Interleukin-1β (IL-1β) is a cytokine whose bioactivity is controlled by activation of the inflammasome. However, in response to lipopolysaccharide, human monocytes secrete IL-1β independently of classical inflammasome stimuli. Here, we report that this constituted a species-specific response that is not observed in the murine system. Indeed, in human monocytes, lipopolysaccharide triggered an "alternative inflammasome" that relied on NLRP3-ASC-caspase-1 signaling, yet was devoid of any classical inflammasome characteristics including pyroptosome formation, pyroptosis induction, and K(+) efflux dependency. Genetic dissection of the underlying signaling pathway in a monocyte transdifferentiation system revealed that alternative inflammasome activation was propagated by TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3. Importantly, involvement of this signaling cascade was limited to alternative inflammasome activation and did not extend to classical NLRP3 activation. Because alternative inflammasome activation embraces both sensitivity and promiscuity of TLR4, we propose a pivotal role for this signaling cascade in TLR4-driven, IL-1β-mediated immune responses and immunopathology in humans.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U) tRNA (U enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF oncogene and by resistance to targeted therapy in melanoma. We show that BRAF -expressing melanoma cells are dependent on U enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U enzymes. Mechanistically, U enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U enzymes and HIF1α. Together, these results demonstrate that U enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

Here we describe a lineage reprogramming system consisting of a B cell line with an estradiol-inducible form of C/EBPalpha where cells can be converted into macrophage-like cells at 100% efficiency within 2 to 3 days. The reprogrammed cells are larger, contain altered organelle and cytoskeletal structures, are phagocytic, and exhibit an inflammatory response. Time-lapse experiments showed that the cells acquire a macrophage morphology and increased migratory activity as early as 10 hr. During induction, thousands of genes become up- or downregulated, including several dozen transcription and chromatin-remodeling factors. Time-limited exposure of cells to the inducer showed that the reprogrammed cells become transgene independent within 1 to 2 days. The reprogramming can be inhibited, at least partially, by perturbation experiments with B cell and macrophage transcription factors. The tightness, robustness, and speed of the system described make it a versatile tool to study biochemical and biological aspects of lineage reprogramming.

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF V600E mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF V600E induces a chronic ER stress status directly increasing basal cell autophagy. BRAF V600E -mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF V600E -mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF V600E melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.