STING activation reprograms the microenvironment to sensitize NF1-related malignant peripheral nerve sheath tumors for immunotherapy.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase-activating protein. Inactivating NF1 mutations cause hyperactivation of RAS-mediated signaling, resulting in the development of multiple neoplasms, including malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in patients with NF1. MPNSTs are difficult to resect and refractory to chemo- and radiotherapy, and no molecular therapies currently exist. Immune checkpoint blockade (ICB) is an approach to treat inoperable, undruggable cancers like MPNST, but successful outcomes require an immune cell-rich tumor microenvironment. While MPNSTs are noninflamed "cold" tumors, here, we converted MPNSTs into T cell-inflamed "hot" tumors by activating stimulator of IFN genes (STING) signaling. Mouse genetic and human xenograft MPNST models treated with a STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNSTs.

Context-dependent ciliary regulation of hedgehog pathway repression in tissue morphogenesis.

A fundamental problem in tissue morphogenesis is identifying how subcellular signaling regulates mesoscale organization of tissues. The primary cilium is a paradigmatic organelle for compartmentalized subcellular signaling. How signaling emanating from cilia orchestrates tissue organization-especially, the role of cilia-generated effectors in mediating diverse morpho-phenotypic outcomes-is not well understood. In the hedgehog pathway, bifunctional GLI transcription factors generate both GLI-activators (GLI-A) and GLI-repressors (GLI-R). The formation of GLI-A/GLI-R requires cilia. However, how these counterregulatory effectors coordinate cilia-regulated morphogenetic pathways is unclear. Here we determined GLI-A/GLI-R requirements in phenotypes arising from lack of hedgehog pathway repression (derepression) during mouse neural tube and skeletal development. We studied hedgehog pathway repression by the GPCR GPR161, and the ankyrin repeat protein ANKMY2 that direct cAMP/protein kinase-A signaling by cilia in GLI-R generation. We performed genetic epistasis between Gpr161 or Ankmy2 mutants, and Gli2/Gli3 knockouts, Gli3R knock-in and knockout of Smoothened, the hedgehog pathway transducer. We also tested the role of cilia-generated signaling using a Gpr161 ciliary localization knock-in mutant that is cAMP signaling competent. We found that the cilia-dependent derepression phenotypes arose in three modes: lack of GLI-R only, excess GLI-A formation only, or dual regulation of either lack of GLI-R or excess GLI-A formation. These modes were mostly independent of Smoothened. The cAMP signaling-competent non-ciliary Gpr161 knock-in recapitulated Gpr161 loss-of-function tissue phenotypes solely from lack of GLI-R only. Our results show complex tissue-specific GLI-effector requirements in morphogenesis and point to tissue-specific GLI-R thresholds generated by cilia in hedgehog pathway repression. Broadly, our study sets up a conceptual framework for rationalization of different modes of signaling generated by the primary cilium in mediating morphogenesis in diverse tissues.

Interactions between TULP3 tubby domain and ARL13B amphipathic helix promote lipidated protein transport to cilia.

The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. It was previously shown that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of nonintegral, membrane-associated proteins and whether such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.

Malignant peripheral nerve sheath tumor: models, biology, and translation.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, invasive cancer that comprise around 10% of all soft tissue sarcomas and develop in about 8-13% of patients with Neurofibromatosis Type 1. They are associated with poor prognosis and are the leading cause of mortality in NF1 patients. MPNSTs can also develop sporadically or following exposure to radiation. There is currently no effective targeted therapy to treat MPNSTs and surgical removal remains the mainstay treatment. Unfortunately, surgery is not always possible due to the size and location of the tumor, thus, a better understanding of MPNST initiation and development is required to design novel therapeutics. Here, we provide an overview of MPNST biology and genetics, discuss findings regarding the developmental origin of MPNST, and summarize the various model systems employed to study MPNST. Finally, we discuss current management strategies for MPNST, as well as recent developments in translating basic research findings into potential therapies.

Studying Hedgehog Signaling During Mouse Neural Tube Development.

The identity of ventral neural progenitors in the neural tube is largely dependent on Hedgehog (Hh) signaling. Variations in staining patterns are excellent indicators of aberrant Hh signaling. Here we describe the basic protocol to stain for progenitor populations based on transcription factor expression. We also provide an overview of ciliary and centrosomal staining in the neural tube.

Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner.

The role of compartmentalized signaling in primary cilia during tissue morphogenesis is not well understood. The cilia localized G protein-coupled receptor, Gpr161, represses hedgehog pathway via cAMP signaling. We engineered a knock-in at the Gpr161 locus in mice to generate a variant (Gpr161mut1), which was ciliary localization defective but cAMP signaling competent. Tissue phenotypes from hedgehog signaling depend on downstream bifunctional Gli transcriptional factors functioning as activators or repressors. Compared to knockout (ko), Gpr161mut1/ko had delayed embryonic lethality, moderately increased hedgehog targets, and partially down-regulated Gli3 repressor. Unlike ko, the Gpr161mut1/ko neural tube did not show Gli2 activator-dependent expansion of ventral-most progenitors. Instead, the intermediate neural tube showed progenitor expansion that depends on loss of Gli3 repressor. Increased extraciliary receptor levels in Gpr161mut1/mut1 prevented ventralization. Morphogenesis in limb buds and midface requires Gli repressor; these tissues in Gpr161mut1/mut1 manifested hedgehog hyperactivation phenotypes-polydactyly and midfacial widening. Thus, ciliary and extraciliary Gpr161 pools likely establish tissue-specific Gli repressor thresholds in determining morpho-phenotypic outcomes.

Ankmy2 Prevents Smoothened-Independent Hyperactivation of the Hedgehog Pathway via Cilia-Regulated Adenylyl Cyclase Signaling.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia. Mice lacking Ankmy2 are mid-embryonic lethal. Knockout embryos have increased Hedgehog signaling and completely open neural tubes showing co-expansion of all ventral neuroprogenitor markers, comparable to the loss of the Hedgehog receptor Patched1. Ventralization in Ankmy2 knockout is completely independent of the Hedgehog pathway transducer Smoothened. Instead, ventralization results from the reduced formation of Gli2 and Gli3 repressors and early depletion of adenylyl cyclase III in neuroepithelial cilia, implicating deficient pathway repression. Ventralization in Ankmy2 knockout requires both cilia and Gli2 activation. These findings indicate that cilia-dependent adenylyl cyclase signaling represses the Hedgehog pathway and promotes morphogenetic patterning.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Tulp3 Regulates Renal Cystogenesis by Trafficking of Cystoproteins to Cilia.

Polycystic kidney disease proteins, polycystin-1 and polycystin-2, localize to primary cilia. Polycystin knockouts have severe cystogenesis compared to ciliary disruption, whereas simultaneous ciliary loss suppresses excessive cyst growth. These data suggest the presence of a cystogenic activator that is inhibited by polycystins and an independent but relatively minor cystogenic inhibitor, either of which are cilia dependent. However, current genetic models targeting cilia completely ablate the compartment, making it difficult to uncouple cystoprotein function from ciliary localization. Thus, the role of cilium-generated signaling in cystogenesis is unclear. We recently demonstrated that the tubby family protein Tulp3 determines ciliary trafficking of polycystins in kidney collecting duct cells without affecting protein levels or cilia. Here, we demonstrate that embryonic-stage, nephron-specific Tulp3 knockout mice developed cystic kidneys, while retaining intact cilia. Cystic kidneys showed increased mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), mTOR, and persistently high cyclic AMP (cAMP) signaling, suggesting contribution of multiple factors to cystogenesis. Based on kidney-to-body-weight ratio, cystic index, and epithelial proliferation in developing tubules or cysts, the severity of cystogenesis upon Tulp3 deletion was intermediate between that caused by loss of polycystin-1 or cilia. However, concomitant Tulp3 loss did not inhibit cystogenesis in polycystin-1 knockouts, unlike ciliary disruption. Interestingly, ciliary trafficking of the small guanosine triphosphatase (GTPase) Arl13b, loss of which causes cystogenic severity similar to ciliary loss, was reduced prior to cyst initiation. Thus, we propose that cystogenesis in Tulp3 mutants results from a reduction of ciliary levels of polycystins, Arl13b, and Arl13b-dependent lipidated cargoes. Arl13b might be the ciliary factor that represses cystogenesis distinct from polycystins.

Basal Suppression of the Sonic Hedgehog Pathway by the G-Protein-Coupled Receptor Gpr161 Restricts Medulloblastoma Pathogenesis.

Sonic hedgehog (Shh) determines cerebellar granule cell (GC) progenitor proliferation and medulloblastoma pathogenesis. However, the pathways regulating GC progenitors during embryogenesis before Shh production by Purkinje neurons and their roles in tumorigenesis remain unclear. The cilium-localized G-protein-coupled receptor Gpr161 suppresses Shh-mediated signaling in the neural tube. Here, by deleting Gpr161 in mouse neural stem cells or GC progenitors, we establish Gpr161 as a tumor suppressor in Shh subtype medulloblastoma. Irrespective of Shh production in the cerebellum, Gpr161 deletion increased downstream activity of the Shh pathway by restricting Gli3-mediated repression, causing more extensive generation and proliferation of GC progenitors. Moreover, earlier deletion of Gpr161 during embryogenesis increased tumor incidence and severity. GC progenitor overproduction during embryogenesis from Gpr161 deletion was cilium dependent, unlike normal development. Low GPR161 expression correlated with poor survival of SHH subtype medulloblastoma patients. Gpr161 restricts GC progenitor production by preventing premature and Shh-dependent pathway activity, highlighting the importance of basal pathway suppression in tumorigenesis.

The G protein-coupled receptor Gpr161 regulates forelimb formation, limb patterning and skeletal morphogenesis in a primary cilium-dependent manner.

The role of basal suppression of the sonic hedgehog (Shh) pathway and its interaction with Indian hedgehog (Ihh) signaling during limb/skeletal morphogenesis is not well understood. The orphan G protein-coupled receptor Gpr161 localizes to primary cilia and functions as a negative regulator of Shh signaling by promoting Gli transcriptional repressor versus activator formation. Here, we show that forelimb buds are not formed in Gpr161 knockout mouse embryos despite establishment of prospective limb fields. Limb-specific deletion of Gpr161 resulted in prematurely expanded Shh signaling and ectopic Shh-dependent patterning defects resulting in polysyndactyly. In addition, endochondral bone formation in forearms, including formation of both trabecular bone and bone collar was prevented. Endochondral bone formation defects resulted from accumulation of proliferating round/periarticular-like chondrocytes, lack of differentiation into columnar chondrocytes, and corresponding absence of Ihh signaling. Gpr161 deficiency in craniofacial mesenchyme also prevented intramembranous bone formation in calvarium. Defects in limb patterning, endochondral and intramembranous skeletal morphogenesis were suppressed in the absence of cilia. Overall, Gpr161 promotes forelimb formation, regulates limb patterning, prevents periarticular chondrocyte proliferation and drives osteoblastogenesis in intramembranous bones in a cilium-dependent manner.

Using Primary Neurosphere Cultures to Study Primary Cilia.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

Trafficking to the primary cilium membrane.

The primary cilium has been found to be associated with a number of cellular signaling pathways, such as vertebrate hedgehog signaling, and implicated in the pathogenesis of diseases affecting multiple organs, including the neural tube, kidney, and brain. The primary cilium is the site where a subset of the cell's membrane proteins is enriched. However, pathways that target and concentrate membrane proteins in cilia are not well understood. Processes determining the level of proteins in the ciliary membrane include entry into the compartment, removal, and retention by diffusion barriers such as the transition zone. Proteins that are concentrated in the ciliary membrane are also localized to other cellular sites. Thus it is critical to determine the particular role for ciliary compartmentalization in sensory reception and signaling pathways. Here we provide a brief overview of our current understanding of compartmentalization of proteins in the ciliary membrane and the dynamics of trafficking into and out of the cilium. We also discuss major unanswered questions regarding the role that defects in ciliary compartmentalization might play in disease pathogenesis. Understanding the trafficking mechanisms that underlie the role of ciliary compartmentalization in signaling might provide unique approaches for intervention in progressive ciliopathies.

Smoothened determines ß-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves ß-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-ß-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment.

Sec13 Regulates Expression of Specific Immune Factors Involved in Inflammation In Vivo.

The Sec13 protein functions in various intracellular compartments including the nuclear pore complex, COPII-coated vesicles, and inside the nucleus as a transcription regulator. Here we developed a mouse model that expresses low levels of Sec13 (Sec13(H/-)) to assess its functions in vivo, as Sec13 knockout is lethal. These Sec13 mutant mice did not present gross defects in anatomy and physiology. However, the reduced levels of Sec13 in vivo yielded specific immunological defects. In particular, these Sec13 mutant mice showed low levels of MHC I and II expressed by macrophages, low levels of INF-γ and IL-6 expressed by stimulated T cells, and low frequencies of splenic IFN-γ+CD8+ T cells. In contrast, the levels of soluble and membrane-bound TGF-ß as well as serum immunoglobulin production are high in these mice. Furthermore, frequencies of CD19+CD5-CD95+ and CD19+CD5-IL-4+ B cells were diminished in Sec13(H/-) mice. Upon stimulation or immunization, some of the defects observed in the naïve mutant mice were compensated. However, TGF-ß expression remained high suggesting that Sec13 is a negative modulator of TGF-ß expression and of its immunosuppressive functions on certain immune cells. In sum, Sec13 regulates specific expression of immune factors with key functions in inflammation.