SUMMARY Although tyrosine kinase inhibitors (TKIs) have significantly transformed the treatment of EGFR mutant lung adenocarcinoma (LUAD), their effectiveness is often limited by acquired drug resistance. In a significant portion of the cases, resistance arises without detectable mutations or changes in gene copy numbers, suggesting epigenetic mechanisms underlie resistance. Recent evidence indicates that the three-dimensional (3D) folding organization of the genome regulates the cancer cell epigenome, which can activate oncogenes, inactivate tumor suppressor genes, and promote metastasis and stemness. However, it remains unknown whether and how 3D genome alternations contribute to resistance to targeted therapies. To address this knowledge gap, we have developed a chromatin tracing strategy to visualize the 3D genome organization at the single-cell level in the native cancer tissue context. We applied this method to a reliable mouse model of LUAD driven by the oncogene Kras and identified characteristic alterations in the 3D genome during cancer development, including a notable structural bottleneck in LUAD progression. Moreover, we demonstrated that the 3D genome encodes distinct cancer states within individual cells and uncovers novel genetic dependencies in LUAD, highlighting the critical role of 3D genome reorganization in LUAD development. Based on these findings, we propose that structural changes in the 3D genome similarly mediate resistance to targeted therapies. To investigate this hypothesis, we intend to employ our in situ chromatin tracing approach to examine how the 3D genome reorganizes in the context of EGFR inhibitor resistance using human LUAD cancer cell lines, patient biospecimens, and PDX models, extending studies on the epigenetic basis of TKI resistance in the parent P50 award to an entirely new layer of epigenetic information, the 3D genome. By utilizing our established analytical methods, we will decipher the resistance mechanisms encoded by the 3D genome and validate their functional significance through genetic and pharmacological experiments in both cell culture and animal models. These studies will foster the development of novel prognostic and predictive biomarkers of drug resistance based on single-cell 3D genome conformations and aid in the identification of novel therapeutic targets to overcome drug resistance.