Katherine H. Fisher

Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation.

Although many signal transduction pathways have been implicated in the development of human disease, the identification of pathway targets and the biological processes that mediate disease progression remains challenging. One such disease‐related pathway is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade whose constitutive misactivation by the JAK2 V617F mutation underlies most human myeloproliferative disorders. Here, we use transcript profiling of Drosophila haemocyte‐like cells to identify JAK/STAT target genes, combined with an in vivo model for JAK‐induced blood cell overproliferation, to identify the main effectors required for haematopoietic tumour development. The identified human homologues of the Drosophila effectors were tested for potential V617F‐mediated transcriptional regulation in human HeLa cells and compared with small interfering RNA‐derived data, quantify their role in regulating the proliferation of cancer‐derived cell lines. Such an inter‐species approach is an effective way to identify factors with conserved functions that might be central to human disease.

Methotrexate Is a JAK/STAT Pathway Inhibitor

Background The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. Methods & Findings We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin. Conclusions Aminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, we suggest that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential.

Cellular interpretation of the long-range gradient of Four-jointed activity in the Drosophila wing

To understand how long-range patterning gradients are interpreted at the cellular level, we investigate how a gradient of expression of the Four-jointed kinase specifies planar polarised distributions of the cadherins Fat and Dachsous in the Drosophila wing. We use computational modelling to test different scenarios for how Four-jointed might act and test the model predictions by employing fluorescence recovery after photobleaching as an in vivo assay to measure the influence of Four-jointed on Fat-Dachsous binding. We demonstrate that in vivo, Four-jointed acts both on Fat to promote its binding to Dachsous and on Dachsous to inhibit its binding to Fat, with a bias towards a stronger effect on Fat. Overall, we show that opposing gradients of Fat and Dachsous phosphorylation are sufficient to explain the observed pattern of Fat–Dachsous binding and planar polarisation across the wing, and thus demonstrate the mechanism by which a long-range gradient is interpreted. DOI: http://dx.doi.org/10.7554/eLife.05789.001

Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway

BackgroundGenome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated.ResultsHere we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols.ConclusionsOur work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens.

Effect of methotrexate on JAK/STAT pathway activation in myeloproliferative neoplasms

BACKGROUND The myeloproliferative neoplasms are a group of haematological malignancies characterised by pathological activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) intracellular signalling pathway. 50-95% of patients have an acquired mutation (JAK2V617F) causing constitutive activation of JAK2. Our aim was to find new treatments for myeloproliferative neoplasms by identifying compounds that suppress JAK/STAT pathway activation. METHODS We used a luciferase-based transcriptional assay in the low complexity Drosophila model system to screen a library of 2000 small molecules for modulators of JAK/STAT pathway activation. Screen hits were validated with western blotting in the HDLM-2 Hodgkins lymphoma cell line. The HEL cell line, in which constitutive JAK/STAT pathway activation is caused by JAK2V617F, was used to determine the relevance of screen hits for treatment of myeloproliferative neoplasms. FINDINGS Methotrexate and the chemically similar drug aminopterin were independently identified as strong inhibitors of the Drosophila JAK/STAT pathway, an effect conserved to human cells. Methotrexate did not affect protein phosphorylation in other intracellular signalling pathways. Methotrexate caused significant suppression of JAK/STAT activation in HEL cells at a concentration equivalent to that seen in patients taking low-dose oral methotrexate (p≤0·001). INTERPRETATION Our results suggest that methotrexate is a promising treatment for myeloproliferative neoplasms that could be translated into clinical trials after assessment in primary cells. These results are particularly relevant in myelofibrosis. Inhibitors of JAK1/2 improve symptoms and prolong life in myelofibrosis, but their use is limited by cost. Other existing therapies for myelofibrosis appear no more effective than placebo. Methotrexate might bring the benefits of JAK/STAT pathway inhibition at a lower cost. FUNDING Cancer Research UK, Yorkshire Cancer Research, UK Medical Research Council, Wellcome Trust, EU Framework Cancer Pathways.

Designing RNAi Screens to Identify JAK/STAT Pathway Components

The JAK/STAT signaling pathway has essential roles in multiple developmental processes, including stem cell maintenance, immune responses, and cellular proliferation. As a result, it has been extensively studied in both vertebrate systems and lower complexity models, such as Drosophila. Given its connection with such a wide range of biological functions, it is no surprise that pathway misregulation is frequently associated with multiple human diseases including cancer. While the core components of the pathway, and a number of negative regulators, are well known and conserved in many organisms, more subtle levels of regulation and inter-pathway crosstalk are less well understood. With the emergence of RNA interference (RNAi) as a tool to knock down gene expression and so evaluate protein function, high-throughput screens have been developed to identify pathway regulators on a genome-wide scale. Here we discuss the approaches and methods employed thus far for identification of pathway regulators using RNAi in Drosophila. Furthermore, we discuss possible approaches for future screens and the significant potential for applying RNAi technology in vertebrate models.

Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran.

The short receptor Et/Lat negatively regulates Drosophila JAK/STAT signaling. It binds to intracellular components and the Domeless receptor but cannot bind ligands, thus generating a signaling-incompetent complex. Et/Lat is also more stable than Dome. The study provides insights into how short receptors negatively regulate signaling.

A Dual Function for Prickle in Regulating Frizzled Stability during Feedback-Dependent Amplification of Planar Polarity

Summary The core planar polarity pathway coordinates epithelial cell polarity during animal development, and loss of its activity gives rise to a range of defects, from aberrant morphogenetic cell movements to failure to correctly orient structures, such as hairs and cilia. The core pathway functions via a mechanism involving segregation of its protein components to opposite cells ends, where they form asymmetric intracellular complexes that couple cell-cell polarity. This segregation is a self-organizing process driven by feedback interactions between the core proteins themselves. Despite intense efforts, the molecular pathways underlying feedback have proven difficult to elucidate using conventional genetic approaches. Here we investigate core protein function during planar polarization of the Drosophila wing by combining quantitative measurements of protein dynamics with loss-of-function genetics, mosaic analysis, and temporal control of gene expression. Focusing on the key core protein Frizzled, we show that its stable junctional localization is promoted by the core proteins Strabismus, Dishevelled, Prickle, and Diego. In particular, we show that the stabilizing function of Prickle on Frizzled requires Prickle activity in neighboring cells. Conversely, Prickle in the same cell has a destabilizing effect on Frizzled. This destabilizing activity is dependent on the presence of Dishevelled and blocked in the absence of Dynamin and Rab5 activity, suggesting an endocytic mechanism. Overall, our approach reveals for the first time essential in vivo stabilizing and destabilizing interactions of the core proteins required for self-organization of planar polarity.

A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability

ABSTRACT Cytokine receptors often act via the Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway to form a signalling cascade that is essential for processes such as haematopoiesis, immune responses and tissue homeostasis. In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are poorly understood. In order to better understand the processes regulating cytokine receptor levels, and their activity and dimerisation, we analysed the highly conserved JAK/STAT pathway in Drosophila, which acts via a single receptor, known as Domeless. We performed a genome-wide RNAi screen in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. We show that MASK is able to regulate receptor levels and JAK/STAT signalling both in vitro and in vivo. We also show that its human homologue, ANKHD1, is also able to regulate JAK/STAT signalling and the levels of a subset of pathway receptors in human cells. Taken together, our results identify MASK as a novel regulator of cytokine receptor levels, and suggest functional conservation, which may have implications for human health. This article has an associated First Person interview with the first author of the paper. Summary: A genome-wide RNAi screen in Drosophila identifies MASK as a positive regulator of the JAK/STAT signalling via stabilisation of the pathway receptor. This function is conserved in human cells.

Information Flow in Planar Polarity

In developing tissues, sheets of cells become planar polarised, enabling coordination of cell behaviours. It has been suggested that ‘signalling’ of polarity information between cells may occur either bidirectionally or monodirectionally between the molecules Frizzled (Fz) and Van Gogh (Vang). Using computational modelling we find that both bidirectional and monodirectional signalling models reproduce known non-autonomous phenotypes derived from patches of mutant tissue of key molecules, but predict different phenotypes from double mutant tissue, which have previously given conflicting experimental results. Consequently, we re-examine experimental phenotypes in the Drosophila wing, concluding that signalling is most likely bidirectional. Our modelling suggests that bidirectional signalling can be mediated either indirectly via bidirectional feedbacks between asymmetric intercellular protein complexes, or directly via different affinities for protein binding in intercellular complexes, suggesting future avenues for investigation. Our findings offer insight into mechanisms of juxtacrine cell signalling and how tissue-scale properties emerge from individual cell behaviours.