PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer, with a five-year survival of only 10%. There is an urgent need to develop new therapeutic strategies for this disease. Oncogenic KRAS mutations occur in most patients and co-occur with alterations in several different tumor suppressor genes, including TP53, CDKN2A, SMAD4, ARID1A and others. Multiple different transcriptional subtypes of PDAC have also been observed, such as the classical and basal-like programs, which define distinct subsets of disease with differing prognosis and therapeutic response. The recent development of new small molecule inhibitors of KRAS that target KRAS mutations frequently observed in PDAC has the potential to transform the treatment of this disease. We and others have shown that primary and acquired resistance mechanisms can limit the clinical benefit of KRAS inhibitor monotherapy in cancer; thus, understanding the mechanisms of response and resistance to KRAS inhibition in PDAC will be critical to maximize the potential of these therapies. This proposal will use novel mutant-selective KRAS and pan-RAS inhibitors, unique human organoid and mouse models of PDAC, and innovative single-cell and functional genomic approaches to define the genetic, transcriptional and microenvironmental factors that impact response to KRAS inhibition in PDAC. In Aim 1 we will investigate how tumor suppressor genotype can modify response to KRAS inhibition using CRISPR-Cas12a tumor suppressor gene knockout screens in both in vitro and in vivo systems to simultaneously model multiple PDAC genotypes and systematically define genetic biomarkers and mechanisms of sensitivity or resistance to KRAS inhibition. In Aim 2, we will define the role of PDAC transcriptional cell state in modifying response to KRAS inhibition using novel isogenic murine PDAC organoids and human patient-derived PDAC organoids representing the basal-like, classical and neuronal-like subtypes of PDAC. We will characterize subtype-specific adaptive mechanisms of response to KRAS inhibition and evaluate subtype plasticity with a goal to develop combination therapy strategies with KRAS inhibition to target each subtype. In Aim 3, we will build on preliminary single-cell RNA sequencing (scRNA-seq) data from human PDAC biopsies showing that the tumor microenvironment (TME) shapes the transcriptional phenotype and therapeutic response of PDAC cells. We will examine response to oncogenic Kras inhibition in new mouse models of the basal-like, classical and neuronal-like subtypes of PDAC and will interrogate the role of paracrine signaling mechanisms from the TME in modifying malignant cell state and response to KRAS inhibition using ex vivo scRNA-seq and drug sensitivity profiling assays. Collectively, these studies will form a foundation for development of new biomarkers and combination therapies with KRAS inhibition that can be evaluated in future clinical trials for PDAC patients.