Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.

RNA interference (RNAi) has become an important technique for loss-of-gene-function studies in mammalian cells. To achieve reliable results in an RNAi experiment, efficient and specific silencing triggers are required. Here we present genome-wide data sets for the production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. We created a database, RiDDLE, for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective and specific alternative to presently available mammalian RNAi resources.

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The inhibition of NAD synthesis or salvage pathways has been proposed as a novel target for antitumoral drugs. Two molecules with this mechanism of action are at present undergoing clinical trials. In searching for similar novel molecules, we exploited copper-catalyzed [3 + 2] cycloaddition between azides and alkynes (click chemistry) to synthesize 185 novel analogues. The most promising compound displays an IC(50) for cytotoxicity in vitro of 3.8 +/- 0.3 nM and an IC(50) for NAD depletion of 3.0 +/- 0.4 nM. Herein, we strengthen previous data suggesting that this class of compounds induces autophagic cell death. In addition to characterizing this compound and providing a rationale via molecular docking, we reinforce the excellent potential of click chemistry for rapidly generating structure-activity relationships and for drug screening.

High plasma levels of nicotinamide phosphoribosyltransferase (NAMPT), traditionally considered an intracellular enzyme with a key role in NAD synthesis, have been reported in several oncological, inflammatory and metabolic diseases. We now show that eNAMPT can be actively released by melanoma cells in vitro. We analysed the mechanisms of its release, and we found both classical and non-classical pathway involvement. eNAMPT released by melanoma cells, in our hands, has paracrine and autocrine effects: it activates MAPK, AKT and NF-κB pathways and increases colony formation in anchorage-independent conditions. eNAMPT also induces M1 polarization in human monocytes. Last, we demonstrate, for the first time in any cancer type, that eNAMPT levels in plasma of tumour-bearing mice increase and that this increase can be reconducted to the tumour itself. This provides an important cue on previous observations that eNAMPT is increased in patients with cancer. Moreover, silencing NAMPT in melanoma cells leads to a reduction in the tumour growth rate. Our findings extend the basis to consider eNAMPT as a cytokine involved in tumour progression.