PROJECT SUMMARY Lung cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T-cells, allowing tumors to directly suppress T cell activity. Anti-PD-1/PD-L1 antibodies induce potent anti-tumor immunity and have been approved as a first-line therapy for lung cancer. However, only ~20% of all non-small cell lung cancers (NSCLCs) benefit from checkpoint blockade. A further understanding of the mechanisms that regulate the immune checkpoint in lung cancer is therefore needed. To address this, my laboratory used CRISPR-based screening to identify regulators of PD-L1 in lung cancer cells, revealing potent induction of PD-L1 upon activation of the integrated stress response (ISR) pathway. Mechanistically, ISR activation resulted in enhanced PD-L1 translation and suppression of anti-tumor immunity. We further demonstrated that ISR- dependent translation of PD-L1 requires the alternative translation initiation factor eIF5B. The canonical role of this GTPase is to catalyze ribosomal subunit joining, ensuring 80S ribosome assembly and efficient start codon selection. eIF5B overexpression and/or amplification is frequent in human lung cancers and is associated with poor prognosis. Remarkably, eIF5B overexpression is sufficient to induce PD-L1 protein levels even in the absence of ISR activation. These findings uncovered a new mechanism of immune checkpoint activation and suggested that eIF5B may be a novel target for lung cancer intervention. We further demonstrated that enforced expression of eIF5B accelerates proliferation in lung cancer cells, in mouse syngeneic models, and in human bronchial epithelial cells suggesting that it also promotes tumor growth in a cell-autonomous manner. We propose to elucidate the role of eIF5B in lung tumorigenesis by testing the following central hypothesis: EIF5B functions as an oncogene in human lung cancer by inducing PD-L1 translation and by driving a tumor-promoting translational program. Three Specific Aims will be pursued in order to test this hypothesis: In Aim 1, we will dissect the mechanisms through which eIF5B promotes translation of PD-L1 and additional oncogenic mRNAs in lung cancer. In Aim 2, we will functionally evaluate the oncogenic activity of eIF5B using a novel transgenic mouse model with conditional eIF5B overexpression. In Aim 3, we will characterize the effects of eIF5B loss of function on cell autonomous tumor growth versus its effect on T cell responses in the KrasLSL-G12D; Tp53 fl/fl mouse model using a newly generated conditional floxed knockout allele and a heterozygous germline knockout mouse. These aims will take advantage of our expertise, and that of our collaborators, to evaluate eIF5B oncogenic activity, and elucidate the mechanisms through which this translation initiation factor promotes lung tumorigenesis. We anticipate that these studies will provide new insights into mechanisms of translational control in tumor progression and immune evasion.