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.
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.
|Dillon, Christopher P; Weinlich, Ricardo; Rodriguez, Diego A et al. (2014) RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3. Cell 157:1189-202|
|Linkermann, Andreas; Skouta, Rachid; Himmerkus, Nina et al. (2014) Synchronized renal tubular cell death involves ferroptosis. Proc Natl Acad Sci U S A 111:16836-41|
|Weinlich, Ricardo; Green, Douglas R (2014) The two faces of receptor interacting protein kinase-1. Mol Cell 56:469-80|
|Gurung, Prajwal; Anand, Paras K; Malireddi, R K Subbarao et al. (2014) FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes. J Immunol 192:1835-46|
|Green, Douglas R; Galluzzi, Lorenzo; Kroemer, Guido (2014) Cell biology. Metabolic control of cell death. Science 345:1250256|
|Green, Douglas R; Levine, Beth (2014) To be or not to be? How selective autophagy and cell death govern cell fate. Cell 157:65-75|
|Newton, Kim; Hildebrand, Joanne M; Shen, Zhirong et al. (2014) Is SIRT2 required for necroptosis? Nature 506:E4-6|
|Linkermann, Andreas; Green, Douglas R (2014) Necroptosis. N Engl J Med 370:455-65|
|Weinlich, Ricardo; Oberst, Andrew; Dillon, Christopher P et al. (2013) Protective roles for caspase-8 and cFLIP in adult homeostasis. Cell Rep 5:340-8|