Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53–HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53–HDM2 inhibitors, such as NVP-CGM097.DOI: http://dx.doi.org/10.7554/eLife.06498.001

Members of the transforming growth factor-␤ (TGF-␤)superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-␤ receptors and suggested to play a role in TGF-␤ signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-␤ and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-␤ signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-␤ signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.

Our data support a greater focus on the impact of pre-existing, drug-reactive autoantibodies on the development of antibody fragments and biotherapeutics targeting cell surface receptors.

E. coli is a gram-negative bacterium rarely found on human skin. We investigated whether direct interaction of E. coli with keratinocytes might induce an innate immune response through recognition by pattern recognition receptors. The capacity of E. coli to activate innate immune responses and IL-8 induction was investigated. We found that E. coli significantly induced human S100A7 and S100A15 transcript abundance and IL-8 release in cultured primary human keratinocytes. S100A15 is a member of the S100 protein family with previously unknown function. E. coli induced effects could be inhibited by neutralizing Toll-like receptor 4 (TLR4) antibodies, suggesting that E. coli-induced IL-8 and S100A15 expression in human keratinocytes are TLR4 dependent. TLR4-/- mice lacked elevated mS100A15 expression after infection with E. coli in contrast to wild-type mice. In vitro, human S100A15 displayed antimicrobial activity against E. coli. Our findings suggest that E. coli modulates S100A15 and IL-8 expression of keratinocytes by recognition through TLR4.

We have investigated the role of Smad family proteins, known to be important cytoplasmic mediators of signals from the transforming growth factor–β (TGF-β) receptor serine/threonine kinases, in TGF-β–dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-β–dependent differentiation of these cells to monocytes, but not retinoic acid–dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2 and Smad3. Vitamin D3 also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-β1. Simultaneous treatment of these cells with TGF-β1 and all-trans-retinoic acid (ATRA), which leads to almost equal numbers of granulocytes and monocytes, significantly reduced the level of phospho–Smad2/3 and its nuclear accumulation, compared with that in cells treated with TGF-β1 alone. TGF-β1 and ATRA activate P42/44 mitogen-activated protein (MAP) kinase with nearly identical kinetics, ruling out its involvement in these effects on Smad phosphorylation. Addition of the inhibitor-of-protein serine/threonine phosphatases, okadaic acid, blocks the ATRA-mediated reduction in TGF-β–induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor–α (RAR-α), ATRA is unable to reduce levels of TGF-β–induced phospho-Smad2/3, coincident with its inability to differentiate these cells along granulocytic pathways. Together, these data suggest a new level of cross-talk between ATRA and TGF-β, whereby a putative RAR-α–dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-β, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-β–dependent differentiation of the cells to monocytic end points.