College of Pharmacy, Keimyung Univ. Programmed Necrosis Friend? Foe? College of Pharmacy, Keimyung Univ. Young Sik Cho

Life or Death

What if programmed cell death might be blocked? Cell Responses to Stress Proliferation Differentiation Senescence What if programmed cell death might be blocked?

Apoptosis Necrosis Yasuhara et al., JHC 2003; 51: 873-885 Normal

APOPTOSIS VS. NECROSIS (I) Programmed Cell Death Uncontrolled, Accidental Death Naturally occurred Caused by external factor Beneficial effects Detrimental effects

APOPTOSIS VS. NECROSIS (II) Cell morphology Shrinkage Swelling Membrane integrity Preservation Loss DNA Fragmentation Random digestion Caspase activation Caspase dependent No involvement of caspase RIP1 requirement Not necessary Required Immune system No inflammatory response Significant inflammatory response Apoptosis Necrosis

Programmed Necrosis Extensive network of genes involved Executed by regulated mechanism Non-Apoptotic Cell Death Caspase independent Cell Death Necroptosis Necrostatin-1, specific inhibitor of PN Ischemic brain injury, myocardial infarction and septic shock

NECROSIS vs. PROGRAMMED NECROSIS NecroX Mitochondria ROS scavenger Inhibits chemical-induced necrosis LG 생명과학 RIP1 specific inhibitor Protects from TNF-induced PN Junying Yuan (HMS)

Putative Necrosis-inducing agents DNA alkylating agent Heavy metal Shikonin Death Receptors Nec-1

Tumor Necrosis Factor alpha Manipulation of Cell Death Tumor Necrosis Factor alpha (TNF α) 185 AA, 17 kDa forms homotrimer Signal transduction through binding TNF  to TNFR Pleiotropic inflammatory cytokine Survival CHX zVAD TNF alpha Nec-1 Apoptosis Necrosis

TNF-mediated cell death via two sequential complex DD RIP1 TRAF2 DD TNFR-1 RIP1 DD DD TRADD TRAF2 RIP1 DD DD C-8&FADD NFkB Survival Necrosis Apoptosis Micheau and Tschopp, Cell, 2003

Programmed necrosis regulator Topics for discussion DD Unknown RIP1 P P Programmed necrosis regulator Physiological role of Programmed Necrosis?

Identification of RIP3 as a hit for regulation of PN Silencing genes TNF  Apoptosis PN Survival

Receptor interacting protein kinase 3 (RIPK3) RIP family of serine/threonine protein kinases Unique C-terminal domain distinct from other RIP family members Component of TNF R-I signaling complex Induce apoptosis and weakly activate the NFkB transcription factor

RIP1 and RIP3 are specifically required for PN, but not apoptosis

RIP3 -/- MEFs are resistant to TNF-induced PN, but not apoptosis Apoptosis Necrosis

The Kinase and RHIM domains of RIP3 are crucial for programmed necrosis

Pronecrotic complex formation under condition of PN Nec-1: Specific inhibitor of RIP1

Apoptosis Programmed Necrosis RIP3 as a regulator of cell death TNFα

Role of RIP3 for TNF-stimulated PN when caspase inactivated B Uninfected C VV-NS WO/CHX zVAD surrogate VV-TNF

RIP3-dependent PN & Inflammation during VV infection Fat Liver

Viral titers in tissues and survival plot of VV-infected RIP3+/+ and RIP3 -/- mice

Model of the role of RIP3-dependent PN in the innate immune defense against VV infections

Programmed necrosis: Innate immune response Take-Home Message P DD RIP3 RIP1 P P Receptor interacting protein-3 (RIP3) as a regulator of programmed necrosis Programmed necrosis: Innate immune response

Physiological consequences of Programmed Necrosis Diseases Host defensive machinery Stroke Heart attack Sepsis

RIP3 as a therapeutic target?

Interface between apoptosis and programmed necrosis Galluzzi & Kromer, Cell 2008, 135: 1161-1163 Wang et al., Cell 2012, 148, 228-243

Drug development targeting pronecrotic complex RIP1 DD RIP3 P ① ② RIP1-RIP3 interaction Kinase inhibtion Protein substrate ③

Chemicals against programmed necrosis Mol Structure EC50 (µM) Apoptosis TNF zVAD I 2.8 - II 5.4 3.6 III 6.0 IV 5.0 2.0 V 3.0 VI 4.8 VII 12.0 VIII 6.2 0.6 XI 0.1 0.3 1.0 -: not protected

Chemical-induced Programmed Necrosis Ongoing Project Chemical-induced Programmed Necrosis Dissection of Programmed Necrosis Cell Host & Microbe 7, 302-313, 2010

Chemical-Induced Necrosis Shikonin (SKN) IP : RIP3 WB : RIP1 Nec-1 10 uM TNF-induced PN 50 uM CHX - WB : RIP3 + SKN CONT.

Differential roles of RIP1 and RIP3 for programmed necrosis (B) mRIP1/mRIP3 Mock mRIP1 mRIP3 siRNA RIP3 RIP1 (C) β-actin L929 cell line Fibrosarcoma cell line Caspases not activated Gene silencing

Caspase Necrosis zVAD TNFα Nec I Proposed model for distinctive role s of RIP1 and RIP3 Caspase zVAD TNFα DD Nec I RIP3 RIP1 Necrosis

세포사멸 통합 시스템 구축 세포 괴사 자살 자가 포식 심근경식 뇌경색 폐혈성 쇼크 항암 치료 퇴행성 뇌질환 자가면역 질환 당뇨병

Therapeutic Use of Programmed Necrosis for cancer Chemotherapy BCR-ABL positive leukemia by Gleevec (imatinib) DNA alkylating agent Photosensitising molecules Host-Microbes: To kill or To be killed Virus Intracellular bacteria Acute and Chronic Diseases Neurodegenerative disease Septic shock Pancreatitis

Summary of clinical trials for the extrinsic apoptotic pathway Christina et al., Am J Cancer Res 2011, 1: 43-61

Summary of clinical trials for the autophagy pathway Christina et al., Am J Cancer Res 2011, 1: 43-61

Harnessing Cell Death Pathways for Cancer Therapy Boosting apoptotic sensitization to chemotherapy or radiotherapy Harnessing alternative cell death: Drug resistance Autophagy Necrosis PDT

PARP activity switches the cell fate DNA repair PARP Glucose 2 Pyruvate 2NAD+ 2NADH+ 2pyruvate CO2+H2O ADP ATP NAD Alkylating agent Ionizing radiation Amino acids Fatty acids Macro-molecular synthesis Oxidative Phosphorylation Proliferation Cells Programmed Necrosis Vegetative cells Cell survival and

Acknowledgement KMU UMASS Seung Yeon Park Ji Hyun Bae Francis Chan David Moquin Sreerupa Challa Melissa Guildford UMASS Seung Yeon Park Ji Hyun Bae KMU

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