A novel ELP1 mutation impairs the function of the Elongator complex and causes a severe neurodevelopmental phenotype.

BACKGROUND: Neurodevelopmental disorders (NDDs) are heterogeneous, debilitating conditions that include motor and cognitive disability and social deficits. The genetic factors underlying the complex phenotype of NDDs remain to be elucidated. Accumulating evidence suggest that the Elongator complex plays a role in NDDs, given that patient-derived mutations in its ELP2, ELP3, ELP4 and ELP6 subunits have been associated with these disorders. Pathogenic variants in its largest subunit ELP1 have been previously found in familial dysautonomia and medulloblastoma, with no link to NDDs affecting primarily the central nervous system. METHODS: Clinical investigation included patient history and physical, neurological and magnetic resonance imaging (MRI) examination. A novel homozygous likely pathogenic ELP1 variant was identified by whole-genome sequencing. Functional studies included in silico analysis of the mutated ELP1 in the context of the holo-complex, production and purification of the ELP1 harbouring the identified mutation and in vitro analyses using microscale thermophoresis for tRNA binding assay and acetyl-CoA hydrolysis assay. Patient fibroblasts were harvested for tRNA modification analysis using HPLC coupled to mass spectrometry. RESULTS: We report a novel missense mutation in the ELP1 identified in two siblings with intellectual disability and global developmental delay. We show that the mutation perturbs the ability of ELP123 to bind tRNAs and compromises the function of the Elongator in vitro and in human cells. CONCLUSION: Our study expands the mutational spectrum of ELP1 and its association with different neurodevelopmental conditions and provides a specific target for genetic counselling.

Common Regulatory Targets of NFIA, NFIX and NFIB during Postnatal Cerebellar Development.

Transcriptional regulation plays a central role in controlling neural stem and progenitor cell proliferation and differentiation during neurogenesis. For instance, transcription factors from the nuclear factor I (NFI) family have been shown to co-ordinate neural stem and progenitor cell differentiation within multiple regions of the embryonic nervous system, including the neocortex, hippocampus, spinal cord and cerebellum. Knockout of individual Nfi genes culminates in similar phenotypes, suggestive of common target genes for these transcription factors. However, whether or not the NFI family regulates common suites of genes remains poorly defined. Here, we use granule neuron precursors (GNPs) of the postnatal murine cerebellum as a model system to analyse regulatory targets of three members of the NFI family: NFIA, NFIB and NFIX. By integrating transcriptomic profiling (RNA-seq) of Nfia- and Nfix-deficient GNPs with epigenomic profiling (ChIP-seq against NFIA, NFIB and NFIX, and DNase I hypersensitivity assays), we reveal that these transcription factors share a large set of potential transcriptional targets, suggestive of complementary roles for these NFI family members in promoting neural development.

Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction.

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2+ cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.

Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.

Nuclear factor one transcription factors: Divergent functions in developmental versus adult stem cell populations.

Nuclear factor one (NFI) transcription factors are a group of site-specific DNA-binding proteins that are emerging as critical regulators of stem cell biology. During development NFIs promote the production of differentiated progeny at the expense of stem cell fate, with Nfi null mice exhibiting defects such as severely delayed brain and lung maturation, skeletomuscular defects and renal abnormalities, phenotypes that are often consistent with patients with congenital Nfi mutations. Intriguingly, recent research suggests that in adult tissues NFI factors play a qualitatively different role than during development, with NFIs serving to promote the survival and maintenance of slow-cycling adult stem cell populations rather than their differentiation. Here we review the role of NFI factors in development, largely focusing on their role as promoters of stem cell differentiation, and attempt to reconcile this with the emerging role of NFIs in adult stem cell niches.

Patched1 is required in neural crest cells for the prevention of orofacial clefts.

Defects such as cleft lip with or without cleft palate (CL/P) are among the most common craniofacial birth defects in humans. In many cases, the underlying molecular and cellular mechanisms that result in these debilitating anomalies remain largely unknown. Perturbed hedgehog (HH) signalling plays a major role in craniofacial development, and mutations in a number of pathway constituents underlie craniofacial disease. In particular, mutations in the gene encoding the major HH receptor and negative regulator, patched1 (PTCH1), are associated with both sporadic and familial forms of clefting, yet relatively little is known about how PTCH1 functions during craniofacial morphogenesis. To address this, we analysed the consequences of conditional loss of Ptch1 in mouse neural crest cell-derived facial mesenchyme. Using scanning electron microscopy (SEM) and live imaging of explanted facial primordia, we captured defective nasal pit invagination and CL in mouse embryos conditionally lacking Ptch1. Our analysis demonstrates interactions between HH and FGF signalling in the development of the upper lip, and reveals cell-autonomous and non-autonomous roles mediated by Ptch1. In particular, we show that deletion of Ptch1 in the facial mesenchyme alters cell morphology, specifically in the invaginating nasal pit epithelium. These findings highlight a critical link between the neural crest cells and olfactory epithelium in directing the morphogenesis of the mammalian lip and nose primordia. Importantly, these interactions are critically dependent on Ptch1 function for the prevention of orofacial clefts.

Kif7 regulates Gli2 through Sufu-dependent and -independent functions during skin development and tumorigenesis.

Abnormal activation of Hedgehog (Hh) signaling leads to basal cell carcinoma (BCC) of the skin, the most common human cancer. Gli2, the major transcriptional activator of Hh signaling, is essential for hair follicle development and its overexpression in epidermis induces BCC formation and maintains tumor growth. Despite its importance in skin development and tumorigenesis, little is known about the molecular regulation of Gli2. Sufu and Kif7 are two evolutionarily conserved regulators of Gli transcription factors. Here, we show that Sufu and Kif7 regulate Gli2 through distinct mechanisms in keratinocytes. Sufu restricts the activity of Gli2 through cytoplasmic sequestration. Kif7 possesses Sufu-dependent and -independent regulatory functions in Hh signaling: while it promotes Hh pathway activity through the dissociation of Sufu-Gli2 complex, it also contributes to the repression of Hh target genes in the absence of Sufu. Deletion of both Sufu and Kif7 in embryonic skin leads to complete loss of follicular fate. Importantly, although inactivation of Sufu or Kif7 alone in adult epidermis cannot promote BCC formation, their simultaneous deletion induces BCC. These studies establish Sufu and Kif7 as crucial components in the regulation of Gli2 localization and activity, and illustrate their overlapping functions in skin development and tumor suppression.

MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

Hedgehog pathway inhibitor saridegib (IPI-926) increases lifespan in a mouse medulloblastoma model.

The Sonic Hedgehog (Shh) pathway drives a subset of medulloblastomas, a malignant neuroectodermal brain cancer, and other cancers. Small-molecule Shh pathway inhibitors have induced tumor regression in mice and patients with medulloblastoma; however, drug resistance rapidly emerges, in some cases via de novo mutation of the drug target. Here we assess the response and resistance mechanisms to the natural product derivative saridegib in an aggressive Shh-driven mouse medulloblastoma model. In this model, saridegib treatment induced tumor reduction and significantly prolonged survival. Furthermore, the effect of saridegib on tumor-initiating capacity was demonstrated by reduced tumor incidence, slower growth, and spontaneous tumor regression that occurred in allografts generated from previously treated autochthonous medulloblastomas compared with those from untreated donors. Saridegib, a known P-glycoprotein (Pgp) substrate, induced Pgp activity in treated tumors, which likely contributed to emergence of drug resistance. Unlike other Smoothened (Smo) inhibitors, the drug resistance was neither mutation-dependent nor Gli2 amplification-dependent, and saridegib was found to be active in cells with the D473H point mutation that rendered them resistant to another Smo inhibitor, GDC-0449. The fivefold increase in lifespan in mice treated with saridegib as a single agent compares favorably with both targeted and cytotoxic therapies. The absence of genetic mutations that confer resistance distinguishes saridegib from other Smo inhibitors.

Neogenin1 is a Sonic Hedgehog target in medulloblastoma and is necessary for cell cycle progression.

The canonical Sonic Hedgehog (Shh)/Gli pathway plays multiples roles during central nervous system (CNS) development. To elucidate the molecular repertoire of Shh mediators, we have recently described novel transcriptional targets in response to Shh pathway modulation. Among them, we were able to identify Neogenin1 (Neo1), a death dependence receptor, as a new direct Shh downstream regulator in neural precursor proliferation. As appropriate Shh signaling is required for cerebellar growth and alterations cause Shh-driven medulloblastoma (MB), here we have addressed the role of the Shh/Neogenin1 interaction in the context of cerebellar development and cancer. We demonstrate that the Shh pathway regulates Neogenin1 expression in mouse models that recapitulate the Shh MB subtype. We show that the canonical Shh pathway directly regulates the Neo1 gene acting through an upstream sequence in its promoter both in vitro and in vivo in granule neuron precursor cells. We also identified and characterized a functional Gli-binding site in the first intron of the human NEO1 gene. Gene expression profiling of more than 300 MB shows that NEO1 is indeed upregulated in SHH tumors compared to the other MB subgroups. Finally, we provide evidence that NEO1 is necessary for cell cycle progression in a human MB cell line, because a loss of function of NEO1 arrests cells in the G2/M phase. Taken together, these results highlight Neogenin1 as a novel downstream effector of the Shh pathway in MB and a possible therapeutic target.

Mutations in mouse Ift144 model the craniofacial, limb and rib defects in skeletal ciliopathies.

Mutations in components of the intraflagellar transport (IFT) machinery required for assembly and function of the primary cilium cause a subset of human ciliopathies characterized primarily by skeletal dysplasia. Recently, mutations in the IFT-A gene IFT144 have been described in patients with Sensenbrenner and Jeune syndromes, which are associated with short ribs and limbs, polydactyly and craniofacial defects. Here, we describe an N-ethyl-N-nitrosourea-derived mouse mutant with a hypomorphic missense mutation in the Ift144 gene. The mutant twinkle-toes (Ift144(twt)) phenocopies a number of the skeletal and craniofacial anomalies seen in patients with human skeletal ciliopathies. Like other IFT-A mouse mutants, Ift144 mutant embryos display a generalized ligand-independent expansion of hedgehog (Hh) signalling, in spite of defective ciliogenesis and an attenuation of the ability of mutant cells to respond to upstream stimulation of the pathway. This enhanced Hh signalling is consistent with cleft palate and polydactyly phenotypes in the Ift144(twt) mutant, although extensive rib branching, fusion and truncation phenotypes correlate with defects in early somite patterning and may reflect contributions from multiple signalling pathways. Analysis of embryos harbouring a second allele of Ift144 which represents a functional null, revealed a dose-dependent effect on limb outgrowth consistent with the short-limb phenotypes characteristic of these ciliopathies. This allelic series of mouse mutants provides a unique opportunity to uncover the underlying mechanistic basis of this intriguing subset of ciliopathies.

The Virtual Child.

SUMMARY: We are building the world's first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will "develop cancer" that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life.

Elongator and the role of its subcomplexes in human diseases.

The Elongator complex was initially identified in yeast, and a variety of distinct cellular functions have been assigned to the complex. In the last decade, several research groups focussed on dissecting its structure, tRNA modification activity and role in translation regulation. Recently, Elongator emerged as a crucial factor for various human diseases, and its involvement has triggered a strong interest in the complex from numerous clinical groups. The Elongator complex is highly conserved among eukaryotes, with all six subunits (Elp1-6) contributing to its stability and function. Yet, recent studies have shown that the two subcomplexes, namely the catalytic Elp123 and accessory Elp456, may have distinct roles in the development of different neuronal subtypes. This Commentary aims to provide a brief overview and new perspectives for more systematic efforts to explore the functions of the Elongator in health and disease.

Efficient detection and monitoring of pediatric brain malignancies with liquid biopsy based on patient-specific somatic mutation screening.

BACKGROUND: Brain cancer is the leading cause of cancer-related death in children. Early detection and serial monitoring are essential for better therapeutic outcomes. Liquid biopsy has recently emerged as a promising approach for detecting these tumors by screening body fluids for the presence of circulating tumor DNA (ctDNA). Here we tested the limits of liquid biopsy using patient-specific somatic mutations to detect and monitor primary and metastatic pediatric brain cancer. METHODS: Somatic mutations were identified in 3 ependymoma, 1 embryonal tumor with multilayered rosettes, 1 central nervous system neuroblastoma, and 7 medulloblastoma patients. The mutations were used as liquid biomarkers for serial assessment of cerebrospinal fluid (CSF) samples using a droplet digital PCR (ddPCR) system. The findings were correlated to the imaging data and clinical assessment to evaluate the utility of the approach for clinical translation. RESULTS: We developed personalized somatic mutation ddPCR assays which we show are highly specific, sensitive, and efficient in detection and monitoring of ctDNA, with a positive correlation between presence of ctDNA, disease course, and clinical outcomes in the majority of patients. CONCLUSIONS: We demonstrate the feasibility and clinical utility of personalized mutation-based liquid biopsy for the surveillance of brain cancer in children. However, even with this specific and sensitive approach, we identified some potential false negative analyses. Overall, our results indicate that changes in ctDNA profiles over time demonstrate the great potential of our specific approach for predicting tumor progression, burden, and response to treatment.

Spatial transcriptomic analysis of Sonic hedgehog medulloblastoma identifies that the loss of heterogeneity and promotion of differentiation underlies the response to CDK4/6 inhibition.

BACKGROUND: Medulloblastoma (MB) is a malignant tumour of the cerebellum which can be classified into four major subgroups based on gene expression and genomic features. Single-cell transcriptome studies have defined the cellular states underlying each MB subgroup; however, the spatial organisation of these diverse cell states and how this impacts response to therapy remains to be determined. METHODS: Here, we used spatially resolved transcriptomics to define the cellular diversity within a sonic hedgehog (SHH) patient-derived model of MB and show that cells specific to a transcriptional state or spatial location are pivotal for CDK4/6 inhibitor, Palbociclib, treatment response. We integrated spatial gene expression with histological annotation and single-cell gene expression data from MB, developing an analysis strategy to spatially map cell type responses within the hybrid system of human and mouse cells and their interface within an intact brain tumour section. RESULTS: We distinguish neoplastic and non-neoplastic cells within tumours and from the surrounding cerebellar tissue, further refining pathological annotation. We identify a regional response to Palbociclib, with reduced proliferation and induced neuronal differentiation in both treated tumours. Additionally, we resolve at a cellular resolution a distinct tumour interface where the tumour contacts neighbouring mouse brain tissue consisting of abundant astrocytes and microglia and continues to proliferate despite Palbociclib treatment. CONCLUSIONS: Our data highlight the power of using spatial transcriptomics to characterise the response of a tumour to a targeted therapy and provide further insights into the molecular and cellular basis underlying the response and resistance to CDK4/6 inhibitors in SHH MB.

Patched 1 is a crucial determinant of asymmetry and digit number in the vertebrate limb.

The vertebrate hedgehog receptor patched 1 (Ptc1) is crucial for negative regulation of the sonic hedgehog (Shh) pathway during anterior-posterior patterning of the limb. We have conditionally inactivated Ptc1 in the mesenchyme of the mouse limb using Prx1-Cre. This results in constitutive activation of hedgehog (Hh) signalling during the early stages of limb budding. Our data suggest that variations in the timing and efficiency of Cre-mediated excision result in differential forelimb and hindlimb phenotypes. Hindlimbs display polydactyly (gain of digits) and a molecular profile similar to the Gli3 mutant extra-toes. Strikingly, forelimbs are predominantly oligodactylous (displaying a loss of digits), with a symmetrical, mirror-image molecular profile that is consistent with re-specification of the anterior forelimb to a posterior identity. Our data suggest that this is related to very early inactivation of Ptc1 in the forelimb perturbing the gene regulatory networks responsible for both the pre-patterning and the subsequent patterning stages of limb development. These results establish the importance of the downstream consequences of Hh pathway repression, and identify Ptc1 as a key player in limb patterning even prior to the onset of Shh expression.

Functional divergence of the two Elongator subcomplexes during neurodevelopment.

The highly conserved Elongator complex is a translational regulator that plays a critical role in neurodevelopment, neurological diseases, and brain tumors. Numerous clinically relevant variants have been reported in the catalytic Elp123 subcomplex, while no missense mutations in the accessory subcomplex Elp456 have been described. Here, we identify ELP4 and ELP6 variants in patients with developmental delay, epilepsy, intellectual disability, and motor dysfunction. We determine the structures of human and murine Elp456 subcomplexes and locate the mutated residues. We show that patient-derived mutations in Elp456 affect the tRNA modification activity of Elongator in vitro as well as in human and murine cells. Modeling the pathogenic variants in mice recapitulates the clinical features of the patients and reveals neuropathology that differs from the one caused by previously characterized Elp123 mutations. Our study demonstrates a direct correlation between Elp4 and Elp6 mutations, reduced Elongator activity, and neurological defects. Foremost, our data indicate previously unrecognized differences of the Elp123 and Elp456 subcomplexes for individual tRNA species, in different cell types and in different key steps during the neurodevelopment of higher organisms.

The Hedgehog receptor Patched1 regulates myeloid and lymphoid progenitors by distinct cell-extrinsic mechanisms.

Hedgehog (Hh) ligands bind to the Patched1 (Ptch1) receptor, relieving repression of Smoothened, which leads to activation of the Hh signaling pathway. Using conditional Ptch1 knockout mice, the aim of this study was to determine the effects of activating the Hh signaling pathway in hematopoiesis. Surprisingly, hematopoietic-specific deletion of Ptch1 did not lead to activation of the Hh signaling pathway and, consequently, had no phenotypic effect. In contrast, deletion of Ptch1 in nonhematopoietic cells produced 2 distinct hematopoietic phenotypes. First, activation of Hh signaling in epithelial cells led to apoptosis of lymphoid progenitors associated with markedly elevated levels of circulating thymic stromal lymphopoietin. Second, activation of Hh signaling in the bone marrow cell niche led to increased numbers of lineage-negative c-kit(+) Sca-1(+) bone marrow cells and mobilization of myeloid progenitors associated with a marked loss of osteoblasts. Thus, deletion of Ptch1 leads to hematopoietic effects by distinct cell-extrinsic mechanisms rather than by direct activation of the Hh signaling pathway in hematopoietic cells. These findings have important implications for therapeutics designed to activate the Hh signaling pathway in hematopoietic cells including hematopoietic stem cells.

Genetic and functional interaction network analysis reveals global enrichment of regulatory T cell genes influencing basal cell carcinoma susceptibility.

BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common form of human cancer, with more than 90% of tumours presenting with clear genetic activation of the Hedgehog pathway. However, polygenic risk factors affecting mechanisms such as DNA repair and cell cycle checkpoints or which modulate the tumour microenvironment or host immune system play significant roles in determining whether genetic mutations culminate in BCC development. We set out to define background genetic factors that play a role in influencing BCC susceptibility via promoting or suppressing the effects of oncogenic drivers of BCC. METHODS: We performed genome-wide association studies (GWAS) on 17,416 cases and 375,455 controls. We subsequently performed statistical analysis by integrating data from population-based genetic studies of multi-omics data, including blood- and skin-specific expression quantitative trait loci and methylation quantitative trait loci, thereby defining a list of functionally relevant candidate BCC susceptibility genes from our GWAS loci. We also constructed a local GWAS functional interaction network (consisting of GWAS nearest genes) and another functional interaction network, consisting specifically of candidate BCC susceptibility genes. RESULTS: A total of 71 GWAS loci and 46 functional candidate BCC susceptibility genes were identified. Increased risk of BCC was associated with the decreased expression of 26 susceptibility genes and increased expression of 20 susceptibility genes. Pathway analysis of the functional candidate gene regulatory network revealed strong enrichment for cell cycle, cell death, and immune regulation processes, with a global enrichment of genes and proteins linked to TReg cell biology. CONCLUSIONS: Our genome-wide association analyses and functional interaction network analysis reveal an enrichment of risk variants that function in an immunosuppressive regulatory network, likely hindering cancer immune surveillance and effective antitumour immunity.

Tumor cells generate astrocyte-like cells that contribute to SHH-driven medulloblastoma relapse.

Astrocytes, a major glial cell type in the brain, play a critical role in supporting the progression of medulloblastoma (MB), the most common malignant pediatric brain tumor. Through lineage tracing analyses and single-cell RNA sequencing, we demonstrate that astrocytes are predominantly derived from the transdifferentiation of tumor cells in relapsed MB (but not in primary MB), although MB cells are generally believed to be neuronal-lineage committed. Such transdifferentiation of MB cells relies on Sox9, a transcription factor critical for gliogenesis. Our studies further reveal that bone morphogenetic proteins (BMPs) stimulate the transdifferentiation of MB cells by inducing the phosphorylation of Sox9. Pharmacological inhibition of BMP signaling represses MB cell transdifferentiation into astrocytes and suppresses tumor relapse. Our studies establish the distinct cellular sources of astrocytes in primary and relapsed MB and provide an avenue to prevent and treat MB relapse by targeting tumor cell transdifferentiation.