PROJECT SUMMARY/ABSTRACT Tumors can utilize mechanisms of normal physiological immune regulation to suppress the tumor-specific immune response. Targeted immunotherapies directed against immune checkpoints, like PD-1/PD-L1 and CTLA4, have improved patient outcomes but are only effective in a subset of cancers. The durable responses of some patients, resulting from checkpoint-targeted therapy, has sparked great interest in identifying and targeting other innate and adaptive immune checkpoints. Recent studies have identified the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases as a novel innate immune checkpoint. Ubil et al. (JCI, 2018) show that tumors secrete Pros1, a Tyro3/Mer ligand, which suppresses the pro-inflammatory M1 response of host macrophages. Pros1 binding induces the recruitment of p38 and PTP1b to Mer, preventing nuclear translocation of p38 and subsequent expression of M1 associated genes. Genetic deletion of macrophage Mer or PTP1b restores M1 polarization in the presence of Pros1. By targeting Pros1:macrophage Mer signaling it may be possible to enhance cancer immunotherapy. To better understand and prevent Mer mediated immune suppression the following specific aims are proposed: 1) Determine the roles of Mer kinase activity and adapter protein recruitment in M1 suppression, 2) Identify the basis for variable immune suppression by Pros1 secreting tumors and 3) Ascertain whether PTP1b inhibition as mono- or combination therapy can increase intra-tumoral immune infiltrate and improve survival. To establish the importance of Mer kinase activity, CRISPR will be used to genetically ablate the Mer ATP binding site and RNAseq used to monitor global transcriptional changes during Pros1 induced M1 suppression. Bioinformatics analysis will identify key pathways affected by Mer kinase, which will be validated biochemically. Immunoprecipitation and proteomic analysis will be used to determine the importance of Pros1-induced dynamic adapter protein recruitment and novel protein:protein associations. Subtle genetic alterations can lead to reduced Pros1 protein activity. Changes in tumor Pros1 sequence will be correlated with ability to suppress M1 polarization and a predictive model constructed to identify which tumors will be susceptible to Pros1:Mer targeted therapy. Efficacy of pharmacological PTP1b inhibition (mono- and combination therapy) to increase tumor immune infiltrate and survival will be determined in primary and metastatic tumor models. Together, the proposed study may identify novel therapeutic targets to prevent Pros1:Mer mediated immune suppression, yield predictive criteria as to which patients may benefit from Pros1:Mer targeted therapy and whether pharmacological PTP1b inhibition is a viable strategy to improve the immune response to cancer.