The 8th International RASopathies Symposium: Expanding research and care practice through global collaboration and advocacy.

Germline pathogenic variants in the RAS/mitogen-activated protein kinase (MAPK) signaling pathway are the molecular cause of RASopathies, a group of clinically overlapping genetic syndromes. RASopathies constitute a wide clinical spectrum characterized by distinct facial features, short stature, predisposition to cancer, and variable anomalies in nearly all the major body systems. With increasing global recognition of these conditions, the 8th International RASopathies Symposium spotlighted global perspectives on clinical care and research, including strategies for building international collaborations and developing diverse patient cohorts in anticipation of interventional trials. This biannual meeting, organized by RASopathies Network, was held in a hybrid virtual/in-person format. The agenda featured emerging discoveries and case findings as well as progress in preclinical and therapeutic pipelines. Stakeholders including basic scientists, clinician-scientists, practitioners, industry representatives, patients, and family advocates gathered to discuss cutting edge science, recognize current gaps in knowledge, and hear from people with RASopathies about the experience of daily living. Presentations by RASopathy self-advocates and early-stage investigators were featured throughout the program to encourage a sustainable, diverse, long-term research and advocacy partnership focused on improving health and bringing treatments to people with RASopathies.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

Advancing RAS/RASopathy therapies: An NCI-sponsored intramural and extramural collaboration for the study of RASopathies.

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.

The sixth international RASopathies symposium: Precision medicine-From promise to practice.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.

The receptor tyrosine kinase Pvr promotes tissue closure by coordinating corpse removal and epidermal zippering.

A leading cause of human birth defects is the incomplete fusion of tissues, often manifested in the palate, heart or neural tube. To investigate the molecular control of tissue fusion, embryonic dorsal closure and pupal thorax closure in Drosophila are useful experimental models. We find that Pvr mutants have defects in dorsal midline closure with incomplete amnioserosa internalization and epidermal zippering, as well as cardia bifida. These defects are relatively mild in comparison to those seen with other signaling mutants, such as in the JNK pathway, and we demonstrate that JNK signaling is not perturbed by altering Pvr receptor tyrosine kinase activity. Rather, modulation of Pvr levels in the ectoderm has an impact on PIP3 membrane accumulation, consistent with a link to PI3K signal transduction. Polarized PI3K activity influences protrusive activity from the epidermal leading edge and the protrusion area changes in accord with Pvr signaling intensity, providing a possible mechanism to explain Pvr mutant phenotypes. Tissue-specific rescue experiments indicate a partial requirement in epithelial tissue, but confirm the essential role of Pvr in hemocytes for embryonic survival. Taken together, we argue that inefficient removal of the internalizing amnioserosa tissue by mutant hemocytes coupled with impaired midline zippering of mutant epithelium creates a situation in some embryos whereby dorsal midline closure is incomplete. Based on these observations, we suggest that efferocytosis (corpse clearance) could contribute to proper tissue closure and thus might underlie some congenital birth defects.

Proceedings of the fifth international RASopathies symposium: When development and cancer intersect.

This report summarizes and highlights the fifth International RASopathies Symposium: When Development and Cancer Intersect, held in Orlando, Florida in July 2017. The RASopathies comprise a recognizable pattern of malformation syndromes that are caused by germ line mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. Because of their common underlying pathogenetic etiology, there is significant overlap in their phenotypic features, which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, gastrointestinal and ocular abnormalities, neurological and neurocognitive issues, and a predisposition to cancer. The RAS pathway is a well-known oncogenic pathway that is commonly found to be activated in somatic malignancies. As in somatic cancers, the RASopathies can be caused by various pathogenetic mechanisms that ultimately impact or alter the normal function and regulation of the MAPK pathway. As such, the RASopathies represent an excellent model of study to explore the intersection of the effects of dysregulation and its consequence in both development and oncogenesis.

A neurodevelopmental epigenetic programme mediated by SMARCD3-DAB1-Reelin signalling is hijacked to promote medulloblastoma metastasis.

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin-DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

Regulation of mixed-lineage kinase activation in JNK-dependent morphogenesis.

Normal cells respond appropriately to various signals, while sustaining proper developmental programs and tissue homeostasis. Inappropriate signal reception, response or attenuation, can upset the normal balance of signaling within cells, leading to dysfunction or tissue malformation. To understand the molecular mechanisms that regulate protein-kinase-based signaling in the context of tissue morphogenesis, we analyzed the domain requirements of Drosophila Slpr, a mixed-lineage kinase (MLK), for Jun N-terminal kinase (JNK) signaling. The N-terminal half of Slpr is involved in regulated signaling whereas the C-terminal half promotes cortical protein localization. The SH3 domain negatively regulates Slpr activity consistent with autoinhibition via a conserved proline motif. Also, like many kinases, conserved residues in the activation segment of the catalytic domain regulate Slpr. Threonine 295, in particular, is essential for function. Slpr activation requires dual input from the MAP4K Misshapen (Msn), through its C-terminal regulatory domain, and the GTPase Rac, which both bind to the LZ-CRIB region of Slpr in vitro. Although Rac is sufficient to activate JNK signaling, our results indicate that there are Slpr-independent functions for Rac in dorsal closure. Finally, expression of various Slpr constructs alone or with upstream activators reveals a wide-ranging response at the cell and tissue level.

POSH misexpression induces caspase-dependent cell death in Drosophila.

POSH (Plenty of SH3 domains) is a scaffold for signaling proteins regulating cell survival. Specifically, POSH promotes assembly of a complex including Rac GTPase, mixed lineage kinase (MLK), MKK7, and Jun kinase (JNK). In Drosophila, genetic analysis implicated POSH in Tak1-dependent innate immune response, in part through regulation of JNK signaling. Homologs of the POSH signaling complex components, MLK and MKK7, are essential in Drosophila embryonic dorsal closure. Using a gain-of-function approach, we tested whether POSH plays a role in this process. Ectopic expression of POSH in the embryo causes dorsal closure defects due to apoptosis of the amnioserosa, but ectodermal JNK signaling is normal. Phenotypic consequences of POSH expression were found to be dependent on Drosophila Nc, the caspase-9 homolog, but only partially on Tak1 and not at all on Slpr and Hep. These results suggest that POSH may use different signaling complexes to promote cell death in distinct contexts.

The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

Genetic analysis of slipper/mixed lineage kinase reveals requirements in multiple Jun-N-terminal kinase-dependent morphogenetic events during Drosophila development.

Mixed lineage kinases (MLKs) function as Jun-N-terminal kinase (JNK) kinase kinases to transduce extracellular signals during development and homeostasis in adults. slipper (slpr), which encodes the Drosophila homolog of mammalian MLKs, has previously been implicated in activation of the JNK pathway during embryonic dorsal epidermal closure. To further define the specific functions of SLPR, we analyzed the phenotypic consequences of slpr loss and gain of function throughout development, using a semiviable maternal-effect allele and wild-type or dominant-negative transgenes. From these analyses we confirm that failure of dorsal closure is the null phenotype in slpr germline clones. In addition, there is a functional maternal contribution, which can suffice for embryogenesis in the zygotic null mutant, but rarely suffices for pupal metamorphosis, revealing later functions for slpr as the maternal contribution is depleted. Zygotic null mutants that eclose as adults display an array of morphological defects, many of which are shared by hep mutant animals, deficient in the JNK kinase (JNKK/MKK7) substrate for SLPR, suggesting that the defects observed in slpr mutants primarily reflect loss of hep-dependent JNK activation. Consistent with this, the maternal slpr contribution is sensitive to the dosage of positive and negative JNK pathway regulators, which attenuate or potentiate SLPR-dependent signaling in development. Although SLPR and TAK1, another JNKKK family member, are differentially used in dorsal closure and TNF/Eiger-stimulated apoptosis, respectively, a Tak1 mutant shows dominant genetic interactions with slpr, suggesting potential redundant or combinatorial functions. Finally, we demonstrate that SLPR overexpression can induce ectopic JNK signaling and that the SLPR protein is enriched at the epithelial cell cortex.

Germline clone analysis for maternally acting Drosophila hedgehog components.

Many of the genes of Drosophila melanogaster have their transcripts deposited in developing oocytes. These maternally loaded gene products enable an otherwise homo-zygous mutant embryo to survive beyond the first stage of development for which the gene product is required. Zygotic mutations that disrupt the Hedgehog signal transduction pathway typically yield a segment polarity 'lawn of denticles' cuticle phenotype. However, an embryo homozygous mutant for a gene can achieve normal embryonic segmentation precluding classification of the gene as a component of the Hh pathway, if wild-type transcripts from the mother are present. This chapter discusses the theory and importance of analyzing germline clone embryos for maternally acting genes involved in Hh signal transduction, and describes in detail the method to generate mutant germline clone embryos.

Molecular and phylogenetic characterization of Zyx102, a Drosophila orthologue of the zyxin family that interacts with Drosophila Enabled.

Adherens junctions, which are cadherin-mediated junctions between cells, and focal adhesions, which are integrin-mediated junctions between cells and the extracellular matrix, are protein complexes that link the actin cytoskeleton to the plasma membrane and, in turn, to the extracellular environment. Zyxin is a LIM domain protein that is found in vertebrate adherens junctions and focal adhesions. Zyxin's molecular architecture and binding partner repertoire suggest roles in actin assembly and dynamics, cell motility, and nuclear-cytoplasmic communication. In order to study the function of zyxin in development, we have identified a zyxin orthologue in Drosophila melanogaster that we have termed Zyx102. Like its vertebrate counterparts, Zyx102 displays three carboxy-terminal LIM domains, a potential nuclear export signal, and three proline-rich motifs, one of which matches the consensus for mediating an interaction with Ena/VASP (Drosophila Enabled/Vasodilator-stimulated phosphoprotein) proteins. Here we show that Zyx102 and Enabled (Ena), the Drosophila member of the Ena/VASP family, can interact specifically in vitro and that this interaction does not occur when a particular mutant form of Ena, encoded by the lethal ena210 allele, is used. Lastly, we show that the zyx102 gene and Drosophila Ena are co-expressed during oogenesis and early embryogenesis, indicating that the two proteins may be able to interact during the development of the Drosophila egg chamber and early embryo.

Extensive nonmuscle expression and epithelial apicobasal localization of the Drosophila ALP/Enigma family protein, Zasp52.

This study describes the broad tissue distribution and subcellular localization of Drosophila Zasp52, which is related to the large family of ALP (α-actinin associated protein)/Enigma PDLIM (PDZ and LIM domain) proteins of vertebrates. Results demonstrate that ZCL423 is a protein trap insertion in the Zasp52 locus tagging multiple endogenous splice isoforms with GFP. While Zasp52 has been previously characterized in muscle tissues primarily, visualization of GFP fluorescence in Zasp52 protein trap lines revealed expression in many nonmuscle tissues including the central nervous system, secretory glands, and epithelial tissues constituting the embryonic epidermis, the somatic follicle cell layer encapsulating the germline during oogenesis, and imaginal disc precursors to the adult body. In epithelial cells, Zasp52 typically accumulated basally, adjacent to integrin adhesion sites, and apically along adherens junctions, particularly enriched near junctional vertices of multicellular interfaces. Also Zasp52 showed polarized accumulation at the leading edge of migrating cell populations and morphogenetic boundaries similarly enriched for myosin. As such, Zasp52 GFP protein traps may be useful molecular markers for dynamic epithelial rearrangements. Moreover, the pattern of Zasp52 expression within nonmuscle tissues reveals potential functional roles in cell-cell and cell-matrix adhesion, specifically at sites of increased actomyosin contractile tension. In these contexts, the investigation of Zasp52 may provide insights into the functions of numerous PDLIM proteins of the metazoan lineages.

Domain specificity of MAP3K family members, MLK and Tak1, for JNK signaling in Drosophila.

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-ß activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability.

Drosophila heat shock response requires the JNK pathway and phosphorylation of mixed lineage kinase at a conserved serine-proline motif.

Defining context specific requirements for proteins and pathways is a major challenge in the study of signal transduction. For example, the stress-activated protein kinase (SAPK) pathways are comprised of families of closely related transducers that are activated in a variety of tissues and contexts during development and organismal homeostasis. Consequently, redundant and pleiotropic effects have hampered a complete understanding of the individual contributions of transducers in distinct contexts. Here, we report on the function of a context-specific regulatory phosphorylation site, PXSP, in the Drosophila mixed lineage kinase protein, Slpr, a mitogen-activated protein kinase kinase kinase (MAP3K) in the Jun Kinase (JNK) pathway. Genetic analysis of the function of non-phosphorylatable (PXAP) and phosphomimetic mutant (PXEP) Slpr transgenes in several distinct contexts revealed minimal effects in JNK-dependent tissue closure processes but differential requirements in heat stress response. In particular, PXAP expression resulted in sensitivity of adults to sustained heat shock, like p38 and JNK pathway mutants. In contrast, PXEP overexpression conferred some resistance. Indeed, phosphorylation of the PXSP motif is enriched under heat shock conditions and requires in part, the p38 kinases for the enrichment. These data suggest that coordination of signaling between p38 and Slpr serves to maintain JNK signaling during heat stress. In sum, we demonstrate a novel role for JNK signaling in the heat shock response in flies and identify a posttranslational modification on Slpr, at a conserved site among MAP3K mixed lineage kinase family members, which bolsters stress resistance with negligible effects on JNK-dependent developmental processes.

Rho1 regulates apoptosis via activation of the JNK signaling pathway at the plasma membrane.

Precisely controlled growth and morphogenesis of developing epithelial tissues require coordination of multiple factors, including proliferation, adhesion, cell shape, and apoptosis. RhoA, a small GTPase, is known to control epithelial morphogenesis and integrity through its ability to regulate the cytoskeleton. In this study, we examine a less well-characterized RhoA function in cell survival. We demonstrate that the Drosophila melanogaster RhoA, Rho1, promotes apoptosis independently of Rho kinase through its effects on c-Jun NH(2)-terminal kinase (JNK) signaling. In addition, Rho1 forms a complex with Slipper (Slpr), an upstream activator of the JNK pathway. Loss of Moesin (Moe), an upstream regulator of Rho1 activity, results in increased levels of Rho1 at the plasma membrane and cortical accumulation of Slpr. Together, these results suggest that Rho1 functions at the cell cortex to regulate JNK activity and implicate Rho1 and Moe in epithelial cell survival.

Dissecting JNK signaling, one KKKinase at a time.

Specificity in signal transduction is essential to ensure distinct and appropriate cellular responses to extracellular cues. Determining the mechanisms that mediate specificity is key to understanding complex cell behaviors in development, when multiple pathways fire simultaneously and individual pathways are used recurrently. Jun kinase (JNK) signal transduction exemplifies a pathway that is used multiple times in animal development and homeostasis. Indeed, molecular genetic analysis of JNK signaling in Drosophila has shown that a core signaling module consisting of Hep (JNKK), Bsk (JNK), and Jun regulates various processes, including tissue morphogenesis, wound repair, stress response, innate immune response, and others. Six putative JNKK kinase (JNKKK) family members are present in the fly genome, which could activate the core module in response to distinct stimuli. The diversity of kinases at this level of the signaling hierarchy could substantially increase the number of possible signals that feed into activation of the core module. Recent studies have described the distinct phenotypic consequences of mutations in three of the genes, Slpr (dMLK), Tak1, and Mekk1. These data, together with Drosophila cell culture and genomic array analyses support the contention that the choice of JNKKK may contribute to signaling specificity in vivo. Whether this is achieved by individual JNKKKs or by means of a combinatorial mechanism will require a systematic characterization of compound mutants and a toolbox of transcriptional reporters specific for distinct JNK-dependent processes.

Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, slipper.

The Jun kinase (JNK) pathway has been characterized for its role in stimulating AP-1 activity and for modulating the balance between cell growth and death during development, inflammation, and cancer. Six families of mammalian kinases acting at the level of JNKKK have emerged as upstream regulators of JNK activity (MLK, LZK, TAK, ASK, MEKK, and TPL); however, the specificity underlying which kinase is utilized for transducing a distinct signal is poorly understood. In Drosophila, JNK signaling plays a central role in dorsal closure, controlling cell fate and cell sheet morphogenesis during embryogenesis. Notably, in the fly genome, there are single homologs of each of the mammalian JNKKK families. Here, we identify mutations in one of those, a mixed lineage kinase, named slipper (slpr), and show that it is required for JNK activation during dorsal closure. Furthermore, our results show that other putative JNKKKs cannot compensate for the loss of slpr function and, thus, may regulate other JNK or MAPK-dependent processes.