67Generating syngeneic NF2 models of unclassified renal cell carcinoma

Background Renal cell carcinoma (RCC) comprises of several histological subtypes, with clear cell RCC (∼75%), papillary RCC (∼15%), and chromophobe RCC (∼5%) being the most prevalent. Unclassified RCC (uRCC) accounts for 4–5% of cases that do not align with these major subtypes and is typically aggressive with recurrent mutations in tumor suppressor genes such as NF2, SETD2, CDKN2A, and BAP1. A subset of uRCC defined by NF2 loss exhibits dysregulation of the Hippo–YAP pathway and is associated with poor clinical outcomes. NF2-deficient RCC show limited response to standard therapies, including immune checkpoint inhibitors and VEGFR tyrosine kinase inhibitors. Prior studies using CRISPR-Cas9–based genetically engineered mouse models (GEMMs) have demonstrated that combined loss of CDKN2A/B, NF2, and SETD2 drives tumorigenesis and metastasis. However, the immunologic landscape and therapeutic vulnerabilities of NF2-mutant RCC remain largely uncharacterized. Methods To address this, we developed a versatile and transplantable organoid system on a pure C57BL/6 (B6) background that enables rapid genetic manipulation and faithfully models NF2-deficient RCC. LTL+ proximal tubule cells were isolated from microdissected B6 mouse kidney cortex via flow sorting and used to generate kidney organoids. Using RNP–based CRISPR editing, we engineered sequential knockout lines: CDKN2A/B, CDKN2A/B + SetD2, CDKN2A/B +SetD2 +NF2 with and without c-myc. Bulk–RNA sequencing were performed to confirm the cell of origin. Organoids were orthotopically injected into immunocompetent B6 mice to assess for tumor formation in immunocompetent hosts. In parallel, Ex vivo and in-vivo CRISPR–screens are underway to identify therapeutic vulnerabiltiies in the context of NF2 loss. Results Wild–type B6 kidney organoids demonstrated robust expression of proximal tubule markers and established their transcriptomic fidelity. Efficient gene targeting in organoids was confirmed using CRISPRseq and western blot. Mutant organoid lines exhibited accelerated growth rates compared to WT controls. Bulk RNA–sequencing of mutant organoid demonstrated NF2 and Setd2 specific transcriptional perturbations. Orthotopically injected organoids are being monitored longitudally for tumor formation via ultrasound imaging. Conclusions We have established a tractable, immunocompetent, and transplantable murine organoid-based model of RCC harboring clinically relevant tumor suppressor mutations. This platform enables high-throughput interrogation of NF2-deficient RCC and offers a foundation for discovery of genotype-specific therapeutic targets. Ongoing work includes the completion of customized CRISPR dropout screens—guided by bulk RNA-seq data from both murine organoids and human tumors—to uncover lineage- and mutation-specific dependencies. Hits from these screens will inform downstream therapeutic strategies and be validated through histologic, immunohistochemical, and tumor microenvironmental analyses of harvested tumors and derived organoids.