Abstract 6102: <i>In vivo</i> CRISPR screening of chromatin regulators reveals p53-dependent drivers of lung metastasis in esophageal cancer

Abstract Metastatic esophageal cancer exhibits a strong predilection for dissemination to the lungs. Lung metastases are associated with poor survival outcomes, therapeutic resistance, and limited treatment options, underscoring the urgent need to identify targetable mechanisms driving lung colonization. Yet, these molecular mechanisms driving lung-specific tropism remain poorly understood, which serves as the basis for our novel approaches. Herein, we employed state-of-the-art in vivo CRISPR-Cas9 knockout screens using a sgRNA library targeting chromatin regulators to identify epigenetic modulators of lung metastasis in esophageal cancer. This screen targeted 600 genes with a pooled sgRNA library enriched for epigenetic regulators and chromatin remodelers. The library, containing 6 sgRNAs per gene and appropriate non-targeting controls, was transduced into isogenic cells with either mutant Trp53R172H/- (a frequently detected hotspot Trp53 mutation in the DNA binding domain) or its depletion at a low multiplicity of infection to ensure single sgRNA integration per cell. The transduced cells were injected orthotopically or via tail-vein into mice, and comparative abundance analysis was performed between the pre-implantation pool and lung metastatic lesions. Our screen uncovered key chromatin regulators that selectively promote lung colonization in the presence of mutant p53, including histone methyltransferases and demethylases (Kmt2d, Kdm1b, Kdm4d), histone deacetylases (Hdac4), and additional DNA and chromatin modifiers (Eya2, Prmt8, Parp14, Tox4, Eny2, and Gata2a). To elucidate the mechanisms by which these epigenetic regulators contribute to metastatic potential, we are performing comprehensive histone methylation and acetylation profiling in the corresponding KO clones, in combination with ATAC-seq and RNA-seq. This integrative approach will enable us to correlate histone modification landscapes with chromatin accessibility and gene expression programs, delineating how mutant p53 cooperates with specific chromatin regulators to reprogram enhancer networks and drive pro-metastatic transcriptional states. Collectively, these studies will define epigenetic mechanisms underlying lung tropism in esophageal cancer and identify actionable vulnerabilities for therapeutic targeting, with broader implications for TP53-mutant cancers. Citation Format: Raul Navaridas, Gizem Efe, Ali Iftikhar, Karen J. Dunbar, Katherine Cunningham, Emily Esquea, Noriyuki Noriyuki, Constanza Tapia Contreras, Alice E. Shin, Francisco J. Sánchez-Rivera, Chao Lu, Anil K. Rustgi. In vivo CRISPR screening of chromatin regulators reveals p53-dependent drivers of lung metastasis in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6102.