Metabolic Profiling Reveals Nucleotide Synthesis as a Metabolic Vulnerability in Malignant Rhabdoid Tumors

Tumor Metabolic Reprogramming Identifies Key Vulnerability in Pediatric Malignant Tumors – Comprehensive Research Based on Organoid Models

Background

Malignant Rhabdoid Tumors (MRT) are highly invasive pediatric cancers that primarily affect infants and can develop throughout the body, with the intracranial variant known as Atypical Teratoid/Rhabdoid Tumors (AT/RT). Despite current treatments, including intensive chemotherapy, radiotherapy, and surgical interventions, prognosis for localized or metastatic cases remains exceedingly poor, highlighting the urgent need for new therapeutic strategies. Additionally, metabolic reprogramming is regarded as one of the defining hallmarks of cancer. Targeting the metabolic vulnerabilities of tumors may provide insights into treating these refractory neoplasms. However, the complex metabolic characteristics and potential vulnerabilities in pediatric kidney tumors, especially MRT and AT/RT, remain largely unexplored.

To address this, the authors of this study aimed to use organoid technologies (Tumoroids) to analyze pediatric kidney tumors, particularly MRT. As organoids can faithfully retain the genetic, phenotypic, and metabolic characteristics of patient-derived tissues in culture, they are considered more representative models for disease research. By combining gene expression analysis and metabolomics, the research team identified nucleotide synthesis as a specific metabolic vulnerability in MRT and further validated the efficacy of both novel and approved drugs in vitro and in vivo on MRT models.


Study Source

This study was conducted under the leadership of Marjolein M.G. Kes, Francisco Morales-Rodriguez, and collaborators. The primary institutions involved include the Princess Máxima Center for Pediatric Oncology and Utrecht University, both located in Utrecht, the Netherlands. The study, titled “Metabolic profiling of patient-derived organoids reveals nucleotide synthesis as a metabolic vulnerability in malignant rhabdoid tumors,” was published in Cell Reports Medicine on January 21, 2025.


Research Process

Design and Methodology

  1. Metabolic Gene Expression Profiling

    • Using mRNA sequencing, the team analyzed tumor tissues and matching organoids (45 samples in total, including Wilms tumor, RCC, and MRT subtypes) to identify metabolic pathways associated with MRT.
    • Principal Component Analysis (PCA) and Gene Ontology (GO) enrichment analysis were used.
  2. Metabolomics Analysis

    • LC-MS (Liquid Chromatography-Mass Spectrometry) metabolomics were performed on MRT organoids, Wilms tumor organoids, and normal kidney-derived organoids.
    • Purine (e.g., IMP, GMP) and pyrimidine (e.g., UMP) metabolic changes were highlighted.
  3. Sensitivity Testing of Nucleotide Synthesis Inhibitors

    • Drugs such as Methotrexate (MTX) and Bay-2402234 (Bay) were tested on a range of tumor organoid models, including MRT, AT/RT, Rhabdomyosarcoma (RMS), and normal kidney organoids.
    • Annexin V/DAPI flow cytometry was employed to verify drug-induced apoptosis.
    • IC50 values and rescue experiments using nucleoside and folinic acid supplementation were performed to confirm the mechanisms.
  4. Isotope Tracing Experiments

    • [13C]-labeled glucose was administered to track the metabolic flux and assess changes in purine and pyrimidine nucleotide synthesis pathways.
    • Key metabolic intermediates and isotopologue fractions were quantified.
  5. In Vivo Validation (PDX Models)

    • MRT patient-derived xenografts (PDX) models were established in mice.
    • Tumor dynamics and expression of the proliferation marker Ki67 were analyzed following MTX treatment under folic acid-depleted dietary conditions.

Research Findings

  1. MRT-Specific Metabolic Features

    • PCA revealed significant metabolic differences between the three major kidney tumor types. MRT samples formed a distinct cluster, separate from Wilms tumor and RCC.
    • GO enrichment analysis showed significant upregulation of purine and pyrimidine nucleotide biosynthesis pathways in MRT.
  2. Unique Nucleotide Metabolism

    • LC-MS revealed significantly elevated levels of purine (e.g., IMP, GMP, AMP) and pyrimidine (e.g., UMP, CMP) metabolites in MRT organoids compared to normal kidney-derived organoids.
    • In contrast, Wilms tumor organoids exhibited increased tricarboxylic acid (TCA) cycle metabolites.
  3. Drug Sensitivity

    • Both MTX and Bay demonstrated remarkable efficacy against MRT and AT/RT, with IC50 values in the nanomolar range. Normal kidney organoids and Wilms tumor organoids showed relatively reduced sensitivity.
    • Flow cytometry showed significant induction of apoptosis by MTX (2.7-fold) and Bay (2.5-fold) in MRT organoids.
  4. Isotope Tracing

    • MRT organoids exhibited higher [13C] incorporation into nucleotides, particularly advanced isotopologues (e.g., [m+6]), indicating elevated de novo nucleotide biosynthesis activity.
    • Both MTX and Bay disrupted these pathways, leading to reductions in nucleotide levels.
  5. In Vivo Validation

    • In PDX mouse models, MTX treatments significantly delayed tumor growth and decreased Ki67 expression in MRT tumors.
    • MTX treatment led to >20% weight loss in some mice, indicating potential toxicity with prolonged use.

Conclusions and Implications

This study uncovered a unique dependency on nucleotide biosynthesis in MRT, providing a novel therapeutic target for these aggressive pediatric tumors. Methotrexate (MTX), an already-approved clinical drug, displayed strong potential as a treatment option, raising the possibility of integrating it into existing regimens to improve patient survival. Additionally, Bay-2402234, with its high metabolic specificity, offers an alternative candidate with potentially better safety profiles.


Key Highlights and Contributions

  1. Identification of Metabolic Vulnerabilities
    This study is among the first to precisely map the metabolic landscape of pediatric solid tumors, identifying nucleotide dependence as a critical vulnerability in MRT.

  2. Validation of Drug Efficacy
    The study highlights the potential of MTX and Bay in targeting purine and pyrimidine synthesis pathways, supporting their utility in precision oncology.

  3. Clinical Translational Prospects
    With strong in vivo validation and pre-existing clinical safety data for these drugs, the study opens pathways for their potential application in treating fatal pediatric tumors.


Limitations and Future Directions

The study acknowledged that the efficacy of MTX may be reduced when exogenous nucleotide supply is sufficient, emphasizing the need for future validations under physiological nutrient concentrations. Additionally, further research into combination therapies and resistance mechanisms is warranted. Overall, this research paves the way for deeper insights and innovative treatments for pediatric cancers.