Abstract Renal cell carcinomas (RCCs), which account for about 80% of kidney cancers, comprise diverse subtypes with variable clinical prognoses. Renal medullary carcinoma (RMC) is a rare, but highly aggressive RCC subtype that accounts for 0. 5% of cases and lacks effective therapies. Although metabolic rewiring represents a shared feature across most RCCs, the metabolic plasticity of these tumors poses a challenge for exploiting these changes for cancer therapeutics. Systematic approaches are therefore needed to identify potential therapeutic targets. To address this, we utilize high-throughput functional genomic screening to identify genetic dependencies in patient-derived RCC models. CRISPR knockout screens identified distinct metabolic pathways whose perturbation reduced cell fitness in RMC, highlighting potential therapeutic applications. Additionally, these screens uncovered novel tumor suppressors genes, whose loss increases cell proliferation. To ensure cancer-specificity of our findings, parallel genetic screens in non-transformed kidney cells are critical to evaluate the safety and selectivity of candidate therapeutic targets. Here, we aimed to establish our dual-nuclease combinatorial screening platform, dubbed CHyMErA, in Primary Renal Proximal Tubule Epithelial (RPTEC) and Human Kidney-2 (HK-2) cells. CHyMErA enables highly efficient gene perturbation through co-expression of Cas9 and Cas12a nucleases together with hybrid guide (hg) RNAs, allowing coordinated dual-targeting of individual genes. RPTEC and HK-2 cells were transduced with lentiviral constructs expressing either catalytically active nucleases or nuclease-dead variants fused to a transcriptional repression domain (CRISPRi), enabling gene knockout and transcriptional interference approaches. Expression of the Cas proteins was confirmed by western blotting, and combinatorial editing efficiency was assessed by targeting the expression of cell surface proteins, with genome editing quantified via flow cytometry. We successfully verified expression of Cas9 and Cas12a variants in both RPTEC and HK-2 lines. However, flow cytometry analysis revealed that RPTECs exhibit low editing efficiency, suggesting a possible sensitivity to DNA damage. Consequently, current efforts in RPTEC are focused on optimizing the transcriptional repression systems, while both knockout and CRISPRi-based CHyMErA systems are being explored in HK-2 cells. In summary, we established the CHyMErA screening platform in non-transformed kidney cell models, providing a foundation for systematic combinatorial genetic screening. Future genome-wide screens in these models will facilitate the identification of general kidney tissue dependencies and the validation of RMC-specific vulnerabilities, paving the way to uncovering potential targets for RCC therapeutic interventions. Citation Format: Olga Drozdovitch, Chelsee Holloway, Michael Aregger. Establishing a combinatorial genetic screening platform to uncover cancer-specific dependencies in renal medullary carcinoma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₂): Abstract nr B016.
Drozdovitch et al. (2026) studied this question.