Xu, C.; Ma, H.; Sheng, T.; Lee, M. H.; Abdul Ghani, S. A. B.; Perlaza-Jimenez, L.; Huang, K. K.; Lim, S. K.; Srivastava, S.; Ong, X.; Tay, S. T.; Ho, S. W. T.; Tan, A. L. K.; Zhu, F.; Nasca, V.; Randon, G.; Ashktorab, H.; Cheng, A. S.-L.; Jeyasekharan, A. D.; Li, S.; Teh, M.; Sundar, R.; Powell, D. R.; Rosenbluh, J.; Pietrantonio, F.; Yong, W. P.; So, J. B.-Y.; Tan, P.

Gene expression signatures (\"molecular phenotypes\") are extensively utilized in cancer research. Using direct capture Perturb-seq (dcPerturb-seq), we surveyed how molecular phenotypes in gastric cancer (GC) are shaped by cell-intrinsic genetic alterations interacting with cell-extrinsic therapeutic exposures. Interrogating >200 GC-related genes against 4 distinct epigenetic drug classes across multiple gastric lines, we captured 18.9 million pharmacogenomic expression interactions in 669,065 cells representing baseline and post-therapeutic molecular phenotypes. This single-cell pharmacogenomic compendium confirmed previously known gene-driven molecular phenotypes (TP53, PCNA), elucidated poorly characterized genes (ZBTB41), and uncovered novel gene dosage-molecular phenotype relationships. Molecular phenotypes in post-therapeutic surviving cells revealed distinct gene perturbation-induced pathways causing convergent drug resistance (EMT plasticity, cell cycle alterations, metabolic reprogramming), with MED12, EP300 and KLF4 modulating histone deacetylase inhibitor (HDACi) resistance. Mapping of in vitro molecular phenotypes to primary human GCs imparted prognostic information and insights into spatial heterogeneity and targeted therapy response. Our results illustrate how dcPerturb-seq can systematically and comprehensively map diverse cancer-associated molecular phenotypes across multiple gene-gene and gene-drug interactions, yielding translational insights.