PROJECT SUMMARY / ABSTRACT Metastatic colorectal cancer (mCRC) is the second leading cause of cancer-related death in the US. Roughly 8% of mCRCs harbor a mutation in BRAF, associated with the worst prognosis and poor response to treatment. Limitations of standard of care (SOC) therapies targeting BRAF + EGFR include a response rate of 20% and short time to cancer progression. Results of our original R01 underscore that mCRC is not dominated by individual oncogenes, rather is mediated by the concerted upregulation of multiple signaling pathways. A key discovery was a reversible SRC-relayed COX2-inflammatory program that drives resistance to BRAF + EGFR inhibition (Nature Cancer 2023). Addition of the COX2 inhibitor (COX2i), celecoxib, consistently resulted in more durable suppression of tumor growth in patient derived xenograft (PDX) models. Furthermore, promising orthogonal mechanisms of resilience to SOC therapy emerged: a CDK1/2 pathway identified from the reprogrammed kinome of residual PDX tumors, as well as a BCL-XL driven anti-apoptotic state and a pro-survival IL8/CXCR2 autocrine loop. With our original R01, we discovered a strategy to improve duration of disease control; with our R01 Renewal, our translational goals are to manage BRAFV600E mCRC as a chronic disease, to increase response rate, and trigger tumor cell elimination. Our approach for this proposal is to test the hypothesis that BRAFV600E mCRC tumors engage a specific set of stress response programs that enable cancer cells to adapt and survive under therapeutic pressure and represent druggable vulnerabilities. In Aim 1, we will probe converging phospho-signaling circuits that regulate response to combination therapies using kinome profiling tools (high-throughput kinase-activity mapping, inhibitor-bead mass spectrometry) and advanced computational modeling to map the phospho-signaling circuits of banked, residual PDX tumors treated with SOC ±COX2i ±CDK1/2i. Aim 2 will elucidate core mechanisms of resilience to therapeutic pressure, utilizing BH3-profiling to define the intracellular mechanisms promoting persistence of tumor cells and identify anti- apoptotic factors; ELISA and antibody arrays to characterize the autocrine mechanisms that prime BRAFV600E mCRC to survive drug treatments; and multiplex immuno-fluorescence to quantify the co-expression and subcellular localization of key, pro-survival proteins in therapy-resistant PDX tumors. The purpose of Aims 1 and 2 is to reveal how therapeutic pressure induces adaptive stress responses that converge on a few critical signaling hubs, which can be leveraged as drug targets and tested in in vitro models. In Aim 3, we will test combinations and sequencing strategies with highest clinical potential in PDX models: promising candidates from our original R01 and additional targets uncovered in Aims 1-2 will be evaluated in therapeutic switch approaches (i.e., next-line regimen and de-escalation strategy), and new rational combination...