PROJECT 1: ABSTRACT Autophagy is a self-degradation process whereby cancer cells recycle defective organelles and macromolecules as a nutrient source to support their increased metabolic needs. Autophagy is elevated and essential for the tumorigenic growth of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC), providing the rationale for clinical evaluation of the autophagy inhibitor hydroxychloroquine (HCQ) for PDAC. Disappointingly, when used as monotherapy in combination with standard of care, HCQ has shown limited to no clinical efficacy for PDAC. We recently determined that the treatment of PDAC with inhibitors of the key KRAS effector pathway, the RAF- MEK-ERK mitogenic activated protein kinase cascade, unexpectedly caused further elevation of autophagy, rendering PDAC acutely dependent on autophagy, and hypersensitive to autophagy inhibition. We determined that ERK inhibition impaired other critical metabolic processes (glycolysis, mitochondrial function) that then led to compensatory upregulation of autophagy. Our findings, together with essentially identical conclusions by another independent co-published study, has led to our initiation of two clinical trials evaluating two approved MEK inhibitors (trametinib, binimetinib) in combination with HCQ for PDAC. Since our recent studies suggest that ERK inhibitors will have superior activity in PDAC, this has prompted our initiation of a phase II clinical trial with the ERK inhibitor LY3214996 in combination with HCQ for metastatic PDAC (Aim 1). While early observations from compassionate care utilization of this combination support a significant clinical impact for this combination, our preliminary studies (Aims 2 and 3) support our premise that we can improve upon this therapy. Aim 2 studies are based on our application of a 2,500-gene druggable genome CRISPR-Cas9 genetic-loss-of- function screen to identify genes that modulate HCQ anti-tumor activity. The identified hits that either enhance or reduce HCQ growth inhibitor activity represent candidate combinations or biomarkers for HCQ resistance, respectively. The biomarkers for response can then be applied to tumor biopsies collected in the Aim 1 clinical trial evaluation. Aim 3 studies involve our application of a chemical library screen using a 525-oncology drug set to identify combinations that enhance the cytotoxicity of HCQ. Together, combinations that arise from Aims 2 and 3 studies will then be advanced to Aim 4 studies, where we will apply PDAC organoid or orthotopic mouse tumor models to identify combinations for future clinical evaluation. Since we have found that ERK MAPK inhibition causes tumor-associated gene expression changes that can lead to an improved anti-tumor immune response, we will also evaluate the impact of our combinations on tumor cytokine expression and on tumor- associated immune cells. In summary, our studies will develop novel combination therapies to target autophagy for the treatment of KRAS-mutant PDAC.