Gene editing in the mouse reveals functional mechanisms of ASPS-TFE3 induced translocation renal cell carcinoma

Translocation renal cell carcinoma (tRCC) is an aggressive form of kidney cancer that predominantly affects children and young adults. With no specific FDA-approved therapies, it remains an unmet clinical need. Patients often present with metastatic disease and median overall survival is less than two years. tRCC is driven by oncogenic fusion proteins involving transcription factors of the MiTF family, most commonly TFE3, yet the mechanisms by which these fusion proteins promote tumorigenesis remain poorly understood. To investigate tRCC tumorigenesis, we expressed human ASPS-TFE3, the most prevalent oncogenic fusion, in postnatal renal proximal tubule cells generating the first tRCC mouse model faithfully reproducing the human disease. These mice developed aggressive tRCC with complete penetrance and short latency. In addition, they also developed ASPS and PEComas, illustrating a shared pathogenesis that goes beyond MiTF fusion oncoproteins. Through integrated histologic, ultrastructural, transcriptomic, proteomic and functional analyses, we found that ASPS-TFE3 simultaneously activates mTORC1 signaling and lysosomal pathways. To dissect the role of ASPS-TFE3 in tumor initiation, we employed CRISPR-mediated gene editing. Structural modeling, mutagenesis, and localization studies identified a bHLH domain mutant [ASPS-TFE3(2RA)] that retains nuclear localization but fails to bind DNA and lacks transactivation activity. Unlike wild-type fusions, ASPS-TFE3(2RA) failed to induce tRCC, demonstrating that DNA binding is essential for tumorigenesis. Furthermore, its broader expression in renal epithelial cells compared to ASPS-TFE3 (despite the same driver) illustrates oncogene-induced protective mechanisms expanding our ex vivo studies. Interbreeding experiments coupled with phenotypic and functional studies revealed a context-dependent lineage-specific dominant-negative effect modulating tumor spectrum and latency. Together, these findings provide insight into ASPS-TFE3 tRCC pathogenesis and establish a genetically tractable platform to dissect fusion-driven oncogenesis and evaluate therapeutic strategies.