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We developed a strategy for identifying modulators of juxtacrine signaling, triggered by a cell-surface ligand displayed on synthetic lipid bilayers, via cognate receptors on apposed cells. Using readouts for receptor lateral transport and intracellular signaling, we screened a small interfering RNA (siRNA) library and identified specific receptor tyrosine kinases (RTKs) that directly or indirectly modulate apoptosis signaling by a model death ligand through its cognate death receptors. This approach may be broadly useful for studying juxtacrine cell-cell signaling systems.

Despite the knowledge accumulated during the last two decades about programmed cell death, further investigations of the complex regulatory network of apoptosis, including the extrinsic pathways, are still needed to gain an exhaustive and comprehensive understanding of this critical biological process. In addition, the identification of novel modulators of apoptosis may represent a good opportunity for making new paths into an otherwise heavily investigated area, therefore providing a molecular basis for new therapeutic strategies. In the last decade, RNA interference has become the technology of choice for discovering genes that encode molecules with previously unknown functions in biological pathways of interest. Various RNAi reagents and library formats have been developed and harnessed for high-throughput screening technologies to enable almost limitless investigation to uncover gene functions and networks in the context of basic biology and biomedical research including cancer biology. Although RNAi screening has been demonstrated to be a very powerful tool, various caveats and pitfalls have been progressively uncovered, including, but not limited to the enduring off-target effects. As the novelty of its bells and whistles have begun to diminish, functional genomic screens have morphed into a specialized field within the high-throughput screening community, where expert investigators progressively establish rigorous strategies to mitigate most of its possible flaws. Using various examples of RNAi screens conducted to further understand the extrinsic apoptosis pathway, this chapter describes the different RNAi tools and screening formats available and reviews the parameters one has to critically consider in order to be successful in implementing this technology., (© 2014 Elsevier Inc. All rights reserved.)

Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: HSP90 targeting is a promising therapeutic approach in cancer. 17-AAG is an HSP90 inhibitor with completed Phase I trials in patients with advanced cancer and recently published Phase II trials. The aim of this work was to study the expression of HSP 90 and apoptotic proteins, the effects in culture of 17-AAG on cell survival and apoptosis and to compare Philadelphia-positive (Ph+) ALL to common B cell ALL, in ALL cell lines and in patients' cells collected at ALL diagnosis., Methods: We analysed 2 ALL cell lines and 63 leukaemic samples from patients treated in our institution (44 common B cell ALL and 19 Ph+ ALL). We performed flow cytometry analysis of bone marrow aspiration and cell lines with a combination of anti-HSP90, Bax, Bcl-2 and Bcl-xl antibodies. Apoptosis after cell culture (in presence or not of 17-AAG) was assessed using Annexin V and activated caspase-3 staining., Results: Ph+ ALL cells appeared to be more sensitive to 17-AAG cytotoxicity with a 100 % mortality rate after exposure to 10 μM for 24 h (vs. 62 % for B-common ALL). A high percentage of HSP90-positive cells (in Ph+ ALL samples) was associated with high sensitivity to 17-AAG. 17-AAG induced apoptosis in a dose-dependent manner and was associated with down-regulation of Bcl-2 and Bcl-Xl expression and up-regulation of Bax expression., Conclusion: Considering that Bcr-Abl constitutes HSP 90 substrates, HSP 90 inhibition could be of particular interest for Ph+ ALL disease, even in patients harbouring resistance to tyrosine kinase inhibitor therapy.