Biology subjects

Raleigh, D. R.

Publications and source records attributed to Raleigh, D. R..

16 recordsLinked to original sources

bioRxiv2025

Cell atlas of the developing human meninges reveals a dura origin of meningioma

The vertebrate central nervous system is enveloped by the meninges, consisting of the pia, arachnoid, and dura layers. The arachnoid is hypothesised to give rise to the most common primary intracranial tumours, meningiomas. However, molecular evidence supporting this hypothesis is lacking. There are no effective medical therapies to treat meningiomas that are resistant to local interventions, encumbered by our limited understanding of their cellular origin. To address this limitation in our understanding of meningioma biology, we generated a comprehensive reference single cell and spatial transcriptomic atlas of human fetal meninges at post-conceptional weeks 5-13. We found that the meningeal layers develop concurrently, and identified an inner CDH1-positive dura cell layer expressing tight junction genes consistent with barrier function. We show that transcriptionally, meningioma cells resemble dura-lineage cells, and that common meningioma driver genes were expressed preferentially in the dura lineage. Our findings suggest that meningiomas originate from dura lineage cells. HIGHLIGHTSO_LIscRNA-seq and spatial transcriptomics reveals architecture of human fetal meninges development C_LIO_LIMeningeal layers are formed concurrently, by a gradual refinement of cell states C_LIO_LIA CDH1+ inner dura sublayer expresses tight junction genes like the arachnoid barrier C_LIO_LIMeningioma tumours likely originate from inner dura lineage cells, not arachnoid C_LI

developmental biology↗

bioRxiv2025

Aperiodic neural dynamics define a novel signature of glioma-induced excitation-inhibition dysregulation

Diffuse gliomas remodel neuronal circuits with prognostic and therapeutic significance for patients. Electrophysiologic measures of cortical excitability hold promise for monitoring disease progression and evaluating therapeutic responses. The power law exponent (aperiodic slope) reflects the balance between excitatory and inhibitory activity within neuronal networks, a critical aspect of normal brain function often disrupted in neurological conditions. Despite its potential, the significance of the aperiodic slope in glioma-infiltrated tissue and its underlying cellular processes has not been fully investigated. Here, we integrate multi-modal electrophysiological analysis with transcriptomic profiling to analyze the aperiodic slope in both normal and glioma-infiltrated cortex. We determine that glioma infiltration induces a flattening of the aperiodic slope, indicating a shift toward excitation dominance that varies according to tumor subtype and correlates with impairments in semantic naming. Single-nucleus RNA sequencing revealed that cortical regions with flat aperiodic slope exhibit transcriptional programs enriched in glutamatergic signaling, membrane depolarization, and excitatory synaptic transmission. The aperiodic slope responds to pharmacologically induced changes in cortical inhibition during propofol administration, a GABAA agonist. Our results establish the aperiodic slope as a robust biomarker of glioma-associated excitation-inhibition imbalance, with potential applications in tumor classification and treatment monitoring.

cancer biology↗

bioRxiv2025

IL-6 underlies microenvironment immunosuppression and resistance to therapy in glioblastoma

The glioblastoma tumor immune microenvironment (TIME) is an immunosuppressive barrier to therapy that encumbers glioblastoma responses to immune checkpoint inhibition (ICI). Immunosuppressive cytokines, pro-tumor myeloid cells, and exhausted T-cells are hallmarks of the glioblastoma TIME. Here we integrate spatial and single-cell analyses of patient-matched human glioblastoma samples before and after ICI with genetic, immunologic, single-cell, and pharmacologic studies in preclinical models to reveal that interleukin-6 (IL-6) inhibition reprograms the glioblastoma TIME to sensitize mouse glioblastoma to ICI and radiotherapy. Rare human glioblastoma patients who achieve clinical responses to ICI have lower pre-treatment IL-6 levels compared to glioblastomas who do not respond to ICI. Immune stimulatory gene therapy suppresses IL-6 tumor levels in preclinical murine models of glioblastoma. Furthermore, survival was longer in Il-6 knockout mice with orthotopic SB28 glioblastoma relative to wild-type mice. IL-6 blockade with a neutralizing antibody transiently sensitizes mouse glioblastoma to anti-PD-1 by increasing MHCII+ monocytes, CD103+ migratory dendritic cells (DCs), CD11b+ conventional DCs, and effector CD8+ T cells, and decreasing immunosuppressive Tregs. To translate these findings to a combination treatment strategy for recurrent glioblastoma patients, we show that IL-6 blockade plus ICI durably sensitizes mouse glioblastoma to high-dose radiotherapy.

cancer biology↗

bioRxiv2024

A generative framework for enhanced cell-type specificity in rationally designed mRNAs

mRNA delivery offers new opportunities for disease treatment by directing cells to produce therapeutic proteins. However, designing highly stable mRNAs with programmable cell type-specificity remains a challenge. To address this, we measured the regulatory activity of 60,000 5 and 3 untranslated regions (UTRs) across six cell types and developed PARADE (Prediction And RAtional DEsign of mRNA UTRs), a generative AI framework to engineer untranslated RNA regions with tailored cell type-specific activity. We validated PARADE by testing 15,800 de novo-designed sequences across these cell lines and identified many sequences that demonstrated superior specificity and activity compared to existing RNA therapeutics. mRNAs with PARADE-engineered UTRs also exhibited robust tissue-specific activity in animal models, achieving selective expression in the liver and spleen. We also leveraged PARADE to enhance mRNA stability, significantly increasing protein output and therapeutic durability in vivo. These advancements translated to notable increases in therapeutic efficacy, as PARADE-designed UTRs in oncosuppressor mRNAs, namely PTEN and P16, effectively reduced tumor growth in patient-derived neuroglioma xenograft models and orthotopic mouse models. Collectively, these findings establish PARADE as a versatile platform for designing safer, more precise, and highly stable mRNA therapies.

systems biology↗

bioRxiv2024

Glioblastoma Neurovascular Progenitor Orchestrates Tumor Cell Type Diversity

Glioblastoma (GBM) exhibits developmental programs and marked cellular heterogeneity, yet how these features are organized into connected lineage hierarchies remains unclear. Here we identify a rare tumor-intrinsic population, termed the neurovascular progenitor (NVP), that occupies an intermediate position between the major GBM organizational axes. NVP cells co-express neural progenitor and perivascular transcriptional features, are consistently detected across independent patient cohorts, retain canonical GBM copy-number alterations, and localize in situ in both vessel-associated and parenchymal niches. Using direct-from-patient lineage tracing in a human organoid tumor transplantation system, we show that individual NVP cells clonally generate both neural-like and mesenchymal/vascular-like malignant progeny, providing a concrete lineage link between states that are commonly considered mutually exclusive. Despite comprising [~]1% of tumor cells, NVP-derived lineages account for a majority of observed tumor cell types and disproportionately contribute to cycling compartments. Orthogonally, ablation of NVP-associated programs in an in vivo GBM model remodels tumor composition, elicits compensatory progenitor states, and significantly prolongs survival. Together, these findings position NVP as a fate-restricted yet highly influential lineage intermediate that serves as a functional bridge and organizational nexus within GBM hierarchies, linking population-level lineage architecture to the behavior of a specific progenitor cell type.

cancer biology↗

bioRxiv2024

Multiplatform molecular profiling and functional genomic screens identify prognostic signatures and mechanisms underlying MEK inhibitor response in somatic NF1 mutant glioblastoma

NF1 is recurrently mutated in glioblastoma yet the molecular landscape and efficacy of targeted therapies remain unclear. Here, we combine bulk and single cell genomics of human somatic NF1 mutant, IDH-wildtype glioblastomas with functional genomic analysis of cell lines and mouse intracranial tumor models to identify molecular subgroups within NF1 mutant glioblastomas and mechanisms underlying MEK inhibitor response. Targeted DNA sequencing showed homozygous deletion of the cell cycle regulator CDKN2A/B is a poor prognostic marker in somatic NF1 mutant, but not NF1 wildtype, tumors. DNA methylation array profiling revealed three epigenetic groups highlighted by distinct clinical features, co-mutation patterns, and reference methylation classifier identities. Genome-wide CRISPRi screens in glioblastoma cells revealed cell cycle regulators are conserved mediators of cell growth while response to the MEK inhibitor selumetinib converges on Ras/RAF/MEK pathway activation. Repression of the RAF regulator SHOC2 sensitizes glioblastomas to selumetinib in vitro and in vivo in mouse intracranial glioblastoma models. Single cell RNA-sequencing of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib reveals distinct responses between mesenchymal-like (MES-like) and non MES-like subpopulations suggesting non-MES like cells are intrinsically resistant to MEK inhibition. Finally, single nuclear RNA-sequencing (snRNA-seq) of human NF1 mutant, CDKN2A/B deleted glioblastomas reveals MES-like tumor cells are associated with selumetinib sensitivity signatures while non MES-like cells exhibit increased cell cycle progression and lack selumetinib sensitivity, further supporting the notion that MEK inhibition specifically targets MES-like tumor cell subpopulations. Taken together, our data underscores the heterogeneity between and within somatic NF1 mutant glioblastomas and delineates mechanisms of MEK inhibitor response across distinct tumor subpopulations, guiding the development of future therapeutic strategies that may synergize with MEK inhibition for NF1 mutant tumors. The tumor suppressor NF1 is mutated in 15% of glioblastomas,1-3 the most common malignant brain tumor with poor outcomes and few effective treatments.4 NF1 is a GTPase activating protein (GAP) that negatively regulates Ras, and thus, NF1 loss leads to induction of Ras/RAF/MEK/ERK signaling, driving tumorigenesis and comprising a targetable molecular cascade.5,6 Genomic analysis of glioblastoma demonstrates NF1 mutation is associated with a mesenchymal-like (MES-like) transcriptomic tumor cell subpopulation and altered tumor microenvironment.7,8 More broadly, DNA methylation analysis reveals multiple epigenetic subgroups with overlapping relationships to transcriptomic subtype and DNA alterations, underscoring the complex relationship between genetic drivers and molecular signatures.9 While the updated 2021 Central Nervous System WHO tumor classification incorporates an ever increasing amount of molecular criteria for diffuse astrocytic tumors,10 the existence and clinical significance of molecular subgroups within somatic NF1 mutant, IDH-wildtype glioblastomas based on genetic co-mutations, epigenetic profile, or transcriptomic signatures remain unclear. Preclinical data support the utility of MEK inhibition in NF1 mutant gliomas,11,12 and the MEK inhibitor selumetinib is FDA approved for tumors arising in patients with syndromic neurofibromatosis type 1 (NF-1) harboring a germline NF1 mutation.13,14 In NF-1 associated gliomas, MEK inhibition demonstrates efficacy in a limited case series,15 and combined BRAF/MEK inhibition shows efficacy in BRAF p.V600E mutant gliomas,16 further supporting the translational potential of Ras/Raf/MEK/ERK blockade within genetically defined glioma subtypes. Nevertheless, treatment resistance to molecular monotherapy remains a challenge,17-20 and the mechanisms underlying MEK inhibitor resistance in NF1 mutant glioma are unknown. Here, we integrate targeted DNA sequencing, DNA methylation profiling, and single nuclear RNA-sequencing (snRNA-seq) of human patient somatic NF1 mutant, IDH-wildtype glioblastomas with single cell RNA-sequencing (scRNA-seq), genome-wide clustered regularly interspaced short palindromic repeats interference (CRISPRi) screens, and pharmacologic studies in cell lines and mouse intracranial glioblastoma models to define molecular subgroups and functional mediators of MEK inhibitor response. Targeted DNA sequencing of NF1 mutant, IDH-wildtype glioblastomas (n=186 tumors) revealed CDKN2A/B deletion was associated with poor outcomes in NF1 mutant, but not NF1 wildtype, glioblastomas. DNA methylation profiling (n=129 tumors) demonstrated three epigenetic subgroups with distinct clinical features, co- mutation patterns across cell cycle genes, and reference methylation classifier identities. Genome-wide CRISPRi screens in mouse SB28 and human GBM43 glioblastoma cells identified a conserved cell cycle gene network mediating cell growth, consistent with the clinical importance of additional hits affecting the cell cycle in human somatic NF1 mutant glioblastomas. Moreover, genome-wide mediators of selumetinib response converged upon two Ras pathway effectors mediating selumetinib sensitivity: BRAF and SHOC2. SHOC2 repression in glioblastoma cells significantly improved selumetinib response both in vitro and in intracranial allografts in vivo. Single cell RNA-sequencing (scRNA-seq) of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib revealed MES-like tumor cells correlated with CDKN2A retention and the CRISPRi screen selumetinib sensitivity signature, with selumetinib resistant cells displaying Ras pathway induction. In contrast, non-MES like tumor cells were CDKN2A deficient and lacked expression of the CRISPRi screen selumetinib sensitivity signature, with selumetinib resistant cells inducing a glial de-differentiation program. Finally, snRNA-seq of NF1 mutant, CDKN2A/B deleted, IDH-wildtype glioblastomas (n=9) showed non MES-like tumor cells exhibit increased cell cycle progression and were not associated with the CRISPRi screen selumetinib sensitivity signature. MES-like tumor cells within newly diagnosed, but not recurrent, tumors retained expression of the CRISPRi screen selumetinib sensitivity signature, suggesting resistance can arise both between and within specific transcriptomic glioblastoma cell tumor cell subpopulations. Taken together, our data identifies clinically important subgroups of NF1 mutant, IDH-wildtype glioblastomas and supports a model in which heterogeneity between tumors and within tumor cell subpopulations underlies MEK inhibitor response, supporting the need for additional synergistic therapeutic approaches beyond maximal Ras pathway blockade for NF1 mutant glioblastomas.

genomics↗

bioRxiv2024

Eed controls craniofacial osteoblast differentiation and mesenchymal proliferation from the neural crest

The histone methyltransferase Polycomb repressive complex 2 (PRC2) is required for specification of the neural crest, and mis-regulation of neural crest development can cause severe congenital malformations. PRC2 is necessary for neural crest induction, but the embryonic, cellular, and molecular consequences of PRC2 activity after neural crest induction are incompletely understood. Here we show that Eed, a core subunit of PRC2, is required for craniofacial osteoblast differentiation and mesenchymal proliferation after induction of the neural crest. Integrating mouse genetics with single-cell RNA sequencing and epigenetic profiling, our results reveal that conditional knockout of Eed after neural crest cell induction causes severe craniofacial hypoplasia, impaired craniofacial osteogenesis, and attenuated craniofacial mesenchymal cell proliferation that is first evident in post-migratory neural crest cell populations. We show that Eed drives mesenchymal differentiation and proliferation in vivo and in primary craniofacial cell cultures by epigenetically regulating diverse transcription factor programs that are required for specification of post-migratory neural crest cells. These data enhance understanding of epigenetic mechanisms that underlie craniofacial development, and shed light on the embryonic, cellular, and molecular drivers of rare congenital syndromes in humans.

developmental biology↗

bioRxiv2024

Leveraging Single-Cell Sequencing to Classify and Characterize Tumor Subgroups in Bulk RNA-Sequencing Data

Accurate classification of cancer subgroups is essential for precision medicine, tailoring treatments to individual patients based on their cancer subtypes. In recent years, advances in high-throughput sequencing technologies have enabled the generation of large-scale transcriptomic data from cancer samples. These data have provided opportunities for developing computational methods that can improve cancer subtyping and enable better personalized treatment strategies. Here in this study, we evaluated different feature selection schemes in the context of meningioma classification. While the scheme relying solely on bulk transcriptomic data showed good classification accuracy, it exhibited confusion between malignant and benign molecular classes in approximately ~8% of meningioma samples. In contrast, models trained on features learned from meningioma single-cell data accurately resolved the sub-groups confused by bulk-transcriptomic data but showed limited overall accuracy. To integrate interpretable features from the bulk (n=78 samples) and single-cell profiling (~10K cells), we developed an algorithm named CLIPPR which combines the top-performing single-cell models with RNA-inferred copy number variation (CNV) signals and the initial bulk model to create a meta-model, which exhibited the strongest performance in meningioma classification. CLIPPR showed superior overall accuracy and resolved benign-malignant confusion as validated on n=792 bulk meningioma samples gathered from multiple institutions. Finally, we showed the generalizability of our algorithm using our in-house single-cell (~200K cells) and bulk TCGA glioma data (n=711 samples). Overall, our algorithm CLIPPR synergizes the resolution of single-cell data with the depth of bulk sequencing and enables improved cancer sub-group diagnoses and insights into their biology.

bioinformatics↗

bioRxiv2024

Meningioma transcriptomic landscape demonstrates novel subtypes with regional associated biology and patient outcome.

Meningiomas, the most common intracranial tumor, though mostly benign can be recurrent and fatal. WHO grading does not always identify high risk meningioma and better characterizations of their aggressive biology is needed. To approach this problem, we combined 13 bulk RNA-Seq datasets to create a dimension-reduced reference landscape of 1298 meningiomas. Clinical and genomic metadata effectively correlated with landscape regions which led to the identification of meningioma subtypes with specific biological signatures. Time to recurrence also correlated with the map location. Further, we developed an algorithm that maps new patients onto this landscape where nearest neighbors predict outcome. This study highlights the utility of combining bulk transcriptomic datasets to visualize the complexity of tumor populations. Further, we provide an interactive tool for understanding the disease and predicting patient outcome. This resource is accessible via the online tool Oncoscape, where the scientific community can explore the meningioma landscape.

cancer biology↗

bioRxiv2023

In vivo perturb-seq of cancer and immune cells dissects oncologic drivers and therapy response

Genetic perturbation screens with single cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking. Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery (CED) of sgRNA libraries into models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance, as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune/stromal cells following in vivo CRISPRi perturbations. In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application.

genomics↗

bioRxiv2023

Glioma-neuronal circuit remodeling induces regional immunosuppression

Neuronal activity-driven mechanisms impact glioblastoma cell proliferation and invasion1-7, and glioblastoma remodels neuronal circuits8,9. Distinct intratumoral regions maintain functional connectivity via a subpopulation of malignant cells that mediate tumor-intrinsic neuronal connectivity and synaptogenesis through their transcriptional programs8. However, the effects of tumor-intrinsic neuronal activity on other cells, such as immune cells, remain unknown. Here we show that regions within glioblastomas with elevated connectivity are characterized by regional immunosuppression. This was accompanied by different cell compositions and inflammatory status of tumor-associated macrophages (TAMs) in the tumor microenvironment. In preclinical intracerebral syngeneic glioblastoma models, CRISPR/Cas9 gene knockout of Thrombospondin-1 (TSP-1/Thbs1), a synaptogenic factor critical for glioma-induced neuronal circuit remodeling, in glioblastoma cells suppressed synaptogenesis and glutamatergic neuronal hyperexcitability, while simultaneously restoring antigen-presentation and pro-inflammatory responses. Moreover, TSP-1 knockout prolonged survival of immunocompetent mice harboring intracerebral syngeneic glioblastoma, but not of immunocompromised mice, and promoted infiltrations of pro-inflammatory TAMs and CD8+ T-cells in the tumor microenvironment. Notably, pharmacological inhibition of glutamatergic excitatory signals redirected tumor-associated macrophages toward a less immunosuppressive phenotype, resulting in prolonged survival. Altogether, our results demonstrate previously unrecognized immunosuppression mechanisms resulting from glioma-neuronal circuit remodeling and suggest future strategies targeting glioma-neuron-immune crosstalk may open up new avenues for immunotherapy.

cancer biology↗

bioRxiv2023

NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy

Meningiomas are the most common primary intracranial tumors1-3. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental4,5. Resistance to radiotherapy is common in high-grade meningiomas6, and the cell types and signaling mechanisms driving meningioma tumorigenesis or resistance to radiotherapy are incompletely understood. Here we report NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find NOTCH3+ meningioma mural cells are conserved across meningiomas from humans, dogs, and mice. NOTCH3+ cells are restricted to the perivascular niche during meningeal development and homeostasis and in low-grade meningiomas but are expressed throughout high-grade meningiomas that are resistant to radiotherapy. Integrating single-cell transcriptomics with lineage tracing and imaging approaches across mouse genetic and xenograft models, we show NOTCH3 drives tumor initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. An antibody stabilizing the extracellular negative regulatory region of NOTCH37,8 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival in preclinical models. In summary, our results identify a conserved cell type and signaling mechanism that underlie meningioma tumorigenesis and resistance to radiotherapy, revealing a new therapeutic vulnerability to treat meningiomas that are resistant to standard interventions.

cancer biology↗

bioRxiv2022

Epigenetic reprogramming shapes the cellular landscape of schwannoma

Cell state evolution underlies tumor development and response to therapy1, but mechanisms specifying cancer cell states and intratumor heterogeneity are incompletely understood. Schwannomas are the most common tumors of the peripheral nervous system and are treated with surgery and ionizing radiation2-5. Schwannomas can oscillate in size for many years after radiotherapy6,7, suggesting treatment may reprogram schwannoma cells or the tumor microenvironment. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas. We find schwannomas are comprised of 2 molecular groups distinguished by reactivation of neural crest development pathways or misactivation of nerve injury mechanisms that specify cancer cell states and the architecture of the tumor immune microenvironment. Schwannoma molecular groups can arise independently, but ionizing radiation is sufficient for epigenetic reprogramming of neural crest to immune-enriched schwannoma by remodeling chromatin accessibility, gene expression, and metabolism to drive schwannoma cell state evolution and immune cell infiltration. To define functional genomic mechanisms underlying epigenetic reprograming of schwannomas, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of cancer evolution and establish a paradigm of epigenetic reprograming of cancer in response to radiotherapy.

cancer biology↗

bioRxiv2021

LncRNA Miat and Metadhedrin maintain a treatment resistant stem-like niche of medulloblastoma cells

Medulloblastoma is a pediatric brain tumor arising from the cerebellum and brainstem that is curable with surgery and aggressive cytotoxic therapy including craniospinal irradiation and chemotherapy. Preclinical models indicate that a small pool of de-differentiated, stem cell-like medulloblastoma cells are resistant to cytotoxic treatment and contribute to medulloblastoma relapse after aggressive therapy. Here, we identify Miat as a Shh and Myc regulated long noncoding RNA (lncRNA) that is required for maintenance of a treatment-resistant medulloblastoma stem-like phenotype. Loss of Miat delays medulloblastoma formation in genetically defined mouse models of Shh medulloblastoma and enforces differentiation of tumorigenic stem-like medulloblastoma cells into a non-tumorigenic cell state. Miat facilitates treatment resistance in part by downregulating p53 signaling and impairing radiation induced cell death in stem-like MB cells, which can be reversed by therapeutic inhibition of Miat with antisense oligonucleotides. The RNA binding protein Metadhedrin (Mtdh), which is associated with resistance to cytotoxic therapy in numerous types of cancer, co-localizes with Miat in stem-like medulloblastoma cells. Further, loss of Mtdh activates p53 signaling and reduces tumorigenicity in stem-like medulloblastoma cells. Taken together, these data reveal a critical role for the lncRNA Miat in sustaining a treatment resistant pool of tumorigenic stem-like medulloblastoma cells.

cancer biology↗

bioRxiv2021

CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids

Introductory paragraphThe Hedgehog (HH) pathway is critical for development and adult tissue homeostasis1. Aberrant HH signaling can cause congenital malformations, such as digit anomalies and holoprosencephaly2, and other diseases, including cancer3. Signal transduction is initiated by HH ligand binding to the Patched 1 (PTCH1) receptor on primary cilia, thereby releasing inhibition of Smoothened (SMO), a HH pathway activator4. Although cholesterol and several oxysterol lipids, which are enriched in the ciliary membrane, play a crucial role in HH activation4,5, the molecular mechanisms governing the regulation of these lipid molecules remain unresolved. Here, we identify Canopy 4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that controls membrane sterol lipid levels. Cnpy4-/- embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway at the level of SMO in vitro, and elevates membrane levels of accessible sterol lipids such as cholesterol, an endogenous ligand involved in SMO activation6. Thus, our data demonstrate that CNPY4 is a negative regulator that fine-tunes the initial steps of HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.

biochemistry↗

bioRxiv2020

DYRK2 is a ciliary kinase involved 1 in vertebrate Hedgehog signal transduction

Primary cilia are organelles specialized for signaling. We previously defined the proteomes of sea urchin and sea anemone cilia to identify ciliary proteins that predate the origin of bilateria. This evolutionary perspective on cilia identified DYRK2, a kinase not been previously implicated in ciliary biology. We found that DYRK2 localizes to cilia and that loss of DYRK2 disrupts ciliary morphology. We also found that DYRK2 participates in ciliary Hh signal transduction, communicating between SMO and GLI transcription factors. Mutation of mouse Dyrk2 resulted in skeletal defects reminiscent of those caused by loss of Indian hedgehog (Ihh). Like Dyrk2 mutations, pharmacological inhibition of DYRK2 dysregulates ciliary length control and attenuates Hedgehog signaling. Thus, DYRK2 is required for ciliary morphology, for Hedgehog signaling in vitro, and for skeletal development. We propose that DYRK2 is part of the mechanism that transduces SMO to activate GLI transcription factors within cilia.

developmental biology↗