PROJECT SUMMARY The overall goal of this project is to expand our understanding of the cellular and molecular mechanisms that link translation initiation inhibition to apoptosis and tumor suppression triggered by Interleukin-24 (IL-24). Clinical testing of IL-24 as a gene-based therapeutic for the treatment of solid tumors demonstrated that IL-24 is efficacious and is safe. This proposal is consistent with the current view that modern anti-cancer therapy must be target-specific, inhibit the growth of cancer with minimal effect on normal cells, and that effective anti- cancer agents of low toxicity can be achieved by targeting the fundamental mechanisms responsible for the genesis, maintenance, and/or progression of cancer. We have shown that IL-24 inhibits translation initiation by inducing phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), depletion of the ternary complex, upregulation of expression and activity of ATF4, and inhibition of eIF4F complex in vitro. The working hypothesis of this application is that restoration of physiological restrains on translation initiation by Interleukin 24 (IL-24), an inhibitor of translation initiation, represents a new paradigm in cancer therapy. We have conducted pharmacokinetic, toxicity, and in vivo efficacy studies on IL-24, which established not only its anti-cancer activity, but also that IL-24 translationally down-regulates the expression of PD-L1, a critical anti-tumor immunity factor. In this application we propose to continue our efforts to characterize the cancer cell-specific actions of IL-24 as an inhibitor of translation initiation. Specifically, we will 1) test IL-24 anti- cancer translation initiation inhibition-mediated mechanism of action in vivo (Specific Aim 1), 2) determine the role of activating transcription factor 4 (ATF4) on IL-24-mediated inhibition of translation initiation and apoptosis, (Specific Aim 2), and 3) investigate for the first time the role of PD-L1 down-regulation in the anti- cancer effect of IL-24 and its potential utility as an immune checkpoint inhibitor (Specific Aim 3). For these proposed studies, we will collaborate with Dr. Bertal H. Aktas, from Harvard Medical School, and expert on targeting translation initiation for cancer therapy and Dr. Juan Lafaille, from New York University School of Medicine, and Immunology expert.