Project 1 - Abstract/Summary Oncogenic KRAS (KRAS*) is universally present in >90% of human pancreatic adenocarcinoma (PDAC) and functions as the dominant driver for tumor development. The central theme of Project 1 has been the elucidation of KRAS* function in PDAC and translate of these insights into effective therapies. In our previous grant cycles, Project 1 and our P01 team have established the essential role for KRAS* in PDAC maintenance, prompting the development of KRAS*-targeted exosome-based RNAi therapy from Project 3 that has been translated into a first-in-human trial. Collaborative work from Project 1 and Project 2 has not only elaborated the regulation of anabolic metabolism and nutrient salvage pathways that are essential for KRAS*-driven tumor maintenance, but also identified molecular and metabolism mechanisms leading to the resistance to KRAS* depletion. While these studies elucidated the cancer cell-autonomous regulation of KRAS*-dependency in PDAC, recent studies from Projects 1 and 3, further demonstrated the profound impact of KRAS* on the remodeling of the tumor microenvironment (TME), a major hallmark of PDAC and barrier for effective therapies, including immunotherapy. Specifically, we discovered a shift of myeloid infiltration in TME toward macrophages following KRAS* extinction, which produce TGFBeta to support Kras*-independent cancer cell survival growth. In depth analysis of the TME further identified alternative immune checkpoints and demonstrated the importance of myeloid cells in immune suppression, leading to the development of novel immunotherapy regimens with unprecedented prolongation of survival in preclinical models. In this treatment study, we discovered a similar shift toward monocytic myeloid lineage which may contribute to the resistance to combinatory immune checkpoint inhibition. Therefore, in this next cycle, Project 1 will focus on elucidating the crosstalk between KRAS* and the TME, focusing on monocytic myeloid cells, with the goal of translating these mechanistic insights into effective new therapies. To achieve this goal, we will work closely with our innovative Cores, which have extensive expertise in pathology, mouse modeling and computational biology, to conduct a systematic in vivo genetic validation of the myeloid-dependent KRAS* escape mechanism. These studies will be followed by experiments to characterize the myeloid-mediated resistance to the alternative immune checkpoint inhibition regimen we have discovered. In the end, we will evaluate the anti-tumor potential of blocking such myeloid- mediated resistance mechanisms in combination with novel immune checkpoint inhibitions. Our study will be highly integrated with Project 2 ...