AGENTS FOR THE TREATMENT OF PATIENTS WITH NSCLC AND METHODS TO PREDICT RESPONSE

Abstract

Agents for the treatment of patients having non-small cell lung cancer and methods of diagnostics related include an inhibitor of miR 24 3p, a locked nucleic acid, including methods of predicting response to platinum-based chemotherapy, for patients having, or suspected of having, non-small cell lung cancer.

Claims

1. An inhibitor of miR-24-3p, comprising a locked nucleic acid, for use in the treatment of a human patient with lung cancer.

2. An inhibitor of miR-24-3p for use according to claim 1, wherein said patient has non-small cell lung cancer (NSCLC), lung adenocarcinoma (LUAD) and stage III or IV NSCLC and/or LUAD.

3. An inhibitor of miR-24-3p for use according to claim 1, for use in combination with a platinum-based chemotherapeutic agent.

4. An inhibitor of miR-24-3p of claim 1, for use in improving the response to platinum-based chemotherapy in a human patient with lung cancer, a human patient with non-small cell lung cancer (NSCLC) and/or lung adenocarcinoma (LUAD) and stage III or IV NSCLC and/or LUAD.

5. A combination of compounds comprising an inhibitor of miR-24-3p of claim 1, and a platinum-based chemotherapy agent.

6. A combination of compounds according to claim 5, wherein said combination of compounds is suitable for simultaneous administration of the miR-24-3p inhibitor and of the chemotherapeutic agent.

7. A combination of compounds according to claim 5, wherein said combination of compounds is suitable for administration separated in time of the miR24-3p inhibitor and of the chemotherapeutic agent.

8. A combination of compounds according to claim 5, for use in the treatment of a human patient with lung cancer, a human patient with NSCLC and/or LUAD and stage III or IV NSCLC and/or LUAD.

9. An in vitro method of determining likelihood of response to platinum-based chemotherapy in a human patient with lung cancer, comprising the step of assessing the level of expression of at least one miRNA selected from the group consisting of miR-24-3p, miR-23a-3p and miR-27a-3p in a biological sample previously obtained from a patient.

10. A method according to claim 9, wherein the patient has non-small cell lung cancer (NSCLC), lung adenocarcinoma (LUAD).

11. A method according to claim 10, wherein the patient has stage III or IV NSCLC and/or LUAD.

12. A method according to claim 9, wherein said biological sample is a sample from a tumour of said patient or wherein said biological sample is a blood sample from said patient.

13. A method according to claim 9, wherein the expression level is assessed for one, two, or three miRNAs selected from the group consisting of miR-24-3p, miR-23a-3p and miR-27a-3p.

14. A method according to claim 9, wherein the expression level of said miRNA(s) is measured using at least one method selected from the group consisting of quantitative PCR (qPCR), real-time qPCR and expression microarrays.

15. A method according to claim 9, wherein the expression level of said miRNA(s) is compared to a preestablished reference level, said reference level being the average expression level or the median expression level measured in a group of patients having LUAD cancer, wherein the patients in said group are either unselected for response to platinum-based chemotherapy or are selected non-responder patients.

16. A method according to claim 9, wherein the expression level of said miRNA(s) in a biological sample from a tumour of said patient is compared with a reference level which is the expression level in a biological sample from non-tumour tissue of said patient.

17. A method according to claim 9, wherein the expression level of at least two miRNAs selected from the group consisting of miR-24-3p, miR-23a-3p and miR-27a-3p, and optionally wherein the average expression level of the miRNAs is used for comparisons.

18. A method according to claim 9, additionally comprising the step of concluding that the patient has an increased likelihood of resistance to platinum-based chemotherapy if the expression level measured in the biological sample of said patient is equal to or higher than said reference level.

19. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0057] FIG. 1. High Throughput screen for miRNAs whose increased expression induces cisplatin resistance in A549 cells.

[0058] A549 cells were transfected with the pre-miR library for two days and the transfected cells were then exposed to cisplatin at 60 μM for three days. Cell viability was measured in single well using Cell Titer Glow assay.

[0059] (A): Plot of normalized viability versus −log.sub.10 FDR-adjusted P-value for all miRNAs overexpressed in A549 cells. Vertical dashed lines denote the selected normalized viability cutoff, while the Horizontal line denotes the 0.01 selected FDR-adjusted P-value cutoff. Each dot represents a miRNA. Dots surrounded in the upper right quadrant represent miRNAs associated with cisplatin resistance at the chosen cutoffs. The standardized residual viability of the miR-24-3p miRNA was estimated at 4.9512.

[0060] (B) Venn diagram showing the overlap between miRNAs identified in the gain-of-function screen and those expressed in A549 cells. Expression was determined by small RNA Seq analysis on SOLiD 5500 WFTechnology). The threshold of expression was a minimum of 100 reads/millions of total mature miRNAs reads in at least one experimental condition.

[0061] FIG. 2. Effects of miR-24-3p overexpression in A549 cells on either cisplatin- or vinorelbin-induced cell death.

[0062] A549 cells were transfected with pre-miR-24-3p for 2 days and the transfected cells were then exposed to either cisplatin or vinorelbin.

[0063] (A): Annexin V/PI staining of A549 cells overexpressing miR-24-3p and exposed to 60 μM cisplatin for 24 h.

[0064] (B): Vinorelbin dose response analysis of A549 cells overexpressing miR-24-3p. An ATP-based assay was used to assess cell viability.

[0065] FIG. 3. Strategy to identify miR-24-3p targets involved in cisplatine resistance.

[0066] FIG. 4. BIM and PUMA are direct targets of miR-24-3p: Co-transfection of pre-miR-24-3p or pre-miR-Neg and either human BIM 3′UTR- or PUMA 3′UTR-derived psiCHECK-2 constructs in HEK293 cells.

[0067] (A): Sequences cloned in psiCHECK-2 in the XhoI and NotI restrictions sites

[0068] (B): Results of luciferase assays in HEK293 cells transfected with the indicated constructions and pre-miR-24-3p or control miRNA.

[0069] Cells were harvested two days after transfection and luciferase activity were analysed. All renilla luciferase activities were normalized with firefly luciferase activity. **p<0.01.

[0070] FIG. 5. DNA damage response induced by cisplatin of A549 cells overexpressing miR-24-3p.

[0071] A549 cells overexpressing miR-24-3p were exposed to cisplatin for 7 h and nuclear foci of (i) phosphorylated histone H2A.X at SER139 (y-HAX) and (ii) phosphorylated ATM at Ser1981 (p-ATM) were assessed by confocal microscopy.

[0072] (A): Representative immunofluorescence microphotographs of y-HAX,

[0073] (B): Representative immunofluorescence microphotographs of p-ATM.

[0074] (C): percentage of cells (n=3) exposed to cisplatin and containing more than five nuclear foci of y-HAX and phosphor-ATM, respectively after miR-24-3p overexpression.

[0075] FIG. 6. Western Blot showing the effects of miR-24-3p overexpression in A549 cells on (A) BIM and PUMA, (B) PNPO an PDXK, (C) the caspase 3 dependent apoptotic response induced by cisplatin.

[0076] FIG. 7. Effect of (A) BIM, (B) PUMA, (C) PDXK and (D) PNPO silencing on caspase 3 dependent apoptotic response induced by cisplatin.

[0077] FIG. 8. Effect of miR-24-3p inhibition on (A) BIM and PUMA, (B) PNPO an PDXK, (C) the caspase 3 dependent apoptotic response induced by cisplatin.

[0078] FIG. 9. Effect of TP53 mutational status on cisplatin resistance induced by miR-24-3p.

[0079] (A) Kill curve of H1299 cells (with a homozygous partial deletion of the TP53 gene) overexpressing miR-24-3p or a miRNA control and exposed to increasing dose of cisplatin.

[0080] (B) Validation of the effects of cisplatin in two LUAD cell lines, H1299 (with a homozygous partial deletion of the TP53 gene) and H1975 (homozygous for the c.818G<A mutation of the TP53 gene).

[0081] FIG. 10. In vivo experiments. miR-24-3p inhibition by LNA-modified oligonucleotides (SEQ ID Nos: 11 and 12). The results were obtained after RT-qPCR on lung total RNAs.

[0082] FIG. 11. Effects of miR-24-3p, miR-27a-3p and miR-23a-3p overexpression in A549 cells on cisplatin sensitivity.

[0083] FIG. 12. Networks of miR-24-3p, miR-27a-3p and miR-23a-3p predicted targets. Venn diagram summarizing the genes and predicted targets (in bold*) significantly down-regulated by miR-24-3p, miR-23a-3p and miR-27a-3p.

[0084] FIG. 13. Expression of miR-23a-27a-24-2 cluster and Kaplan-Meier survival curve from the TCGA data base.

[0085] Panels A, C and E: Box-plot diagram showing the expression levels of mir-24-2* (A), miR-23a (C) and miR-27a (E) measured by Small RNA-Seq in early-stages LUAD patients depending on their survival status 5 years following the diagnosis (n=293). For each plot a corresponding P value is shown. A: Alive; D: Dead.

[0086] Panels B, D, F and G: Kaplan-Meier curves for overall survival for patients with LUAD stratified by median value, according to expression of mir-24-2* (B), miR-23a (D), miR-27a (E) and the mean expression of the 3 mature miRNAs (F). The overall survival of patients with a normalized value lower than the median value is higher in all cases. For each plot the P value is indicated: (B) p-value=0.073; (D) p-value=0.0052; (F) p-value=0.02; (G) p-value=0.0365.

[0087] (*The name miR-24-2 refers to the gene, whereas the name miR-24-3p refers to the miRNA)

[0088] FIG. 14. Set up of a qPCR assay for the detection of circulating miR-23a-27a-24-2 cluster in biofluids.

[0089] (A). Summary of the miRNAs analyzed in plasmas from control patients.

[0090] (B). Correlation between the normalized expression of the selected miRNAs from the same sample in 2 independent assay plates.

[0091] (C). Correlation of miR-24 normalized expression values between the same series of 14 control plasmas analyzed in 2 independent experiments.

[0092] (D). Histogram of miR-24 normalized expression values in the series of control plasmas.

[0093] (E). Box plot showing the expression of miR-21-5p, miR-23a-3p, miR-24-3p and miR-27a-3p in the plasmas from control patients.

[0094] FIG. 15. Comparison of circulating miRNA levels in plasma from healthy donors and LUAD patients. Box plot showing the normalized expression of miR-21-5p (A), miR-23a-3p (B), miR-24-3p (C) and miR-27a-3p (D) in the plasmas from healthy donors (n=14) and LUAD patients (n=20).

EXAMPLES

Example 1. Identification of miRNA Associated with Cisplatin Resistance of Lung Adenocarcinoma Using Functional Genetic Screening

[0095] Functional genetic screen is an extremely powerful approach for cancer drug resistance gene discovery and validation (Iorns et al., 2007). Therefore the inventors used this approach to comprehensively identify miRNAs whose increased expression induce cisplatin resistance of lung adenocarcinoma cells using a library of miRNA mimics corresponding to miRbase version 16. The gain-of-function data indicates that overexpression of about 10 miRNAs reproducibly induces cisplatin resistance (2 independent screens using mimic library performed in duplicate) (FIG. 1A). Importantly, none of the miRNA identified in the screen and whose increased expression is the most associated with cisplatin resistance are currently described to affect cisplatin response of LUAD. In addition, among the 10 miRNA identified in the screen, 5 were also expressed in A549 cells (FIG. 1B). Therefore, the inventors chose to first focus on miR-24-3p, a miRNA expressed in A549 cells, and identified as the best miRNA candidate according to statistics and degree of resistance conferred by its overexpression. Importantly, miR-24-3p is also known to be upregulated in NSCLC (Zhao et al., 2015).

[0096] To ensure reliability of these findings, miR-24-3p effects on cisplatin sensitivity were independently validated in A549 cell lines (FIG. 2). These results showed that in A549 cells transfected with the pre-miR-24-3p (and so overexpressing miR-24-3p), the response to cisplatin was lesser than in cells which did not overexpress miR-24-3p. In addition, A549 cells overexpressing miR-24-3p are specifically (comparison with vinorelbin treatment) and strongly resistant to platinum based-compounds including the 2nd generation platinum analog: oxaliplatin (i.e., resistant to cisplatin, carboplatin and oxaliplatin; FIG. 2 and data not shown).

Example 2. Identification of Relevant miR-24-3p Target Genes

[0097] To identify relevant miR-24-3p target genes, the inventors used two distinct complementary approaches (FIG. 3):

[0098] 1. miR-24-3p target candidates were identified using a combination of bioinformatics and experimental approaches. For this, the inventors determined the whole gene expression profile of A549 cells overexpressing or not this miRNA and performed a bioinformatic search for genes (i) whose 3′UTR contain one (or more) miR-24-3p binding site(s) and (ii) whose annotation corresponds to GO terms associated with cell death, apoptosis, cell survival, cell cycle or senescence using tools developed by the inventors. This led the inventors to the identification and validation using luciferase constructs of BIM and PUMA, two pro-apoptotic BH3-only proteins, as miR-24-3p targets (FIG. 4).

[0099] 2. A comparative analysis of data obtained through a genomewide siRNA screen study (Galluzzi et al., 2012) to identify genes (i) whose decreased expression induces cisplatin resistance in A549 cells and (ii) whose 3′UTR contains one (or more) miR-24-3p binding sites. Among the 32 genes revealed by the genomewide siRNA screening approach, only one contains miR-24-3p binding sites in its 3′UTR: PDXK, a kinase that phosphorylates vitamin B6, a central regulator of cisplatin responses (Galluzzi et al., 2012). Interestingly, another gene involved in vitamin B6 metabolism, PNPO, was also found to contain a potential miR-24-3p site in its 3′UTR and was also found repressed in the miR-24-3p signature profiling described above.

Example 3. miR-24-3p is a Potent Regulator of Cisplatin-Induced Cell Death of LUAD

[0100] As cisplatin initiated cell death by inducing DNA lesions, the inventors explored whether overexpression of miR-24-3p in A549 cells exposed to cisplatin influenced the DNA damage response mediated by ATM before apoptosis occurred. The results of the inventors showed that A549 cells overexpressing miR-24-3p exhibited less DNA damage (assessed by phospho-yH2AX nuclear foci) as well as decreased ATM signaling 7 h following cisplatin exposure, without affecting cisplatin disposition in tumor cells (FIG. 5).

[0101] To gain further insights into the anti-apoptotic effects mediated by miR-24-3p, the inventors further studied the impact of miR-24-3p modulation on these 4 targets and the caspase 3 dependent apoptotic response induced by cisplatin (FIGS. 6-8). Altogether, these results suggest that miR-24-3p strongly inhibits caspase 3 dependent apoptotic response induced by cisplatin likely through targeting 4 genes: BIM, PUMA, PDXK and PNPO.

[0102] To assess the contribution of each miR-24-3p targets on the sensitivity of A549 cells to cisplatin, these cells were transfected with individual siRNA against either BIM, PUMA, PDXK, PNPO or a siRNA control. Expression of cleaved caspase 3 was used to evaluate the apoptotic response induced by cisplatin. Results shown on FIG. 7 demonstrated that siRNA-mediated depletion of each transcript decreased cisplatin-induced apoptosis, and thus, demonstrated that miR-24-3p promoted cisplatin resistance through direct targeting of BIM, PUMA, PDXK and PNPO.

[0103] A549 cells were transfected with miR-24-3p inhibitor or control for 24 hours and then exposed or not to cisplatin for 24 hours. The results showed that the inhibition of miR-24-3p increased the cisplatin induced apoptotic response in A549 cells, demonstrated by an increase expression of both cleaved caspase 3 and cleaved PARP, two characteristic markers of the apoptotic process (FIG. 8). These results demonstrated the possibility of using inhibitors of miR-24-3p to increase the sensitivity of resistant cells to cisplatin.

[0104] Interestingly, this data was independently validated in two other LUAD cell lines, H1299 (with a homozygous partial deletion of the TP53 gene), and H1975 (homozygous for the c.818G>A mutation of the TP53 gene) suggesting that miR-24-3p contributed to cisplatin resistance independently of p53 (FIG. 9).

Example 4. In Vivo EXPERIMENTS

[0105] In vivo experiments were performed using 9-12 weeks old male C57BL/6 mice and LNA-modified oligonucleotides designed against miR-24-3p. These inhibitors of miR-24-3p were purchased from Exiqon: LNA miR-24-3p inhibitor n° 1 whose sequence is TGCTGAACTGAGCC (SEQ ID NO: 11) and LNA miR-24-3p inhibitor n° 2 whose sequence is GCTGAACTGAGCC (SEQ ID NO: 12).

[0106] As shown in FIG. 10, both LNA anti-miR-24-3p oligonucleotides efficiency inhibited miR-24-3p when they were administrated on the local route (intratracheal administration) but also when they were used in a systemic route of administration (intraperitoneal administration). These results demonstrated the feasibility of the in vivo miR-24-3p inhibition and suggested that these inhibitors could be used to improve the cisplatin activity in vivo.

Example 5. Expression Levels of miRNAs of the miR-24-2 Cluster and Prognosis in LUAD Cancer Patients

[0107] The inventors collected 293 cases of LUAD patients with tumor small RNA-seq data from the TCGA database. The inventors analysed the expression of miR-24-3p in 2 subgroups of patients according to their survival status 5 years after the diagnosis and found that the overall expression differed significantly between alive and dead patients (FIG. 13, panel A). Interestingly, the inventors found similar data with the 2 other mature miRNAs expressed from the same cluster (FIG. 13, panels B and C). Kaplan-Meier survival analysis revealed that the group of patients with high expression of each of these 3 miRNAs had shorter survival compared to the low expression group (FIG. 13, panels D, E and F). These data were also confirmed using the mean expression value of the 3 miRNAs of the cluster (FIG. 13, panel G).

[0108] These data showed that a high expression level of miR-24-3p was associated with a poor prognosis in LUAD cancer patients. It might be thus clinically relevant to measure the expression levels of miR-24-3p (and of the two other members of the cluster) to stratify patients and to select patients at risk for a poor therapeutic response to cisplatin treatment.

[0109] Such an assay would comprise contacting a biological sample obtained from the subject to detect the levels of at least one (e.g., one, two, three) of miR-23a-3p, miR-24-3p and miR-27a-3p (or the mean level of these 3 miRNAs), wherein the level of expression of these miRNAs above a predefined reference level identified the subject predicted to be at risk of a poor therapeutic response.

[0110] The level of at least one miR gene product could be measured in cells of a biological sample obtained from the subject. For example, a tissue sample could be removed from a subject after lung surgery or by conventional biopsy techniques. In another example, a blood sample could be removed from the subject, and circulating RNA could be isolated by standard techniques. The blood or tissue sample could be obtained from the subject prior to initiation of any therapeutic treatment (radiotherapy or chemotherapy), preferably at time of diagnosis. A corresponding control lung tissue sample could be obtained from unaffected tissues of the subject (matched normal adjacent tissue in case of lung surgery). The control tissue or blood sample was then processed along with the sample from the subject, so that the levels of mature miRNA from the subject's sample could be compared to the corresponding levels from the control sample. An increase in the level of expression of miR-23a-3p/miR-24-3p/miR-27a-3p in the sample obtained from the subject, relative to the level of the corresponding miRNA gene product in a control sample, could be indicative of tumor aggressiveness and cisplatin resistance. The threshold used to identify LUAD patients at risk from the other could be obtained by performing expression analysis on a prospective cohort of LUAD patients with known initial response to chemotherapy (including platinum-based chemotherapeutic reagent) following the RECIST criteria. The selection of 100 patients samples (tumor tissue and matched normal adjacent tissue and plasma) is currently ongoing with the support of the Nice Hospital Tumor Biobank (Pr Paul Hofman) and expression levels of miR-23a-3p/miR-24-3p/miR-27a-3p is performed using methods described below in order to consolidate the data obtained on the TCGA cohort.

[0111] The level of a miRNA gene product in a sample could be measured using any technique that was suitable for detecting RNA expression levels in a biological sample. Suitable techniques for determining RNA expression levels in cells from a biological sample (e.g., qRT-PCR, in situ hybridization, small RNA-Seq, microarrays) are well known to those skilled in the art.

Example 6. The miR-23a-27a-24-2 Cluster Promotes Cisplatin Resistance of LUAD

[0112] As miR-24-3p was part of a miRNA cluster transcribed as a single primary miRNA and subsequently processed into three mature miRNAs: miR-23a-3p, miR-27a-3p and miR-24-3p, the inventors evaluated whether miR-23a-3p and/or miR-27a-3p also contributed to cisplatin resistance. The results of the inventors, performed in A549 LUAD cell line, showed that, albeit to a lesser extent than miR-24-3p, overexpression of miR-23a-3p was able to induce cisplatin resistance of A549 cells (FIG. 11). Interestingly the inventors found several common predicted targets between these miRNAs, several of them having been associated with apoptotic/necrotic cell death and DNA damage such as PPIF and NEK6 (FIG. 12).

Example 7. miR-24-3p as a Biomarker for the Prediction of Cisplatin Resistance

[0113] Analysis of data from TCGA indicated that high expression level of miR-24-3p and of the other members of the cluster was associated with a poor prognosis in LUAD cancer patients (FIG. 13). The inventors built a local prospective cohort (P. Hofman and C-H Marquette, CHU Nice) to specifically assess the predictive value of miR-24-3p expression in lung tissue or plasma from LUAD patients for the prediction of cisplatin resistance. For this goal, a multiplex qPCR protocol was set up for the detection of circulating miR-23a-27a-24-2 cluster in biofluids (FIG. 14). Data indicated that the inventors could reproducibly measure the levels of more than 60 miRNA candidates including the miR-23a-27a-24-2 cluster in plasmas from a cohort of 14 healthy donors. Preliminary data obtained on a first set of 20 serums from LUAD patients (at different stages of diagnosis) indicated that circulating levels of the miR-23a-27a-24-2 cluster were upregulated in LUAD compared to healthy donors (FIG. 15).

Methods

[0114] RNA isolation. Total RNAs were extracted from lung tissue and cell samples with TRIzol solution (Invitrogen). Integrity of RNA was assessed by using an Agilent BioAnalyser 2100 (Agilent Technologies) (RIN above 7).

[0115] Mature miRNA expression. MiR-24-3p expression was evaluated using TaqMan MicroRNA Assay (Applied Biosystems) as specified in their protocol. Real-time PCR was performed using Universal Master Mix (Applied Biosystems) and ABI 7900HT real-time PCR machine. Expression levels of mature microRNAs were evaluated using comparative CT method (2-deltaCT). A multiplex qPCR assay on miRNA-purified plasmas was performed on a Biomark machin (Fluidigm) using the miScript Microfluidics PCR Kit (Qiagen). Normalization was performed using an external spike-in control added before RNA extraction from 200 μl of plasma.

[0116] Expression microarrays. For gene expression arrays RNA samples were labeled with Cy3 dye using the low RNA input QuickAmp kit (Agilent) as recommended by the supplier. 825 ng of labeled cRNA probe were hybridized on 8×60K high density human Agilent microarrays. Two (biological replicates were performed for each comparison. Data was log 2 transformed and normalized using a cyclic loess algorithm in the R programming environment.

[0117] Transfection. Pre-miR-24-3p, and control miRNA (miR-Neg #1) were purchased from Life technologies. For miR-24-3p knockdown experiments, LNA anti-miR-24-3p and LNA negative control were ordered from Exiqon. siRNA directed against BIM, PUMA, PNPO and PDXK were purchased from Life technologies. A549 and H1299 cells were grown in 10% FCS in DMEM and transfected at 30 to 40% confluency in 6-12- or 96 well plates using Lipofectamin RNAi MAX™ (Life technologies) with pre-miRNA, siRNAs LNA inhibitors at a final concentration of 5 nM unless indicated.

[0118] Luciferase assay. Molecular constructs were made in psiCHECK-2 (Promega) by cloning behind the Renilla luciferase in the XhoI and NotI restrictions sites, annealed oligonucleotides derived from BIM, PUMA 3′ UTR. HEK293 cells were plated into 96-well and cotransfected using lipofectamin 2000 (Invitrogen) with 0.2 μg of psiCHECK-2 plasmid construct and pre-miR-24-3p or control miRNA at different concentrations. 48 hours after transfection, Firefly and Renilla Luciferase activities were measured using the Dual-Glo Luciferase assay (Promega).

[0119] Annexin V assay. Annexin-V-FITC apoptosis detection kit (Life technologies) was used to detect apoptotic activity. Cells were collected and resuspended in binding buffer, and incubated with annexin-V-FITC and propidium iodide in the dark for 15 minutes. Annexin-V-FITC binding was determined by flow cytometry (excitation wavelength of 488 nm; emission wavelength of 530 nm) using the FITC signal detector (FL1), and propidium iodide staining was detected by the phycoerythrin emission signal detector (FL2).

[0120] Functional genetic screen. Mimics were transfected in authenticated A549 lung adenocarcinoma cell line in 96 well plate format. 48 h following transfection, cells were exposed to cisplatin at 60 μM (2 times DL50; DL50 corresponding to the drug concentration lethal to 50% of the tumor cells) for three days. Viability was assessed using the cell titer glo assay (Promega). Normalization was carried out by dividing each sample value by the median of all samples on the plate (the majority of sample wells will thus serve as a reference). Hits were identified using the rank-product method. Normalization and statistics will be performed using the R software environment.

[0121] miRNA candidate selection. miRNA candidates were selected for further analysis based on statistical significance (p<0.01) and degree of resistance induced (assessed by viability after drug exposure, normalized viability above 3).

[0122] Functional genetic screen validation for miR-24-3p. Data were confirmed independently on a distinct lung adenocarcinoma cell line (H1299). Specificity to cisplatin resistance was assessed using docetaxel and vinorelbine.

[0123] MiRNA targets analysis. MiRonTop is an online java web tool (available athttp://www.microarray.fr:8080/miRonTop/index) that integrates DNA microarrays data to identify the potential implication of miRNAs on a specific biological system (Lebrigand et al., 2010). Briefly, MiRonTop ranks the transcripts into 2 categories (‘Upregulated’ and ‘Downregulated’), according to thresholds for expression level and for differential expression. It then calculates the number of predicted targets for each miRNA, according to the prediction software selected (Targetscan, MiRBase, PicTar, exact seed search: 2-7 or 1-8 first nucleotides of the miRNA, TarBase v1), in each set of genes. Enrichment in miRNA targets in each category is then tested using the hypergeometric function.

[0124] Protein extraction and immunoblotting. Cells were lysed in lysis buffer (M-PER protein extraction reagent) and protease inhibitors cocktail (Pierce). The lysates were quantified for protein concentrations using the Bradford assay (Biorad). Proteins (10 μg per sample) were separated by SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (GE Healthcare). The membranes were blocked with 5% fat free milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and subsequently incubated with primary antibodies overnight at 4° C. After washing with TBS-T for 30 minutes at room temperature, the membrane was further incubated with horseradish peroxidase-conjugated secondary antibodies for 1.5 hours, followed by 30 minutes of washing with TBS-T. Protein bands were visualized with Amersham ECL substrates (GE Healthcare).

[0125] Immunofluorescence analysis. A549 cells were grown on a Round Glass Coverslips Ø16 mm (thermo scientific) placed inside a 12 Multiwell Plate. Coverslips slides were washed in phosphate-buffered saline and fixed in 4% paraformaldehyde for 15 min, cells were then permeated using 0.1% Triton X-102 (Agilent Technologies) for 10 min. and blocked with PBS solution containing BSA (3%) for 30 min. Incubation with primary antibodies was performed in a blocking solution BSA (1%) at 37° C. for 1 h. After three washes with PBS, cells were incubated with secondary antibodies. Forty five min later, Coverslips slides were fixed on microscope slides using ProLong Gold Antifade Reagent with DAPI (Invitrogen). Fluorescence was viewed with an FV10i Olympus confocal scanning microscope.

[0126] Quantification of intracellular platinum. For total cell platinum accumulation experiments, cells were lyzed in RIPA lysis buffer after extensive washing with ice-cold PBS and allowed to settle in the incubator overnight. Intracellular quantification of platinum was determined by atomic absorption spectrometry (AA spectrometer). Platinum levels were normalized to protein levels.

[0127] Detection of cisplatin-GG DNA adducts. Immunofluorescence staining and measurement of specific DNA platination products was performed essentially as previously described (Liedert et al., Nucleic Acids Res, 2006, 34, e47) using a rat primary antibody that specifically recognizes CDDP-GG DNA adducts (RC-18).

[0128] In vivo experiments were performed using 9-12 weeks old male C57BL/6 mice purchased from Charles River. LNA-modified oligonucleotides designed against miR-24-3p were purchased from Exiqon: LNA miR-24-3p inhibitor no 1 whose sequence is TGCTGAACTGAGCC (SEQ ID NO: 11) and LNA miR-24-3p inhibitor no 2 whose sequence is GCTGAACTGAGCC (SEQ ID NO: 12). Said LNA miR-24-3p inhibitors were dissolved in PBS and then injected intratracheally using a MicroSprayer Aerosolizer (single dose of 5 mg/kg) or intraperitoneally using an insulin syringe (single dose of 10 mg/kg). Three days after miR-24-3p inhibitor injection, lungs were collected and total RNAs were extracted using the phenol-chloroform method.

[0129] RT qPCR: miRNA expression was assessed using TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assays (Thermo Fisher Scientific) as specified by the manufacturer. Real-time PCR was performed using Universal Master Mix II (Thermo Fisher Scientific) and ABI 7900HT real-time PCR machine. Expression levels of mature microRNAs were evaluated using comparative CT method. For normalization, transcript levels of SNO251 (mouse samples) were used as endogenous control for miRNA real time PCR.

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