Abstract B045: Breaking the Warburg code: RD3 redefines energetics in metastatic Neuroblastoma

Abstract Tumor cell metabolome intricately shapes its identity and therapeutic vulnerabilities. Cancer cells dynamically adapt to the tumor microenvironment (TME) – ‘Warburg effect’, a classic example of metabolic plasticity wherein cancer cells preferentially convert glucose to lactate despite availability of oxygen to thrive challenging environments. While metabolic profiling has advanced tumor biology, spatially resolved rewiring of metabolome remains poorly defined. Neuroblastoma (NB), a pediatric cancer with high propensity for hematogenous metastasis exhibits diverse underscored metabolic phenotypes. Our recent studies uniquely identified the predictive, prognostic relevance and function of Retinal Degeneration protein 3 (RD3) in NB pathogenesis including its role in shaping the tumor immune environment. Herein, we investigated whether and how RD3 affects metabolic plasticity in metastatic NB, offering new therapeutic avenues for high-risk (HR) NB. For this, global untargeted metabolomics was performed in bone marrow (BM) derived clones (CHLA-90, CHLA-140) from HR-NB patients and compared with their RD3 stably reinstated counterparts. Data preprocessing, mass detection, EIC building, feature resolving and grouping, ion identity networking and spectral library search was performed using Mzmine4.7 followed by compound identification using Compound discoverer. RD3-driven shift in metabolites were identified using univariate analysis with stringent criteria (log2fold change) coupled with false discovery rate (FDR, log10PadJ). Pathway enrichment analysis was acquired using MetaboAnalyst6.0. Functional validation of RD3-dependent metabolic shift was performed by assessing mitostress (Seahorse Xfe Analyzer) in RD3 expressing clone (CHLA-15) derived from primary (retroperitoneal) site, RD3-null CHLA-90 (BM) and compared with their RD3 silenced and expressed clones respectively. Compared to RD3-/- clones, we observed definitive conserved (cell-line independent) rearrangement of 289 metabolites (288, downregulated; 1, upregulated) in RD3+/+ clones. Predominantly, the metabolites were highly associated in amino acid metabolism (p= 6.61*10-3; FDR, 2.1*10-1) followed by fatty acid and steroid metabolism (p= 3.7*10-2; FDR, 3.02*10-1). Notably, metabolites involved in aerobic glycolysis (Warburg effect, Enrichment score, 1.953) were among the least active in RD3+/+ reinstated clones indicating a significant shift in cell energetics. Consistently, mitostress test further revealed that RD3-loss in HR-NB favors a glycolytic phenotype whereas RD3 expression promoted a more quiescent phase. Our findings explicitly demonstrate that RD3 expression in metastatic NB suppresses glycolysis and reprograms cellular metabolism. Furthermore, the conserved shifts in amino acid and fatty acid metabolism highlight RD3 as a potential metabolic gatekeeper. These RD3-driven alterations could unveil novel therapeutic vulnerabilities in HR-NB, offering new avenues for targeted intervention. Funding: This work was funded by the DoD CA-210339; OCAST-HR19-045; and the NIH P20GM103639 Citation Format: Poorvi Subramanian, Natarajan Aravindan, Sivaroopan Aravindan. Breaking the Warburg code: RD3 redefines energetics in metastatic Neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B045.