Journal: The EMBO Journal
Article Title: METTL3/MYCN cooperation drives neural crest differentiation and provides therapeutic vulnerability in neuroblastoma
doi: 10.1038/s44318-024-00299-8
Figure Lengend Snippet: ( A ) HOXC8 (red), and Flag (green) IF were performed in Flag- MYCN overexpressed (Dox + , from day 5 onwards) SAP after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added on day 9 of differentiation. Box-whisker plots show HOXC8 signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 2200 cells and data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar represents 10 μm. ( B ) HOXC9 (green), and MYCN (red) IF were performed in Flag- MYCN overexpressed (Dox + , from day 5 onwards) SAP after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added on day 9 of differentiation. Box-whisker plots show HOXC9 intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 1500 cells and data from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar represents 10 μm. ( C ) PRPH (green), and MYCN (red) IF were performed in Flag- MYCN overexpressed (Dox + , from day 5 onwards) SN-stage cells after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added from day 9 of differentiation. Box-whisker plots show quantification of PRPH signal intensity and neurite length. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 50 μm. ( D ) Representative IF images of TUBB3 (green) in SK-N-BE(2) cells that were pretreated with either DMSO or STM2457 (10 μM) for 24 h, followed by RA treatment for another 3 days. Box-whisker plots show the quantification of neurite length. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 50 μm. ( E ) RPA32 (red) [top] and gamma-H2AX (green) [bottom] IF were performed in Flag-MYCN overexpressed (Dox + , from day 5 onwards) SN-stage cells after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added from day 13 of differentiation. Box-whisker plots show either RPA32 or gamma-H2AX signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 90 cells and data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 10 μm. ( F ) RPA32 (red) IF was performed in SK-N-BE(2) cells with TetO shCtrl or TetO shM3-1 after 48 h Dox induction. Box-whisker plots show RPA32 signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 800 cells and data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 5 μm. ( G ) Left: Representative IF showing expression of RPA32 (red) in SK-N-BE(2) cells treated either with DMSO, STM2457 (10 μM), doxorubicin (1 μM), or a combination of STM2457 with doxorubicin for 24 h. Scale bar is 100 μm. Middle: Box-whisker plots show RPA32 signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 1900 cells. Data are from three independent experiments. Right: Bar plots show relative cell viability in SK-N-BE(2) cells treated for 72 h with DMSO, STM2457, Doxorubicin, and a combination of STM2457 with doxorubicin. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was conducted using a one-way ANOVA with Tukey’s post hoc test. ( H ) Left: Cartoon demonstrating the experimental strategy used for the mouse in vivo experiment performed with patient-derived xenograft (PDX) cells. MNA COG-N-415x, PDX cells were injected into NSG mice. Once tumors reached 170 mm 3 mice were randomly allocated into four treatment groups ( n = 4–6 mice per group) and treated for 14 days with either vehicle (20% hydroxypropyl-beta cyclodextrin) daily, STM2457 (50 mg/kg in vehicle) daily, doxorubicin (0.2 mg/kg in vehicle) every three days or a combination of STM2457 and doxorubicin at the same doses. Line plots show tumor volume (middle) and body weight (right) in the treatment groups. Data are presented as mean ± SEM. Statistical analysis was conducted using a two-way ANOVA with Tukey’s post hoc test. .
Article Snippet: TetO shCtrl (pLKO-Tet-On-shRNA-Control) , Addgene , Cat# 98398.
Techniques: Whisker Assay, Two Tailed Test, Expressing, In Vivo, Derivative Assay, Injection
![( A ) Schematic diagram showing key steps involved in the differentiation process of human embryonic stem cells (hESC) into trunk neural crest cells (tNCC), followed by their further differentiation into sympathoadrenal progenitors (SAP) and, ultimately, into sympathetic neurons (SN). ( B ) Representative immunofluorescence (IF) images illustrating the expression of distinct lineage markers at different stages of differentiation: HOXC9 for tNCC, PHOX2B for SAP, and PRPH for SN. The scale bar is indicative of 50 µm. ( C ) Representative immunoblot shows the levels of METTL3, METTL14 RBM15, and WTAP across various stages of differentiation, including hESC, tNCC, SAP, and SN. Vinculin, A-tubulin, and GAPDH were loading controls. The values below the blots indicate the fold change (normalized to loading control) in the levels of METTL3, METTL14, RBM15, and WTAP. The experiments were repeated three times. ( D ) The total number of m 6 A peaks in hESC and tNCC, the p value was calculated using a permutation test, and the number of permutations was set to 1000. ( E ) Identified motifs from de novo motif analysis of m 6 A peaks enriched in hESC and tNCC and P values were obtained using the HOMER tool. ( F ) Metagene analysis showing relative m 6 A peak density at genes in hESC and tNCC. ( G ) Venn diagram showing overlap of the m 6 A positive (m 6 A + ) [containing at least one m 6 A peak] genes in hESC and tNCC. ( H ) Left: Venn diagram comparison of differentially expressed genes (DEGs) [hESC vs. tNCC] and m 6 A + . Right: Top enriched terms associated with m 6 A-containing DEGs (hESC vs. tNCC) were identified using enrichGO, with p values obtained through Fisher’s exact test. ( I ) Representative immunoblot shows the levels of METTL3, in hESC and tNCC with control <t>(shCtrl)</t> or stable METTL3 KD (shMETTL3-1, shMETTL3-2). Vinculin and GAPDH were loading control. The values below the blots indicate the fold change (normalized to loading control) in the levels of METTL3. The experiments were repeated three times. ( J ) Top enriched terms associated with DEGs (shCtrl vs. shMETTL3-1) that are m 6 A+ in tNCC were identified using enrichGO, with P values obtained through Fisher’s exact test. ( K ) Genome browser screenshots of HOXC8 and HOXC9 3´UTR, showing the presence of m 6 A in tNCC, neural crest stem cells (NCSC) at day 7 and at day 14. ( L ) RT-qPCR data showing the relative expression of HOXC8 and HOXC9 in SAP following METTL3 KD. GAPDH was used to normalize the qPCR data. Data are shown as mean ± SEM of three independent biological replicates. Two-way ANOVA with Šídák’s multiple comparisons test was used. ( M ) shCtrl, shMETTL3-1, and shMETTL3-2 hESC were differentiated to SN, followed by IF with PRPH antibody to assess neurite length and PRPH signal intensity. Data are represented by box-whisker plots where the median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. This analysis was conducted across three independent biological replicates and statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. Scale bar represents 100 µm. .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9786/pmc11649786/pmc11649786__44318_2024_299_Fig1_HTML.jpg)
![( A ) Left: Co-IP of METTL3 or MYCN from lysates of Flag-MYCN overexpressing SHEP cells (SHEP MYCN ) after Dox induction for 24 h, blotted with MYCN or METTL3 antibodies. IgG served as a negative control. Right: Proximity ligation assay (PLA) in tNCC and 24 h Dox-induced SHEP MYCN cells depicting METTL3 and MYCN PLA signal (green) in the nucleus (marked by DAPI). The negative control shows PLA with only the METTL3 antibody. Scale bar is 5 μm. The experiments were repeated three times. ( B ) Co-IP of METTL3 or MYCN from lysates of SHEP MYCN after 24 h of Dox induction and 1 h treatment with 300 nM flavopiridol (FP), followed by western blotting with MYCN or METTL3 antibodies. IgG was used as a negative control. The experiments were repeated three times. ( C ) Venn diagram comparison of METTL3 and MYCN binding sites determined from ChIP-seq experiments performed in tNCC. ( D ) Distribution and heatmaps of normalized ChIP-seq reads for METTL3, MYCN, and H3K27ac over the MYCN and METTL3 overlapping peak coordinates. The data is centered on MYCN peaks (− 4 kb to +4 kb). ( E ) Distribution of METTL3 ChIP signal in a metagene profile. The data is centered at the transcription start site (TSS) [−1 kb to +1 kb], at genes that are co-bound by METTL3 and MYCN or bound by METTL3 only in tNCC. ( F ) Genome browser screenshot showing METTL3, MYCN, and H3K27ac ChIP-seq signals in hESC and tNCC over the HOXC8 and HOXC9 gene locus. ( G ) Venn diagram comparison of METTL3 and MYCN binding sites determined from ChIP-seq experiments performed in SHEP MYCN cells before and after Dox induction for 24 h. ( H ) Distribution and heatmaps of normalized ChIP-seq reads for METTL3 and MYCN overlapping peaks centered on MYCN peaks (− 4 kb to +4 kb) in SHEP MYCN cells after Dox induction. ( I ) Left: Browser screenshot showing m 6 A RIP-seq tracks at 3´UTR of HOXC8 and HOXC9 genes in SHEP MYCN cells before and after Dox induction for 24 h. Right: m 6 A RIP-qPCR data showing enrichment of both HOXC8 and HOXC9 in SHEP MYCN cells before and after Dox induction for 24 h. Data are represented as a percentage of input. IgG was used as a negative control. Data are from three independent experiments and shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test was used. ( J ) RT-qPCR data showing the expression of HOXC8 and HOXC9 in SHEP MYCN cells Left: SHEP cells Right: with either control (TetO shCtrl) or METTL3 KD (TetO shM3-1) after Dox induction for 6 days. GAPDH was used to normalize the qPCR data. Data are shown as mean ± SD of three independent biological replicates. Two-way ANOVA with Šídák’s multiple comparisons test was used. ( K ) Left: Distribution of METTL3 ChIP signal in a metagene profile. The data are centered at the transcription start site (TSS) [−1 kb to +1 kb], at genes that are co-bound by METTL3 and MYCN or bound by METTL3 only in SHEP MYCN cells after 24 h Dox induction. ( L ) Venn diagram comparing m 6 A+ and METTL3/MYCN co-bound genes SHEP MYCN cells after Dox 24 h induction. METTL3 and MYCN co-bound regions were determined using the ChIP-seq experiments. ( M ) Metagene analysis showing relative m 6 A peak density at genes co-bound by METTL3 and MYCN or the rest of m 6 A-containing genes in SHEP MYCN cells after Dox induction. ( N ) Box-whisker plots showing the number of m 6 A peaks/genes that are co-bound by METTL3 and MYCN (median = 2) or the rest of m 6 A-containing genes (median = 1) in SHEP MYCN cells after Dox induction. The number of co-bound peaks and Rest peaks used for this analysis are 3127 and 3349, respectively. Whiskers indicate the 1st to 99th percentiles, and any outliers beyond this range are shown as individual dots. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test. ( O ) PLA in SHEP MYCN cells with or without Dox induction for 24 h depicting METTL14 and H3K36me3 PLA signal (green) in the nucleus (marked by DAPI). The negative control shows PLA with only the H3K36me3 antibody. Signal intensity measurements were taken from over 50 cells. Data are from three independent experiments and presented as box-whisker plots where the median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles and any outliers beyond this range are displayed as individual dots. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar represents 10 μm. .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9786/pmc11649786/pmc11649786__44318_2024_299_Fig4_HTML.jpg)
![( A ) HOXC8 (red), and Flag (green) IF were performed in Flag- MYCN overexpressed (Dox + , from day 5 onwards) SAP after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added on day 9 of differentiation. Box-whisker plots show HOXC8 signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 2200 cells and data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar represents 10 μm. ( B ) HOXC9 (green), and MYCN (red) IF were performed in Flag- MYCN overexpressed (Dox + , from day 5 onwards) SAP after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added on day 9 of differentiation. Box-whisker plots show HOXC9 intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 1500 cells and data from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar represents 10 μm. ( C ) PRPH (green), and MYCN (red) IF were performed in Flag- MYCN overexpressed (Dox + , from day 5 onwards) SN-stage cells after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added from day 9 of differentiation. Box-whisker plots show quantification of PRPH signal intensity and neurite length. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 50 μm. ( D ) Representative IF images of TUBB3 (green) in SK-N-BE(2) cells that were pretreated with either DMSO or STM2457 (10 μM) for 24 h, followed by RA treatment for another 3 days. Box-whisker plots show the quantification of neurite length. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 50 μm. ( E ) RPA32 (red) [top] and gamma-H2AX (green) [bottom] IF were performed in Flag-MYCN overexpressed (Dox + , from day 5 onwards) SN-stage cells after DMSO or STM2457 (10 μM) treatment. STM2457 or DMSO was added from day 13 of differentiation. Box-whisker plots show either RPA32 or gamma-H2AX signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 90 cells and data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 10 μm. ( F ) RPA32 (red) IF was performed in SK-N-BE(2) cells with TetO shCtrl or TetO shM3-1 after 48 h Dox induction. Box-whisker plots show RPA32 signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 800 cells and data are from three independent experiments. Statistical analysis was performed using a two-tailed unpaired t test. Scale bar is 5 μm. ( G ) Left: Representative IF showing expression of RPA32 (red) in SK-N-BE(2) cells treated either with DMSO, STM2457 (10 μM), doxorubicin (1 μM), or a combination of STM2457 with doxorubicin for 24 h. Scale bar is 100 μm. Middle: Box-whisker plots show RPA32 signal intensity normalized to DAPI intensity. The median is indicated by a horizontal line, the boxes represent the 25th to 75th percentiles, the whiskers show the 10th to 90th percentiles, and any outliers beyond this range are displayed as individual dots. Signal intensity measurements were taken from over 1900 cells. Data are from three independent experiments. Right: Bar plots show relative cell viability in SK-N-BE(2) cells treated for 72 h with DMSO, STM2457, Doxorubicin, and a combination of STM2457 with doxorubicin. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was conducted using a one-way ANOVA with Tukey’s post hoc test. ( H ) Left: Cartoon demonstrating the experimental strategy used for the mouse in vivo experiment performed with patient-derived xenograft (PDX) cells. MNA COG-N-415x, PDX cells were injected into NSG mice. Once tumors reached 170 mm 3 mice were randomly allocated into four treatment groups ( n = 4–6 mice per group) and treated for 14 days with either vehicle (20% hydroxypropyl-beta cyclodextrin) daily, STM2457 (50 mg/kg in vehicle) daily, doxorubicin (0.2 mg/kg in vehicle) every three days or a combination of STM2457 and doxorubicin at the same doses. Line plots show tumor volume (middle) and body weight (right) in the treatment groups. Data are presented as mean ± SEM. Statistical analysis was conducted using a two-way ANOVA with Tukey’s post hoc test. .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9786/pmc11649786/pmc11649786__44318_2024_299_Fig6_HTML.jpg)
