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Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.).

A number of pathogens cause host cell death upon infection, and Yersinia pestis, infamous for its role in large pandemics such as the "Black Death" in medieval Europe, induces considerable cytotoxicity. The rapid killing of macrophages induced by Y. pestis, dependent upon type III secretion system effector Yersinia outer protein J (YopJ), is minimally affected by the absence of caspase-1, caspase-11, Fas ligand, and TNF. Caspase-8 is known to mediate apoptotic death in response to infection with several viruses and to regulate programmed necrosis (necroptosis), but its role in bacterially induced cell death is poorly understood. Here we provide genetic evidence for a receptor-interacting protein (RIP) kinasecaspase-8-dependent macrophage apoptotic death pathway after infection with Y. pestis, influenced by Toll-like receptor 4-TIR-domain-containing adapter-inducing interferon-β (TLR4-TRIF). Interestingly, macrophages lacking either RIP1, or caspase-8 and RIP3, also had reduced infection-induced production of IL-1β, IL-18, TNF, and IL-6; impaired activation of the transcription factor NF-κB; and greatly compromised caspase-1 processing. Cleavage of the proform of caspase-1 is associated with triggering inflammasome activity, which leads to the maturation of IL-1β and IL-18, cytokines important to host responses against Y. pestis and many other infectious agents. Our results identify a RIP1-caspase-8/RIP3-dependent caspase-1 activation pathway after Y. pestis challenge. Mice defective in caspase-8 and RIP3 were also highly susceptible to infection and displayed reduced proinflammatory cytokines and myeloid cell death. We propose that caspase-8 and the RIP kinases are key regulators of macrophage cell death, NF-κB and inflammasome activation, and host resistance after Y. pestis infection.

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In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 orthologue CG3711 resulted in a RAS-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.

SummaryBackgroundPatients with refractory or relapsed haematological malignancies have few treatment options and short survival times. Identification of effective therapies with genomic-based precision medicine is hampered by intratumour heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Ex-vivo drug-response profiling in patient biopsies might aid effective treatment identification; however, proof of its clinical utility is limited.MethodsWe investigated the feasibility and clinical impact of multiparametric, single-cell, drug-response profiling in patient biopsies by immunofluorescence, automated microscopy, and image analysis, an approach we call pharmacoscopy. First, the ability of pharmacoscopy to separate responders from non-responders was evaluated retrospectively for a cohort of 20 newly diagnosed and previously untreated patients with acute myeloid leukaemia. Next, 48 patients with aggressive haematological malignancies were prospectively evaluated for pharmacoscopy-guided treatment, of whom 17 could receive the treatment. The primary endpoint was progression-free survival in pharmacoscopy-treated patients, as compared with their own progression-free survival for the most recent regimen on which they had progressive disease. This trial is ongoing and registered with ClinicalTrials.gov, number NCT03096821.FindingsPharmacoscopy retrospectively predicted the clinical response of 20 acute myeloid leukaemia patients to initial therapy with 88·1% accuracy. In this interim analysis, 15 (88%) of 17 patients receiving pharmacoscopy-guided treatment had an overall response compared with four (24%) of 17 patients with their most recent regimen (odds ratio 24·38 [95% CI 3·99–125·4], p=0·0013). 12 (71%) of 17 patients had a progression-free survival ratio of 1·3 or higher, and median progression-free survival increased by four times, from 5·7 (95% CI 4·1–12·1) weeks to 22·6 (7·4–34·0) weeks (hazard ratio 3·14 [95% CI 1·37–7·22], p=0·0075).InterpretationRoutine clinical integration of pharmacoscopy for treatment selection is technically feasible, and led to improved treatment of patients with aggressive refractory haematological malignancies in an initial patient cohort, warranting further investigation.FundingAustrian Academy of Sciences; European Research Council; Austrian Science Fund; Austrian Federal Ministry of Science, Research and Economy; National Foundation for Research, Technology and Development; Anniversary Fund of the Austrian National Bank; MPN Research Foundation; European Molecular Biology Organization; and Swiss National Science Foundation.

SummaryLipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems.

Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific molecules contribute to the stimulatory activity. QS-21 is a defined, highly purified, and soluble saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. We observed QS-21 to elicit caspase-1-dependent IL-1␤ and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, our data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1␤/IL-18 release. At higher concentrations, QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixture of saponins also induced IL-1␤ in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, we have identified QS-21 as a nonparticulate single molecular saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigenspecific responses in vivo.Because many protein antigens do not elicit strong immune responses on their own, vaccines often contain stimulatory adjuvants that enhance cell-mediated and humoral immune responses to help confer stronger protection. However, despite widespread use, there is little known regarding the pathways affected by many adjuvants. A better understanding of the mechanisms involved in adjuvant-generated protection can assist in the design of better vaccines against infections that currently lack effective immunization.Adjuvants activate an innate immune response, which in turn determines the strength and quality of the adaptive immune response. This response is first mediated by activation of antigen-presenting cells (APCs) such as dendritic cells and macrophages. Engagement of pattern recognition receptors, such as extracellular, membrane-bound Toll-like receptors (TLRs) 2 and cytosolic inflammasome-stimulating Nod-like receptors (NLRs) by their ligands elicits an inflammatory milieu and can eventually lead to a honed adaptive immune response.The NLR inflammasomes are multiprotein complexes that upon activation license the proteolytic processing of the zymogen pro-caspase-1 into mature caspase-1 (1). caspase-1 can then activate pro-forms of the inflammatory cytokines IL-1␤ and IL-18 into mature proteins, which are then secreted through unknown pathways. IL-1␤ and IL-18 are potent proinflammatory cytokines that can, for instance, promote T helper 17 ...