, the RNA binding-deficient mutant ENO1down is as active as the wild-type protein (FIG. 8a), showing that the K343A mutation does not incapacitate the enzyme. In contrast, ENO1up fails to rescue the knock-down-induced inhibition of lactate accumulation (FIG. 8a), although it is fully active when tested in the absence of RNA in vitro (FIGS. 8b and c, FIG. 9a). The activity measurements were controlled with a mutant lacking enzymatic activity (ENO1as, E295A/D320A/K394A, FIG. 9a). These results are consistent with the notion that ENO1's RNA binding interferes with its enzymatic activity in cells.

[0174] The inventors complemented these experiments by nucleofection of target and control synthetic 35-mer RNAs into HeLa cells. The results unambiguously confirm specific riboregulation of lactate production by the ENO1 target RNAs tested (FIGS. 9b and c). Accordingly, the inventors showed that RNA ligands inhibit ENO1's enzymatic activity in vitro, and ENO1's enzymatic substrates specifically compete with its RNA binding. Increasing the concentration of RNA ligands in cultured cells inhibits glycolysis.

Example 3: Riboregulation of ENO1 Affects Mouse Embryonic Stem Cell Differentiation

[0175] To explore physiological functions of the ENO1-RNA interaction, the inventors chose mouse embryonic stem cells (mESCs). Like many cancer cells, mESCs utilizes glucose as a major energy source in the undifferentiated state. Removal of the leukaemia inhibitory factor (LIF) from the culture medium induces differentiation, accompanied by a decrease in glycolysis and increased respiration (FIGS. 10a and b). Interestingly, the decrease in glycolysis correlates with increased ENO1 RNA binding after LIF withdrawal (FIG. 10c).

[0176] To directly test the effect of RNA on ENO1 in mESCs, the inventors nucleofected control or target RNAs and measured lactate accumulation in the medium. Confirming the results from HeLa cells (FIGS. 9b and c), all three specific ENO1-binding RNAs significantly reduce lactate accumulation, in stark contrast to the control RNAs (FIG. 8d).

[0177] Unfortunately, the RNA nucleofection protocol is incompatible with meaningful mESC differentiation analyses. For this reason, the inventors used unperturbed mESCs, withdrew LIF for a period of seven days, and sorted cells that were positive for the expression of Brachyury (BFP-positive), which is primarily found in cells differentiating towards the primitive streak, or Eomes (mCherry-positive), which is predominantly expressed in the definitive endoderm. The inventors detected that lactate accumulation in the medium of Eomes+cells significantly exceeds that of Brachyury+cells (FIG. 11a), suggesting that the differentiation to the definitive endoderm may require sustained glycolysis in comparison to the primitive streak. Of note, ENO1 binding to RNA correlates inversely (compare FIGS. 11a and b).

[0178] To examine whether this correlation reflects a causal requirement for riboregulation of ENO1 during ESC differentiation, the inventors knocked out endogenous ENO1 and introduced murine versions of the ENO1 variants, characterized above, into the Rosa26 locus by CRISPR/Cas9 genome editing. The different heterologous forms of ENO1 are expressed at similar levels and below the expression level of endogenous ENO1 (FIG. 10d). As one would expect, lactate accumulation in the medium of ENO1wt cells is somewhat less than seen in control cells (FIG. 11d). As previously observed in HeLa cells, ENO1up displays increased RNA binding and mediates decreased lactate production; likewise, ENO1down shows a decrease in RNA binding compared to ENO1wt (FIGS. 11c and d).

[0179] Independent clones of these cell lines were subjected to LIF withdrawal and analysed for differentiation to the different germ layers. Engineered mESCs expressing ENO1wt differentiated normally into the distinct germ layers, as assessed by qPCR analysis of the respective expression of marker genes (FIG. 11e). By contrast, ENO1up-expressing cells fail profoundly in their differentiation to definitive endoderm and neuroectoderm (FIG. 11f), while the expression of primitive streak and mesodermal markers was quite variable and statistically not significantly affected. The inventors also noticed that ENO1down cells, where ENO1's activity escapes riboregulation, show increased differentiation towards the definitive endoderm (FIG. 11g).

[0180] To corroborate that the phenotypic changes of ENO1up-expressing cells is a consequence of diminished ENO1 activity, the inventors fused an auxin-inducible degron tag to the C-terminus of endogenous ENO1 of both alleles in mESCs carrying the OsTir1 receptor in the TIGRE locus. The inventors then triggered ENO1 degradation by the addition of auxin for 48 hours at the previously determined critical point of differentiation of four days, where the inventors detected an increase in ENO1's RNA association. When depleting ENO1 from differentiating mESCs at this point, the inventors observe specific, defective differentiation towards neuroectoderm and definitive endoderm, phenocopying cells expressing ENO1up (FIGS. 12A and B).

[0181] Taken together, the inventors' results disclose a physiological ENO1 riboregulation in the course of stem cell differentiation, such as mESC differentiation, and its requirement for the formation of specific germ layers, especially for the formation of the endodermal germ layer. Importantly, pluripotent stem cells expressing an ENO1 mutant that is hyper-inhibited by RNA are severely impaired in their glycolytic capacity and in endodermal differentiation, whereas cells with an RNA binding-deficient ENO1 mutant display disproportionately high endodermal marker expression. As such, these results represent a novel form of regulated cell differentiation.

Example 4: Analysis of ENO1 Ubiquitination

[0182] Interestingly, TRIM21 (E3 ligase) co-immunoprecipitates with ENO1 (FIG. 13a). Based on Mass Spectrometry data, the inventors discovered that K89 and K92 of ENO1 are ubiquitinated, which overlap with the amino acids mutated for ENO1up ENO1-K89A/K92A/K105A mutant). Also, immunoprecipitation (IP) of ubiquitin revealed enhanced ubiquitination of ENO1wt compared to ENO1up (FIG. 13b). Therefore, the inventors analyzed whether riboregulation of ENO1 is (at least partially) controlled by at least one posttranslational modification of ENO1, such as ubiquitination. According to this data, the transcriptome as a whole bearing thousands of relevant ENO1-binding regions would serve a very specific regulatory function, and influencing, e.g., ubiquitination can modulate RNA binding to ENO1, and, thereby, allow therapeutic intervention.

Example 5: Acetylation Augments ENO1's RNA Binding

[0183] The inventors questioned what may explain the difference between the enhanced RNA binding of the ENO1up mutant in cellulo and the normal RNA binding of the recombinant protein in vitro. Considering that the ENO1up mutant represents a change of three lysine residues to alanine, the inventors hypothesised that a post-translational lysine modification such as ubiquitination or acetylation in cellulo could activate ENO1's RNA binding. Interestingly, treatment of HeLa cells with sodium butyrate, an inhibitor of protein deacetylases, profoundly induced ENO1's acetylation and RNA binding (FIG. 14A).

[0184] SIRT2 had previously been implicated in the deacetylation of ENO1. Thus, the inventors knocked down SIRT2′s expression with siRNAs and assessed the consequences on the RNA-binding of wild-type ENO1 and the ENO1 mutants that the inventors had generated. RNA binding of ENO1wt and ENO1down increased when knocking down ENO1's putative deacetylase, while ENO1up remained unaffected, suggesting that the ENO1up mutation mimics acetylated ENO1 (FIGS. 14B and C). To test this possibility more directly, the inventors mutated the lysines that are changed to alanine in ENO1up (K89, K92 and K105) to the more conventionally used acetylation-mimic, glutamine (ENO1KtoQ). In support of the above results and interpretation, ENO1KtoQ shows the same enhancement of RNA binding as ENO1up compared to ENO1wt (FIGS. 14D and E). While these lysines might not be in direct contact with RNA, acetylation at these positions may cause a conformational change opening the protein for RNA binding.

Example 6: ENO1's RNA Association Increases During Differentiation

[0185] To temporally resolve the increase in RNA binding over the course of mESC differentiation, the inventors performed RIP-RT-qPCR experiments for the ligand and control mRNAs previously validated in HeLa cells. The inventors detected only minimal changes in ENO1's RNA association during the first three days, a modest increase in its binding to some of the ligands after five days (FIG. 15A, FTH1, PABPC1 and PPIA), and a pronounced, significant enrichment for four of the five ligands after seven days without LIF (FIG. 15A). This rise in RNA binding during mESC differentiation is accompanied and may be caused by an increase in ENO1's acetylation (FIG. 15C).

REFERENCES

[0186] The references are: [0187] [1] Richardson, C. C. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc. Natl. Acad. Sci. 54, 158-165 (1965). [0188] [2] Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat. Methods 13, 508-514 (2016). [0189] [3] Zhang, W., Xie, M., Shu, M.-D., Steitz, J. A., and DiMaio, D. (2016). A proximity-dependent assay for specific RNA-protein interactions in intact cells. RNA 22, 1785-1792.