PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is characterized by resistance to current therapies, a high degree of desmoplasia, and an immunosuppressive tumor microenvironment (TME). This scientific proposal focuses on cyclic AMP Response Element Binding protein 1 (CREB) as a transcriptional factor downstream of KRAS that promotes disease aggressiveness and poor survival. Genetic loss or pharmacological inhibition of CREB leads to significant attenuation of PDAC tumor burden and improved survival in multiple murine PDAC models. Utilizing high throughput sequencing approaches, we have identified tumor cell-derived leukemia inhibitory factor (LIF) as a key CREB-regulated immunomodulatory cytokine, serving as a possible paracrine mediator of tumor- macrophage crosstalk in the TME. We hypothesize that targeting the CREB-LIF signaling axis can remodel the immunosuppressive TME by significantly impacting tumor-associated macrophages (TAM)s and reinvigorating the T cell-based adaptive anti-tumor immune response to improve the efficacy of checkpoint immunotherapy. Based on our preliminary data, the critical role of the CREB regulated LIF signaling will be investigated through three specific aims. 1) Elucidate the molecular mechanism and impact of CREB regulated LIF in PDAC oncogenesis. Here, we will assess the CREB-mediated regulation and functional roles of tumor cell-derived LIF by exploring the nucleo-cytoplasmic localization, interaction with transcriptional complexes, and gene regulatory networks. We identify the CREB-LIF axis requirement in the tumor cell growth using CRISPR/Cas9 genome editing with CREB on or off in vivo pancreas tissues and evaluate the expression of LIF relative to CREB in patient PDAC tissues, patient-derived xenografts, and primary tumor derivative organoids. 2) Determine how CREB-dependent LIFR signaling in macrophages impacts the tumor immune landscape. We hypothesize that the CREB modulates LIF release and promotes TAM infiltration/polarization towards a tumor-promoting M2-like phenotype via activating STAT3 in macrophages. We will determine the effect of CREB-regulated LIF release on TAM activity using CREB on or off with LIF knockout tumor cells and assess the impact of CREB-regulated LIF signaling on the adaptive immune response and TME during PDAC progression in myeloid-specific knockout models of Lifr and Stat3, and 3) Determine if CREB inhibition and conventional immunotherapeutic approaches reduce tumor growth and improve overall survival. We hypothesize that the tumor cell-intrinsic CREB-LIF activation is a pivotal switch that drives immune suppression in the pancreatic TME, which can synergize with immune checkpoint inhibition. We will use CREB inhibitor with checkpoint inhibitor to establish the safety of the therapies and study tumor growth and overall survival in genetic mouse models, patient-derived xenografts, and organoids to further complement these preclinical studies. Together, these results wil...