RIPK-dependent necrosis in development and cancer

描述（由申请人提供）：虽然半胱天冬酶在细胞凋亡中的作用已经确立，但这些蛋白酶在程序性坏死过程中的作用知之甚少。这一应用是基于一些惊人的发现，这些发现表明caspase-8或其接合蛋白FADD的消融可以通过删除程序性坏死所需的激酶RIPK3而完全恢复胚胎致命性。我们的研究表明，FADD、caspase-8和FLIP（一种缺乏催化半胱氨酸的caspase样分子）的复合物可以防止ripk依赖性坏死。我们发现FADD-FLIP-RIPK3 TKO小鼠发育正常，这进一步支持了这一点。我们提出以下研究来描述这些蛋白在发育和癌症中的功能：目的1。FADD-caspase-8-FLIPL复合体保护的发育靶标是什么？caspase- 8、FADD和FLIPL基因敲除的表型都显示胚胎在e10.5左右死亡，这与卵黄囊血管化缺陷有关。在这个目标中，我们将测试血管内皮和造血细胞的早期祖细胞作为这种发育缺陷的最早和最重要的靶点的想法。这样，我们将确定ripk3坏死的其他靶点，并研究参与这种胚胎致死的信号通路。目标2。肿瘤发生中ripk依赖性坏死是如何调控的？Caspase-8在人类中存在于2q33上，在人类神经母细胞瘤、小细胞肺癌和其他癌症中经常被沉默或缺失。然而，这代表了一个悖论，因为许多细胞类型的这种损失使细胞对ripk依赖性坏死敏感。在这里，我们探讨caspase-8的缺失如何不能使肿瘤细胞系对ripk依赖性坏死敏感。我们的研究包括RIPK3转录如何在原代和转化组织中受到控制，以及RIPK1和肿瘤抑制因子CYLD在控制ripk依赖性坏死中的作用。目标3。ripk依赖性坏死是否代表一种潜在的治疗途径？许多癌症治疗方法寻求促进细胞凋亡，这可能会或可能不会促进辅助抗肿瘤免疫。通过将信号转移到ripk坏死，我们可以a)防止抵抗这种死亡形式的组织（例如肝脏）的医源性损伤，同时b)促进肿瘤细胞死亡的炎症模式。我们将建立“纯”rik3诱导的坏死与细胞凋亡的模型，以检验抗肿瘤的结果，并将探索一种反直觉的方法，通过体内死亡受体结联在原位和移植物肿瘤中触发ripk依赖性坏死。肿瘤新血管被靶向的可能性（基于Aim 1的考虑）也将被探讨。这些研究为我们提出的新模型提供了大量的测试和探索，并有可能大大增加对正常发育和癌症中控制生命细胞和死亡的基本过程的理解。

DESCRIPTION (provided by applicant): While the role of caspases in apoptosis is well established, little is known about the role of these proteases in the process of programmed necrosis. This application is based on startling findings that show that embryonic lethality as a result of ablation of caspase-8 or its adapter protein, FADD, is fully rescued by deletion of RIPK3, a kinase required for programmed necrosis. Our studies indicate that a complex of FADD, caspase-8, and FLIP, a caspase-like molecule that lacks a catalytic cysteine, protects against RIPK-dependent necrosis. This is further supported by our finding that the FADD-FLIP-RIPK3 TKO mouse develops normally. We propose the following studies to delineate the functions of these proteins in development and cancer: Aim 1. What is the developmental target protected by the FADD-caspase-8-FLIPL complex? The phenotypes of the caspase- 8, FADD, and FLIPL knockouts all show embryonic lethality around e10.5, associated with a defect in yolk sac vascularization. In this aim, we will test the idea that early progenitors of vascular endothelium and hematopoietic cells serve as the earliest and most important targets of this developmental defect. In so doing, we will identify additional targets of RIPK3-necrosis and investigate the signaling pathways engaged in this embryonic lethality. Aim 2. How is RIPK-dependent necrosis regulated in oncogenesis? Caspase-8, which in humans is present on 2q33, is often silenced or deleted in human neuroblastoma, small cell lung carcinoma, and other cancers. This represents a paradox, however, as such loss in many cell types sensitizes cells to RIPK-dependent necrosis. Here we explore how loss of caspase-8 can fail to sensitize tumor lines to RIPK-dependent necrosis. Our studies include how RIPK3 transcription is controlled in primary and transformed tissues and the role of RIPK1 and the tumor suppressor, CYLD, in controlling RIPK-dependent necrosis. Aim 3. Does RIPK-dependent necrosis represent a potential avenue for therapy? Many approaches to cancer therapy seek to promote apoptosis, which may or may not promote ancillary anti-tumor immunity. By shifting signals to RIPK-necrosis we may a) prevent iatrogenic damage in tissues resistant to this form of death (e.g., liver) while b) promoting an inflammatory mode of tumor cell death. We will model "pure" RIK3-induced necrosis versus apoptosis to examine the anti-tumor consequences, and will explore a counter- intuitive approach to triggering RIPK-dependent necrosis in autochthonous and grafted tumors by death receptor ligation in vivo. The possibility that tumor neo-vasculature is targeted (based on considerations from Aim 1) will also be explored. These studies provide a number of tests and explorations of the new model we propose, and hold the potential to greatly increase of understanding of this fundamental process controlling life cell and death, both in normal development and in cancer. PUBLIC HEALTH RELEVANCE: Cell death is crucial for normal homeostasis, and defects in this process underlie many human diseases. This project explores how cell death is controlled at the level of precise molecular interactions, amenable to pharmacologic manipulation, testing a new model of this process in development and cancer.