PROJECT SUMMARY Tumor-Associated immune Macrophages (TAMs) are a metabolically and functionally heterogeneous population of cells that play critical roles in both anti-tumor immunity and tumor growth.Importantly, the type of TAM infiltrate within a neoplasm has been shown to be a prognostic factor for tumor growth and sensitivity to cancer immunotherapies. Hence, elucidating the mechanisms that determine the differentiation of pro-tumor and anti- tumor TAMs within the tumor microenvironment (TME) can lead to novel therapeutic approaches. For nearly 40 years, it has been known that inhibition of the mitochondrial electron transport chain (ETC), which is composed of five multiprotein complexes, is critical for the anti-tumor activity of TAMs. However, the mechanisms by which the activity of the ETC is controlled in TAMs and how this process determines the functions of these immune cells within the TME are largely unknown. Interestingly, the terminal enzyme of the ETC, Complex IV (CIV), is the only ETC complex in which its core protein subunits are replaced by closely related isoforms to tune its activity in response to signals from the tissue microenvironment. Using single-cell transcriptomics and 7 novel mouse strains that we generated, we demonstrated that type I and II interferons (IFNs) potently induce a single transcript encoding the peptide NDUFA4L3 and the microRNA miR-147 in human and mouse TAMs from melanoma tumors. Notably, we also showed that NDUFA4L3 and miR-147 work in concert to remodel CIV protein subunit composition in TAMs through the degradation and subsequent replacement of a core component of CIV, NDUFA4. Importantly, NDUFA4 degradation as a result of NDUFA4L3 and miR-147 induction by IFNs, or its genetic deletion, blocks tumor growth and leads to a dramatic accumulation of anti-tumor TAMs. Therefore, we hypothesize that regulation of CIV subunit composition and activity in TAMs through the induction of NDUFA4L3 and miR-147 by IFNs is a key evolutionarily conserved metabolic checkpoint by which TAM differentiation is controlled, which could be targeted in the context of cancer immunotherapies. Thus, in aim 1 of this project, we will use two novel conditionally knock-out strains for NDUFA4 and NDUFA4L3 that we generated, single-cell and spatial transcriptomics, and the humanized mouse MISTRG-6 that supports the development of human TAMs, to establish how CIV subunit composition in TAMs regulate anti-tumor immunity and responses to checkpoint blockade inhibitors. In aim 2, we will use a molecular model of CIV with an atomic resolution that we generated, metabolomics, and novel technologies to measure cellular metabolism and mitophagy rates at the single cell level to determine how CIV protein subunit composition and its activity contribute to the metabolic and functional states of TAMs. As recent reports demonstrate that macrophages from patients with loss of function mutations in NDUFA4 show a striking pro-inflammatory gene sign...