Mechanisms Promoting Copper Dependent Cell Death in Cancer - PROJECT SUMMARY Copper is an essential co-factor for enzymes across the animal kingdom yet even modest intracellular concentrations can be toxic, resulting in cell death. The toxicity of copper prompted the development of copper ionophores such as elesclomol as potential cancer therapeutics. However, lack of clarity regarding their mechanism of action has prevented their clinical development despite a favorable tolerability profile in clinical trials. We have recently described that copper ionophores promote a surprising new form of regulated cell death, distinct from other known and well annotated cell death pathways (such as apoptosis, ferroptosis and necroptosis). This new form of regulated cell death is copper dependent (hence termed “cuproptosis”) and is regulated by mitochondrial FDX1 and cellular protein lipoylation, a conserved lysine post-translational modification regulating only 4 key mitochondrial enzymes. Despite this essential role in enabling cuproptosis, little is known about the natural function of FDX1 in the cell, and the regulation and manifestation of protein lipoylation in different cancer types. Establishing a coherent mechanism explaining FDX1 and protein lipoylation regulation of copper ionophore induced cell death is crucial for any future attempts to repurpose these molecules as cancer therapeutics. As such, we will combine multidisciplinary approaches that include genomic and proteomic perturbation strategies with metabolite profiling and multiplexed imaging in cell culture and distinct mouse tumor models to determine the natural function of the key regulators of cuproptosis (FDX1 and protein lipoylation) in cancer cell growth and tumor formation, enabling a mechanistic understanding of how they regulate cuproptosis. Specifically, in Aim 1, we will focus on elucidating the natural function of the key regulator of cuproptosis, FDX1, in cells. We will take a focused approach to determine if it regulates Fe-S cluster biosynthesis or protein lipoylation, we will use proteomic approaches to characterize its protein-protein interactions, and we will perform deep mutational scanning to map the structure-function interactions of FDX1. In Aim 2, we will specifically focus on the unique regulatory role of lipoylated DLAT in promoting cuproptosis. We will use different image-based approaches to define the biophysical properties of lip-DLAT aggregates and their protein composition identifying links to multiple other cellular features. In Aim 3 we will first explore the levels of protein lipoylation across hundreds of cancer cell lines and establish the cell state associated with high lipoylation cancer cells. Then, we will determine the manifestation and dependency on FDX1 and protein lipoylation in both classic xenograft tumor models and in models of metastatic tumors growing in different organs. Lastly, we will use these in vivo models to establish the efficacy and on-target engagement of copper ionophores in cancer cell models that are predicted to be highly sensitive to these compounds. Together, this proposal will advance our understanding of the role of FDX1 and protein lipoylation in cancer and how these key regulators promote cuproptosis in cells and tumors.
