Anti-tumor immunity controlled through mRNA m 6 A and YTHDF1 in dendritic cells
Abstract
Emerging evidence revealed important roles of tumor neoantigens in generating spontaneous antitumor immune responses and predicting clinical responses to immunotherapies 1 , 2 . Despite the presence of numerous neoantigens, complete tumor elimination rarely occurs in many patients, due to failures in mounting a sufficient and lasting antitumor immune response 3 , 4 . Here, we show that durable neoantigen-specific immunity is regulated by messenger RNA (mRNA) N 6 -methyadenosine (m 6 A) methylation through the m 6 A-binding protein YTHDF1 5 . In contrast to wild-type mice, Ythdf1 -deficient ( Ythdf1 −/− ) mice exhibit an elevated antigen-specific CD8 + T cell antitumor response. Loss of YTHDF1 in classical dendritic cells (cDCs) enhanced the cross-presentation of tumor antigen and the cross-priming of CD8 + T cells in vivo . Mechanistically, transcripts encoding lysosomal proteases are marked by m 6 A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts elevates translation of lysosomal cathepsins in DCs, with the inhibition of cathepsins markedly enhancing cross-presentation of the wild-type DCs. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1 −/− mice, implicating YTHDF1 as a new potential therapeutic target in anticancer immunotherapy.
METHODS
Mice
Ythdf1 −/− mice were generated as previously described 15 . Founder mice with mutant alleles were backcrossed to C57BL/6J for two generations. Mice used for experiments are further backcrossed to C57BL/6J for seven generations, totally 9 generations. To ensure the comparability in genetic background, mice were maintained by crossing heterozygous and heterozygous. Ythdf1 −/− mice or their littermates control WT mice were used in all experiments. Littermates were co-housing during experiments to reduce variants in their microbiome and environment. Primers used for genotyping of Ythdf1 −/− mice: CACCTGAGTTCAGATCATTAC and GCTCCAGACTGTTCATCC. Female Rag2 −/− mice, 2C CD8 + T cell receptor (TCR)-Tg, CD11c-Cre and Zbtb46-DTR mice were purchased from Jackson laboratory. Female CD11c-Cre Mettl14 f/f conditional knockout mice were generated in house. All mice were used at 6–12 weeks of age. All the mice were maintained under specific pathogen-free conditions and used in accordance with the animal experimental guidelines set by the Institute of Animal Care and Use Committee. This study has been approved by the Institutional Animal Care and Use Committee of The University of Chicago.
Cell lines
MC38 is a murine colon adenocarcinoma cell line that was provided by D. Bartlett (University of Pittsburgh, Pittsburgh). B16-OVA, an OVA-transfected clone derived from the murine melanoma cell line B16, were provided by Dr. Yang-Xin Fu (UT Southwestern). B16F10 cell line was purchased from ATCC. MC38-zsGreen-OTIp (MC38-OZ) and B16F10-zsGreen-OTIp (B16-OZ) were selected for a single clone after being transduced by lentivirus expressing zsGreen-OTIp (SIINFEKEL). MC38-SIY is an EGFR-SIY-transfected clone derived from the murine colon cell line MC38. Cells were maintained either in DMEM (Invitrogen) supplemented with 10% FBS and 1% penicillin-streptomycin supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 0.1 mM non-essential amino acid at 37 °C in 5% CO 2 .
Primary cell cultures
Single-cell suspensions of bone marrow cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, supplemented with 20 ng/ml GM-CSF (Biolegend). Fresh media with GM-CSF was added into culture on days 3 and 5. On day 6, CD11c + DCs were purified using EasySep Mouse CD11c Positive Selection Kit II (STEMCELL). To culture Flt3L-DCs, single-cell suspensions of bone marrow cells were cultured in IMDM medium containing 10% fetal bovine serum at the concentration of 1×10 6 /ml. Cells were supplemented with 100 ng/ml Flt3-Ligand (PEPROTECH) for 9–10 days to obtain the Flt3L-DCs.
Tumor growth and treatments
1 × 10 6 B16-OVA or MC38 tumor cells were injected s.c. into the flank of mice. Tumor volumes were measured by length (a) and width (b) and calculated as tumor volume = ab 2 /2. Mice with tumor volumes less than 200 mm 3 are considered to be survival. For in vivo depletion study, 200 μg of anti-CD8 antibody (clone YTS169.4) or anti-NK1.1 (clone PK136) was injected i.p. three days after tumor inoculation. To block cathepsins in vivo , mice were inoculated with 1× 10 6 B16-OVA cells. On day 11, mice with established tumors were treated with E64 intratumorally. For anti-PDL1 treatment, 1× 10 6 B16-OVA tumor cells were s.c. injected into the flank of mice. Tumors were allowed to grow for seven days and treated i.p. by anti-PDL1 (clone10F.9G2) or Rat Ig. Tumor-free mice after treatment were monitored over time and the percentage of tumor-regression was calculated. To block IFNγ, tumor-bearing mice were treated with 50 μg anti-IFNγ m 6 Ab (clone XMG1.2) intratumorally and PD-L1 expression on tumor cells was evaluated by flow cytometry. All antibodies were ‘InVivoMAb’ from BioXCell. For adoptive transfer of T cells, Rag mice were inoculated with 5×10 5 B16-OVA on day 0. On the same day, T cells were purified from WT or Ythdf1 −/− mice using T cell negative isolation Kit (STEM CELL). 5×10 6 T cells were i.v. injected into Rag2 −/− mice. Tumor-bearing mice were sacrificed before the diameter of tumor reached to 2 cm and tumor size limit has been approved by the Institutional Animal Care and Use Committee of The University of Chicago.
Generation of bone marrow chimera
To generate bone marrow chimeric mice, C57BL/6 mice were exposed to 800 rads of X-ray. After 24 hours, 5×10 6 bone marrow cells, consisting of 2.5×10 6 WT or Ythdf1 −/− BM cells and 2.5×10 6 Zbtb46-DTR BM cells, were i.v. injected into irradiated mice. Six weeks after reconstitution, Zbtb46-DTR : Ythdf1 +/+ and Zbtb46-DTR : Ythdf1 −/− mixed BM chimera mice were inoculated with 10 6 B16-OVA cells and treated with 400 ng diphtheria toxin (sigma) or PBS every other day for sixteen days.
Flow cytometry and cell sorting
For flow cytometric analysis and cell sorting, tumors, lymph nodes and spleens were collected from mice and digested with 0.26 U/ml Liberase TM and 0.25 mg/ml DNase I at 37°C for 30 min. Samples were then filtered through a 70 μm cell strainer and washed twice with staining buffer. Cells were re-suspended in staining buffer (PBS with 2% FCS and 0.5 M EDTA). Cells were incubated with Fc Block (clone 2.4G2; BioX Cell) for 10 min. Subsequently, specific antibodies were added and staining was continued for 30 min on ice. Information for all the antibodies used are provided in Supplementary Table 1 . OT-I specific T cells were stained using iTAg Tetramer/H-2K b OVA (SIINFEKEL) (MBL). After a washing step, cells were either analyzed on a BD Fortessa (BD) or sorted by AriaIIIu (BD). For the staining of cathepsins, splenocytes were stained with CD11c, B220, MHCII, CD8 and CD11b and then fixed with 4% PFA (Biolegend) for 30 minutes. Fixed cells were then washed twice with the 1x intracellular staining perm and wash buffer (Biolegend). Antibody against CTSA, CTSB, CTSD or CTSH was added and incubated overnight respectively. Alexa Fluor 568 Goat-anti-Rabbit IgG was added as the secondary antibody. CD11c + MHCII + B220 − was gated and the expression of cathepsins was evaluated by the fluorescence intensity. Analysis of flow cytometry data was performed using Flowjo (Treestar).
Measurement of IFNγ-secreting CD8 + T cells by ELISPOT assay
For antigen-specific CD8 + T cell functional assay in the B16-OVA model, 12 days after tumor inoculation, 3×10 5 lymphocytes were re-stimulated with 1μg/ml SIINFEKEL or MC38 tumor cells (lymphocyte:MC38 = 50:1) for 48 hours. 96-well HTS-IP plate (Millipore) was pre-coated with anti-IFNγ antibody (BD Bioscience) with a 1:250 dilution overnight at 4 °C. After co-culture, cells were removed. 2 mg/ml biotinylated anti-IFNγ antibody (BD Bioscience) with a 1:250 dilution was added and incubated for 2 h at room temperature or overnight at 4°C. Avidin-horseradish peroxidase (BD Bioscience) with a 1:1000 dilution was then added and the plate was incubated for 1h at room temperature. The cytokine spots of IFN-γ were developed according to product protocol (BD Bioscience).
Antigen-presentation assay
For cross-presentation of tumor neoantigen, CD11b + or CD8 + DC were purified from draining lymph node of WT or Ythdf1 −/− mice six days after inoculating with B16-OVA, MC38-OTIp or MC38-EGFR-SIY. OT-I or 2C naive CD8 + T cells were isolated from lymph nodes and spleen of 6 to 12-week-old mice. Negative selection was carried out with a negative CD8 isolation kit (StemCell Technologies, Inc.) following manufacturer’s instruction. DCs were co-cultured with OT-I naive CD8 T cells at the ratio of 1:10 for three days with or without 1 μg/ml SIINFEKEL peptide. For cross-presentation of soluble OVA, splenic DCs were sorted and stimulated with 100 ng/ml LPS overnight. DCs were then pulsed with different concentration of OVA (endotoxin free, Sigma) for 5 hours. Cells were washed and co-cultured with OT-I naive CD8 + T cells for three days. For in vitro cross-presentation of tumor neoantigen, Flt3L-DCs were collected on day 9–10 and co-cultured with necrotic B16-OVA tumor cells overnight. B220 − CD11c + cells were subsequently purified. GMDCs from Mettl14 f/f or CD11c-Cre Mettl14 f/f mice were harvested on day 6 and co-cultured with necrotic B16-OVA tumor cells for 16 hours. To inhibit cathepsins, GMDCs were pre-treated with E64 (sigma) for two hours followed by co-culturing with tumor cells. CD11c + cells were then purified and incubated with naive CD8 + T cells from OT-I mice for three days. IFN-γ production was detected by IFN-γ Flex Set CBA assay (BD Bioscience). To inhibit cathepsins in ex vivo cDCs, WT or Ythdf1 −/− mice were inoculated with 5×10 5 MC38. 36 hours after tumor inoculation, spleens were collected and digested, and CD11c + DCs were purified using EasySep Mouse CD11c Positive Selection Kit II (STEMCELL). CD11 + cDCs were then treated with 0.04 μM E64 (Sigma) overnight followed by co-culturing with OVA protein for 4 hours. Any free OVA protein was then removed from the culture medium, and CD11c + cells were incubated with CTV-labelled OT-I cells for three days. The cross-priming capacity of DC was analyzed by the dilution of CTV in CD8 + T cells. For cathepsin inhibition assay in vitro , Flt3L-DCs were treated with 5μg/ml CA-074 methyl ester (Selleck), 5μg/ml cathepsin L inhibitor III(Sigma) or the combination (5μg/ml CA-074 methyl ester and 5μg/ml cathepsin L inhibitor III) for 2 h followed by co-culturing with necrotic B16-OVA for 16 h, and then Flt3L-DCs were purified using EasySep Mouse CD11c Positive Selection Kit II (STEMCELL). The purified cells were incubated with OT I cells at the ratio of 1:20 for three days. The cross-priming capacity of DCs was then evaluated by the IFN-γproduction. To detect the MHC-H2K b -SIINFEKEL, mice were inoculated with B16-OVA. After 12 days, tumors were collected and tumor infiltrating DCs (CD45 + CD11b + ly6c − MHCII + CD24 + CD11c + ) were stained with monoclonal antibody 25.D1.
Cell trace violet labelling
10 million splenocytes from naive OT- I mouse were re-suspended in 1 ml PBS followed by incubating with 5 uM CellTraceViolet Dye(CTV, ThermoFisher) at 37°C for 20 minutes. 5 ml RPMI-1640 medium was added to the cells and incubated for 5 minutes to remove the free dye in the solution. These cells were then centrifuged and incubated with pre-warmed RPMI- for at least 10 minutes at room temperature for subsequent analysis.
RIP-seq
20 million GMDCs were harvested and co-cultured with or without necrotic B16-OVA overnight. The procedure was adapted from the previous report 10 . Five million Flt3L-DCs were harvested. DCs were then purified and pelleted by centrifuge for 5 min. Cells were washed twice with cold PBS and the cell pellet was re-suspended with 2 volumes of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/mL RNase inhibitor). Lysate was incubated on ice for 5 min and centrifuged for 15 min to clear the lysate. 1/10 volume of cell lysate was saved as input and total RNA was extracted by Trizol. The rest of cell lysate was incubated with 5 μg anti-YTHDF1 (Proteintech) at 4°C overnight with gentle rotation followed by incubation with 40μl protein G beads for 1 hour at 4°C. The beads were then washed five times with 1 mL ice-cold washing buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/mL RNase inhibitor). The IP complex was resuspended in 400 μl 1xProteinase K and digested with 2 mg Proteinase K at 55°C for 1 hour. RNA was then extracted by RNA isolation kit (Zymo). Input and IP RNA of each sample were used to generate the library using TruSeq stranded mRNA sample preparation kit (Illumina).
m 6 A-seq
Total RNA was isolated from DCs. Polyadenylated RNA was further enriched from total RNA by using Dynabeads® mRNA Purification Kit (Invitrogen). RNA samples were fragmented into ~100-nucleotide-long fragments with sonication. Fragmented RNA (100 ng mRNA or 5 μg total RNA) was performed m 6 A-IP following EpiMark N 6 -methyladenosine enrichment kit (NEB E1610S) protocol. RNA was enriched through RNA Clean&Concentration-5 (Zymo Research) and used for library generation with SMARTer Stranded Total RNA-Seq Kit (Takara). Sequencing was performed at the University of Chicago Genomics Facility on an Illumina HiSeq4000 machine in single-read mode with 50 bp per read. Sequencing reads were aligned to the mouse genome mm9 by STAR (version 2.6.0c) 29 . The m 6 A-enriched regions (peaks) in each m 6 A-IP sample were detected by MACS2 (version 2.1.1.20160309) 30 with q value less than 0.01 and corresponding m 6 A-Input sample was used as the control. Peaks that were detected by both replicates were considered as high confident peaks. The peaks annotation and binding motif were analyzed by HOMER (version 4.9) 31 .
Ribosome profiling
5×10 6 DCs were treated with 100 μg/ml cycloheximide (CHX) for 7 minutes. The cells were then harvested by the cell lifter. The cell suspension was spun at 400g for 5 min and the cell pellet was washed twice with 5 ml cold PBS with CHX (100 μg/ml). 200 μl lysis buffer (10 mM Tris, pH 7.4, 150 mM KCl, 5 mM MgCl 2 , 100 μg/ml CHX, 0.5% Triton-X-100, freshly added 1:100 protease inhibitor, 40 U/ml SUPERasin) was added to the cell pellet and lysed on ice for 15 minutes with rotating. 10% clarified lysate was saved as INPUT and the rest lysate was separated through a 5 ml 10%−50% sucrose gradient and centrifuged at 4 °C for 2 h at 28,000 r.p.m. Fractions were collected separately and analyzed by Qubit™ RNA HS Assay Kit (Invitrogen). The fractions corresponding to monosome or polysome were combined resepectively and concentrated on Amicon-Ultra 100K columns (Millipore). Two A260 units of ribosome fractions were digested with 60 U RNase I (Ambion) at room temperature for 30 minutes. RNA was extracted by RNA Clean&Concentrate (Zymo) and ribosomal RNA were deleted prior to size selection. RNA fragments (26–32 nt) were isolated by 15% denaturing Urea-PAGE gel. RNA was eluted from gel in elution buffer (300 mM sodium acetate pH 5.2, 1 mM EDTA) followed by phenol-chloroform extract and ethanol precipitation. RNA fragments were dephosphorylated and prepared into libraries by SMARTer® smRNA-Seq Kit (Clontech). The first 3 bases of sequencing reads were removed fastx_trimmer (version 0.0.14). The adapter sequences and polyA tails were firstly trimmed from sequencing reads by using cutadapt (version 1.15) with --minimum-length 18 -n 3 -a “AAAAAAAAAAAAAA” -a “AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC” parameters 32 . Trimmed reads were filtered for mitochondrial DNA and ribosomal RNA by Bowtie2 (version 2.3.4) 33 . All remaining reads were mapped to the mouse genome mm9 with STAR (version 2.6.0c) 29 . Uniquely mapped reads were selected by using SAMtools (version 1.7) 34 with mapping quality >= 20, and then removing duplication. The raw counts of coding regions were calculated by HOMER (version 4.9) 31 . The differentially TE (translational efficiency 35 , Equation 1 ) genes were detected by Bioconductor DESeq2 package (version 1.18.1) 36 with p.adj <= 0.1 and |log2FoldChange| >= 0.5.
Measurement of RNA lifetime
DCs were seeded in 24-well plate at 50% confluency. After 2 h, actinomycin D was added to 5 mg/ml at 3 h, 1 h and 0 h before collection. The total RNA was purified by RNeasy kit with an additional DNase-I digestion step on column. RNA quantities were determined by RT-qPCR. The specific primers used are as follows: Ctsb _Forward: CTGCTTACCATACACCAT, Ctsb _Reverse: TCCTTCACACTGTTAGAC; Ctsd _Forward: GGCAAGAGGTATCAAGGT, Ctsd _Reverse: CAGGTAGAAGGAGAAGATGT; Ctsl _Forward: GAGTTCGCTGTGGCTAAT, Ctsl _Reverse: GAGGTTCTTGCTGCTACA; Gapdh _Forward: ACCTGCCAAGTATGATGA, Gapdh_ Reverse: GGAGTTGCTGTTGAAGTC.
Immunohistochemistry of human biopsies
All samples encompassing tumor biopsies from 22 colorectal cancer patients were obtained with informed consent under a protocol approved by the University of Chicago Institutional Review Board. We have complied with all relevant ethical regulations. Information about the patient sex, age, and tumor characteristics are given in Supplementary Table 2 . To stain YTHDF1 and CD8, antigen retrieval was performed with 10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH9. Slides were processed with the VECTASTAIN Elite ABC HRP kit and DAB Substrate Kit (Vector Laboratories). Slides were counterstained with hematoxylin and dehydrated through graded alcohols and xylene. A total of 22 tumor samples had sufficient tissue for unambiguous analyses; For IHC quantification, DAB stains of IHC images were separated by color deconvolution algorithms47 in Fiji, a derivative of ImageJ. The mean DAB intensity of 3 random images at 795×650 pixels was calculated and converted into optical density (OD). CD8 positive cells were analyzed by Image J cell counter. The average infiltration of CD8 + cell and average expression of YTHDF1 were assessed within the surrounding stroma tissues.
Western blot analysis
To detect the expression of cathepins, GMDCs were harvested on day 6 and co-cultured with necrotic B16-OVA cells at the ratio of 1:1 for 16 hours. CD11c + DCs were then purified. Equal numbers of cells were lysed on ice for 15 min using 1xlysis buffer (CST) supplemented with a protease inhibitor cocktail (Calbiochem). Cell lysis was centrifuged at 16,100g at 4°C for 15 min. Clarified supernatant was loaded into 4–12% NuPAGE Bis-Tris gel and transferred to PVDF membranes (Life Technologies). Membranes were blocked for 1 hour in 5% milk TBST and then incubated with primary antibodies in the blocking buffer overnight at 4°C. After 5 times washing, membranes were incubated with secondary antibodies for 1 hour at room temperature. The information for all the antibodies used are provided in Supplementary Table 1 .
Degranulation of tumor infiltrating NK cells
Tumor infiltrating leukocytes were resuspended at 5×10 5 /ml and stimulated with phorbol-12-myristate-13-acetate (PMA) (2.5 μg/ml) and ionomycin (0.5 μg/ml) in 96-well plate. CD107α-PE antibody and 1xbredeldin A (Biolegend) were added directly to the well and incubated for 4 h at 37 °C in 5% CO 2 . Cells were stained for CD45 and NK1.1 (BD Biosciences) for 30 min. Samples were washed and then fixed in 1% paraformaldehyde.
Phagocytosis in vivo
5 × 10 5 B16F10 cells expressing zsGreen-OTI were injected s.c. into WT and Ythdf1 −/− mice. Tumor tissues were harvested and digested.
Maturation of DC
GMDCs were harvested and co-cultured with 100 ng/ml LPS overnight. The cytokine production was measured by mouse inflammation kit (BD).
Identification of off-target site and T7EI assay
Identified two off-target loci for each sgRNA site with highest score were selectively amplified by primers listed below: YTHDF1_For: TGACATTGGTGGCCATATCTGTC YTHDF1_Rev: TGTCTGCCCATCAACAACTGTGC Tex52_For: AGGATGAGAGGTGTTCAGCTAGAC Tex52_Rev: TCTGTAGGCCCAGAGTCCTCAG Nrp2_For: AGGGTAATACTACCACACATCAACCG Nrp2_Rev: AGAGCTGGGGTCTAATTGAATTTGGG Eme1_For: TGCTGTCTCGCCTCGCAATAGC Eme1_Rev: TGCGTACACTTAAGTCTGCCTGG MED20_For: TCAAGGGCTTCTTCCAGAGTGCC MED20_Rev: AGGCACCACACAAACCAGGCAAG HiPure Tissue DNA Mini Kit (Magen, D3121–03) was used to extract genomic DNA from the tails of WT and Ythdf1 −/− mice. The PCR reactions to amplify 350 bp fragment (for Y1 mouse) and 510 bp fragment (for WT mouse) were carried out in 30 μl reaction, using 15 μl of 2x EasyTaq PCR SuperMix (AS111 TransGen Biotech), 0.75 μM each of forward and reverse primers and 1 μl genomic DNA. The reaction products were subjected to 1.5% agarose gel electrophoresis. For T7E1 cleavage assay, equal volume PCR products of Ythdf1 −/− and WT mouse were mixed and then denatured and annealed in NEBuffer 2 (NEB) using a thermal cycler. Hybridized PCR products were digested with T7 endonuclease I (NEB, M0302L) or ddH2O (as control) for 20 minutes at 37°C and subjected to 1.5% agarose gel electrophoresis.
Statistical analysis and reproducibility
No statistical method was used to predetermine sample size. Mice were assigned at random to treatment groups for all mouse studies and, where possible, mixed among cages. There were no mice excluded from experiments. Blinded staining and blinded analysis were performed for IHC experiments. Experiments were independently repeated two to three times. Data were analyzed using Prism 5.0 software (GraphPad) and presented as mean values ± s.e.m. The P values were assessed using one-tailed or two-tailed unpaired Student’s t-test. For survival curve, statistics were done with the log-rank (Mantel-Cox) test. For translational efficiency, P values were calculated by likelihood ratio test and adjusted by Benjamini & Hochberg method. For cumulative distribution, two-sided Kolmogorov-Smirnov test was used to calculate the P values.
Data processing and analysis
Illumina reads were post-processed and aligned to the mouse mm9 assembly using STAR 29 program (version 2.6.0c) with default parameters. To visualize sequencing signals in the genome browser, we generated RIP-seq and m 6 A-seq bigwig files with bamCoverage function from deepTools (version 3.0.1) 37 with ‘-bs=1 --normalizeUsing BPM’. For RIP-seq, Piranha software (version 1.2.1) 38 was to detect the binding sites of YTHDF1 with “-b 100 -i 100”. Metagene plots were performed by the Bioconductor GUITAR 39 package (version 1.16.0). Peaks that were detected by both replicates were considered as high confident peaks. GO term analyses were performed by metascape 40 .
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. RIP-seq, Ribo-seq and m 6 A-seq data sets have been deposited in Gene Expression Omnibus under the accession number GSE115106. A summary of sequencing experiments is provided in Supplementary Table 3 . The differential translational efficiency results provided in Supplementary Table 4 . Source data for bar graphs and box-plots in Figures and Extended Data Figures are provided in separate excel files.
Extended Data
Supplementary Material
Footnotes
Competing interests C.H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics, Inc. A patent application on YTHDF1 has been filed by the University of Chicago.