Project Summary Lung cancer is a leading cause of death and the second most common cancer in the United States. Application of immunotherapies has led to significant improvement in survival of cancer patients. However, there is further need to achieve better responses to current immunotherapies. The most promising results had been achieved when combining anti-PD-L1/PD-1 with treatments that activate immune function. We propose that exogenous carbon monoxide (COex) applied at low non-toxic doses is a potential adjuvant for patients with lung cancer treated with immunotherapy. CO is generated endogenously by heme oxygenase-1 (HO-1) that acts during homeostasis to break down heme to three active metabolites. We have demonstrated that COex applied at 250 ppm for 1 hour per day (that corresponds to the dose of CO obtained from burning 2 cigarettes) blocks progression of lung and prostate cancers. COex can block tumor growth via targeting metabolism of cancer cells. However, since COex blocks growth of different tumor types and at various metabolic rates as well as is more efficient in vivo than in vitro in inducing apoptosis of cancer cells, we reasoned COex has a broader effect in the TME that includes immune cells. Our preliminary data suggest that COex promotes more ‘immuno-receptive’ tumor microenvironment (TME). We propose that reprograming of the TME by regulating heme metabolism regulates responses to immunotherapy. Our aim is to: To determine the role of COex in redirecting immune responses to increase efficacy of immunotherapy. Specifically, we intent to: i) Determine effectiveness of the combination of COex and anti-CTLA-4, anti-PD-L1 or anti-PD-1 treatment in a mouse model of lung cancer. ii) Determine COex-mediated induction of CD86 via mTOR-Notch1 signaling during response to immunotherapy in the lung cancer model. iii) Define the importance of endogenous HO-1-derived CO during response to immunotherapy. The investigations proposed in this application will delineate the role of CO and HO-1 in activation of the host immune responses via selective signaling pathways leading to increase in myeloid function in the TME. Our findings will have fundamental and therapeutic implications for patients with lung cancer, especially that COex is in clinical trials. This study is well-aligned with the mission of the NCI and this RFA “NCI Clinical and Translational Exploratory/Developmental Studies (R21 Clinical Trial Optional)” (PAR-19-356).