PMCID string | Title string | Sentences string |
|---|---|---|
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Statistical comparisons to CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Insets, magnified graphs highlight the additional difference in polyamine levels induced by ProArg-free diet over DFMO only. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Data are mean ± s.e.m. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD: n = 5; CD + DMFO: n = 5; ProArg-free: n = 6; ProArg-free + DFMO: n = 4. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Two-tailed t-test. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | n denotes the number of mice measured by metabolomics. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Source data a, Schematic of arginine, proline and glutamine metabolism and its direct link to polyamines via ornithine. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | GSAL, glutamate-γ-semialdehyde; P5C, pyrroline-5-carboxylate. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | b, Differential serum metabolite levels comparing ProArg-free diet with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Blue dots highlight metabolites that are significantly depleted (FDR < 0.05) and the rose dot indicates a metabolite that was upregulated compared with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD: n = 8; ProArg-free: n = 7. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | c, Serum arginine, proline, glutamine and ornithine across groups. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Statistical comparisons to CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Data are mean ± s.e.m. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD: n = 8; CD + DMFO: n = 10; ProArg-free: n = 7; ProArg-free + DFMO: n = 7. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | d, Tumour arginine, proline, glutamine and ornithine levels reveal dysregulation of arginine and proline metabolism with combined ProArg-free diet plus DFMO treatment. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Average age at end point is eight weeks. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Statistical comparisons to CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Data are mean ± s.e.m. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD: n = 5; CD + DMFO: n = 5; ProArg-free: n = 8; ProArg-free + DFMO: n = 4. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | e, A ProArg-free diet enhances polyamine depletion in tumour tissue induced by DFMO in prolonged treatment. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Average age at end point is 12 weeks. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Statistical comparisons to CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Insets, magnified graphs highlight the additional difference in polyamine levels induced by ProArg-free diet over DFMO only. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Data are mean ± s.e.m. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | CD: n = 5; CD + DMFO: n = 5; ProArg-free: n = 6; ProArg-free + DFMO: n = 4. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Two-tailed t-test. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | n denotes the number of mice measured by metabolomics. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Source data Targeted liquid chromatography–mass spectrometry (LC–MS/MS) measurements of tumour polyamines revealed that DFMO treatment decreased putrescine, the direct product of ornithine decarboxylation by ODC, and its derivatives such as spermidine. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The ProArg-free diet potentiated the DFMO effect to further decrease polyamine levels, achieving more than tenfold reduction in spermidine compared with CD and more than twofold reduction compared with DFMO monotherapy (Fig. 3e). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | N-acetylspermidine and N-acetyl-putrescine were also decreased by a ProArg-free diet plus DFMO, consistent with reduced catabolic flux (Fig. 3e and Extended Data Fig. 5f). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Combined dietary intervention with DFMO treatment resulted in superior spermidine depletion compared with co-treatment with the polyamine uptake inhibitor AMXT1501 (Fig. 3e and Extended Data Fig. 5g), highlighting the primacy of intratumoral polyamine synthesis. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Reducing proline or arginine substrate intake individually was insufficient to enhance polyamine depletion beyond DFMO monotherapy (Extended Data Fig. 5h,i). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The dependency on intracellular polyamine biosynthesis of neuroblastoma via ODC and its substrates was further confirmed in an ex vivo neuroblastoma cell model, with depletion of proline and arginine from the medium synergizing when ODC was inhibited (DFMO) or downregulated (via short hairpin RNA (shRNA)) (Supplementary Fig. 2). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Growth rescue was observed from natural and synthetic polyamines across neuroblastoma lines upon polyamine biosynthesis inhibition by DFMO or combined with proline and arginine depletion medium (Supplementary Fig. 2h–j). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Thus, dual dietary amino acid restriction depletes the key polyamine precursor ornithine and, combined with DFMO, leads to enhanced tumour polyamine depletion. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Polyamines stimulate translation and cell growth. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Arginine and proline also feed directly into translation as proteinogenic amino acids. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Combining proline and arginine depletion with DFMO in vitro enhanced translation inhibition by DFMO (Supplementary Fig. 3a–c). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | To disentangle the amino acid and polyamine effects of the ProArg-free diet and DFMO on translation, we carried out ribosome profiling (Ribo-seq) (Fig. 4a; quality control presented in Supplementary Fig. 3d–h). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | This approach identifies transcriptome-wide mRNA loading with ribosomes and which codons are decoded by ribosomes at the moment of cell lysis. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Increased ribosome density can indicate sites of stalled translation, due either to uncharged tRNAs or a defect in the translation machinery. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | With ProArg-free diet, DFMO or ProArg-free diet plus DFMO, ribosome occupancy was shifted slightly towards the start codon and decreased at early elongation of the protein-encoding transcript. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Exclusively under ProArg-free diet plus DFMO treatment, ribosomes accumulated at stop codons, indicating defective ribosome release (Supplementary Fig. 3i,j). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Such elongation and termination defects have been reported in cell models functionally deficient in eIF5A, a translation factor that is post-translationally modified by the polyamine spermidine. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | As this suggested in vivo eIF5A dysfunction, we probed eIF5A hypusination status (that is, spermidine modification of eIF5A) across treatment groups. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Whereas all tumours from the CD and ProArg-free groups without drug demonstrated complete eIF5A hypusination, reflecting sufficient spermidine for this purpose, two out of eight tumours treated with DFMO alone and five out of eight tumours from the ProArg-free diet plus DFMO group had reduced eIF5A hypusination and increased K47 eIF5A acetylation, indicative of attenuated hypusination. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | We also observed modestly reduced hypusination in these tumours using hypusine-specific antibodies (Extended Data Fig. 6a–e).Fig. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | 4Ribo-seq reveals defective decoding of codons with adenosine in the third position following polyamine depletion.a, For functional evaluation of translation, tumours were lysed in the presence of a translation inhibitor for preparation of RNA-seq and Ribo-seq libraries. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Ribosome-protected RNA fragments were isolated and sequenced to assess translation. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | b, Normalized ribosome depth at positions encoding three or more consecutive proline residues. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Decoding of these polyproline tracts is affected by combining DFMO (1% in the drinking water) with proline and arginine-free diet. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | c, Proline translation defects are codon-specific. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Relative ribosome density centred around proline codons across treatment groups relative to CD (zero line). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Left, density of ribosomes at polyproline tracts. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Right, codon occupancy on proline codons outside of polyproline tracts. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Increased occupancy manifests at CCA and less at CCC. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | d, Codons with adenosine in the third position show specific translation defects induced by the combined ProArg-free diet plus DFMO treatment compared with CD diet when comparing the transcriptome-wide relative ribosome occupancy. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Codons that require tRNAs with modifications at position 34 for decoding are highly enriched in codon pausing following ProArg-free diet plus DFMO treatment compared with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Relative pausing of codons in the ribosomal P site. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | GalQ, galactosyl-queuosine; I, inosine; manQ, mannosyl-queuosine; mchmU, 5-methoxycarbonyl-hydroxymethyluridine; mcmsU, 5-methoxycarbonylmethyl-2-thiouridine; ncmU, 5-carbamoylmethyluridine; Q, queuosine. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | e, Schematic showing two mechanisms of polyamine depletion therapy. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Only the combined treatment induces mild hallmarks of eIF5A hypusination deficiency and boosts the codon-specific translation defect induced by polyamine depletion. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | As described in Fig. 3, data in b–e are from the Th-MYCN mouse model. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | For all mean, n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Panels a and e created in BioRender. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Morscher, R. (2025) https://BioRender.com/75ofpvn (a); https://BioRender.com/iydko99 (e).Source data a, For functional evaluation of translation, tumours were lysed in the presence of a translation inhibitor for preparation of RNA-seq and Ribo-seq libraries. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Ribosome-protected RNA fragments were isolated and sequenced to assess translation. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | b, Normalized ribosome depth at positions encoding three or more consecutive proline residues. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Decoding of these polyproline tracts is affected by combining DFMO (1% in the drinking water) with proline and arginine-free diet. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | c, Proline translation defects are codon-specific. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Relative ribosome density centred around proline codons across treatment groups relative to CD (zero line). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Left, density of ribosomes at polyproline tracts. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Right, codon occupancy on proline codons outside of polyproline tracts. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Increased occupancy manifests at CCA and less at CCC. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | d, Codons with adenosine in the third position show specific translation defects induced by the combined ProArg-free diet plus DFMO treatment compared with CD diet when comparing the transcriptome-wide relative ribosome occupancy. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Codons that require tRNAs with modifications at position 34 for decoding are highly enriched in codon pausing following ProArg-free diet plus DFMO treatment compared with CD. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Relative pausing of codons in the ribosomal P site. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | GalQ, galactosyl-queuosine; I, inosine; manQ, mannosyl-queuosine; mchmU, 5-methoxycarbonyl-hydroxymethyluridine; mcmsU, 5-methoxycarbonylmethyl-2-thiouridine; ncmU, 5-carbamoylmethyluridine; Q, queuosine. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | e, Schematic showing two mechanisms of polyamine depletion therapy. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Only the combined treatment induces mild hallmarks of eIF5A hypusination deficiency and boosts the codon-specific translation defect induced by polyamine depletion. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | As described in Fig. 3, data in b–e are from the Th-MYCN mouse model. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | For all mean, n = 5. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Panels a and e created in BioRender. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Morscher, R. (2025) https://BioRender.com/75ofpvn (a); https://BioRender.com/iydko99 (e). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Source data In addition to its global functions, hypusinated eIF5A has been implicated in facilitating peptide bond formation involving repetitive instances of the amino acid proline, termed polyproline tracts. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Owing to its reactive amine localized within a ring structure, proline is a poor peptidyl acceptor. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | We thus evaluated relative ribosome occupancy at polyproline tracts, with high occupancy indicating slow decoding by ribosomes, due either to uncharged proline tRNA or to eIF5A deficiency. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Increased occupancy was observed at polyproline tracts in ProArg-free diet plus DFMO-treated tumours, but not at proline codons with ProArg-free diet alone (Fig. 4b). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Collectively, these data indicate that the combined ProArg-free diet and DFMO therapy impairs translation beyond tumour amino acid levels and tRNA charging by depleting spermidine to levels low enough to also impair eIF5A hypusination in more than half of the tumours. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | As the role of polyamine levels in ribosomal decoding at codon resolution is currently unknown, we next sought to differentiate its effects from eIF5A hypusination-related roles in translational reprogramming. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Further analysis of the Ribo-seq data revealed a surprising aspect of the stalling at polyproline tracts induced by combined ProArg-free diet and DFMO therapy: ribosome stalling was observed predominantly at only one of the four proline codons (CCA). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | There was less stalling at CCC and no stalling at CCG or CCT. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | This indicated an additional unanticipated level of translation regulation at the codon, rather than the amino acid level. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Extending the analysis to all proline codons (that is, including those that were not in polyproline tracts) revealed the same phenomenon: selected stalling at CCA codons (Fig. 4c). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | This phenotype contrasts with the genetic modulation of eIF5A hypusination, which preserves normal polyamine levels, yet affects all proline codons (Extended Data Fig. 6f,g). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Conversely, supplementing neuroblastoma cell lysates with polyamines in an in vitro translation assay preferentially facilitated the translation of CCA codons over CCG codons (Extended Data Fig. 6h,i). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Together, these results highlight an effect of polyamine depletion that is not amino acid-specific or recapitulated by genetic ablation of hypusination as the dominant driver of codon-specific translational reprogramming under ProArg-free diet plus DFMO treatment. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | We next assessed the global effect of combined ProArg-free diet plus DFMO treatment across individual codons at high resolution. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Across all amino acids, ribosome pausing was highly dependent on the codon type (as opposed to amino acid identity). |
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