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.

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.

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.

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.

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.

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.

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.

SOX9 Defines Distinct Populations of Cells in SHH Medulloblastoma but Is Not Required for Math1-Driven Tumor Formation.

Medulloblastoma is the most common malignant pediatric brain tumor and there is an urgent need for molecularly targeted and subgroup-specific therapies. The stem cell factor SOX9, has been proposed as a potential therapeutic target for the treatment of Sonic Hedgehog medulloblastoma (SHH-MB) subgroup tumors, given its role as a downstream target of Hedgehog signaling and in functionally promoting SHH-MB metastasis and treatment resistance. However, the functional requirement for SOX9 in the genesis of medulloblastoma remains to be determined. Here we report a previously undocumented level of SOX9 expression exclusively in proliferating granule cell precursors (GCP) of the postnatal mouse cerebellum, which function as the medulloblastoma-initiating cells of SHH-MBs. Wild-type GCPs express comparatively lower levels of SOX9 than neural stem cells and mature astroglia and SOX9low GCP-like tumor cells constitute the bulk of both infant (Math1Cre:Ptch1lox/lox ) and adult (Ptch1LacZ/+ ) SHH-MB mouse models. Human medulloblastoma single-cell RNA data analyses reveal three distinct SOX9 populations present in SHH-MB and noticeably absent in other medulloblastoma subgroups: SOX9 + MATH1 + (GCP), SOX9 + GFAP + (astrocytes) and SOX9 + MATH1 + GFAP + (potential tumor-derived astrocytes). To functionally address whether SOX9 is required as a downstream effector of Hedgehog signaling in medulloblastoma tumor cells, we ablated Sox9 using a Math1Cre model system. Surprisingly, targeted ablation of Sox9 in GCPs (Math1Cre:Sox9lox/lox ) revealed no overt phenotype and loss of Sox9 in SHH-MB (Math1Cre:Ptch1lox/lox;Sox9lox/lox ) does not affect tumor formation. IMPLICATIONS: Despite preclinical data indicating SOX9 plays a key role in SHH-MB biology, our data argue against SOX9 as a viable therapeutic target.

Systems pharmacogenomics identifies novel targets and clinically actionable therapeutics for medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Thus, there is an urgent need for improved, targeted therapies that minimize these harmful side effects. METHODS: We identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB. RESULTS: Our analyses identified drugs targeting CDK4, CDK6 and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses, we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing patient-derived xenograft tumours of the Sonic Hedgehog and Group 3 subtype, providing the first demonstration of its efficacy in MB. CONCLUSIONS: Our findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype.

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.

Vincristine-induced peripheral neuropathy is driven by canonical NLRP3 activation and IL-1ß release.

Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1ß from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.

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.

Patient-derived orthotopic xenograft models of medulloblastoma lack a functional blood-brain barrier.

BACKGROUND: Novel targeted therapies for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, are urgently required. A major hurdle in the development of effective therapies is the impaired delivery of systemic therapies to tumor cells due to a specialized endothelial blood-brain barrier (BBB). Accordingly, the integrity of the BBB is an essential consideration in any preclinical model used for assessing novel therapeutics. This study sought to assess the functional integrity of the BBB in several preclinical mouse models of MB. METHODS: Dynamic contrast enhancement magnetic resonance imaging (MRI) was used to evaluate blood-brain-tumor barrier (BBTB) permeability in a murine genetically engineered mouse model (GEMM) of Sonic Hedgehog (SHH) MB, patient-derived orthotopic xenograft models of MB (SHH and Gp3), and orthotopic transplantation of GEMM tumor cells, enabling a comparison of the direct effects of transplantation on the integrity of the BBTB. Immunofluorescence analysis was performed to compare the structural and subcellular features of tumor-associated vasculature in all models. RESULTS: Contrast enhancement was observed in all transplantation models of MB. No contrast enhancement was observed in the GEMM despite significant tumor burden. Cellular analysis of BBTB integrity revealed aberrancies in all transplantation models, correlating to the varying levels of BBTB permeability observed by MRI in these models. CONCLUSIONS: These results highlight functional differences in the integrity of the BBTB and tumor vessel phenotype between commonly utilized preclinical models of MB, with important implications for the preclinical evaluation of novel therapeutic agents for MB.

Correction: Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source.

[This corrects the article DOI: 10.1371/journal.pone.0065818.].

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.

Granule neuron precursor cell proliferation is regulated by NFIX and intersectin 1 during postnatal cerebellar development.

Cerebellar granule neurons are the most numerous neuronal subtype in the central nervous system. Within the developing cerebellum, these neurons are derived from a population of progenitor cells found within the external granule layer of the cerebellar anlage, namely the cerebellar granule neuron precursors (GNPs). The timely proliferation and differentiation of these precursor cells, which, in rodents occurs predominantly in the postnatal period, is tightly controlled to ensure the normal morphogenesis of the cerebellum. Despite this, our understanding of the factors mediating how GNP differentiation is controlled remains limited. Here, we reveal that the transcription factor nuclear factor I X (NFIX) plays an important role in this process. Mice lacking Nfix exhibit reduced numbers of GNPs during early postnatal development, but elevated numbers of these cells at postnatal day 15. Moreover, Nfix-/- GNPs exhibit increased proliferation when cultured in vitro, suggestive of a role for NFIX in promoting GNP differentiation. At a mechanistic level, profiling analyses using both ChIP-seq and RNA-seq identified the actin-associated factor intersectin 1 as a downstream target of NFIX during cerebellar development. In support of this, mice lacking intersectin 1 also displayed delayed GNP differentiation. Collectively, these findings highlight a key role for NFIX and intersectin 1 in the regulation of cerebellar development.

Elongator mutation in mice induces neurodegeneration and ataxia-like behavior.

Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of diverse neurological disorders.

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.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.