Adenosine Methylation Level of miR-125a-5p Promotes Anti-PD-1 Therapy Escape through the Regulation of IGSF11/VSIG3 Expression.

BACKGROUND: Despite encouraging anti-tumour activity in lung cancer, anti-PD-1 therapy has encountered increasing resistance to treatment. Several companion diagnostic assays have been performed to identify patients who may benefit from this immunotherapy and to adapt this therapy in case of acquired resistance. METHODS: A large panel of methods was used for the analysis of expression and methylation levels of miRNAs (qPCR, MemiRIP, …), protein/miRNA interactions (CLIP, oligo pull-down, …), and protein-protein interactions (CoIP) in cells and/or blood samples. RESULTS: Our work highlights that the saturation of PD-1 by anti-PD1 therapies induces an immune escape phenomenon due to the overexpression of IGSF11 following adenosine methylation of miR-125a-5p. Mechanistically, we identify METTL3/KHDRBS3 and HuR as two crucial players in the methylation and the loss of the repressive function of this miRNA. Finally, our work shows that the adenosine methylation of miR-125a-5p is analyzable from EVs/exosomes from longitudinal blood samples and that such EVs/exosomes modulate the IGSF11/VSIG3 expression in lung cancer cells to promote an immune escape phenomenon. CONCLUSIONS: Our data provide a biomarker (m6A-miR-125a-5p level) and two therapeutic solutions (anti-IGSF11 antibody and METTL3 inhibitor) that could potentially address the anti-PD1 therapy failure in the context of precision and personalized medicine.

Anti-PD1 therapy induces lymphocyte-derived exosomal miRNA-4315 release inhibiting Bim-mediated apoptosis of tumor cells.

Anti-PD1 immunotherapy, as a single agent or in combination with standard chemotherapies, has significantly improved the outcome of many patients with cancers. However, resistance to anti-PD1 antibodies often decreases the long-term therapeutic benefits. Despite this observation in clinical practice, the molecular mechanisms associated with resistance to anti-PD1 antibody therapy have not yet been elucidated. To identify the mechanisms of resistance associated with anti-PD1 antibody therapy, we developed cellular models including purified T cells and different cancer cell lines from glioblastoma, lung adenocarcinoma, breast cancer and ovarian carcinoma. A murine model of lung cancer was also used. Longitudinal blood samples of patients treated with anti-PD1 therapy were also used to perform a proof-of-concept study of our findings. We found that anti-PD1 exposure of T-cell promotes an enrichment of exosomal miRNA-4315. We also noted that exosomal miRNA-4315 induced a phenomenon of apopto-resistance to conventional chemotherapies in cancer cells receiving exosomal miRNA-4315. At molecular level, we discern that the apopto-resistance phenomenon was associated with the miRNA-4315-mediated downregulation of Bim, a proapoptotic protein. In cellular and mice models, we observed that the BH3 mimetic agent ABT263 circumvented this resistance. A longitudinal study using patient blood showed that miRNA-4315 and cytochrome c can be used to define the time period during which the addition of ABT263 therapy may effectively increase cancer cell death and bypass anti-PD1 resistance.This work provides a blood biomarker (exosomal miRNA-4315) for patient stratification developing a phenomenon of resistance to anti-PD1 antibody therapy and also identifies a therapeutic alternative (the use of a BH3 mimetic drug) to limit this resistance phenomenon.

N6-Adenosine Methylation of miRNA-200b-3p Influences Its Functionality and Is a Theranostic Tool.

MicroRNAs (miRNAs or miRs) play crucial roles in biological and pathological processes. Some miRNAs also appear as promising biomarkers and therapeutic tools. However, the epitranscriptomic regulation of miRNAs is not yet fully elucidated in all of their fields of application. We report that adenosine methylation of miR-200b-3p inhibits its repressive function toward its mRNA targets such as XIAP by blocking the formation of the miRNA/3' UTRmRNA duplex. Our data indicate that the adenosine methylation of miR-200b-3p is associated with the survival of glioblastoma patients. Collectively, our data support the idea that the adenosine methylation of miR-200b-3p can be used as a prodrug having a selective cytotoxicity against cancer cells (while being harmless to peripheral blood mononuclear cells [PBMCs], astrocytes, neurons, and hepatocytes).

Radiotherapy-induced overexpression of exosomal miRNA-378a-3p in cancer cells limits natural killer cells cytotoxicity.

Aim: We here hypothesized that tumor-derived exosomal miRNA (TexomiR) released from irradiated tumors may play a role in the tumor cells escape to natural killer (NK) cells. Materials & methods: Our study included the use of different cancer cell lines, blood biopsies of xenograph mice model and patients treated with radiotherapy. Results: The irradiation of cancer cells promotes the TET2-mediated demethylation of miR-378 promoter, miR-378a-3p overexpression and its loading in exosomes, inducing the decrease of granzyme-B (GZMB) secretion by NK cells. An inverse correlation between TexomiR-378a-3p and GZMB was observed in murine and human blood samples. Conclusion: Our work identifies TexomiR-378a-3p as a molecular signature associated with the loss of NK cells cytotoxicity via the decrease of GZMB expression upon radiotherapy.

Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme.

BACKGROUND: Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). METHODS: RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. RESULTS: Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. CONCLUSION: Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.

Identification of a transient state during the acquisition of temozolomide resistance in glioblastoma.

Drug resistance limits the therapeutic efficacy in cancers and leads to tumor recurrence through ill-defined mechanisms. Glioblastoma (GBM) are the deadliest brain tumors in adults. GBM, at diagnosis or after treatment, are resistant to temozolomide (TMZ), the standard chemotherapy. To better understand the acquisition of this resistance, we performed a longitudinal study, using a combination of mathematical models, RNA sequencing, single cell analyses, functional and drug assays in a human glioma cell line (U251). After an initial response characterized by cell death induction, cells entered a transient state defined by slow growth, a distinct morphology and a shift of metabolism. Specific genes expression associated to this population revealed chromatin remodeling. Indeed, the histone deacetylase inhibitor trichostatin (TSA), specifically eliminated this population and thus prevented the appearance of fast growing TMZ-resistant cells. In conclusion, we have identified in glioblastoma a population with tolerant-like features, which could constitute a therapeutic target.

Diuron exposure and Akt overexpression promote glioma formation through DNA hypomethylation.

BACKGROUND: Diuron is an environmental component listed as a likely human carcinogen. Several other studies report that diuron can be oncogenic for bladder, urothelial, skin, and mammary cells. No study mentions the putative effect of diuron on the glioma occurrence. OBJECTIVES: We here wanted to investigate the effects of diuron exposure on the glioma occurrence while wishing to incriminate a putative implication of DNA methylation modulation in this process. METHODS: In in vivo model of glioma, diuron exposure was firstly compared or combined with oncogenic overexpressions already known to promote gliomagenesis. ELISA quantifying the 5-methylcytosine level on DNA was performed to examine the global DNA methylation level. Quantitative real-time polymerase chain reaction and proximity ligation in situ assay were performed to identify the molecular causes of the diuron-induced changes of DNA methylation. The signatures diuron-induced changes of DNA methylation were analyzed in a cohort of 23 GBM patients. RESULTS: Diuron exposure is not sufficient to promote glioma, such as the oncogenic overexpression of Akt or Ras. However, the combination of diuron exposure and Akt overexpression promotes glioma. We observed that the diuron/Akt-induced glioma is characterized by three phenotypic signatures characterizing cancer cells: a global DNA hypomethylation, a loss of sensitivity to cell death induction, and a gain of signals of immune escape. Our data associated these phenotypes with three aberrant DNA methylation signatures: the LLT1, PD-L1, and Bcl-w hypomethylations. Strikingly, we observed that these three concomitant hypomethylations were only observed in GBM patients having a potential exposure to diuron via their professional activity. CONCLUSIONS: As single player, diuron is not an oncogenic of glioma, but it can participate to the glioma formation in association with other events (also devoid of oncogenic property as single player) such as Akt overexpression.

Glyphosate Primes Mammary Cells for Tumorigenesis by Reprogramming the Epigenome in a TET3-Dependent Manner.

The acknowledgment that pollutants might influence the epigenome raises serious concerns regarding their long-term impact on the development of chronic diseases. The herbicide glyphosate has been scrutinized for an impact on cancer incidence, but reports demonstrate the difficulty of linking estimates of exposure and response analysis. An approach to better apprehend a potential risk impact for cancer is to follow a synergistic approach, as cancer rarely occurs in response to one risk factor. The known influence of glyphosate on estrogen-regulated pathway makes it a logical target of investigation in breast cancer research. We have used nonneoplastic MCF10A cells in a repeated glyphosate exposure pattern over 21 days. Glyphosate triggered a significant reduction in DNA methylation, as shown by the level of 5-methylcytosine DNA; however, in contrast to strong demethylating agent and cancer promoter UP peptide, glyphosate-treated cells did not lead to tumor development. Whereas UP acts through a DNMT1/PCNA/UHRF1 pathway, glyphosate triggered increased activity of ten-eleven translocation (TET)3. Combining glyphosate with enhanced expression of microRNA (miR) 182-5p associated with breast cancer induced tumor development in 50% of mice. Culture of primary cells from resected tumors revealed a luminal B (ER+/PR-/HER2-) phenotype in response to glyphosate-miR182-5p exposure with sensitivity to tamoxifen and invasive and migratory potentials. Tumor development could be prevented either by specifically inhibiting miR 182-5p or by treating glyphosate-miR 182-5p-cells with dimethyloxallyl glycine, an inhibitor of TET pathway. Looking for potential epigenetic marks of TET-mediated gene regulation under glyphosate exposure, we identified MTRNR2L2 and DUX4 genes, the hypomethylation of which was sustained even after stopping glyphosate exposure for 6 weeks. Our findings reveal that low pressure but sustained DNA hypomethylation occurring via the TET pathway primes cells for oncogenic response in the presence of another potential risk factor. These results warrant further investigation of glyphosate-mediated breast cancer risk.

miR-370-3p Is a Therapeutic Tool in Anti-glioblastoma Therapy but Is Not an Intratumoral or Cell-free Circulating Biomarker.

In the last decade, microRNAs (miRs) have been described as biomarkers and therapeutic agents. Based on this finding, our aim here is to know if (1) miRNA-370-3p can be used as a biomarker associated with a favorable survival and if (2) miRNA-370-3p can be used as a therapeutic tool that increases the efficiency of standard anti-GBM treatment. A first approach using the data available on the "Prognostic miRNA Database" indicated that the expression level of miRNA-370-3p in GBM (T-miR-370-3p) is not associated with a prognosis value for survival. A second approach quantifying the expression level of cell-free circulating miRNA-370-3p (cfc-miR-370-3p) also indicated that cfc-miR-370-3p is not associated with a prognosis value for survival. To investigate whether miR-370-3p can be used in vivo to increase the anti-GBM effect of TMZ, we then used the model of LN18-induced GBMs in mice. Our data indicated that the miRNA-370-3p/TMZ treatment was two times more efficient than the TMZ treatment for decreasing the tumor volume. In addition, our study correlated the decrease of tumor volume induced by the miRNA-370-3p/TMZ treatment with the decrease in FOXM1 and MGMT (i.e., two targets of miR-370-3p). Our data thus support the idea that miR-370-3p could be used as therapeutic tool for anti-glioblastoma therapy, but not as a biomarker.

Specific Inhibition of DNMT3A/ISGF3γ Interaction Increases the Temozolomide Efficiency to Reduce Tumor Growth.

DNA methylation is a fundamental feature of genomes and is a candidate for pharmacological manipulation that might have important therapeutic advantage. Thus, DNA methyltransferases (DNMTs) appear to be ideal targets for drug intervention. By focusing on interactions existing between DNMT3A and DNMT3A-binding protein (D3A-BP), our work identifies the DNMT3A/ISGF3γ interaction such as a biomarker whose the presence level is associated with a poor survival prognosis and with a poor prognosis of response to the conventional chemotherapeutic treatment of glioblastoma multiforme (radiation plus temozolomide). Our data also demonstrates that the disruption of DNMT3A/ISGF3γ interactions increases the efficiency of chemotherapeutic treatment on established tumors in mice. Thus, our data opens a promising and innovative alternative to the development of specific DNMT inhibitors.

Histone H3 phosphorylation in GBM: a new rational to guide the use of kinase inhibitors in anti-GBM therapy.

Histones post-translational modifications (PTMs) are crucial components of diverse processes that modulate chromatin. Among the histones PTMs, the histones phosphorylation appears such crucial since it plays a significant role into DNA repair structure, transcription and chromatin compaction during cell division and apoptosis. However, little is known about the prognostic value of the histone phosphorylation in human cancer. This point could be considerate such as an important gap in anti-cancer therapy since the use of adequate kinase inhibitors could remedy to the aberrant histone phosphorylation associated with a poor prognosis factor. To remedy at this situation, we analyzed the phosphorylation level of histone H3 at the residues T3, T6, S10, S28, Y41 and T45 in a collection of 42 glioblastoma multiformes (GBM). Our data indicated that the high level of pH3T6, pH3S10 and pH3Y41 are signatures associated with a poor prognosis of overall survival (OS) of GBM treated with the "temozolomide and irradiation standard" treatment of GBM (named TMZ+Irad treatment). Our data also showed that these signatures are correlated with the high activity of kinases already described as writers of the pH3T6, pH3S10 and pH3Y41 i.e. the PKC, Aurora-B and JAK2, respectively. Finally, our analysis revealed that the use of Enzastaurin, AZD1152, and AZD1480 abrogated the high level of pH3T6, pH3S10 and pH3Y41 while increasing the sensitivity to the "temozolomide and irradiation"-induced cell death. To conclude, it appears that this work provides biomarkers for patient stratification for a therapy including kinase inhibitors.

Specific inhibition of DNMT1/CFP1 reduces cancer phenotypes and enhances chemotherapy effectiveness.

AIM: DNA methylation is a fundamental biologic process of genomes and is a candidate for pharmacological manipulation that might have important therapeutic advantages. Thus, DNA methyltransferases (DNMTs) appear to be ideal targets for drug intervention. MATERIALS & METHODS: To develop a new generation of DNMT inhibitor, we analyzed the ability of peptides to selectively inhibit certain DNMT1-incuding complexes. RESULTS: Our study demonstrates that the disruption of DNMT1/CFP1-including complexes increases the efficiency of chemotherapeutic treatment on established tumors in mice. CONCLUSION: Our data opens a promising and innovative alternative to the development of DNMT inhibitors.

Identification of TET1 Partners That Control Its DNA-Demethylating Function.

Several recent reports have identified TET1 as the main enzyme modulating DNA methylation and gene transcription via hydroxylation of 5-methylcytosine. However, little is known about the protein network that controls TET1 activity. By using a new proximity ligation in situ assay, we identified MeCP2, HDAC1/6/7, EZH2, mSin3A, PCNA, and LSD1 as TET1-interacting proteins. We also discerned that TET1/PCNA acts as a demethylator of the cyclical methylation/demethylation process, the perturbation of which promotes the aberrant methylation hallmarks frequently observed in cancer cells.

Specific inhibition of one DNMT1-including complex influences tumor initiation and progression.

BACKGROUND: Reactivation of silenced tumor suppressor genes by DNMT inhibitors has provided an alternative approach to cancer therapy. However, DNMT inhibitors have also been shown to induce or enhance tumorigenesis via DNA hypomethylation-induced oncogene activation and chromosomal instability. To develop more specific DNMT inhibitors for efficient cancer therapy, we compared the effects of peptides designed to specifically disrupt the interaction of DNMT1 with different proteins. FINDINGS: Our data indicated that the use of an unspecific DNMT inhibitor (5aza-2deoxycytidine), a DNMT1 inhibitor (procainamide) or peptides disrupting the DNMT1/PCNA, DNMT1/EZH2, DNMT1/HDAC1, DNMT1/DNMT3b and DNMT1/HP1 interactions promoted or enhanced in vivo tumorigenesis in a mouse glioma model. In contrast, a peptide disrupting the DNMT1/DMAP1 interaction, which per se did not affect tumor growth, sensitized cancer cells to chemotherapy/irradiation-induced cell death. Finally, our data indicated that the peptide disrupting the DNMT1/DMAP1 interaction increased the efficiency of temozolomide treatment. CONCLUSION: Our data suggest that the DNMT1/DMAP1 interaction could be an effective anti-cancer target and opens a new avenue for the development of new strategies to design DNMT inhibitors.

Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycle.

BACKGROUND: The clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during the cellular division is a crucial biological process controlled by the DNA methyltransferase Dnmt1, mainly. To investigate possible dynamic mechanisms of DNA methylation inheritance during the cell cycle, we used a Proximity Ligation In Situ Assay (P-LISA) to analyze the kinetic of formation and DNA recruitment of Dnmt1-including complexes. RESULTS: P-LISA, sequential chromatin immunoprecipitation and quantitative methylation specific PCR revealed that the Dnmt1/PCNA/UHRF1-including complexes are mainly formed and recruited on DNA during the S-phase of cell cycle, while the formation and the DNA recruitment of several Dnmt1/transcription factors-including complexes are not S-phase dependent but are G0/G1 and/or G2/M phases dependent. CONCLUSION: Our data confirm that DNA methylation inheritance occurs in S-phase, and demonstrate that DNA methylation inheritance can also occur in G0/G1 and G2/M phases of the cell cycle.

Synthesis and antioxidant activity evaluation of new hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones obtained by Biginelli reaction in two steps.

New hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones were prepared in two steps from ethyl 4-phenyl-6-methyl-2-oxo tetrahydropyrimidine-5-carboxylates or 4-phenyl-6-methyl-2-thioxotetrahydropyrimidine-5-carboxylates, previously prepared by Biginelli reaction using appropriate aldehyde, urea derivatives and ethyl acetoacetate. Their antioxidant properties were evaluated by two methods: scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and scavenging effect on hydroxyl radicals. The results show that the compounds containing thiourea moiety have better activity.

5-amino salicylic acid bound nanoparticles for the therapy of inflammatory bowel disease.

Nanoparticles (NP) are known for their specific accumulation in the inflamed tissues in the colon and may therefore allow a selective delivery to the site of inflammation including a reduction of adverse effects. 5-amino salicylic acid (5ASA) loaded NP were designed in order to investigate their therapeutic potential in the treatment of inflammatory bowel disease. 5ASA was covalently bound to poly(caprolactone) prior to all formulation steps. Oil/water emulsification or nanoprecipitation methods were used for the NP formulation. Particle diameters were either 200 or 350 nm for emulsification or nanoprecipitation, respectively. In-vitro drug release demonstrated a significant drug retention inside the NP formulation. Toxicity of the different formulations was evaluated on Caco-2 and HEK cell culture which was slightly increased for 5ASA grafted NP in comparison to blank NP (Me5ASA-NP: 75 microg/l; blank NP: 210 microg/l). In-vivo, clinical activity score and myeloperoxidase activity decreased after administration of all 5ASA containing formulations (untreated control: 28.0+/-5.6 U/mg; 5ASA-NP (0.5 mg/kg): 15.2+/-5.6 U/mg; 5ASA solution (30 mg/kg): 16.2+/-3.6 U/mg). NP formulations allowed to lower significantly the dose of 5ASA. These oral NP formulations demonstrated their therapeutic potential and appear to be an interesting approach for the therapy of inflammatory bowel disease.