PMCID string | Title string | Sentences string |
|---|---|---|
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. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Source data Given the essential role of MYCN in the development and maintenance of neuroblastoma and its positive feedback loop with ODC1 and eIF5A, we explored whether the loss of MYC targets indicates a disruption of the core oncogenic regulatory circuit. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Both MYCN mRNA expression and MYCN protein were preferentially downregulated in tumours under combined ProArg-free diet plus DFMO treatment (Fig. 6b and Extended Data Fig. 9c). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Other core transcription factors were also suppressed, supporting a broad disruption of the MYCN-driven core regulatory circuit (Extended Data Fig. 9d,e). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Despite MYCN downregulation, its transcriptional targets in the polyamine pathway remained unchanged or were upregulated in response to polyamine depletion in vivo (Extended Data Fig. 9f–h) or pharmacological inhibition in vitro (Extended Data Fig. 9i). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Further, TP53 expression remained unaffected on the protein or ribosome level (Extended Data Fig. 9j,k). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | On the mechanistic level, modulating MYC or MYCN activity by genetic or pharmacological means showed no pausing of ribosomes at codons with adenosine in the third position, suggesting a MYC-independent translation phenotype as a driver (Supplementary Fig. 7a–h). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Evaluating tumour differentiation status according to clinical pathological criteria showed that tumours in the CD and ProArg-free groups were uniformly undifferentiated (<5% cytologic differentiated with absent neuropil). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | By contrast, we observed a strong differentiation phenotype upon polyamine-depleting treatment, with one-third of CD plus DFMO-treated tumours being differentiated (more than 5% cytologic differentiated with absent neuropil) and two thirds of ProArg-free diet plus DFMO-treated tumours differentiating or partially differentiating with abundant neuropil (a feature of neural differentiation; Fig. 6c and Extended Data Fig. 10a). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Thus, combining a ProArg-free diet with DFMO therapy led to marked reductions in polyamines, inducing selective translation defects that suppressed tumour cell proliferation and induced tumour differentiation (Fig. 6d). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The therapeutic relevance of combining dietary amino acid depletion with DFMO was further emphasized in a model using established patient-derived neuroblastoma cell line xenografts in mice (Extended Data Fig. 10b–e). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Replicating the genetic model, long-term survival beyond 100 days and apparent cures were observed in one-quarter of the ProArg-free diet plus DFMO-treated mice and treatment was well tolerated without weight changes (Extended Data Fig. 10f,g). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Whereas on the gene-expression level MYCN and ODC1 were unchanged, MYCN protein expression was decreased independently of ODC1 (Extended Data Fig. 10h,i). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Mechanistically decreased proliferation was confirmed by histology, along with a pro-differentiation phenotype (Extended Data Fig. 10j,k), highlighting the role of enhanced polyamine depletion inducing differentiation for treatment of neuroblastoma. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Here we find that neuroblastoma, a highly malignant childhood cancer, is vulnerable to polyamine depletion achieved by the combination of a diet free of proline and arginine to deplete the polyamine precursor ornithine, and pharmacological inhibition of ODC, the committed step of polyamine synthesis, with high-dose DFMO. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The diet plus drug combination markedly enhanced polyamine depletion and exerted a strong anti-cancer effect in a highly lethal transgenic mouse neuroblastoma model, and a human neuroblastoma mouse xenograft model, with durable complete responses in both. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Use of defined diet and drug combinations is emerging as a clinically viable strategy for cancer treatment. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Ketogenic diet can synergize with classical chemotherapy and targeted agents, and can be achieved, with proper support, by patients making careful food choices (NCT05300048 and NCT01535911). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Diets lacking certain amino acids require laboratory formulation, but show acceptable taste and desired metabolic effects in humans, and are also entering cancer efficacy trials (NCT05078775). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Such diets show effects resembling enzyme-based treatments that catabolize particular amino acids, such as asparaginase, a long-standing standard of care for treating childhood leukaemias. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | In some cases, however, diets have important advantages. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | For example, dietary arginine depletion decreases both arginine and ornithine, whereas arginase therapy depletes arginine by converting it into the polyamine precursor ornithine. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Conversely, refeeding has been shown to induce polyamine biosynthesis in intestinal stem cells, triggering tumorigenicity. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Thus, preventing intestinal interconversion of substrates through a dietary approach or alternative interventions suitable for achieving ornithine depletion offer an effective combination with DFMO to deplete polyamines. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Arrested cellular differentiation by retained embryonal gene-expression circuits is a hallmark of paediatric cancers. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Neuroblastoma is a prime example, with hyperactive MYCN driving embryonal programmes. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Induction of differentiation through transcriptional reprogramming is a validated therapeutic strategy, exemplified by the nuclear hormone receptor agonist retinoic acid. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Here we provide evidence for the feasibility of triggering differentiation in paediatric cancers through proteome reprogramming. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Specifically, we find that polyamine depletion promotes the translation of pro-differentiation proteins and suppresses that of cell cycle proteins, leading to neuronal differentiation of neuroblastoma. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Whether similar benefits could be achieved in other MYC-driven cancers merits investigation. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The selective effect of polyamine depletion on translation of certain genes was unexpected, and was not driven by genetic ablation of eIF5A hypusination. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The underlying biochemistry involves polyamine deficiency shifting codon optimality by impairing translation of codons with adenosine at the wobble base by position 34-modified tRNAs, and codons being thus preferentially enriched (or depleted) in different gene sets. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | This mechanism raises the possibility that codon usage has evolved in tandem with metabolism, such that metabolic limitation acts on translation of specific codons to rewire the proteome. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Specifically, our data point to polyamine depletion suppressing proliferation and promoting differentiation via utilization of codons with adenosine in the third position. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | This programme may have evolved to support proper developmental decisions, but using diet and pharmacology, it has therapeutic potential in treating neuroblastoma. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Flash-frozen primary neuroblastoma tumour samples were provided by the Children’s Oncology Group (COG) under study number ANBL16B2 Q. International Neuroblastoma Pathology Classification histologic parameters, MYCN amplification status, age and stage for every patient was obtained centrally via the COG Statistics and Data Center. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Tumour cell content of samples was confirmed over 80% percent. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Patient and tumour characteristics are given in Supplementary Table 1. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Water-soluble metabolites were extracted and analysed as described below. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Animal studies followed protocols approved by Princeton University and Children’s Hospital of Philadelphia Institutional Animal Care and Use Committees. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | For xenografts used for metabolomics, cancer cell lines were grown in RPMI supplemented with 10% FBS and 0.01% insulin/transferrin solution. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Cell lines were provided by the COG Cell Culture Repository: LA-N-5, SMS-SAN, CHLA-90 and SK-N-SH. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | All cell lines repeatedly tested negative for Mycoplasma. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Subcutaneous xenografts were established on 6-week-old female CD1-nu mice by injection of 100 μl 50/50 RPMI/Matrigel solution containing 10 cells of the respective cell line. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | For xenografts used in therapeutic trials, tumours were established on 4- to 6-week-old female NCr-nu mice (Charles River) by injection of 100 μl 50/50 RPMI/Matrigel solution containing 3 × 10 IMR5 cells (MYCN amplified, ALK amplified). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Mice were randomized to specific treatment when tumours were ≥200 mm. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Mice were sacrificed when tumours were 2,000 mm. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Tumour mass was inferred using tumour volume using volume to mass of xenograft conversion described in McLean et al.. The Th-MYCN mouse model was used to investigate the functional changes of metabolism driven by MYCN. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | 129×1/SvJ mice transgenic for the Th-MYCN construct were originally obtained from B. Weiss. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Th-MYCN hemizygous mice were bred and litters randomized to assigned therapy. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Mice were genotyped from tail-snip-isolated DNA using quantitative PCR and only transgene-homozygous mice (Th-MYCN) were included in these studies. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | In this model, MYCN expression is targeted to the mouse neural crest under the tyrosine hydroxylase promoter, recapitulating hallmark features of human neuroblastoma. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Tumours arise at autochthonous sites in an immunocompetent host with histologic, genomic, and immune similarities to human neuroblastoma. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Tumours are fully penetrant with onset prior to day 14 in >75% based on histologic audits and are lethal by 7 weeks. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Mice were maintained with 12 h of dark daily (18:00 to 06:00). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The rodent holding rooms were maintained at a temperature range of 18.9 °C–25.6 °C with an ideal setpoint of 22.2 °C. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The humidity was maintained within a range of 30–70% with an ideal setpoint of 50%. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Tissues and tumours were collected from mice in fed state and immediately clamped into liquid nitrogen using Wollenberger clamp. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | All tissues were stored in −80 °C. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Frozen tissues were transferred to 2 ml Eppendorf tubes, which were precooled on dry ice, and then pulverized using Cyromill. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The resulting tissue powder was weighed (around 10 mg) and mixed well by vortexing in extraction buffer (40 μl extraction buffer per mg tissue). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The extraction solution was neutralized with NH4HCO3 as above and centrifuged in a microfuge at maximum speed for 30 min at 4 °C. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Supernatant was transferred to LC–MS vials for analysis. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Blood samples were drawn from mouse tail veins using a microvette and kept on ice. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | After centrifugation (10 min, benchtop microfuge maximum speed, 4 °C), serum was collected in a 1.5 ml tube and stored at −80 °C. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Five microlitres of serum were mixed with 200 μl extraction buffer (40:40:20 acetonitrile: methanol: water with 0.5% formic acid) and neutralized with 15% NH4HCO3. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | After centrifugation (30 min, benchtop microfuge maximum speed, 4 °C), supernatant was transferred to LC–MS vials for analysis. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Metabolomics was performed on the following systems. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | A quadrupole-orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific), operating in positive or negative mode was coupled to hydrophilic interaction chromatography (HILIC) via electrospray ionization. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Scans were performed from m/z 70 to 1,000 at 1 Hz and 140,000 resolution. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Liquid chromatography separation was on a XBridge BEH Amide column using a gradient of solvent A (20 mM ammonium acetate, 20 mM ammounium hydroxide in 95:5 water:acetonitrile, pH 9.45) and solvent B (acetonitrile). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Flow rate was 150 μl min. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The liquid chromatography gradient was: 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B. Autosampler temperature was 5 °C, and injection volume was 5–10 μl. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Complementary, primary samples analysed on an Exactive (Thermo Fisher Scientifc) operating in negative ion mode. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Liquid chromatography separation was achieved on a Synergy Hydro-RP column (100 mm × 2 mm, 2.5 μm particle size, Phenomenex), using reversed-phase chromatography with the ion pairing agent tributylamine in the aqueous mobile phase to enhance retention and separation. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | An adaptive scan range was used with an m/z from 85–1,000. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Resolution was 100,000 at 1 Hz. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The total run time was 25 min with a flow rate at 200 μl min. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Solvent A is 97:3 water/methanol with 10 mM tributylamine and 15 mM acetic acid; solvent B is methanol. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The gradient is 0 min, 0% B; 2.5 min, 0% B; 5 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; 25 min, 0% B. Metabolomics data analysis was performed using ElMaven software (https://github.com/ElucidataInc/ElMaven). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | For labelling experiments, correction for natural abundance of C was performed using Accucor (https://github.com/XiaoyangSu/AccuCor). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Th-MYCN mice were housed in groups and food was supplied without restriction to guarantee sufficient supply. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Mice weights were recorded every day. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | During experiments mice were freely moving and tissues and serum were analysed following the above-mentioned method. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Tumour and inter organ cooperativity in proline, arginine and ornithine biosynthesis was analysed on the whole-body level. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The mice were on normal light cycle (06:00–18:00). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | In vivo infusion was performed on 6- to 7-week-old normal Th-MYCN mice pre-catheterized in the right jugular vein and C metabolite tracers were infused for 2.5–5 h to achieve isotopic pseudo-steady state. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | The mouse infusion setup included a tether and swivel system, connecting to the button pre-implanted under the back skin of mice. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Mice were fasted from 09:00 to 14:00 and infused from 14:00 to 16:30. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Tracers were dissolved in saline and infused via the catheter at a constant rate (0.1 μl min per g mouse weight) using a Just infusion Syringe Pump. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | One-hundred millimolar [U-C]glutamine was dissolved and infused for 2.5 h, 40 mM [U-C]arginine was infused for 5 h, 200 mM [U-C]glucose was infused for 5 h, 10 mM [U-C]proline for was infused 5 h and 5 mM [U-C]ornithine was infused for 5 h. At the end of infusion, mice were dissected and tissues were clamped in aluminium foil and stored in liquid nitrogen. |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | ArgPro-free diet was purchased from TestDiet Baker (1812426 (5CC7) for CD, 1816284-203 (5WYF) for ProArg-free diet, 1819015-203 (5WZ3) for arginine-free diet and 1816284-203 (5BDL) for proline-free diet). |
PMC12527938 | Reprogramming neuroblastoma by diet-enhanced polyamine depletion | Detailed makeup is given in Supplementary Table 2. |
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