Mechanisms promoting copper dependent cell death in cancer

项目摘要 铜是动物界酶的一种重要辅助因子， 浓度可能是有毒的，导致细胞死亡。铜的毒性促使了铜的发展 离子载体如elesclomol作为潜在的癌症治疗剂。然而，由于缺乏明确的 尽管在临床上具有良好的耐受性，但其作用机制阻止了其临床开发 审判我们最近描述了铜离子载体促进了一种令人惊讶的新形式的调节性细胞死亡， 不同于其他已知的和充分注释的细胞死亡途径（例如细胞凋亡、铁凋亡和铁凋亡）。 坏死性凋亡）。这种新形式的调节性细胞死亡是铜依赖性的（因此称为“铜中毒”）， 由线粒体FDX 1和细胞蛋白脂酰化调节，保守的赖氨酸翻译后 修饰仅调节4种关键线粒体酶。尽管这在使铜中毒中起着重要作用， FDX 1在细胞中的天然功能，以及蛋白质的调节和表现形式知之甚少， 不同癌症类型的脂肪酰化。建立解释FDX 1和蛋白质脂酰化的连贯机制 调控铜离子载体诱导的细胞死亡对于任何未来尝试重新利用这些分子都是至关重要的 as cancer癌症therapeutics治疗.因此，我们将结合联合收割机多学科的方法，包括基因组和 蛋白质组干扰策略与细胞培养中的代谢物分析和多重成像以及不同的 小鼠肿瘤模型，以确定铜中毒的关键调节因子（FDX 1和蛋白质）的天然功能。 脂酰化）在癌细胞生长和肿瘤形成中的作用，从而能够从机制上理解它们如何调节 铜中毒具体来说，在目标1中，我们将重点阐明关键调节因子的自然功能， 铜中毒，FDX 1，在细胞中。我们将采取集中的方法来确定它是否调节Fe-S簇生物合成 或蛋白质脂酰化，我们将使用蛋白质组学方法来表征其蛋白质-蛋白质相互作用， 将进行深度突变扫描，以绘制FDX 1的结构-功能相互作用。在目标2中，我们将 特别关注脂酰化DLAT在促进铜中毒中的独特调节作用。我们将使用不同的 基于图像的方法来定义lip-DLAT聚集体及其蛋白质的生物物理特性 识别与多个其他细胞特征的链接的组合物。在目标3中，我们将首先探索蛋白质水平 在数百种癌细胞系中进行脂酰化，并建立与高脂酰化癌症相关的细胞状态 细胞然后，我们将确定两种经典的FDX 1和蛋白质脂酰化的表现和依赖性， 异种移植肿瘤模型和在不同器官中生长的转移性肿瘤模型。最后，我们将使用这些 体内模型，以建立癌细胞模型中铜离子载体的功效和靶向接合 对这些化合物高度敏感的细胞这项提案将共同推动我们的 了解FDX 1和蛋白质脂酰化在癌症中的作用，以及这些关键调节因子如何促进 细胞和肿瘤中的铜中毒。

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.