METHOD FOR TREATING AND PROGNOSING CANCER LIKE GLIOBLASTOMA

Abstract

The present invention relates the treatment and prognostic of cancer like glioblastoma. Here, the inventors focused their study on the impact of presence of N6-adenosine methylation in miRNA-200b-3p in samples of patients suffering from glioblastoma multiforme (GBM). Their study was particularly focused on the impact of miRNA-200b-3p and its adenosine methylation on the expression of XIAP. XIAP acts as an anti-apoptotic protein via the inhibition of caspase-3 and -7 activation and high XIAP expression is associated with a poor survival in several solid tumors. Thus, the miR-200b-3p-mediated repression of XIAP mRNA expression appears as a mechanism governing the caspase-3 and -7 activity and the apoptosis. In theory, in the presence of miR-200b-3p, XIAP mRNA expression is repressed and caspase-3 and -7 can be activated to promote apoptosis. Thus, the present invention relates to an in vitro method for determining the prognosis of the survival time of a patient suffering from a cancer comprising the steps consisting of i) determining the expression level of the miR-200b-3p and/or the N6-adenosine methylated miRNA-200b-3p (miR-200b-3p m6A) in a sample from said patient and to the N6-adenosine methylated miRNA-200b-3p (miR-200b-3p m6A) for use in the treatment of a cancer in a subject in need thereof.

Claims

1. An in vitro method for determining the prognosis of the survival time of a patient suffering from a cancer and then treating the patient comprising i) determining the expression level of miR-200b-3p and/or N6-adenosine methylated miRNA-200b-3p (miR-200b-3p m6A) in a sample from said patient, ii) determining that said expression level of miR-200b-3p m6A is inferior to 10% of a miR-200b-3p m6A predetermined reference value and/or that the expression level of the miR-200b-3 is higher than an miR-200b-3 predetermined reference value and iii) treating the patient determined to have an expression level of miR-200b-3p m6A that is inferior to 10% of the miR-200b-3p m6A predetermined reference value and/or an expression level of miR-200b-3 that is higher than the miR-200b-3 predetermined reference value with a therapeutically effective amount of N6-adenosine methylated miRNA-200b-3p (miR-200b-3p m6A).

2. The in vitro method according to the claim 1 wherein the expression level of the miR-200b-3p and the expression level of N6-adenosine methylated miRNA-200b-3p (miR-200b-3p m6A) are both determined.

3. The in vitro method according to claim 1 wherein the cancer is a glioblastoma multiforme (GBM).

4. The in vitro method according to claim 1, wherein the sample according to the invention is blood, plasma, serum sample or a cancer biopsy.

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. A method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of N6-adenosine methylated miRNA-200b-3p (miR-200b-3p m6A).

10. (canceled)

11. The method of claim 9, wherein the N6-adenosine methylated miRNA-200b-3p (miR-200b-3p m6A) is a prodrug.

12. The method of claim 9, wherein the cancer is a glioblastoma multiforme (GBM).

13. The method of claim 9, wherein the miR-200b-3p m6A has the nucleic acid sequence SEQ ID NO:1 with a methylation on the second to last nucleic acid of the 3′ end.

Description

FIGURES

[0099] FIG. 1: The N6-adenosine methylation of miR-200b-3p limits its translational repressor function toward anti-apoptotic players and confers poor prognosis in GBM patients.

[0100] A. Samples were stratified according to the miR-200b-3pexp and miR-200b-3p % m6A parameters in order to distinguish the 3 indicated groups. Each box represents a sample/patient. For each group, the average of XIAP expression was analyzed with Human XIAP ELISA Kit (Abcam, France) was calculated and represented on the graph.

[0101] B. Kaplan-Meier representation of survival curves for GBM patients those tumors are characterized by a miR-200b-3pm6A>10% or a miRNA-200b-3pexp-low and by a miR-200b-3pm6A<10% and a miRNA-200b-3pexp-high.

[0102] FIG. 2: The N6-adenosine methylation of miR-200b-3p selectively induces apoptosis in cancer cells and has an anti-tumor growth effect.

[0103] A. miR-200b-3p promotes cell death by itself in cancerous and non-cancerous cells (excepted neuron RN33b), while miR-200b-3b induced apoptosis by itself in U87 cells, only. The LDH-Cytotoxicity Assay Kit (Abcam, France) is used to estimate the cell death 24h after the m6A-miR-200b-3b incubation.

[0104] B. Impact of the adenosine-methylated form of miR-200b-3p on the tumor growth in mice model.

[0105] FIG. 3: miR-200b-3p could also be used as a therapeutic tool in other cancer types.

[0106] In cell lines transfected with m6A-miR-200b-3p, cell death is induced in several cancer cell line types, when cell lines are able to demethylate this miR.

Example

[0107] Material & Methods

[0108] miRNA Extraction

[0109] miRNA extractions were performed using the NucleoSpin® miRNA kit (Macherey Nagel, France) according to the manufacturer's instructions.

[0110] miRNA and siRNA Transfection

[0111] Briefly, 6×105 cells were seeded in each well of 4-well plates. Transfection was performed using HiPerFect Transfection Reagents (Qiagen, France) and 10 ng miR (Qiagen, France) or 10 nm of Silencer® siRNA (Thermo Fisher, France), according to the manufacturer's recommendations. For siRNA controls, transfection control (HiPerfect Transfection Reagent only) and a negative control (Silencer® Negative control #1 siRNA) had been used. For miR controls, transfection control (HiPerfect Transfection Reagent only) and an oligo (miScript Inhibitor Negative Control; Qiagen, France) had been used.

[0112] Acellular METTL3 Methylation Assay

[0113] METTL3-including complexes were immunoprecipitated from cellular lysate obtained after sonication and the use of CHAPS buffer (40 mM HEPES, pH 7.4, 120 mM NaCl, 1% CHAPS, 1 mM EDTA, supplemented with protease and phosphatase inhibitors). Immunoprecipitations were performed using Catch and Release v2.0 Reversible Immunoprecipitation System (Merck, France) and anti-METTL3 (Abcam, France). IgG (Abcam, France) was used as control. Elutions from IP were performed using the non-denaturing Elution Buffer according to the manufacturer's instructions. Then 30 μL of elution were used in METTL3 enzymatic assay. METTL3 enzymatic assay was conducted in reaction buffer (20 mM Tris pH 7.5, 1 mM DTT, 0.01% Triton X-100, 40U/100 ml buffer RNaseOUT). The reaction mixture contained unmethylated mimic miR-200b-3p with biotin tag and SAM. Enzymatic assay reactions were incubated overnight at room temperature on shaker. After streptavidin isolation, the presence of N6-adenosine methylation was determined by dot blot. Dots were then incubated with anti-m6A and anti-adenosine (as loading control) antibodies overnight. For signal detection secondary HRP antibodies were used and signal was detected on ChemiDoc MP (Bio-Rad, France).

[0114] RNA-Immunoprecipitation for miRNA

[0115] For immunoprecipitation of RNA, two rounds using 5 μg of anti-m6A antibody (Abcam, France) and 5 μg of small RNA were performed. The reaction was carried out using Dynabeads Protein G Immunoprecipitation kit with some modifications (ThermoFisher Scientific, France) such as described by Berulava et al. (2015) [6]. As a control, immunoprecipitation was 15 performed using IgG (Abcam, France) instead of anti-m6A antibody. miRs obtained from m6A immunoprecipitation were reverse transcribed using miRScript II RT kit (Qiagen, France) and analyzed using the miScript miRNA PCR Array Human Cancer Pathway kit (Qiagen, France) according to the manufacturers' instructions. Fold enrichment was next calculated using Ct value obtained from RT-qPCR performed with input miR, IP-IgG and IP-m6A and the 2-ΔΔCt formula.

[0116] Cross-Linking Immunoprecipitation (CLIP)

[0117] CLIP were performed using RiboCluster Profiler RIP-Assay (CliniScience, France) from 10 millions per sample of UV crosslinked cells (150 mJ/cm2 of UVA (365 nm) according to the manufacturer's instructions. IP were performed in presence of 15 g of anti-GW182 (#RN033P, CliniScience, France) and anti-TNRC6B (#9913, Merck-Millipore, France) for overnight at 4° C.

[0118] Quantitative PCR of miRNA

[0119] For miRNA expression analysis and detection from product of RIP performed with anti-m6A antibody, RNA was reverse transcribed using miRScript II RT kit and analyzed by qPCR with the miScript SYBR Green PCR Kit using the specific hsa-miR miScript Primer Assays (Qiagen, France) according to the manufacturers' instructions.

[0120] ELISA

[0121] Proteins extracts were obtained by using RIPA Lysis and Extraction Buffer (Thermo Scientific, France) in accordance with the manufacturer's instructions. XIAP (Human) Cell-Based ELISA Kit (Abnova, Taiwan), Alpha Ketoglutarate (alpha KG) Assay Kit (ab83431) (Abcam, France) Human FTO ELISA Kit (68ELH-FTO) (Tebu-Bio, France) Methyltransferase like 3 (METTL3), ELISA Kit (MBS9326769) (My BioSource, USA), CST-PathScan® Total Ezh2 Sandwich ELISA Kit (Ozyme, France), EpiQuik Dnmt1 Assay Kit (EpiQuik Dnmt1 Assay Kit, Euromedex/EpiGentek, France), Human Bcl-2 ELISA Kit (Abcam, France), Caspase-2 ELISA Kit (Tebu-Bio, France) and PathScan® Total PD-L1 Sandwich ELISA Kit (Ozyme, France) were performed according to the manufacturer's instructions.

[0122] Tumor Xenografts in Nude Mice

[0123] Cells were harvested by trypsinization, washed and resuspended in saline buffer. Cell suspensions were injected s.c. into the flank of 7-8-week-old mice (Janvier, France) in 100 μl of sterile PBS. Tumor volume based on caliper measurements was calculated using the modified ellipsoidal formula (Tumor volume=½(length×width2)).

[0124] The experimental procedures with animals were in accordance with the guidelines of Institutional Animal Care and the French National Committee of Ethics. In addition, all experiments were conducted according to the Regulations for Animal Experimentation at the “Plateforme Animalerie” in the “Institut de Recherche en Sante de l'Université de Nantes (IRS-UN)” and approved by the French National Committee of Ethics.

[0125] Cell Lines

[0126] U87, U87IDH1mut, RN33b and A549 cells were obtained from the American Type Culture Collection (ATCC, Molsheim, France). HASTR040/astrocytes were obtained from Clonexpress (Gaithersburg, USA). OE21 cells were obtained from Sigma (France). HEP10 cells were obtained from ThermoFisher (France). MCF7 and T47D cells were provided by the Dr P. Juin's lab. SKOV3 cells were provided by the Dr E. Scottet's lab. OV90 cells were provided by the Dr R. Spisek's lab.

[0127] Results

[0128] The m6A Methyltransferase METTL3, the m6A Demethylase FTO and Alpha-Ketoglutarate Regulate the N6-Adenosine Methylation of miR-200b-3p

[0129] Literature reports that miR-200 and particularly miR-200b-3p play a role in GBM [17][18][19][20]. Berulava et al. (2015) have identified the presence of m6A in certain miRNAs such as miR-200b-3p [6]. In agreement with these findings, we have investigated the miR-200b-3p level expression (miR-200b-3pexp) and the percentage of miRNA-200b-3p containing m6A (miR-200b-3p % m6A) in a collection of 32 GBM samples. RT-qPCR experiments indicated a high level of heterogeneity in miR-200b-3pexp with a max/min ratio equal to 37.6 (data not shown). RNA immunoprecipitation performed with an anti-m6A antibody followed by qPCR analysis (miRIPm6A-qPCR) indicated that 10/32 tumors contained a miR-200b-3p % m6A>10% (data not shown). In addition, we observed a correlation between miR-200b-3p % m6A and miR-200b-3pexp (p=0.0022) (data not shown).

[0130] In order to identify the molecular mechanisms governing the N6-adenosine methylation of miR-200b-3p in GBM patients, we first focused our analyses on FTO and αKG, since FTO is an adenosine demethylase that requires alpha-ketoglutarate (αKG) to catalyze the adenosine demethylation [11]. In our collection of 32 GBMs, Pearson's correlation tests show an absence of significant correlation FTO expression level with miR-200b-3p % m6A (p=0.0824) (data not shown) and between αKG and miR-200b-3p % m6A (p=0.0668) (data not shown). To consider these two parameters, we isolated GBM samples harboring a low FTO expression level (lower than median) and a low αKG level (lower than median) (FTOLow/αKGLow) from the other GBM samples (data not shown). Based on this subdivision, we noted that GBM samples harboring FTOLow/αKGLow were more m6A-methylated than other GBM samples (p=0.0042) (data not shown). Thus, we conclude that both FTO and αKG affect the m6A-methylation level of miR-200b-3p: the N6-adenosine methylation level of miR-200b-3p is elevated when FTO and αKG levels are lower. The involvement of FTO and αKG in the N6-adenosine methylation of miR was also supported by the fact that siRNA directed against FTO increased miR-200b-3p % m6A (data not shown), αKG treatment decreased miR-200b-3p % m6A (data not shown), Meclofenamic Acid (MA, a selective FTO inhibitor [21]) increased the miR-200b-3p % m6A (data not shown). In addition, we noted that the knock-down of ALKBH5 (a RNA adenosine demethylase [10]) did not changed the miR-200b-3p % m6A (data not shown). Thus, all these results support the idea that FTO and αKG act in concert to decrease the adenosine methylation of miR-200b-3p.

[0131] Alarcón et al. (2015) having identified that methyltransferase-like 3 (METTL3) methylates pri-miRNA in mammalian cells [5], we hypothesized that METTL3 could be implicated in the adenosine methylation of miR-200b-3p. To support this hypothesis, we first observed a significant correlation between miR-200b-3p % m6A and the METTL3 expression level (p=0.0010) (data not shown). Secondly, acellular experiments indicated that the immunoprecipitate of METTL3 (i.e. METTL3-including complexes) methylates miRNA-200b-3p in vitro (data not shown). Thirdly, METTL3 knock-down (siRNA method) decreased the level of m6A in miR-200b-3p (data not shown). To conclude, these three distinct experiments implicate METTL3 as a writer of N6-adenosine methylation of miR-200b-3p.

[0132] All the above results suggest that αKG, FTO and METTL3 collectively influence the presence of m6A in miR-200b-3p. In order to take into consideration the influence of these three parameters on the level of adenosine methylation of miR-200c-3p, we have calculated what we called the αFMscore. For each GBM samples, +1 was affected when the expression of αKG, FTO and METTL3 is predicted to increase the N6-adenosine methylation i.e. when the αKG and FTO expressions are lower or equal to the median value of our cohort and when METTL3 expression is higher than the median value of our cohort. −1 was affected when the expression of αKG, FTO and METTL3 is predicted to decrease the N6-adenosine demethylation i.e. when the αKG and FTO expressions are higher than the median value of our cohort and when METTL3 expression is lower or equal to the median value of our cohort. For example, a GBM harboring a high level of αKG and FTO and a low level of METTL3 has a αFMscore equal to +1, while another GBM harboring a low level of αKG and FTO and a low level of METTL3 has a αFMscore equal to +3. Thus, we noted that the αFMscore and the percentage of presence of m6A in miR-200b-3p were significantly correlated in our collection of 32 GBM (p=0.0006) (data not shown).

[0133] Taken together, our data support the idea that METTL3, FTO and αKG are involved in the regulation of the N6-adenosine methylation of miR-200b-3p.

[0134] The N6-Adenosine Methylation of miR-200b-3p Limits its Translational Repressor Function Towards Anti-Apoptotic Players and Confers Poor Prognosis in GBM Patients

[0135] XIAPmRNA being identified as a target of miR-200b-3p (according to the miRTarBase website), we next investigated whether there is a link between miR-200b-3pexp, miR-200b-3p % m6A and the XIAP expression in our collection of 32 GBM samples.

[0136] Our study did not correlate miR-200b-3pexp and the XIAP expression when all GBM samples were considered (p=0.8803) (data not shown).

[0137] We then extended our study by dividing our samples in 3 groups by taking into consideration the adenosine methylation percentage of miR-200b-3p (FIG. 1A). Group #1 included samples with miR-200b-3p % m6A>10%. Group #2 included samples with a percentage miR-200b-3p % m6A<10% and miR-200b-3pexp inferior to the median (miR-200b-3pexp-low). Group #3 included samples with miR-200b-3p % m6A<10 and an expression level of miR-200b-3p superior to the median (miR-200b-3pexp-high).

[0138] For all samples having miR-200b-3p % m6A<10 (group #2 and #3), we noted that XIAP expression is inversely correlated with miR-200b-3pexp (FIG. 1A). This data is consistent with the dogma saying that miRNA is a post-transcriptional repressor.

[0139] Surprisingly, we noted that the average of XIAP expression of group #1's samples is higher than the ones of the two other groups (FIG. 1A). These results suggest that miR-200b-3p regulates XIAP expression when its sequence does not contain m6A (or a level inferior to 10%) and that the m6A presence in miR-200b-3p could abrogate the post-transcriptional repressor function of this miRNA.

[0140] To investigate this hypothesis, U251 cells were treated with an unspecific oligonucleotide (negative control), miR-200b-3pmimetic or m6A-modified miR-200b-3pmimetic. As expected, we did not observe any change in XIAP expression when cells were treated with unspecific oligonucleotide, while XIAP expression strongly decreased when cells were treated with miR-200b-3pmimetic (data not shown). Interestingly, we noted that this decrease is less efficient when cells were treated with the same quantity of m6A-modified miR-200b-3pmimetic (data not shown). Thus, it appears that the presence of m6A in miR-200b-3p abrogates the post-transcriptional repressor function of this miRNA toward XIAPmRNA.

[0141] We next performed Cross-Linking Immunoprecipitation and qPCR (CLIP-qPCR) analyses to determine whether the adenosine-methylation of miR-200b-3p influences the endogenous formation of 3′UTR-mRNA-XIAP/miR-200b-3p duplex. In our assays, immunoprecipitation is performed via an antibody directed against GW182 and TNRC6B (i.e. two proteins of the RISC complex having a central role in miRNA-mediated silencing), and qPCRs were performed to detect the enrichment/presence of miRNA and 3′UTRmRNA on the GW182- and TNRC6B-mediated co-immunoprecipitation products. CLIP-qPCRs were performed from samples with knock-down of METTL3 in order to estimate the impact of the loss of adenosine-methylation on the GW182- and TNRC6B-mediated co-immunoprecipitation of miRNAs and mRNAs. The miR-150-5p/3′UTR-mRNA-EP300 duplex was considered as a control. The choice of this control was dictated by the fact that miR-150-5p is not adenosine-methylated and the fact that miR-150-5p targets 3′UTR-mRNA-EP300.

[0142] We first noted that miR-150-5p and 3′UTR-mRNA-EP300 were present in GW182- and TNRC6B-mediated co-immunoprecipitation products, and this independently of the METTL3 knock-down (data not shown). Secondly, we noted that the METTL3 knock-down increased the presence of miR-200b-3p and 3′UTR-XIAP in the GW182- and TNRC6B-immunoprecipitates (data not shown). Thus, these last results indicate that the METTL3-mediated adenosine-methylation status of miR-200b-3p influences the endogenous formation of 3′UTR-mRNA-XIAP/miR-200b-3p duplex.

[0143] By affecting the expression of XIAP, an apoptotic player, our data suggest that the expression level and the N6-adenosine methylation level of miR-200b-3p could affect the intrinsic apoptosis level of tumors. To investigate this hypothesis, we analyzed the Caspase/DEVDase activity as a marker of the intrinsic apoptosis level of tumors. Our work indicates that tumors harboring the miRNA-200b-3pexp-low signature or the miR-200b-3p % m6A>10% signature have a lower intrinsic apoptosis level (data not shown).

[0144] Finally, we observed that patients whose tumors harbored the miRNA-200b-3pexp-low signature or the miR-200b-3p % m6A>10% signature have a lower survival outcome than the other GBM patients (FIG. 1B).

[0145] m6A-miR-200b-3p Appears as a Promising Tool in Anti-GBM Therapy

[0146] Based on the fact that the miR-200b-3p affects the intrinsic apoptosis level, we extended our study by investigating whether miR-200b-3p and m6A-miR-200b-3p could be used as a therapeutic tool. For this purpose, the miR-200b-3p- and m6A-miR-200b-3p-induced cell death was measured from a panel of cells representing human brain cells (astrocytes (HAST40), neurons (RN33b) and astrocytoma (U87). We included in this panel U87IDH1mut cells since IDH1 mutation is observed in GBM. Besides, we observed that the presence of IDH1 mutation decreased αKG and increased the adenosine methylation of miR-200b-3p in a context of the FTO and METTL3 expression level being unchanged (data not shown). Meclofemalic acid was also used as a FTO inhibitor [21]. Because peripheral blood is the place where exposure to chemicals occurs, PBMC (peripheral blood mononuclear cells) were also included in our study. Firstly, our data indicated that miRNA-200b-3p induced cell death in all cells with the exception of neuron (RN33b cell line) (FIG. 2A). Secondly, we observed that m6A-miR-200b-3p induced cell death in U87 cells, but not in U87IDH1mut, U87Meclofemalic, PBMC, neurons and astrocytes (FIG. 2A). In other terms, these data suggest that the ability of m6A-miR-200b-3p to induce cell death occurs in cancer cells and not in non-cancerous cells like PMBC, neurons and astrocytes. Based on our knowledge, the absence of massive m6A-miR-200b-3p-induced cell death in U87IDH1mut could be associated to the fact that these cells have a lower quantity of αKG, i.e. a lower quantity of the enzyme co-factor (FTO) catalyzing the adenosine demethylation of miR-200b-3p. Besides, the fact that the meclofemalic acid treatment abrogated the m6A-miR-200b-3p-induced cell death in U87 cells confirmed the involvement of FTO in this process (FIG. 2A).

[0147] We have then investigated the putative anti-GBM effect of m6A-miR-200b-3p in an in vivo model of GBM. For this purpose, U87-induced GBMs were generated by xenograft in mice. When the volume of the U87-induced GBMs was close to 100 mm3, three mice were randomly untreated, treated with temozolomide (TMZ) and/or with m6A-miR-200b-3p (data not shown). The option to use TMZ is due to the fact that this alkylating agent is the chemotherapeutic agent included in the current standard care protocol in GBM treatment [22].

[0148] By comparing the effect of the TMZ treatment with the effect of the m6A-miR-200b-3p treatment, we could clearly see that the m6A-miR-200b-3p treatment has similar efficiency than the TMZ-25 mg/kg treatment (FIG. 2B). We also noted that the m6A-miR-200b-3p+TMZ-25 mg/kg treatment has the same efficiency than the TMZ-50 mg/kg treatment (FIG. 2B).

[0149] miR-200b-3p could Also be Used as a Therapeutic Tool in Other Cancer Types

[0150] The above data are focused on the XIAP regulation by miR-200b-3p, but it is well known that one miRNA has multiple targets. Consequently, we next investigated whether the adenosine methylation of miR-200b-3p could abrogate its translational repressor function towards other putative protein targets than XIAP. Among the putative protein targets of miR-200b-3p (according to the miRTarBase website [23]), we focused our study on two other apoptotic players (Bcl-2 (B-cell lymphoma 2, Uniprot #P10415) and Caspase-2 (cysteine-dependent aspartate-directed proteases 2, Uniprot #P42575)), two epigenetic players (EZH2 (Enhancer of zeste homolog 2, Uniprot #Q15910) and DNMT1 (DNA (cytosine-5)-methyltransferase 1, Uniprot #P26358)) and a negative immune checkpoint PD-L1 (Programmed cell death 1 ligand 1, Uniprot #Q2NZQ7). Our data indicated that the presence of m6A in miRNA-200b-3p also abrogated the translational repressor function of miR-200b-3p toward Bcl-2 and PD-L1 (data not shown).

[0151] Finally, we investigated whether the ability of m6A-miR-200b-3p to induce cell death was specific of U87 cells. For this purpose, cancerous cell lines representative of several cancers were transfected with m6A-miR-200b-3p (U251 and T98G for glioblastoma, A549 and H1975 for lung, MCF7 and T47D for breast, OE21 for esophagus, OV90 and SKOV3 for ovaries). Four non-cancerous cell lines were also included in our study. Four hours after cells transfection, we noted that all cells were transfected with similar quantity of m6A-miR-200b-3p since the range of increase of miR-200b-3p expression was homogeneous (10-13 fold induction) (FIG. 3). Then, we noted that cell death occurred in cells having the ability to adenosine-demethylate miR-200b-3b i.e. in U251, A549, T47D and SKOV3 (FIG. 3). The absence of cell death in other cell lines and particularly in non-cancerous cell lines was explained by the inability of these cells to adenosine-methylate miR-200b-3b (m6A enrichment transfected/control being equal to 1) (FIG. 3).

[0152] Taken together, all these last results are consistent with the fact that m6A-miR-200b-3p appears as a promising tool in anti-GBM therapy.

[0153] Conclusion:

[0154] Recent investigations concerning the description of the molecular mechanisms of bases modification of miRNAs have provided meaningful progresses in the understanding of regulation of the miRNAs biogenesis and functionality. Thus, after the studies of Alarcón et al. (2015), Berulava et al. (2015) and Konno et al. (2019), our study reports the presence of m6A in miRNAs via the realization of RNA immunoprecipitation with an anti-m6A-antibody followed by RT-qPCR [5] [6] [7]. Despite these posterior studies, our investigation harbors several innovative points.

[0155] First, the work of the inventors indicates that the adenosine methylation of miR-200b-3p abrogates its translational repressor function towards its putative targets such as XIAP, Bcl-2 and PD-L1. The works published by Alarcón et al. (2015) and Berulava et al. (2015) report the existence of 2 different consensus sequences for the m6A methylation in pri-miRNAs (UGAC) and in mature miRNAs (ADRA) [5][6]. Interestingly, the inventors noted that the miRNA-200b-3p sequence contains a sequence matching one of the consensus. They also noted that the miR-200b-3p sequence contains a sequence matching the consensus sequence binding by METTL3/WTAP defined by Ping et al. (2014) [12]. From a certain perspective, this last point can also constitute an argument supporting the role of METTL3 in the adenosine methylation of miRNAs.

[0156] The work of Berulava et al. (2015) indicate that FTO plays a crucial role in the demethylation of miRNAs [6]. The data of the inventors complete this by indicating that the presence of αKG also acts as a non-negligible player in the demethylation of miRNAs.

[0157] In addition to these 2 initial reports, this study shows that the presence of m6A acts as an inhibitor of the post-transcriptional repressor function of miRNAs. Mechanistically, these data indicate that the presence of m6A limits the formation of miRNA/mRNA duplex. This study is also distinguished from the first two studies by its clinical translational study effort using a cohort of cancer patients. Indeed, this study is the first to mention that the level of N6-adenosine methylation of a miRNA (in association with the expression level of this miRNA) acts as a biomarker characterizing GBM patients with a poor survival. This study is also distinct to the one recently published by Konno et al. (2019) since Konno and colleagues considerate the adenosine methylation of miR as a tool to distinguish early pancreatic cancer patients from healthy controls with an extremely high sensitivity and specificity; while in our article the adenosine methylation of miR-200b-3p is associated with a prognosis value of response for GBM patients [7] and could have a therapeutic function.

[0158] The work of Berulava et al. (2015) and the one of Yuan et al. (2014) introduce a debate about the impact of the adenosine methylation of miRNA on their stability [6] [24]. These data focusing on miR-200b-3p seems to indicate that the adenosine methylation of this miRNA does not impact on its expression. Indeed, the modulation of its adenosine methylation level via siRNA directed against FTO and METTL3 or via chemical components does not affect its expression. However, this finding being obtained on one miRNA, it is not possible to generalize a rule about the impact of the adenosine methylation on the miRNA stability.

[0159] By observing that the adenosine methylated miR-200b-3p was not recruited to the RISC complex, these data reinforces the idea that the adenosine methylation of miRNA appears as molecular mechanism governing the miRNA functionality via the regulation of the duplex formation between miRNA and mRNA. More generally, these data support the idea that nucleotide modification occurring in miRNA or in 3′UTR-mRNA alters the formation of miR/3′UTR-mRNA duplex, such as reported by Lockhart et al. (2019) [25].

[0160] By reporting that m6A methylation of miRNAs could act as a biomarker characterizing GBM patients with a poor survival, our data open the idea that the molecular actor writing this epitranscriptomic signature (METTL3 according to our data) could be used as a target for the development of epidrugs. Indeed, this point of view is already discussed since METTL3 promotes oncogene translation [26].

[0161] During the last decade, miRNA mimics and molecules targeting miRNAs (anti-miRs) have shown promising results in preclinical development [27][28]. Four arguments strongly support the idea that the adenosine-methylated form of miR-200b-3p could be used as a promising therapeutic tool. First, m6A-miR-200b-3p is apoptogenic by itself via the repression of XIAP, an anti-apoptotic protein. Secondly, these data indicate that m6A-miR-200b-3p promotes cell death in cancerous cells such as U87 (but also in other cancer cell lines) and not in non-cancerous cells such as neurons, PBMC, astrocytes and hepatocytes. Thirdly, the in vivo data of the inventors indicate that m6A-miR-200b-3p has an anti-tumor growth effect in an in vivo model of GBM. Fourthly, these in vivo data also indicate that the m6A-miR-200b-3p/TMZ combination permits to limit the dose of TMZ since the m6A-miR-200b-3p/TMZ-25 mg/kg combination has the same anti-tumor growth effect than the use of the TMZ-50 mg/kg treatment. Thus, all these arguments define the adenosine-methylated form of miR-200b-3p as the prodrug form of this miRNA. More interestingly, these data indicate that its conversion under an active form occurs in cancer cells but not in non-cancerous cells. This observation is highly promising since it can be translated such as the fact that only cancerous cells have the “tools” (FTO and αKG) to activate the prodrug form of miR-200b-3p. Thus, the adenosine-methylated form of miRNAs could be considered such as a manner to limit the off-targets effect of miRNA therapy associated with the relative lack of addressing of miRNA-based therapy against the cancer cells [29]. These data also introduce the idea that the presence of IDH1 mutations could be considered such as a biomarker excluding the use of adenosine-methylated form of miRNAs since cells presenting IDH1 mutations have a low level of αKG. Concretely, the first reading of this idea might exclude the use of m6A-miR-200b-3p treatment in less than 10% of primary GBM and in 6-10% of de novo AML, as example [30] [31]. However, this point is available when the m6A-miR-200b-3p treatment is envisioned as single treatment since its combination with BAY1436032 (a pan-mutant IDH1 inhibitor [32]) restored its ability to promote cell death (data not shown).

[0162] In conclusion, the results of the inventors opens a new area in the understanding of epigenetic modifications concerning miRNA and in the development of innovative epidrugs. Indeed, since several years chemical modifications of RNAs (i.e. epitranscriptomic) are defined such as central players in the control of messenger and ncRNA activity [33]. Our data reinforce this idea by showing that the adenosine methylation of miRNAs abrogates their post-transcriptional repressive function. By initiating the idea that adenosine-methylated miRNA could be used as a prodrug, our work provides the base for the development of a new pathway of anti-cancer therapeutic strategies targeting miRNA. Thus, in the future years, the understanding of the mechanisms involved in the epigenetic regulation of miRNA could improve patient stratification and the development of successful miRNA-based therapeutic strategies.

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