ABSTRACT Resistance to targeted therapy is a major challenge in oncology and barrier improving patient survival. As a paradigm-defining example, EGFR tyrosine kinase inhibitors (TKIs) are effective in many EGFR-mutated non small-cell lung cancer patients. However, promising initial responses in these patients are always followed by the development of acquired resistance, most often a lethal event. The cellular basis for this stems from an incomplete initial response forming a reservoir of residual disease caused by tumor cell persistence and drug tolerance, through poorly understood mechanisms. In extensive preliminary data we discovered that the persistence and drug tolerance of EGFR-mutant lung cancer cells is dependent on a mitotic stress response elicited by drug treatment. Drug tolerant tumor cells are dependent on continued mitotic stress signaling, and are vulnerable to Aurora Kinase inhibitors in vitro and in vivo. Tumor samples from patients progressing on EGFR inhibitors frequently displayed evidence of ongoing stress signaling, often co-occurring with other genetic changes commonly associated with drug resistance. Here we seek to mechanistically dissect how this stress signaling aids in tumor cell persistence and acquired resistance and the role it plays in shaping tumor evolution after therapy. We will test the hypothesis that stress from acute oncogene withdrawal drives the unexplained and key features of drug tolerance and residual disease during EGFR TKI treatment that is: cellular adaptation and resistance to apoptosis (Aim 1) and the catalysis of genetic evolution leading to the de novo gain of resistance causing mutations (Aim 2). Finally, this knowledge will be used to identify new therapeutic strategies to forestall tumor evolution by limiting stress signaling (Aim 3). To address this hypothesis, our team consists of experts in lung cancer, systems biology and clinical translation and will use innovative new single cell approaches, live cell imaging, state-of-the-art animal and organoid models and patient samples. Our goal is to lay the mechanistic groundwork that shifts the paradigm from the current reactionary approach of targeting acquired resistance after it emerges to a proactive approach that targets sources of residual disease to prevent acquired resistance.