Hepatoblastoma, the most prevalent pediatric liver cancer, almost always carries a WNT-activating CTNNB1 mutation, yet exhibits notable molecular heterogeneity. To characterize this heterogeneity and identify novel targeted therapies, we perform comprehensive analysis of hepatoblastomas and tumor-derived organoids using single-cell RNA-seq/ATAC-seq, spatial transcriptomics, and high-throughput drug profiling. We identify two distinct tumor epithelial signatures: hepatic ‘fetal’ and WNT-high ‘embryonal’, displaying divergent WNT signaling patterns. The fetal group is enriched for liver-specific WNT targets, while the embryonal group is enriched in canonical WNT target genes. Gene regulatory network analysis reveals enrichment of regulons related to hepatic functions such as bile acid, lipid and xenobiotic metabolism in the fetal subtype but not in the embryonal subtype. In addition, the dichotomous expression pattern of the transcription factors HNF4A and LEF1 allows for a clear distinction between the fetal and embryonal tumor cells. We also perform high-throughput drug screening using patient-derived tumor organoids and identify sensitivity to HDAC inhibitors. Intriguingly, embryonal and fetal tumor organoids are sensitive to FGFR and EGFR inhibitors, respectively, indicating a dependency on EGF/FGF signaling in hepatoblastoma tumorigenesis. In summary, our data uncover the molecular and drug sensitivity landscapes of hepatoblastoma and pave the way for the development of targeted therapies.

肝母细胞瘤是最常见的儿科肝癌，几乎总是携带 WNT 激活的 CTNNB1 突变，但表现出显着的分子异质性。为了表征这种异质性并确定新的靶向疗法，我们使用单细胞 RNA-seq/ATAC-seq、空间转录组学和高通量药物分析对肝母细胞瘤和肿瘤衍生的类器官进行全面分析。我们确定了两个不同的肿瘤上皮特征：肝脏“胎儿”和 WNT 高“胚胎”，显示出不同的 WNT 信号模式。胎儿组富集肝脏特异性 WNT 靶标，而胚胎组富集经典 WNT 靶基因。基因调控网络分析显示，在胎儿亚型中，与胆汁酸、脂质和外源性代谢等肝功能相关的调节子富集，但在胚胎亚型中则不然。此外，转录因子 HNF4A 和 LEF1 的二分类表达模式允许明确区分胎儿和胚胎肿瘤细胞。我们还使用患者来源的肿瘤类器官进行高通量药物筛选，并确定对 HDAC 抑制剂的敏感性。有趣的是，胚胎和胎儿肿瘤类器官分别对 FGFR 和 EGFR 抑制剂敏感，表明肝母细胞瘤肿瘤发生依赖于 EGF/FGF 信号传导。总之，我们的数据揭示了肝母细胞瘤的分子和药物敏感性景观，并为靶向治疗的开发铺平了道路。