Roshan J. Thapa

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases

Significance The interferons are small secreted proteins with powerful antiviral and cytotoxic properties. Here, we outline a signaling pathway activated by interferons that results in the precipitous necrotic death of susceptible cells. Interferon-induced necrosis proceeds via a novel, progressive mechanism that requires RNA transcription, as well as the sequential activity of three serine-threonine kinases: PKR, RIP1, and RIP3. This pronecrotic kinase cascade is normally held in check by FADD and caspases. As FADD can be disabled by phosphorylation during mitosis, our findings suggest the existence of a putative cell cycle-dependent checkpoint that licenses interferon-induced necrosis. Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1–RIP3 “necrosome” complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition

Significance The protein kinase receptor interacting protein 1 controls signaling via death receptors, Toll-like receptors, and retinoic acid-inducible gene 1-like receptors, dictating inflammatory outcomes as broad as cytokine activation and cell death. RIP1 makes a vital contribution during development, evident from the fact that RIP1-deficient mice die soon after birth. Here, we show that a kinase-independent function of RIP1 dampens the consequences of innate immune cell death. During parturition, RIP1 prevents the lethal consequences of RIP3-dependent necroptosis as well as caspase 8 (Casp8)-dependent apoptosis. In contrast to the RIP1-deficient phenotype, mice lacking a combination of RIP1, RIP3, and Casp8 are born and mature into viable, fertile, and immunocompetent adults. These results demonstrate the important protective role of RIP1 against physiologic and microbial death cues encountered at birth. The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1−/− mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like–mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1−/−Casp8−/−Rip3+/− mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.

Inactivation of ribosomal protein L22 promotes transformation by induction of the stemness factor, Lin28B

Ribosomal protein (RP) mutations in diseases such as 5q- syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.

Requirement of FADD, NEMO, and BAX/BAK for Aberrant Mitochondrial Function in Tumor Necrosis Factor Alpha-Induced Necrosis

ABSTRACT Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-xL protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.

NF-κB Protects Cells from Gamma Interferon-Induced RIP1-Dependent Necroptosis

ABSTRACT Interferons (IFNs) are cytokines with well-described immunomodulatory and antiviral properties, but less is known about the mechanisms by which they promote cell survival or cell death. Here, we show that IFN-γ induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-κB signaling. Induction of necroptosis by IFN-γ was found to depend on Jak1 and partially on STAT1. We also demonstrate that IFN-γ activates IκB kinase β (IKKβ)-dependent NF-κB to regulate a transcriptional program that protects cells from necroptosis. IFN-γ induced progressive accumulation of reactive oxygen species (ROS) and eventual loss of mitochondrial membrane potential in cells lacking the NF-κB subunit RelA. Whole-genome microarray analyses identified sod2, encoding the antioxidant enzyme manganese superoxide dismutase (MnSOD), as a RelA target and potential antinecroptotic gene. Overexpression of MnSOD inhibited IFN-γ-mediated ROS accumulation and partially rescued RelA-deficient cells from necroptosis, while RNA interference (RNAi)-mediated silencing of sod2 expression increased susceptibility to IFN-γ-induced cell death. Together, these studies demonstrate that NF-κB protects cells from IFN-γ-mediated necroptosis by transcriptionally activating a survival response that quenches ROS to preserve mitochondrial integrity.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

Distinct Roles for the NF-κB RelA Subunit during Antiviral Innate Immune Responses

Production of type I interferons (IFNs; prominently, IFN-α/β) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-β proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnβ promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnβ expression. Here, we show that RelA instead sustains autocrine IFN-β signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnβ induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnβ expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-β signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.ABSTRACT Production of type I interferons (IFNs; prominently, IFN-α/β) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-β proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnβ promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnβ expression. Here, we show that RelA instead sustains autocrine IFN-β signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnβ induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnβ expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-β signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.

Targeted Blockade of JAK/STAT3 Signaling Inhibits Ovarian Carcinoma Growth

Ovarian carcinoma is the fifth leading cause of death among women in the United States. Persistent activation of STAT3 is frequently detected in ovarian carcinoma. STAT3 is activated by Janus family kinases (JAK) via cytokine receptors, growth factor receptor, and non–growth factor receptor tyrosine kinases. Activation of STAT3 mediates tumor cell proliferation, survival, motility, invasion, and angiogenesis, and recent work demonstrates that STAT3 activation suppresses antitumor immune responses and supports tumor-promoting inflammation. We hypothesized that therapeutic targeting of the JAK/STAT3 pathway would inhibit tumor growth by direct effects on ovarian carcinoma cells and by inhibition of cells in the tumor microenvironment (TME). To test this, we evaluated the effects of a small-molecule JAK inhibitor, AZD1480, on cell viability, apoptosis, proliferation, migration, and adhesion of ovarian carcinoma cells in vitro. We then evaluated the effects of AZD1480 on in vivo tumor growth and progression, gene expression, tumor-associated matrix metalloproteinase (MMP) activity, and immune cell populations in a transgenic mouse model of ovarian carcinoma. AZD1480 treatment inhibited STAT3 phosphorylation and DNA binding, and migration and adhesion of cultured ovarian carcinoma cells and ovarian tumor growth rate, volume, and ascites production in mice. In addition, drug treatment led to altered gene expression, decreased tumor-associated MMP activity, and fewer suppressor T cells in the peritoneal TME of tumor-bearing mice than control mice. Taken together, our results show pharmacologic inhibition of the JAK2/STAT3 pathway leads to disruption of functions essential for ovarian tumor growth and progression and represents a promising therapeutic strategy. Mol Cancer Ther; 14(4); 1035–47. ©2015 AACR.

NF-YA Underlies EZH2 Upregulation and Is Essential for Proliferation of Human Epithelial Ovarian Cancer Cells

Epithelial ovarian cancer (EOC) accounts for the most gynecologic malignancy–associated deaths in the United States. Enhancer of zeste homolog 2 (EZH2), which silences gene expression through generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3), is often overexpressed in EOCs and has been suggested as a therapeutic target. However, the mechanism underlying EZH2 overexpression in EOCs is unknown. Here, we show that EZH2 is upregulated at the transcription level, and two CCAAT boxes in the proximal regions of the human EZH2 gene promoter are critical for its transcription in EOC cells. Indeed, NF-YA, the regulatory subunit of the CCAAT-binding transcription factor NF-Y, is expressed at higher levels in human EOCs than in primary human ovarian surface epithelial (HOSE) cells. In addition, there is a positive correlation between expression of NF-YA and EZH2 in EOCs. Notably, high NF-YA expression predicts shorter overall survival in patients with EOCs. The association of NF-YA with the promoter of the human EZH2 gene is enhanced in human EOC cells compared with primary HOSE cells. Significantly, knockdown of NF-YA downregulates EZH2, decreases H3K27Me3 levels, and suppresses the growth of human EOC cells both in vitro and in a xenograft mouse model. Notably, NF-YA knockdown induces apoptosis of EOC cells and ectopic EZH2 expression partially rescues apoptosis induced by NF-YA knockdown. Together, these data reveal that NF-Y is a key regulator of EZH2 expression and is required for EOC cell proliferation, thus representing a novel target for developing EOC therapeutics. Mol Cancer Res; 11(4); 360–9. ©2013 AACR.

Identification of STAT2 Serine 287 as a Novel Regulatory Phosphorylation Site in Type I Interferon-induced Cellular Responses

Background: STAT2 is a key transcription factor that mediates the protective role of type I interferons in host defense. Results: Type I interferons induce the phosphorylation of STAT2 at serine 287. Conclusion: Serine 287-STAT2 is a regulatory site involved in modulating the transcriptional and cellular responses to type I interferons. Significance: Deregulated STAT2 signaling may contribute to heightened type I interferon responses and susceptibility to many diseases. STAT2 is a positive modulator of the transcriptional response to type I interferons (IFNs). STAT2 acquires transcriptional function by becoming tyrosine phosphorylated and imported to the nucleus following type I IFN receptor activation. Although most STAT proteins become dually phosphorylated on specific tyrosine and serine residues to acquire full transcriptional activity, no serine phosphorylation site in STAT2 has been reported. To find novel phosphorylation sites, mass spectrometry of immunoprecipitated STAT2 was used to identify several phosphorylated residues. Of these, substitution of serine 287 with alanine (S287A) generated a gain-of-function mutant that enhanced the biological effects of IFN-α. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-α. In contrast, a phosphomimetic STAT2 mutant (S287D) produced a loss-of-function protein that weakly activated IFN-induced ISGs. Our mechanistic studies suggest that S287A-STAT2 likely mediates its gain-of-function effects by prolonging STAT2/STAT1 dimer activation and retaining it in transcriptionally active complexes with chromatin. Altogether, we have uncovered that in response to type I IFN, STAT2 is serine phosphorylated in the coiled-coil domain that when phosphorylated can negatively regulate the biological activities of type I IFNs.