Fig 2: YTHDF2-mediated CDKN2B and SPOCK2 mRNA degradation contributes to mutant p53-associated malignancy.a In vivo mouse xenograft study demonstrates that knockdown of YTHDF2, SVIL, or MLL1 hampers LNZ308-p53(G245D) tumor growth (n = 4 biologically independent mice). The sizes of the tumors were measured at the indicated time. b Expression of CDKN2B or SPOCK2 inhibits cell proliferation of LFS astrocytes (n = 6 biologically independent samples). c In vitro AIG assay demonstrates ectopic expression of CDKN2B or SPOCK2 leading to decreased colony numbers of LFS astrocytes. All colonies are counted and measured after 2-month culture (n = 6 biologically independent samples). d In vivo mouse xenograft study shows ectopic expression of CDKN2B or SPOCK2 abrogating LNZ308-p53(G245D) tumor growth (n = 3 biologically independent mice). The sizes of the tumors were measured at the indicated time. e Knockdown of CDKN2B or SPOCK2 rescues YTHDF2 depletion-induced growth inhibition in LFS astrocytes (n = 6 biologically independent samples). f In vitro AIG assay demonstrates knockdown of CDKN2B or SPOCK2 rescuing in vitro colony formation of YTHDF2-depleted LFS astrocytes. All colonies are counted and measured after 2-month culture (n = 6 biologically independent samples). g Xenograft study indicates that knockdown of CDKN2B or SPOCK2 rescues YTHDF2 knockdown-induced LNZ308-p53(G245D) tumor growth inhibition in nude mice (n = 4 biologically independent mice). The sizes of the tumors were measured at the indicated time. h Transcriptome analysis of CDKN2B-restored LFS astrocytes. Bubble plot for visualizing enriched GO and KEGG pathway analyses of differentially upregulated genes in CDKN2B-restored LFS astrocytes. X axis label represents the enrichment factor (number of differentially expressed genes enriched in the pathway/total number of genes in the pathway) and Y axis label represents GO annotation and KEGG pathway. Size and color of the bubble represent number of differentially expressed genes enriched in the GO or KEGG pathways and enrichment significance (P value), respectively. The data are presented as the mean ± SEM; two-way ANOVA with Bonferroni’s multiple comparison test (a–g). *P < 0.05, ***P < 0.001. Source data and exact P values are provided in the Source Data file.

Fig 3: Identification of YTHDF2 targets via m6A MeRIP-seq, eCLIP-seq, and RNA-seq in LFS astrocytes.a m6A MeRIP-seq indicates distribution of m6A peaks in different regions (5’UTR, first exon, other exon, and 3’UTR) of transcripts. Pie chart shows the percentage of m6A peaks within distinct regions of transcripts in LFS astrocytes. b Violin plot demonstrates significant elevation of highly m6A-marked transcripts upon YTHDF2 depletion. c Examination of YTHDF2 IP enrichment by eCLIP-seq. d Metagene plot of YTHDF2 eCLIP-seq indicates enrichment of YTHDF2-interacting mRNA peaks in the 3’UTR clustered around stop codons. e Motif analysis demonstrates that YTHDF2 binding motifs are similar to the consensus m6A motif RRACH. f Venn diagram identifying 84 YTHDF2-targeted m6A transcripts validated by a combination of m6A MeRIP-seq, YTHDF2 eCLIP-seq, and RNA-seq in LFS astrocytes. These transcripts include CDKN2B and SPOCK2 mRNAs. g IGV track views of m6A peaks located on CDKN2B and SPOCK2 transcripts in LFS astrocytes. h RT-qPCR indicates decreased expression of YTHDF2-targeted CDKN2B and SPOCK2 transcripts in LFS astrocytes (n = 3 biologically independent samples). i Low expression of YTHDF2-targeted CDKN2B and SPOCK2 is correlated with poor overall survival of LGG/GBM patients. Log-rank (Mantel–Cox) test is performed to compute significance. j Immunostaining demonstrates lower CDKN2B in engrafted LFS cerebral organoids (upper panel) and increased CDKN2B in YTHDF2-depleted engrafted LFS cerebral organoids (lower panel), Scale bar, 100 µm. Anti-CDKN2B antibodies only recognize human but not mouse CDKN2B proteins. k mRNA stability assay demonstrates that YTHDF2 knockdown leads to an increased half-life of CDKN2B and SPOCK2 mRNAs. shCtrl-LFS and shYTHDF2-LFS astrocytes are treated with actinomycin D and total RNAs are isolated at 0, 30, and 60 min. (n = 3 biologically independent samples). l RT-qPCR demonstrates elevated expression of YTHDF2 targets CDKN2B and SPOCK2 upon depletion of p53, YTHDF2, or SVIL as well as inhibition of MLL1 function by OICR-9429 or MI-2-2 in LFS astrocytes (n = 3 biologically independent samples). The results are representative of at least three independent experiments (c, j). The data are presented as the mean ± SEM); two-way ANOVA with Bonferroni’s multiple comparison test (l); unpaired two-tailed Student’s t test (h); multiple t test (k); **P < 0.01, ***P < 0.001. Source data and exact P values are provided in the Source Data file.