TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.

The ubiquitin-editing protein A20 (TNFAIP3) is a known key player in the regulation of immune responses in many organs. Genome-wide associated studies (GWASs) have linked A20 with a number of inflammatory and autoimmune disorders, including psoriasis. Here, we identified a previously unrecognized role of A20 as a pro-apoptotic factor in TNF-induced cell death in keratinocytes. This function of A20 is mediated via the NF-κB-dependent alteration of cIAP1/2 expression. The changes in cIAP1/2 protein levels promote NIK stabilization and subsequent activation of noncanonical NF-κB signaling. Upregulation of TRAF1 expression triggered by the noncanonical NF-κB signaling further enhances the NIK stabilization in an autocrine manner. Finally, stabilized NIK promotes the formation of the ripoptosome and the execution of cell death. Thus, our data demonstrate that A20 controls the execution of TNF-induced cell death on multiple levels in keratinocytes. This signaling mechanism might have important implications for the development of new therapeutic strategies for the treatment of A20-associated skin diseases.

CFLIP is required for epidermal integrity and skin inflammation silencing via protection from TNF-induced keratinocyte apoptosis. Here, we generated and analyzed cFLIP epidermal KO mice with additional TNF deficiency. Intriguingly, the ablation of TNF rescued the pathological phenotype of epidermal cFLIP KO from characteristic weight loss and increased mortality. Moreover, the lack of TNF in these animals strongly reduced and delayed the epidermal hyperkeratosis and the increased apoptosis in keratinocytes. Our data demonstrate that TNF signaling in cFLIP-deficient keratinocytes is the critical factor for the regulation of skin inflammation via modulated cytokine and chemokine expression and, thus, the attraction of immune cells. Our data suggest that autocrine TNF loop activation upon cFLIP deletion is dispensable for T cells, but is critical for neutrophil attraction. Our findings provide evidence for a negative regulatory role of cFLIP for TNF-dependent apoptosis and partially for epidermal inflammation. However, alternative signaling pathways may contribute to the development of the dramatic skin disease upon cFLIP deletion. Our data warrant future studies of the regulatory mechanism controlling the development of skin disease upon cFLIP deficiency and the role of cFLIP/TNF in a number of inflammatory skin diseases, including toxic epidermal necrolysis (TEN).

The necrosome is the key macromolecular signaling platform initiating necroptosis, i.e., a RIPK1/RIPK3-dependent program of cell death with an important role in the control of inflammation in multicellular organisms. However, the composition and structure of the necrosome remain incompletely understood. Here we use biochemical assays, quantitative mass spectrometry, and AlphaFold modeling to decipher the composition and derive a structural model of the CD95L/BV6-induced necrosome. We identify RIPK1 as the central component of the necrosome, forming the core of this complex. In addition, AlphaFold modeling provides insights into the structural mechanisms underlying RIPK1 oligomerization, highlighting the critical role of type-II interactions between the Death Domains (DDs) of FADD and RIPK1 in the assembly of RIPK1-mediated complexes. The role of type-II DD interactions in necroptosis induction is further validated through structure-guided site-directed mutagenesis. Our findings could be useful for the pharmacological targeting of the necroptosis network to treat diseases associated with dysregulated cell death and inflammation.

Abstract 3614 Despite well-described anti-cancer effects and promising results in clinical trials, little is known regarding the selectivity of histone deacetylase (HDAC) inhibitors in killing malignant and sparing normal cells. Using Down syndrome associated myeloid leukemia (DS-AMKL) as a model for an exceptionally sensitive form of acute myeloid leukemia (AML), we elucidated a role of HDAC inhibitors in blocking autophagy, which exploits a specific vulnerability of several types of cancer. We could show that HDAC1/2 inhibition efficiently inversed the DS-AMKL gene signature, resulting in p53-independent cell cycle arrest (BrdU incorporation and CFSE assay) and apoptosis (Annexin V/ 7-AAD and Nicoletti staining, caspase 3/7 activity assay) of DS-AMKL cell lines (CMK and CMY) and primary DS-AMKL leukemic blast. In contrast, control cell lines K562 and M07 were resistant to VPA-induced apoptosis. DS-AMKL is characterized by the high activation of the insulin-like growth factor (IGF) signaling cascade, which in turn results in the constitutive activation of mammalian target of rapamycin (mTOR). mTOR has been characterized as a major suppressor of autophagy, a lysosomal degradation process that takes place constitutively at a basal level and protects cells against damaged or unnecessary organelles and protein aggregates. Measuring endogenous LC3-I to LC3-II conversion levels by Western blot, a posttranslational modification during autophagy, we confirmed a lower basal autophagic activity in DS-AMKL cells compared to the control cell lines, K562 and M07 (LC3B-II/actin [RU]: 1.5 CMK, 1.0 CMY, 6.0 K562 and 3.0 M07). Studying the effect of HDAC inhibition on autophagy, we detected an initial, dose-dependent increase in LC3-II band formation, punctae formation and decrease in total LC3-GFP cellular abundance (i.e., autophagy activation) 12h after VPA treatment in all cell lines (CMK, CMY, K562 and M07), indicative for induction of autophagy. However, prolonged exposure to VPA for up to 24h led to a block of autophagic flux, as shown by a gradual accumulation of LC3-GFP (fold increase in LC3-GFP MFI compared to untreated control: 1.3 CMK**, 1.1 CMY*, 2.0 K562** and 2.0 M07** at 2mM VPA; **p<0.01; *p<0.05). Autophagy participates in the removal of damaged mitochondria, which prevents the initiation of intrinsic apoptotic pathway or DNA damage by increased ROS generation. Our results demonstrated a dose-dependent relationship between VPA-associated accumulation of mitochondrial mass and the intrinsic autophagic activity of the respective leukemic cell lines (fold increase in MitoTracker green MFI: 4.7 CMK**, 4.8 CMY**, 1.3 K562 and 1.2 M07 at 10mM VPA; **p<0.01). Likewise, the VPA-mediated mitochondrial mass accumulation correlated with ROS formation (fold increase in CM-H₂DCFDA MFI: 3.3 CMK**, 2.7 CMY**, 1.5 K562** and 1.2 M07** at 10mM VPA; **p<0.01) and reflects the sensitivity to VPA induced apoptosis. In addition, we detected DNA-double strand breaks as indicated by an increase of phosphorylated H2AX. The effects of HDAC inhibitors on DS-AMKL cells could be recapitulated by pharmacologic (autophagosome-lysosome fusion using vinblastine and nocodazole; autophagolysosomal degradation using ammonium chloride, chloroquine and hydroxychloroquine) and genetic (shRNA-mediated knockdown of ATG5 or ATG7) inhibition of autophagy, underlining dependency on their low basal level of autophagy. Inversely, induction of autophagy by starvation reversed the effects of HDAC inhibition on cell lines and primary DS-AMKL cells (i.e. induction of apoptosis, accumulation of mitochondria and ROS-production).

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