m6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade

Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.


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Data analysis
For Statistical analysis: GraphPad Prism 5 ( Ver. 6.07 ), Prism 7 (Ver. 7.0d), and Microsoft excel 2010 For Flow Cytometry analysis : BD CellQuest (TM)-Ver. 5.2 and FlowJo software (version 10.5.3; FlowJo LLC) For Soft agar colony formation and 3D on-top culture in Matrigel: ImageJ ver 1.46r Sequencing analysis: the adapters were removed by using cutadapt for m6A-seq, reads were aligned to the reference genome (hg38) using Tophat v2.0.14 with parameter -g 1 --library-type=fr-firststrand. RefSeq Gene structure annotations were downloaded from UCSC Table Browser. For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data have been deposited in the GEO repository with the accession numbers GSE112902 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112902). Microarray data are accessible at the GEO repository, under accession number GSE128961 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE128961).

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Life sciences study design
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Sample size
Generally no statistical analysis was performed to predetermine the sample size. Sample size and number of animals are selected based on our previous experiences of carrying out similar experiments and published work. Sample size and number of independent experiments are clearly stated in the figure legend or in the Methods section. Three to more independent replicates were used to perform statistical analyses.
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Replication
Experiments were repeated two to three times independently. Replication were described in the figure legends.
Randomization For the xenograft model, animals were randomly assigned into groups receiving various cell line injections. Randomization (formal or otherwise) was not relevant for other data included in the manuscript.

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For immunofluorescence (IF) experiment, blind staining and blind analysis were carried out. For other experiments, the investigators were not blinded to group allocation during data collection and/or analysis.

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Validation
The commercial antibodies were validated based on the information of the manufacturers' instructions and additionally the antibodies were validated by the use of negative control and/or positive control (such as knockdown or overexpression) for FTO, PD-1, METTL3, METTL14 and GFP antibodies. The concentration recommended from antibody's date sheet was used for western blot, dot blot and immunostaining.

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All cell lines were tested to be mycoplasma negative. All lines were routinely tested for mycoplasma contamination.
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No wild animal was used in this research.

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All animal procedures used were approved by the University of Chicago institutional animal care and use committee Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology
Sample preparation For apoptosis assay, Mel624 melanoma cells were dissociated in 0.25% trypsin--EDTA (Gibco) at 37ºC for 5 min and the trypsin was inactivated with 10% FBS DMEM. Then the cells were was determined using the annexin V-FITC apoptosis detection kit (eBioscience, San Diego), according to the manufacturer's instructions. Cell samples were then analyzed by BD FACSCalibur flow cytometer (BD Biosciences).
For Tumor infiltrating Lymphocytes analysis, Tumor tissue from B16F10 tumor-bearing mice (Day 14 after tumor cell inoculation)

Software
For data collection and analysis, the software which is "BD CellQuest (TM)-Ver. 5.2" and "FlowJo software (version 10.5.3; FlowJo LLC)" Cell population abundance N/A

Gating strategy
Gating strategy was performed using positive and negative samples with single staining and combination. For Annexin V staining, positive Annexin V was defined for cells incubated with Annexin V-FITC. Negative Annexin V control was defined for cell non-incubated with Annexin V-FITC (non-stained). Same strategy is used for PI staining. Statistical analysis was performed on the double positive population.
Live cells (Zombie NIR negative) were gated using Zombie-violet (Catalog:423105) staining. FSC-A and FSC-A to exclude doublets. Lymphocytes were gated on SSC-A and FSC-A. CD4+ and CD8+ TILs gated on CD45+CD3+ cells. Gating strategies are shown in supplementary Figure S12A.
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