Multi-omics analysis identifies stemness-driven molecular subtypes, prognostic signature, epigenetic target APCDD1, and drug candidate Leflunomide in Wilms tumor

Wilms tumor (WT), the most common pediatric renal malignancy, continues to present therapeutic challenges in refractory and relapsed patients. Stemness plays a crucial role in the development and progression of WT, yet the specific mechanisms involved are not yet fully understood. This study systematically investigated the molecular basis of stemness features in WT using multi-omics data and computational biology approaches. Single-cell RNA sequencing combined with the CytoTRACE algorithm revealed that the blastemal cells exhibited the highest stemness score, which correlated significantly with worse prognosis in WT patients. Consensus clustering based on prognostic stemness-related genes stratified WT samples into two distinct molecular subtypes (C1 and C2). The C1 subtype exhibited higher stemness, an immunosuppressive tumor microenvironment characterized by dysfunctional NK cells, and significantly worse clinical outcomes. We subsequently developed and validated a robust prognostic risk signature using Lasso-Cox regression. Mechanistic investigations uncovered that the tumor-suppressive effect of APCDD1 was mediated by promoter hypermethylation, and its expression could be restored by the demethylating agent Decitabine. Two-sample Mendelian randomization analysis indicated that elevated APCDD1 expression had a protective causal effect on WT susceptibility. Through integrative drug repositioning analysis, Leflunomide was identified as a promising candidate for high-risk WT patients. In vitro functional assays suggested that Leflunomide potently inhibited the proliferation, migration, and invasion of WT cells, and induced apoptosis in a dose-dependent manner, as validated by CCK-8, EdU, wound healing and Transwell assays, and flow cytometry with Annexin V/PI staining, respectively. In conclusion, this study proposes a preliminary discovery framework for the stemness-driven molecular landscape for WT, defining relevant subtypes, a prognostic signature, and an epigenetic regulator ( APCDD1 ). We further propose Leflunomide as a novel therapeutic strategy for high-risk WT patients. These findings advance our understanding of WT biology and provide actionable insights for risk stratification and targeted intervention.