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Inhibitor of Apoptosis proteins (IAPs) are a group of E3 ligases involved in tumor necrosis factor (TNF) signalling. Upon TNF stimulation IAPs are needed for proper activation of NF-κB signalling, thereby protecting cells from cytotoxic activity of TNF. IAPs have emerged as attractive targets for anti-cancer therapeutics since it has been shown that overexpression of the IAP antagonist Smac sensitises tumour cells to TNF driven apoptosis as do the synthetic counterparts, Smac mimetics. We demonstrated that cell death induced by inhibition of IAPs through Smac mimetics is not strictly TNF dependent. The combined treatment of Interferon γ (IFNγ), a potent pro-inflammatory cytokine, and Smac mimetics shows potent synergistic killing of a wide range of tumour cell lines as well as primary and transformed cells. Further investigation showed that IFNγ and Smac mimetic induced killing requires NF-κB as well as Jak/STAT signalling and displayed classic apoptotic hallmarkers. Whereas killing of several cell lines could be blocked by the inhibition of caspases, other cell lines like HT29, KATOIII, mouse dermal fibroblasts (MDFs) and keratinocytes remained sensitive. In order to elucidate the involvement of the necroptotic pathway in this apoptosis independent IFNγ and Smac mimetic combined killing we performed knock down experiments and analysed primary and transformed knock out MDFs and keratinocytes lacking members of the necroptotic pathway like RIPK3 −/− and MLKL −/− . The results suggest that IFNγ and Smac mimetic combined treatment can activate both apoptotic and necroptotic death pathways, involving members of the necroptotic pathway. This may provide new insights into yet unknown cell death mechanisms and extend the clinical use of Smac mimetics.

Tumor Necrosis Factor (TNF) is an important factor in initiation and development of inflammation, and anti TNF therapy is well established for treatment of inflammatory diseases such as psoriasis, rheumatoid arthritis and Crohn’s disease.1 However, anti TNF drugs are not always efficient and developing new effective anti-inflammatory drugs is in the interest of many pharmaceutical companies.1 Therefore, We think that better understanding of TNF signalling pathway helps to discover new targets and design more effective drugs for inflammatory diseases. We have previously showed that Lymphotoxin? signals similar to TNF and suggested to be an alternative factor to induce inflammation in the absent of TNF 2 and both TNF and Lymphotoxin are not blockable by newly proposed inhibitor called Progranulin 3. These results encouraged us to investigate downstream regulator of TNF signalling pathway. Surprisingly, after 30 years of research on TNF, role of many components of TNF pathway are still unclear. TNF Receptor Associated Factor-2 (TRAF2) is one of those molecules which its role is controversial and not well defined yet 4. Since TRAF2 knock-out mice are lethal at birth, we have established various conditional and tissue specific TRAF2 knock-out to study the role of TRAF2 in different tissues. Interestingly, we found that loss of TRAF2 in keratinocytes disrupts TNF signalling and causes cell death, epidermal hyperplasia and skin inflammation. Surprisingly and contradictory to other models, additional loss of TNF did not completely rescue this phonotype. We have found that constitutive activation of non-canonical NF κ B and excessive production of TNF and other cytokines are responsible for this TNF independent inflammation. We propose that this novel model of skin inflammation could be a better model and targeting non-canonical NF κ B signalling might be a more effective target for treatment of inflammatory diseases.