Martin Leverkus

cIAPs Block Ripoptosome Formation, a RIP1/Caspase-8 Containing Intracellular Cell Death Complex Differentially Regulated by cFLIP Isoforms

Summary The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either apoptosis or necroptosis. This 2 MDa intracellular complex that we designate “Ripoptosome” is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIPL prevents Ripoptosome formation, whereas, intriguingly, cFLIPS promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the “rheostat” that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.

NFκB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFκB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFκB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFκB was activated by overexpressed FLIPL and FLIPS in a cell type–specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFκB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFκB target gene IL8. As NFκB signaling is inhibited during apoptosis, FasL-induced NFκB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFκB-related response.

Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment

cIAPs keep RIP1 from getting to the DISC complex and complex II; when cIAPs are repressed, signaling is modulated by the cFLIP isoform.

Cutaneous, gastrointestinal, hepatic, endocrine, and renal side-effects of anti-PD-1 therapy

BACKGROUND Anti-programmed cell death receptor-1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma as well as for other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects affect skin, gastrointestinal tract, liver, endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential. METHODS AND FINDINGS In total, 496 patients with metastatic melanoma from 15 skin cancer centers were treated with pembrolizumab or nivolumab; 242 side-effects were described in 138 patients. In 116 of the 138 patients, side-effects affected the skin, gastrointestinal tract, liver, endocrine, and renal system. Rare side-effects included diabetes mellitus, lichen planus, and pancreas insufficiency due to pancreatitis. CONCLUSION Anti-PD1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity.

Neurological, respiratory, musculoskeletal, cardiac and ocular side-effects of anti-PD-1 therapy

BACKGROUND Anti-programmed cell death 1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma and other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects can involve skin, gastrointestinal tract, liver, the endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential. METHODS AND FINDINGS In total, 496 patients with metastatic melanoma from 15 skin cancer centres were treated with pembrolizumab or nivolumab. Two hundred forty two side-effects in 138 patients have been analysed. In 77 of the 138 patients side-effects affected the nervous system, respiratory tract, musculoskeletal system, heart, blood and eyes. Not yet reported side-effects such as meningo-(radiculitis), polyradiculitis, cardiac arrhythmia, asystolia, and paresis have been observed. Rare and difficult to manage side-effects such as myasthenia gravis are described in detail. CONCLUSION Anti-PD-1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity.

Elevated serum levels of interleukin-10 in patients with metastatic malignant melanoma

Interleukin-10 (IL-10), originally described as a product of TH2 cell clones, has been recognized as a potential immunosuppressive cytokine. To investigate the relevance of IL-10 in melanoma patients in vivo, we studied IL-10 serum levels in 104 untreated patients in different stages of the disease; 20 healthy subjects and 22 patients with inflammatory dermatoses served as controls. Serum levels were measured by ELISA. Only one of 31 patients with stage I melanoma (3%) and one of 16 stage II patients (6%) showed detectable IL-10 levels. Interestingly, six of 17 patients with lymph node metastases (stage III, 35%) and 29 of 40 patients with widespread disease (stage IV, 73%) revealed IL-10 levels of 15-480 pg/ml. No healthy person and only one control patient had a detectable IL-10 serum level. The data suggest that IL-10 in melanoma patients may contribute to down-modulation of anti-tumour responses in vivo.

Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation

ABSTRACT Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-κB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-κB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.

MHC class II and CD40 play opposing roles in dendritic cell survival

In contrast to very immature dendritic cells (DC), mature DC are largely resistant to death by CD95 (CD95/APO‐1) ligation. Investigation of other potential death‐inducing ligands showed that mature DC were instead highly susceptible to apoptosis induced by cross‐linking of MHC class II. Thus, increasing DC maturity correlates with increased resistance to CD95 killing, but an increased susceptibility to class II‐mediated killing. Anti‐I‐A/I‐E monoclonal antibodies (mAb) induced rapid (<2 h) apoptotic cell death in mature epidermal, spleen and bone marrow‐derived DC, as determined by annexin/propidium iodide staining, morphological changes, decreased diploidy and loss in mitochondrial membrane potential. Although full class II‐mediated killing required DC cytoskeletal motion, divalent cations and phosphatase activity, neither caspase activation, respiration, RNA or protein synthesis, NO production, nor CD95:CD95L interactions were required. Strikingly, DC pretreated by CD40 mAb cross‐linking, but not by lipopolysaccharide or TNF‐α , were completely resistant to class II‐mediated killing. CD40‐mediated protection was reduced in the presence of the SB202190 inhibitor of the mitogen‐activated protein kinase p38 pathway, but appeared to be independent of p42/44 extracellular signal‐related kinase or NF‐K B activation. Our findings show that in addition to its role as an activator of antigen‐presenting cell function, CD40 provides an important counter‐signal against class II‐induced apoptosis. Thus, these data point to an important role of the T cell in regulating DC survival.

Crystal Violet Assay for Determining Viability of Cultured Cells

Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere.