Abstract 4065: Unveiling drivers of esophageal adenocarcinoma with CRISPR pooled screen and single-cell RNA-sequencing
Authors: Ebtihal H. Mustafa, Julia Milne, Ka Meng Wu, Katherine Papastratos, Niko Thio, Wayne Phillips, Nicholas Clemons
Journal: Cancer Research (2025)

Abstract Esophageal adenocarcinoma (EAC) is an aggressive malignancy with a rising incidence and limited treatment options. It develops from Barrett’s esophagus, a pre-cancerous metaplastic condition, yet the molecular mechanisms driving its progression remain poorly understood. Genomic studies have identified ∼76 candidate genes as potential drivers of EAC. In our previous work, we demonstrated that in the context of mutant TP53, the inactivation of one such candidate tumour suppressor, SMAD4, was sufficient to drive tumorigenesis in non-tumorigenic CP-B Barrett’s esophagus xenografts. Building on this, we combined our EAC tumorigenesis model with Perturb-Seq (CRISPR-mediated gene editing coupled with single-cell RNA sequencing (scRNA-Seq)) to enable high-throughput functional characterization of EAC driver genes. The study specifically aimed to: (i) identify the molecular perturbations driving tumorigenesis in EAC, (ii) understand the transcriptional changes underlying the oncogenic effects of these perturbations, and (iii) tackle the genetic heterogeneity of EAC by categorizing driver genes according to shared transcriptional phenotypes. In vivo loss-of-function CRISPR screen was conducted using a boutique pooled CRISPR/Cas9 sgRNA library (6 sgRNAs per gene) targeting 57 known and putative tumour suppressor genes. To analyze the gene knockout-specific effects on the transcriptome at single-cell resolution, capture sequences compatible with 10X Feature Barcoding were incorporated into the sgRNAs. The library was transduced into two independent esophageal pre-malignant cell models, CP-B and CP-D, followed by xenotransplantation in NSG immunodeficient mice. We established that the knockout of 40 out of the 57 candidate tumour suppressors were independently sufficient to induce tumour formation in vivo. By integrating the scRNA-Seq data corresponding to each tumour suppressor knockout, we found that depletion of these tumour suppressor genes inhibited the proteasomal degradation of oncogenes and anti-apoptotic proteins, such as cyclin E, c-Myc, and Mcl-1. Additionally, this depletion led to the upregulation of genes involved in cell cycle progression, DNA repair, and lipid and carbohydrate metabolism. Perturb-seq also showed that driver-dependent transcriptional changes can be categorized into a smaller number of functional pathways allowing us to potentially consider groups of drivers as functional units. Collectively, the findings of this study enhance our understanding of the molecular events driving EAC tumorigenesis and offer a comprehensive view of the transcriptional phenotypes that can be targeted in future research. These insights lay the foundation for the development of novel therapeutic strategies for this highly heterogeneous disease. Citation Format: Ebtihal H. Mustafa, Julia Milne, Ka Meng Wu, Katherine Papastratos, Niko Thio, Wayne Phillips, Nicholas Clemons. Unveiling drivers of esophageal adenocarcinoma with CRISPR pooled screen and single-cell RNA-sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4065.