PMCID string | Title string | Sentences string |
|---|---|---|
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | When sorting individual codons by relative occupancy, a primary factor determining translation speed was the nucleotide at the third position. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Whereas codons with adenosine in the third position showed increased occupancy (a proxy for stalling) in ProArg-free diet plus DFMO-treated tumours, occupancy at codons with guanosine in the third position was markedly decreased (Fig. 4d and Extended Data Fig. 6j). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The same phenotype, with a reduced effect size, was shown for the DFMO monotherapy group, suggesting a previously unknown role of polyamines (rather than low arginine or proline) in the decoding of codons with adenosine at the third position (also referred to as the ‘wobble’ position) (Fig. 4e and Extended Data Fig. 6k–n). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Similarly, at the global level this phenotype was not identified following genetic or pharmacological inhibition of hypusination (Supplementary Fig. 4). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | On the contrary, ribosome pausing appeared to be driven by disrupted codon–anticodon interactions with tRNAs exhibiting complex biochemical modifications at the anticodon wobble base position (tRNA base 34) of all highly paused codons with adenosine in the third position, as well as the two highly paused codons with thymidine in the third position (Fig. 4d). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | This was further supported by the pausing phenotype affecting all three ribosome sites (A, P and E; Extended Data Fig. 6j). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Whereas this was mostly independent of the tRNA modification status at position 34, the ProArg-free diet itself increased queuosine-related modifications, putatively further adding to the polyamine induced codon-specific translation imbalance (Supplementary Fig. 5a–d). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Although interactions of polyamines with ribosomes and tRNAs have been reported and documented to stimulate translation in vitro, to our knowledge, this codon resolution phenotype has not previously been described (Fig. 4e). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | To better understand the phenotypic consequences induced by this specific translation defect, we integrated RNA-sequencing (RNA-seq), Ribo-seq and proteomics data (Fig. 5a and Supplementary Fig. 6a–d). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The main driver of transcriptional changes was DFMO. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The ProArg-free diet itself induced only minor such changes, and transcriptional changes strongly overlapped between DFMO alone and the combined ProArg-free diet plus DFMO treatment. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | By contrast, Ribo-seq and proteomics revealed distinct changes in tumours under combined ProArg-free diet plus DFMO therapy. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The effect was independent of proline or polyproline abundance in the proteins (Supplementary Fig. 6e–h), and instead correlated with genes that were enriched in codons with adenosine in the third position. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Fig. 5Regulation of translation by polyamine depletion is driven by fractional codon content.a, Gene set enrichment analysis (GSEA) of protein biosynthesis using omics layers: gene-expression (RNA-seq), gene translation (Ribo-seq) and protein (proteomics) levels. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | GSEA compares ProArg-free plus DFMO to CD using the Reactome gene sets. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | All pathways are depicted, ranked by significance (Benjamini–Hochberg correction) and signed by normalized enrichment score (NES). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The most downregulated and upregulated sets at the protein level are cell cycle and neuronal system, respectively. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Lines connect gene sets across the omics layers. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | b, Mean fraction of codons with adenosine in the third position (A-ending codons) across all pathways and significantly changed pathways identified in a. Pathways taken from Reactome gene sets. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | c, The percentage of codons with adenosine in the third position correlates with the average protein level across Reactome pathways. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Pathways with an increasing fraction of codons with adenosine in the third position have lower protein levels in ProArg-free DFMO compared with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | FC, fold change. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | d, Fold change across omics layers of top downregulated cell cycle proteins indicates that differences between ProArg-free diet plus DFMO and CD occur predominantly on the protein level. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | e, Percentage of codons with the respective nucleotide at the third position in the Itgb3bp gene (encoding CENPR protein) compared with the transcriptome background. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | In a,c,d, RNA-seq, ProArg-free + DFMO: n = 5; CD: n = 4. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Ribo-seq, n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Proteomics, ProArg-free + DFMO: n = 6; CD: n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Panels a and b created in BioRender. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Morscher, R. (2025) https://BioRender.com/ygrgncb (a); https://BioRender.com/566gynw (b).Source data a, Gene set enrichment analysis (GSEA) of protein biosynthesis using omics layers: gene-expression (RNA-seq), gene translation (Ribo-seq) and protein (proteomics) levels. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | GSEA compares ProArg-free plus DFMO to CD using the Reactome gene sets. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | All pathways are depicted, ranked by significance (Benjamini–Hochberg correction) and signed by normalized enrichment score (NES). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The most downregulated and upregulated sets at the protein level are cell cycle and neuronal system, respectively. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Lines connect gene sets across the omics layers. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | b, Mean fraction of codons with adenosine in the third position (A-ending codons) across all pathways and significantly changed pathways identified in a. Pathways taken from Reactome gene sets. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | c, The percentage of codons with adenosine in the third position correlates with the average protein level across Reactome pathways. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Pathways with an increasing fraction of codons with adenosine in the third position have lower protein levels in ProArg-free DFMO compared with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | FC, fold change. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | d, Fold change across omics layers of top downregulated cell cycle proteins indicates that differences between ProArg-free diet plus DFMO and CD occur predominantly on the protein level. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | e, Percentage of codons with the respective nucleotide at the third position in the Itgb3bp gene (encoding CENPR protein) compared with the transcriptome background. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | In a,c,d, RNA-seq, ProArg-free + DFMO: n = 5; CD: n = 4. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Ribo-seq, n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Proteomics, ProArg-free + DFMO: n = 6; CD: n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Panels a and b created in BioRender. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Morscher, R. (2025) https://BioRender.com/ygrgncb (a); https://BioRender.com/566gynw (b). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Source data Notably, upon combined ProArg-free diet plus DFMO treatment, gene sets linked to neuronal differentiation and neuronal cell identity were the most upregulated at the protein level. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | By contrast, ‘cell cycle’ was the most downregulated gene set, despite exhibiting unchanged RNA expression (Fig. 5a and Extended Data Fig. 7a). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | We next assessed whether the slow translation of codons with adenosine at the third position relates to proteome reprogramming. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Indeed the cell cycle gene set contained a higher frequency of codons with adenosine in the third position that were affected by ribosome pausing, compared with the average for the exome. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Exome-wide, pathways associated with cell cycle programmes were the most enriched in codons with adenosine in the third position. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Neuronal differentiation-associated gene sets were at the opposite end of the spectrum, with the guanosine content showing limited variation in both gene sets (Fig. 5b and Extended Data Fig. 7b–d). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | A higher fraction of codons with adenosine in the third position within a pathway was linked with a lower protein fold change or gene set enrichment (Fig. 5c and Extended Data Fig. 7e). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Beyond the general reduction of cell cycle proteins, four mitosis-related proteins stood out with the strongest downregulation at the protein level under combined ProArg-free diet plus DFMO treatment (Extended Data Fig. 7f,g). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | All four showed at least fourfold downregulation at the protein level, without changes in gene expression (Fig. 5d), in contrast to the regulation in neuronal proteins (Extended Data Fig. 7h,i). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Itgb3bp, encoding CENPR, the most affected protein, showed a remarkably shifted distribution in codon preference, with 38.6% of codons having adenosine in the third position, compared with 18.8% across all protein-coding transcripts (Fig. 5e and Extended Data Fig. 7j,k). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The ribosome distribution along the CENPR coding sequence confirmed preferential pausing at codons with adenosine in the third position upon ProArg-free diet plus DFMO treatment, as indicated by an increased ribosomal pausing sum at codons with adenosine in the third position and reduced pausing at those with guanosine in the third position (Extended Data Fig. 7l–n). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Similar pausing was observed for CEP57 and KIF2C, and the pausing sum correlated to the levels of the most regulated proteins across the two gene sets (Extended Data Fig. 7o,p). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Expression of cell cycle proteins was increased in MYCN-amplified tumours from human patients and correlated to high tumour stage (Extended Data Fig. 8a–c). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Depleting polyamine levels independently of DFMO by ODC knockdown depleted cell cycle protein levels and induced growth defects characterized by cell cycle arrest (Extended Data Fig. 8d–m). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Conversely, external supplementation of polyamines restored cell growth and CENPR levels following enhanced polyamine depletion by DFMO with proline- and arginine-reduced medium. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | This rescue was independent of hypusination status, as indicated by sustained rescue upon Dhps knockdown, preventing spermidine-dependent post-translational modification of eIF5a (Extended Data Fig. 8n). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Thus, polyamine depletion by combined ProArg-free diet and DFMO treatment induces ribosome stalling at codons with adenosine in the third position. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Owing to the selective enrichment of these codons cell cycle genes and their depletion in differentiation genes, this effect of ProArg-free diet and DFMO treatment reprogrammes the proteome in a manner that favours differentiation. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | We hypothesized that the shift away from cell cycle proteins towards neuronal differentiation proteins is likely to reprogramme neuroblastoma tumours in a manner that slows proliferation and induces differentiation. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Ki67 staining confirmed a decrease in actively cycling cells under ProArg-free diet plus DFMO treatment (Extended Data Fig. 9a). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Furthermore, cell growth activation signatures including MYC and E2F targets were impaired (Fig. 6a and Extended Data Fig. 9b).Fig. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | 6Polyamine depletion-mediated proteome rewiring induces neuroblastoma differentiation.a, GSEA across omics layers in all three treatment groups using the Hallmark gene set. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Only the ProArg-free plus DFMO treatment group showed a significant effect compared with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The effect was mainly on the translation and protein level. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Shown are the five top enriched sets (complete sets in Extended Data Fig. 10c). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Size indicates P value (Benjamini–Hochberg correction) and colour represents NES, with red indicating enrichment in the intervention group (CD + DFMO, ProArg-free or ProArg-free + DFMO) and blue indicating enrichment in the CD group. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | b, Western blot analysis of MYCN in tumours from CD and ProArg-free plus DFMO treatment arms. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Negative control (C1), CHLA20 neuroblastoma cell line (MYCN non-amplified, MYC expressing); positive control (C2), IMR5 neuroblastoma cell line (MYCN-amplified). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | GAPDH is used as a loading control. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Blots are representative of two independent experiments yielding similar results. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | c, Representative haematoxylin and eosin (H&E)-stained sections. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD and ProArg-free diet treatments show undifferentiated primitive neuroblasts, absent neuropil and prominent mitotic figures. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD plus DFMO shows poorly differentiated primitive neuroblasts with scant neuropil (arrowhead) and foci of cytodifferentiation (<5% differentiating, arrow). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | ProArg-free diet plus DFMO tumours show high fractions of differentiating neuroblasts (>5% differentiating) with increased cytoplasmic to nuclear ratio (arrow) and abundant neuropil (arrowhead). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Sections are representative of many images with the same observations. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Scale bars, 50 μm. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | d, Summary of treatment effects. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Cell cycle and MYCN programmes are downregulated at the protein level owing to translation inhibition and immature cancer cells are driven into neuronal differentiation. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | In a,c, RNA-seq: ProArg-free DFMO: n = 5; CD: n = 4. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Ribo-seq: n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Proteomics: ProArg-free DFMO: n = 6; CD: n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Panel d created in BioRender. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Morscher, R. (2025) https://BioRender.com/kk9n051.Source data a, GSEA across omics layers in all three treatment groups using the Hallmark gene set. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Only the ProArg-free plus DFMO treatment group showed a significant effect compared with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The effect was mainly on the translation and protein level. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Shown are the five top enriched sets (complete sets in Extended Data Fig. 10c). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Size indicates P value (Benjamini–Hochberg correction) and colour represents NES, with red indicating enrichment in the intervention group (CD + DFMO, ProArg-free or ProArg-free + DFMO) and blue indicating enrichment in the CD group. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | b, Western blot analysis of MYCN in tumours from CD and ProArg-free plus DFMO treatment arms. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Negative control (C1), CHLA20 neuroblastoma cell line (MYCN non-amplified, MYC expressing); positive control (C2), IMR5 neuroblastoma cell line (MYCN-amplified). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | GAPDH is used as a loading control. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Blots are representative of two independent experiments yielding similar results. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | c, Representative haematoxylin and eosin (H&E)-stained sections. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD and ProArg-free diet treatments show undifferentiated primitive neuroblasts, absent neuropil and prominent mitotic figures. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD plus DFMO shows poorly differentiated primitive neuroblasts with scant neuropil (arrowhead) and foci of cytodifferentiation (<5% differentiating, arrow). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | ProArg-free diet plus DFMO tumours show high fractions of differentiating neuroblasts (>5% differentiating) with increased cytoplasmic to nuclear ratio (arrow) and abundant neuropil (arrowhead). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Sections are representative of many images with the same observations. |
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