Project Summary Melanoma is an aggressive and highly metastatic skin cancer. Although modern combination checkpoint inhibitors revolutionized clinical outcomes in advanced cases, over half of all patients are refractory to immunotherapy and require alternative or complementary treatment options. The discovery of ferroptosis provided a novel way to treat cancer. Recently described vulnerability of melanoma cells to ferroptosis offers a new therapeutic opportunity, particularly against the malignant cells which are resistant to current therapies. However, how to exploit such vulnerability is still unclear due to the lack of mechanistic understanding of ferroptosis regulation in melanoma and the tumor-infiltrating immune cells. We discovered that an indiscriminate induction of ferroptosis of the entire tumor tissue has a deleterious impact on protective anti- tumor immune responses, which promotes melanoma progression. Specifically, we found that ferroptotic death of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in tumors is a major mechanism of immune suppression. Therefore, a shift in the current approach to harness ferroptosis for cancer therapy is required. Only by understanding the regulatory mechanisms of ferroptosis in different cellular compartments of the tumor microenvironment (TME) will we be able to engineer effective melanoma therapy based on the differential modulation of ferroptosis. Utilizing cutting-edge redox lipidomics mass spectrometry and single- cell lipidomics imaging methods, this project will uncover critical mechanisms of ferroptosis regulation in the intratumoral PMN-MDSC and melanoma cells. In addition, we will optimize our recently developed therapeutic approach which will promote ferroptosis of the malignant cells while protecting and enhancing anti-tumor immunity. To achieve these goals, we will pursue three specific aims. In Aim 1, we will decipher how the processes of melanogenesis and cell differentiation regulate melanoma cell susceptibility to ferroptosis. The results will provide a strategic approach to maximizing the efficacy of pro-ferroptotic therapy against melanoma cells. Aim 2 will focus on identifying mechanisms of ferroptosis-mediated immune regulation by PMN-MDSC in melanoma TME. The results will reveal how to protect anti-tumor immune responses via targeted ferroptosis inhibition in the myeloid cells of the TME and prevent immune tolerance to cancer. Finally, in Aim 3 we will expand on our preliminary data to investigate therapeutic potential of differentially regulating ferroptosis in the malignant and the myeloid cells of the melanoma TME. This will be accomplished using our previously developed nano-delivery systems based on graphene quantum nanodots and carbon nanotubes. Such approach is highly clinically relevant as it employs both cytotoxic and immunomodulatory strategies against melanoma aimed at reducing immune tolerance to cancer and overcoming modes of cancer resistance to the ...