Mechanistic Decoding of Pharmacodynamic Intratumoral Heterogeneity: A Pan-Cancer Regulatory Framework

Intratumoral heterogeneity (ITH) drives therapy failure through divergent drug response propensities among coexisting subclones - a phenomenon we term pharmacodynamic intratumoral heterogeneity (PharmITH). While single-cell technologies have catalogued transcriptional heterogeneity, which molecular programs shape differential drug responses remains unresolved. Here we decode PharmITH by linking single-cell transcriptomes from 1,279 samples across 24 cancer types with drug-response profiles for 1,218 compounds. We identify 425 hub genes consistently associated with predicted treatment-response heterogeneity and with potential therapeutic relevance and perform multi-omics network analysis in lung adenocarcinoma (LUAD) integrating transcriptional regulation, somatic mutations, cell-cell communication (CCC), and alternative splicing. This analysis identifies four classes of candidate regulatory associations observed in the LUAD case study: 3 transcription factors establishing pre-existing drug-response programs, 41 genomic alterations amplifying pathway signaling through intermediate networks, 5 ligand-receptor pairs coupling microenvironmental cues to intracellular circuits, and 8 genes producing opposing isoforms via differential splicing. These findings converge on 6 core pathways, revealing that PharmITH arises from tumor-intrinsic mechanisms. Experimental validation and an online platform PharmITH for pan-cancer therapy enable state-resolved drug-response analysis and candidate-gene exploration. This approach transforms ITH from descriptive phenotyping to mechanistic dissection, providing actionable targets to overcome therapy resistance.