Fueling Hope: Uncovering Metabolic Vulnerabilities in Childhood Cancer: Targeting Nucleotide Synthesis in Rhabdoid Tumors and Leukemia

Childhood cancer patients face major clinical challenges, including significant therapy-related side effects and limited treatment options for certain high-risk subgroups. In particular, patients with malignant rhabdoid tumors (MRT) or TP53-mutant B-cell precursor acute lymphoblastic leukemia (BCP-ALL) still have poor prognoses, highlighting the urgent need for more effective and less toxic therapies. One promising avenue is targeting cancer metabolism. Metabolic reprogramming is a hallmark of cancer and supports tumor growth and proliferation. Although inhibitors of metabolic enzymes are currently being developed as cancer therapeutics, the metabolic vulnerabilities of pediatric kidney cancers, especially MRT and TP53-mutant BCP-ALL, remain poorly understood. Metabolomics enables the comprehensive study of small-molecule metabolites in biological samples and has become an important approach to investigate disease biology. In addition to conventional metabolomics, stable-isotope tracing techniques allow researchers to study metabolic pathway activity, substrate utilization, and metabolic flux in greater detail. The overall aim of this thesis is to identify tumor-specific metabolic alterations that can be therapeutically targeted to improve outcomes for patients with MRT and BCP-ALL. In addition, this work seeks to characterize the in vivo metabolic environment of MRT in order to develop more physiologically relevant in vitro culture conditions, improving the predictive power of tumor models for metabolism-targeting therapies. Following a general introduction in Chapter 1, Chapter 2 describes a stable-isotope tracing protocol using LC-MS in 3D organoid models grown in Basement Membrane Extract (BME) or Matrigel. In the subsequent chapters, this approach is applied to patient-derived kidney tumoroids, including MRT models, and BCP-ALL cell lines to explore the metabolic landscape of these cancers. In Chapter 3, gene-expression and metabolite profiling of several pediatric kidney tumors, including Wilms tumor, renal cell carcinoma, and MRT, reveals tumor-type-specific metabolic differences. Notably, nucleotide biosynthesis emerges as a key vulnerability in MRT, as inhibition of de novo nucleotide synthesis induces apoptosis in vitro and delays tumor growth in vivo. In Chapter 4, dual inhibition of de novo pyrimidine synthesis and the DNA damage response pathway produces strong anti-leukemic effects in TP53-deficient BCP-ALL. Integrated metabolomic and transcriptomic analyses show that this treatment disrupts oxidative metabolism, increases oxidative stress, and induces ATF4-mediated, TP53-independent cell death. However, translating in vitro metabolic findings to in vivo settings remains challenging because conventional cell culture systems do not fully replicate the tumor microenvironment. Chapter 5 therefore examines how cancer models and culture conditions influence metabolic research and discusses strategies to create more physiologically relevant systems. Building on this, Chapter 6 characterizes the metabolic composition of plasma and tumor interstitial fluid in orthotopic MRT mouse models. These analyses reveal an enrichment of nucleosides and nucleobases in the tumor microenvironment. Culturing MRT cells in physiologic media or supplementing standard media with these metabolites shows that pyrimidines can rescue MRT cells from DHODH inhibitor-induced growth inhibition, a resistance mechanism that can be reversed by blocking nucleoside transport. Finally, Chapter 7 summarizes the findings of this thesis, places them in the context of current literature, and discusses their implications for future research and therapeutic development in pediatric cancer.