PROJECT SUMMARY Immune checkpoint blockade (ICB), for example using anti-PD-L1, is a landmark advance for treating advanced cancers but is nevertheless still effective in all too few patients. Thus, extensive efforts are directed at identifying combination therapeutic strategies to improve response rates. We have discovered that PD-L1, in addition to its role in blocking T cell activation, protects cancer cells against specific chemotherapeutic agents, e.g., cisplatin. Importantly, this mechanism operates independently of T cell recognition of tumor and the cell-extrinsic immune system. We found that PD-L1 inhibits the ability of cancer cells to respond to type I interferon (IFN-I) and that cancer cell-intrinsic responses to IFN-I are critical in increasing cisplatin cytotoxicity. We hypothesize that PD- L1 inhibitors will enhance the ability of IFN-I to sensitize cancer cells to chemotherapy by a cancer-cell intrinsic mechanism, independently of the immune system. Here, we propose to identify chemotherapeutic agents and IFN-I as logical drugs to combine with ICB, to increase response rates in an immune-independent manner. Aim 1. Determine the ability of the anti-PD-L1 ICB to enhance IFN-I responses and cisplatin toxicity in lung cancer cells. FDA-approved anti-PD-L1s (atezolizumab, avelumab, and durvalumab) are currently in clinical use to activate anti-cancer immune responses. However, it has not been assessed how efficiently each FDA-approved anti-PD-L1 blocks the cancer cell-intrinsic immune-independent function of PD-L1. We will determine which antibody against PD-L1 enhances IFNβ responses and cisplatin cytotoxicity in small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) cell lines, using Western analysis and IncuCyte real-time cell monitoring. Aim 2. Define the spectrum of therapeutic agents whose cytotoxicity is enhanced by inhibiting PD-L1. Each chemotherapeutic agent will be affected differently by the cancer cell-intrinsic function of PD-L1, since each induces cell death by different mechanisms. We will determine which chemotherapeutic agent kills cancer cells more efficiently in combination with PD-L1 inhibition (knock-down of PD-L1 or treating with selected antibodies against PD-L1) alone, or by using PD-L1 inhibition plus IFN-I in SCLC cells. Aim 3. Generate pre-clinical in vivo proof of principle for rational combination therapy. After selecting the most efficient combination in vitro in SCLC and NSCLC cell lines, we will evaluate the efficacy of the combination treatment in vivo in NSG mice, which lack most of the immune system, to cleanly evaluate the cancer cell-intrinsic immune-independent PD-L1-mediated effects. Impact. The studies proposed here are highly innovative as they propose a paradigm-shifting approach in which PD-L1 inhibitors are used as reagents to enhance the ability of IFN-I to sensitize cancer cells to chemotherapy in an immune-independent mechanism. This work will lead to improved understa...