Abstract Immune-based therapies are generating considerable excitement due to the success of drugs targeting immune checkpoints. Only small subsets of patients are benefitting from this therapeutic approach, implying that important molecular modifiers of the tumor microenvironment need to be better understood. Endometrioid- type endometrial carcinoma (EEC) accounts for approximately 75% of all endometrial carcinomas, the fourth most common cancer in women in the US. From TCGA data (and validated from our own patient data) we have identified four transcriptome subtypes of EEC with distinct clinicopathologic characteristics and mutation spectra. Cluster II consists of younger, obese patients with low grade EEC yet diminished survival. Although the Cluster II tumors had the lowest overall mutation rate, CTNNB1 exon 3 mutations were very common. These mutations were associated with activation of the Wnt/-catenin signaling pathway. Thus, exon 3 mutations of CTNNB1 are likely a driver for a subset of more aggressive EEC. We hypothesized for the parent SPORE proposal that CTNNB1 mutations reprogram the molecular and cellular landscapes leading to clinically aggressive EEC. In aim 2 of the parent proposal, we will test the hypothesis that CTNNB1 mutation and Wnt/β-catenin pathway activation result in an immunosuppressive tumor microenvironment that would contribute to worse survival. For the expansion proposal, we will focus on 2 other molecular pathways, PTEN and CD73-generated adenosine, that are also important for endometrial carcinoma and may contribute to localized immune suppression. PTEN loss leading to activation of PI3K/AKT signaling in EEC is common, occurring in more than 50% of cases. Recent evidence in melanoma suggests that PTEN loss leads to increased expression of immunosuppressive cytokines, decreased T lymphocyte infiltration into the tumor, and decreased T lymphocyte-mediated tumor cell death. CD73 is the rate-limiting enzyme in adenosine biosynthesis. We have recently shown that CD73 helps to maintain cell-cell contacts via filopodia and membrane zippers that require cortical F-actin polymerization. Loss of CD73 was associated with higher endometrial cancer grade, advanced endometrial cancer stage, and worse survival. Loss of the cell-cell contacts leads to defects in the normal epithelial cell barrier function, which may lead to alterations in the tumor immune microenvironment. The expansion project hypothesis is that PTEN loss and decreased CD73 contribute to endometrial cancer progression by promoting an immunosuppressive tumor microenvironment. Expansion Specific Aim 1. Determine if PTEN protein loss is associated with an immunosuppressive tumor microenvironment in EEC. Expansion Specific Aim 2. Determine if loss of CD73 is associated with an immunosuppressive microenvironment in EEC. For both aims, we will focus on microsatellite-stable EEC and characterize immune cell infiltrates, PD-L1 expression, and tissue cytokines levels.