ABSTRACT Poorly defined fitness features enable a subset of cancer cells to survive therapy, ultimately giving rise to treatment-refractory tumors. The expanded use of sequencing tools have led to the recognition that refractory tumors often lack a genetic mechanism of resistance, and instead co-opt a regenerative program characterized by activation of developmental, inflammatory, mesenchymal and stem cell features. Importantly, these regenerative states are conserved across diverse cancers of epithelial origin, and are potentially reversible. Our preliminary data indicate that therapy resistance in colorectal cancer (CRC) is fueled by preexisting cells via a YAP-driven regenerative program that is strikingly similar to the consensus molecular subtype 4 of CRC, a poor risk phenotype often observed in treatment-refractory tumors. Our central hypothesis is that a preexisting population drives regenerative reprogramming and tumor escape through epigenetic adaptations that can be targeted therapeutically. First we propose characterizing the preexisting (pre-resistant) state using barcode lineage tracing and single-cell technologies. Using patient-derived organoids (PDOs) to isolate the founding clones of resistance, we will study the contribution of preexisting versus actively gained fitness features through whole-exome (WES) and RNA sequencing (RNA-seq). Using pre-clinical models of resistance to KRAS-inhibition (KRASi), we will define the role of genetic and non-genetic drivers of tumor escape. To develop approaches that abrogate regenerative reprogramming, we will perform a focused shRNA screen targeting 50 chromatin regulators. To expedite clinical translation, we will test readily available compounds targeting the top hits of the screen, in combination with standard therapies. We believe that our innovative tools will maximize our opportunity to develop treatment approaches with immediate clinical impact. During the award period, the candidate will conduct research at Weill Cornell Medicine (WCM) and Memorial Sloan Kettering (MSK) under the mentorship of Dr. Lukas Dow and an advisory committee. The candidate will commit at least 9 person-months of his professional effort to the research and career development activities outlined here. With his mentor and advisory committee, the candidate has designed a 5-year plan aimed at expanding his knowledge and expertise in cancer research, including single cell technologies, computational biology, genetic engineering, and functional screens. The goal of the career plan is to launch an independent career as a laboratory investigator focused on therapy resistance and biomarkers of response in CRC.