Mustafa, E. H.; Papastratos, K.; Wu, K.; Thio, N.; Milne, J. V.; Mitchell, N.; Witts, S.; Pechlivanis, M.; Wong, B.; Waddell, N.; Phillips, W. A.; Clemons, N. J.

Esophageal adenocarcinoma (EAC) is a genetically heterogeneous malignancy with few recurrent drivers, limiting effective targeted therapies. Although EAC arises from Barrett's esophagus (BE), mechanisms driving progression from this premalignant state to invasive cancer remain unclear. We combined pooled CRISPR-Cas9 loss-of-function screening, in vivo tumorigenicity assays, and Perturb-seq profiling to define functional drivers of BE transformation. We identified 37 tumor suppressors whose loss promotes progression to EAC, defining a functional landscape of tumor initiation. Despite genetic diversity, these losses converged on four transcriptional programs involving metabolic reprogramming, cell cycle progression, RNA processing, and cellular motility. Furthermore, we identify loss of NIPBL, TGFBR2, and RPL22 as key mediators of resistance to platinum- and taxane-based chemotherapy. Collectively, these findings provide a unifying framework for genomic heterogeneity in EAC, uncover underappreciated tumor suppressor pathways, and establish a resource to guide mechanistic and translational studies aimed at improving treatment strategies in this aggressive cancer.