NF-κB pathway controls mitochondrial dynamics

The Optic atrophy 1 protein (OPA1) is a key element in the dynamics and morphology of mitochondria. We demonstrated that the absence of IκB kinase-α, which is a key element of the nonclassical NF-κB pathway, has an impact on the mitochondrial network morphology and OPA1 expression. In contrast, the absence of NF-κB essential modulator (NEMO) or IκB kinase-β, both of which are essential for the canonical NF-κB pathway, has no impact on mitochondrial dynamics. Whereas Parkin has been reported to positively regulate the expression of OPA1 through NEMO, herein we found that PARK2 overexpression did not modify the expression of OPA1. PARK2 expression reduced the levels of Bax, and it prevented stress-induced cell death only in Bak-deficient mouse embryonic fibroblast cells. Collectively, our results point out a role of the nonclassical NF-κB pathway in the regulation of mitochondrial dynamics and OPA1 expression.Mitochondria perform multiple functions that are critical to the maintenance of cellular homeostasis. Mitochondrial dysfunctions have been linked to the development of degenerative diseases and aging. Damaged mitochondria are removed by mitophagy, a process partially regulated by the PARK2-encoded E3 ubiquitin ligase (Parkin) in a PTEN-induced putative protein kinase 1 (PINK1)-dependent manner.^{1, 2, 3, 4} During mitophagy, the phosphorylation of mitofusin (Mfn) 2 by PINK1 has been suggested to induce the recruitment of Parkin to the mitochondria in cardiomyocytes.⁵ However, previous groups have shown that that Mfn 1 and 2 are dispensable for Parkin-dependent mitophagy in fibroblasts, whereas the Parkin-dependent degradation of these proteins may impair fusion of damaged mitochondria with the healthy network.^{6, 7, 8} PINK1 and Parkin thus act as a quality control machinery on the outer mitochondrial membrane (OMM) to preserve mitochondrial integrity through the ubiquitination of OMM proteins.^{9, 10} Moreover, through its E3 ubiquitin ligase activity,^{11, 12} Parkin was reported to bind to the linear ubiquitin chain assembly complex (LUBAC) and to increase the ubiquitination of NF-κB essential modulator (NEMO),¹³ a component of the classical NF-κB signaling pathway.¹⁴ Müller–Rischart et al. also proposed that Parkin positively regulates the expression of the mitochondrial guanosine triphosphatase Optic atrophy 1 protein (OPA1) through linear ubiquitination of NEMO.¹³ OPA1 is a regulator of mitochondrial inner membrane fusion and cristae remodeling.^{15, 16, 17} A defect in OPA1 expression is associated with mitochondrial network fragmentation and enhanced sensitivity of the cells to undergo apoptosis by promoting cytochrome c release from the mitochondria.^{18, 19, 20} Because NEMO-deficient mouse embryonic fibroblast (MEF) cells display a normal mitochondrial network morphology, we decided to re-examine the role of Parkin in regulating OPA1 expression through the NF-κB signaling pathway.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Intrinsic and chemo-sensitizing activity of SMAC-mimetics on high-risk childhood acute lymphoblastic leukemia

SMAC-mimetics represent a targeted therapy approach to overcome apoptosis resistance in many tumors. Here, we investigated the efficacy of the SMAC-mimetic BV6 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In ALL cell lines, intrinsic apoptosis sensitivity was associated with rapid cIAP degradation, NF-κB activation, TNF-α secretion and induction of an autocrine TNF-α-dependent cell death loop. This pattern of responsiveness was also observed upon ex vivo analysis of 40 primograft BCP-ALL samples. Treatment with BV6 induced cell death in the majority of ALL primografts including leukemias with high-risk and poor-prognosis features. Inhibition of cell death by the TNF receptor fusion protein etanercept demonstrated that BV6 activity is dependent on TNF-α. In a preclinical NOD/SCID/huALL model of high-risk ALL, marked anti-leukemia effectivity and significantly prolonged survival were observed upon BV6 treatment. Interestingly, also in vivo, intrinsic SMAC-mimetic activity was mediated by TNF-α. Importantly, BV6 increased the effectivity of conventional induction therapy including vincristine, dexamethasone and asparaginase leading to prolonged remission induction. These data suggest SMAC-mimetics as an important addendum to efficient therapy of pediatric BCP-ALL.In childhood acute lymphoblastic leukemia (ALL), major improvements in therapy and supportive care have led to increased survival rates.¹ In current treatment protocols, patients are assigned to multi-agent chemotherapy regimens of different intensities based on the patient''s individual risk profile and probability to encounter relapse.² However, 20% of patients relapse or do not respond well to therapy.^{3, 4} Therefore, new treatment strategies are required to overcome resistance mechanisms and enhance outcome. Defects in cell death mechanisms contribute to resistance and treatment failure in many cancers, including leukemia.^{5, 6} We previously described, that deficient apoptosis signaling in primary, patient-derived leukemia cells is indicative for early patient relapse and associated with rapid engraftment in NOD/SCID mice.^{7, 8, 9, 10}Two major apoptosis signaling pathways are known, the death receptor (extrinsic) and mitochondrial (intrinsic) pathway.¹¹ Within the extrinsic pathway, activation of death receptors of the tumor necrosis factor (TNF) superfamily activate caspase-8 via a death-inducing signaling complex (DISC) leading to activation of effector caspases.¹² Activation of intrinsic signaling triggers mitochondrial release of cytochrome c and SMAC (second mitochondria-derived activator of caspase). Released cytochrome c forms a complex together with caspase-9, the apoptosome, which cleaves and activates caspase-3. SMAC antagonizes inhibitor of apoptosis proteins (IAPs), thus enabling apoptosis signaling.¹³IAPs constitute a family of structurally related proteins, defined by the presence of at least one baculoviral IAP repeat (BIR) domain, which can bind to active subunits of caspases, thereby inhibiting their function. Some IAPs (e.g., cIAP) also possess a RING domain conferring E3 ligase activity mediating ubiquitination reactions.¹⁴Since IAPs are often overexpressed and associated with inferior outcome in different malignancies including childhood acute leukemias, they may serve as an attractive target for therapeutic intervention.^{15, 16, 17, 18} Previously, IAP antagonists, binding to selected BIR domains of IAPs and leading to cell death sensitization were designed.^{16, 19, 20, 21} Recently, a new class of molecules mimicking the N-terminal domain of SMAC (SMAC-mimetics) was developed, which bivalently bind to both BIR2 and BIR3 domains, and possess additional intrinsic apoptogenic activity via a TNF-α feed forward loop.^{22, 23} Within this TNF-α loop, SMAC-mimetics stimulate the E3-ubiquitine ligase activity of cIAPs, leading to autoubiquitination and subsequent proteasomal degradation of cIAPs. Depletion of cIAPs enables accumulation of NF-κB-inducing kinase (NIK), resulting in non-canonical activation of NF-κB and NF-κB target gene expression, including TNFA, which stimulates TNFR1 in an autocrine loop.²³ In the absence of cIAP, TNFR1-stimulation triggers the assembly of the secondary RIP1-containing cytoplasmatic complex (complex II), leading to caspase-8 activation and cell death induction.²⁴ SMAC-mimetics may induce cell death dependently or independently of TNF-α,^{25, 26, 27} and we previously showed, that counteracting caspase inhibition by IAP antagonists sensitizes cancer cells for death receptor ligand- and chemotherapy-induced apoptosis.^{28, 29}Here, we analyze the therapeutic potential of the SMAC-mimetic BV6 in pediatric B-cell precursor (BCP)-ALL in vitro, ex vivo and in a preclinical model in vivo, and define the molecular requirements for intrinsic activity.     

Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO^LPC-KO mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. To address potential functional redundancies between death receptors we generated and analysed NEMO^LPC-KO mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO^LPC-KO mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO^LPC-KO mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO^LPC-KO mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.Liver cancer is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide.^{1, 2} Liver cancer predominantly arises in the context of chronic inflammatory conditions, most notably in virus hepatitis (HBV and HCV).^{1, 2} Although infectious agents are the primary cause of liver cancer worldwide, the incidence in western countries is rising due to the increase in obesity and non-alcoholic steatohepatitis.³ The pathogenesis of hepatocellular carcinoma (HCC) is incompletely understood and it is plausible that the different underlying aetiologies determine a distinct context for liver carcinogenesis. However, the prevailing universal concept is that continuous liver parenchymal damage and hepatocyte cell death drive compensatory proliferation and within the context of a chronically inflamed liver tissue mutations and epigenetic changes accumulate eventually transforming hepatocytes into malignant cells. Therefore, understanding the tissue-intrinsic processes that determine cell death and chronic inflammation resulting in hepatocarcinogenesis is a critical need in order to design more effective therapeutic strategies.The nuclear factor κB (NF-κB) pathway is implicated in cancer development in particular in the context of chronic inflammation.^{4, 5} In relation to liver cancer, NF-κB signalling has been implicated in the pathogenesis of hepatitis, liver fibrosis, cirrhosis and HCC.^{6, 7} The IKK complex, composed of two catalytic subunits, IKK1/IKKα and IKK2/IKKβ, and a regulatory subunit termed NEMO/IKKγ, activates NF-κB by phosphorylating inhibitor of NF-κB (IκB) proteins targeting them for degradation by the proteasome and thus allowing the nuclear accumulation of NF-κB dimers.⁵ IKK2 is primarily responsible for targeting and degrading IκBα thus inducing canonical NF-κB activation, although the two kinases show some degree of functional redundancy in controlling canonical NF-κB signalling.^{5, 8} NEMO/IKKγ is indispensable for activation of canonical NF-κB signalling.^{9, 10, 11}NF-κB signalling was proposed to exhibit tumour promoter or tumour suppressor properties in different models of liver cancer. In the Mdr2^−/− mouse model of inflammation-driven liver carcinogenesis, NF-κB inhibition caused by transgenic IκBα super–repressor expression in hepatocytes inhibited HCC progression.¹² Moreover, hepatocyte-restricted ablation of IKK2 prevented hepatitis and liver tumorigenesis induced by overexpression of lymphotoxins α and β in hepatocytes.¹³ However, mice with hepatocyte-specific IKK2 ablation developed more tumours induced by a single injection of the chemical carcinogen diethylnitrosamine,¹⁴ revealing a tumour suppressor role of NF-κB in this context.Studies in mice lacking NEMO specifically in liver parenchymal cells (LPCs) further supported a tumour suppressor function of IKK/NF-κB signalling in liver cancer. NEMO^LPC-KO mice showed spontaneous hepatocyte apoptosis resulting in chronic steatohepatitis and the development of HCC by the age of 1 year.¹⁵ LPC-specific ablation of Fas-Associated with Death Domain (FADD or MORT1), an adapter protein essential for the recruitment of caspase-8 to the Death Inducing Signalling Complex and the induction of death receptor-mediated apoptosis,¹⁶ prevented both spontaneous and LPS-induced apoptosis of NEMO-deficient hepatocytes and the development of steatohepatitis.¹⁵ In addition, LPC-specific knockout of caspase-8 inhibited spontaneous hepatocyte apoptosis and HCC development in NEMO^LPC-KO mice, although it caused non-apoptotic hepatocyte death and cholestasis.¹⁷ Given the essential role of FADD and caspase-8 in mediating apoptosis downstream of death receptors,¹⁶ we hypothesized that death receptor-mediated apoptosis of NEMO-deficient hepatocytes drives the development of hepatitis and HCC in NEMO^LPC-KO mice. The three main death receptors of the TNF receptor superfamily that are capable of inducing caspase-8-mediated apoptosis are TNFR1, Fas/CD95 and TRAIL-R/DR5.¹⁶ To address the role of death receptor-induced apoptosis in triggering the spontaneous death of NEMO-deficient hepatocytes and the development of steatohepatitis and HCC, we generated and analysed NEMO^LPC-KO mice lacking TNFR1, Fas or TRAIL-R specifically in LPCs. Surprisingly, we found that LPC-specific knockout of each of the death receptors alone but also combined deficiency of TNFR1, Fas and TRAIL-R in LPCs did not prevent spontaneous hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. In addition, knockout of TNF in all cells also did not protect NEMO^LPC-KO mice from hepatocyte death, hepatitis and HCC. Collectively, these results demonstrate that TNFR1, Fas and TRAIL-R are not required for the development of chronic liver damage and HCC in NEMO^LPC-KO mice.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34⁺ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.^{1, 2} Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.^{3, 4, 5, 6, 7} p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.^{8, 9} At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).^{3, 5, 6, 7, 10, 11, 12, 13} p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL¹⁴ and Wnt pathways^{15, 16} or by inducing the activation of caspase 8, a pro-death protein.^{17, 18} Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.¹⁰ In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.¹⁹ In contrast, p62/SQSTM1 favors differentiation of osteoclasts,²⁰ osteoblasts,²¹ neurons,²² megakaryocytes²³ and macrophages.²⁴ The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.^{25, 26} Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.^{27, 28} All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.^{29, 30, 31} Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.^{32, 33, 34}Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.³⁵ In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes.     

cFLIP is critical for oligodendrocyte protection from inflammation

Neuroinflammation associated with degenerative central nervous system disease and injury frequently results in oligodendrocyte death. While promoting oligodendrocyte viability is a major therapeutic goal, little is known about protective signaling strategies. We report that in highly purified rat oligodendrocytes, interferon gamma (IFNγ) activates a signaling pathway that protects these cells from tumor necrosis factor alpha (TNFα)-induced cytotoxicity. IFNγ protection requires Jak (Janus kinase) activation, components of the integrated stress response and NF-κB activation. Although NF-κB activation also occurred transiently in the absence of IFNγ and presence of TNFα, this activation was not sufficient to prevent induction of the TNFα-responsive cell death pathway. Genetic inhibition of NF-κB translocation to the nucleus abrogated IFNγ-mediated protection and did not change the cell death induced by TNFα, suggesting that NF-κB activation via IFNγ induces a different set of responses than activation of NF-κB via TNFα. A promising candidate is the NF-κB target cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which is protease-deficient caspase homolog that inhibits caspase-3 activation. We show that IFNγ-mediated protection led to upregulation of cFLIP. Overexpression of cFLIP was sufficient for oligodendrocyte protection from TNFα and short hairpin RNA knockdown of cFLIP-abrogated IFNγ -mediated protection. To determine the relevance of our in vitro finding to the more complex in vivo situation, we determined the impact on oligodendrocyte death of regional cFLIP loss of function in a murine model of neuroinflammation. Our data show that downregulation of cFLIP during inflammation leads to death of oligodendrocytes and decrease of myelin in vivo. Taken together, we show that IFNγ-mediated induction of cFLIP expression provides a new mechanism by which this cytokine can protect oligodendrocytes from TNFα-induced cell death.Interferon gamma (IFN-γ), the only type-II class IFN, has a paradoxical role in modulating cell function. It is critical for innate and adaptive immunity, but has multiple other functions. In the central nervous system (CNS), IFNγ has contrasting effects on the oligodendrocyte progenitor cells (O-2A/OPCs) that generate myelin-producing oligodendrocytes. O-2A/OPCs show suppressed division when exposed to IFNγ.^{1, 2, 3} However, when O-2A/OPCs differentiate into oligodendrocytes, IFNγ becomes pro-apoptotic.^{4, 5, 6, 7} Although IFNγ has a critical role in the pathogenesis of immune-mediated demyelinating disease;^{8, 9} the response of committed oligodendrocytes to IFNγ is more complex. For example, tumor necrosis factor alpha (TNFα) can show enhanced cytotoxicity in oligodendrocytes and transformed human neural cell lines when co-exposed with IFNγ.^{3, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19}In contrast with reported toxic effects of IFNγ on oligodendrocytes, other studies did not see negative effects on mature oligodendrocytes^{5, 9, 20} or saw protection of glial lineage cells. IFNγ protects the Oli-neu oligodendrocyte-like cell line from reactive oxygen and nitrogen species,²¹ and overexpression of IFNγ before the induction of experimental autoimmune encephalomyelitis (EAE) protected oligodendrocytes from immune-mediated damage.⁹ The mechanism of such protection remains elusive.We now report that IFNγ protects purified, committed oligodendrocytes from TNFα-mediated apoptosis via Janus kinase (Jak)-mediated activation of the stress kinase PKR (double-stranded RNA-dependent protein kinase) and NF-κB-induced expression of cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which inhibits caspase activation. Moreover, gain-of-function and loss-of-function experiments show that cFLIP is necessary and sufficient for oligodendrocyte protection from TNFα. These results demonstrate induction of cFLIP in a stress response and NF-κB-dependent manner, leading to inhibition of caspase-mediated apoptosis, and reveal an important role for cFLIP in oligodendrocyte protection in vivo.     

Activation of TNFR2 sensitizes macrophages for TNFR1-mediated necroptosis

Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes.Tumor necrosis factor (TNF) is a pleiotropic cytokine that occurs as a type II transmembrane protein but can be released from the plasma membrane by proteolytic processing.¹ Membrane-bound and soluble TNF both contain the characteristic carboxy-terminal TNF homology domain, which is responsible for self-assembly into trimeric molecules and receptor binding. Membrane-bound and soluble TNF strongly interact with two receptors, TNFR1 and TNFR2, but the two forms of TNF are differentially effective in receptor activation.¹ Whereas membrane-bound TNF activates TNFR1 and TNFR2 efficiently, soluble TNF is sufficient for TNFR1 activation but largely inactive upon binding to TNFR2. TNFR1 belongs to the death receptor subgroup of the TNF receptor family and can trigger apoptosis and necroptosis.^{2, 3, 4} However, cell death induction by TNFR1 is typically efficiently antagonized by concomitant activation of the cytoprotective classical NFκB pathway and/or ubiquitous expression of anti-apoptotic proteins.^{1, 2} The latter involve FLIP proteins which generally inhibit death receptor-induced caspase-8 activation but also complexes containing TRAF2, cIAP1 and cIAP2 which specifically interfere with caspase-8 activation in context of TNFR1 signaling.^{2, 3, 4} Worth mentioning, TRAF2-cIAP1/2 complexes also mediate K63-linked ubiquitination of RIP1 in the TNFR1 signaling complex, thereby facilitating TNFR1-mediated activation of the classical NFκB pathway. Indeed, TNFR1 signaling is predominantly pro-inflammatory as TNFR1-induced cell death is blocked as long as the aforementioned protective mechanisms are not impaired.In contrast to TNFR1, TNFR2 contains no cytoplasmic death domain. Upon ligand binding, TNFR2 recruits TRAF2 and various TRAF2-associated proteins, such as TRAF1, cIAP1 and cIAP2, but also interacts with other signaling proteins independently of TRAF2.^{1, 5} TNFR2 activation has been linked to a variety of immune regulatory functions, which, in contrast to the activities of TNFR1, often result in anti-inflammatory effects.⁶Murine models shed light on the complex interplay of the TNFR1–TNFR2 system in vivo, demonstrating additive, synergistic or even antagonistic effects. At the cellular level, several mechanisms for the cross-talk between TNFR1 and TNFR2 have been identified.¹ Besides the obvious competition for ligand binding, TNFR1 and TNFR2 can induce, for example, autocrine TNF production in a cell type-specific manner.¹ In context of TNFR1 activation by soluble TNF, subsequent induction of membrane-bound TNF results in costimulation of TNFR2, thereby converting the initially transient activation into sustained autocrine signaling. In addition, TNFR1 and TNFR2 compete for the cytoplasmic pool of TRAF2–cIAP1/2 complexes. By depletion and/or degradation of TRAF2, TNFR2 is capable to modulate TNFR1 signaling.¹ Moreover, TNFR2 but not TNFR1, stimulates the alternative NFκB pathway by triggering proteolytic processing of the inactive p100/RelB dimers into active p52/RelB NFκB complexes.⁷ Notably, TNFR2-induced alternative NFκB signaling can be enhanced by TNFR1-mediated induction of p100 and RelB expression via the classical NFκB pathway.⁷In macrophages, the complexity of the TNF-TNFR1/2 system is especially relevant. Macrophages on one hand co-express TNFR1 and TNFR2 and are on the other hand a pathophysiologically important source of TNF, for example, in response to a variety of pathogen-associated molecular patterns (PAMPs). TNF not only acts as a macrophage-derived effector molecule, but in an autocrine fashion also controls macrophage activation and survival, as seen for example during infection with mycobacteria.^{8, 9, 10, 11, 12, 13, 14, 15, 16, 17} However, the molecular mechanisms of TNF-induced cell death in macrophages are incompletely understood and were, therefore addressed in our study. Using macrophages genetically deficient for TNFR1, TNFR2 or TNF together with TNFR1- and TNFR2-specific TNF variants, we show that TNFR2 activation sensitizes macrophages for TNFR1-mediated necroptosis triggered by autocrine produced TNF and provide evidence that this is related to TNFR2-induced depletion/degradation of TRAF2-cIAP1/2 complexes.