Abstract Background/Objective: Enhancers are critical cis-regulatory elements that modulate gene expression, and their dysregulation often drives oncogenic signaling in cancer. Epigenetic profiling in clear-cell Renal Cell Carcinoma (ccRCC) has demonstrated a prominent shift toward enhancer gain in tumors. We hypothesize that these tumor-specific gained enhancers are critical contributors to ccRCC progression. The goal of this study is to identify and functionally delineate the most critical oncogenic enhancers in ccRCC. Methods/Results: To test this, we first deployed a dCas9-KRAB repression screen targeting the top 250 gained enhancers in 786-O and A498 ccRCC cell lines. This screen uncovered a critical oncogenic enhancer, Enh2, whose inhibition significantly suppressed cell proliferation in both cell lines. Through bioinformatic analysis and functional validation, we identified PLXNA1 as the direct target gene of Enh2. Both dCas9-KRAB inhibition and CRISPR-Cas9 deletion of Enh2 resulted in a remarkable reduction of PLXNA1 expression, confirming a causal enhancer-gene relationship. To elucidate the regulatory mechanism by which Enh2 enhances PLXNA1 expression, we utilized the CRISPR affinity purification in situ of regulatory elements (CAPTURE) method to identify protein factors that bind to Enh2. CAPTURE successfully identified HDGF (Hepatoma-Derived Growth Factor) as the key transcription factor that specifically binds to Enh2 to drive PLXNA1 expression. Importantly, the PLXNA1 gene is highly overexpressed in human ccRCC tumors and correlates with poor patient survival. Functional validation confirmed that depletion of PLXNA1 by RNA interference significantly inhibited ccRCC cell growth and proliferation in vitro and in orthotopic mouse models. Conclusion: Our study reveals that PLXNA1 is a novel oncogenic gene driving ccRCC tumor growth, with its expression tightly controlled by the tumor-specific enhancer, Enh2. Mechanistically, the transcription factor HDGF positively regulates PLXNA1 expression by binding to Enh2. These findings define a novel HDGF/Enh2/PLXNA1 oncogenic signaling axis, providing a critical foundation for exploring PLXNA1 as a potential therapeutic target for ccRCC treatment. Citation Format: Tao Wang, Xiaosai Yao, Jeremy Simon, Tyler S. Klan, Charles A. Gersbach, Qing Zhang. CRISPRi screening reveals an enhancer-PLXNA1 axis as a critical regulator in ccRCC 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 B021.
Wang et al. (Fri,) studied this question.