DDX3X suppresses the susceptibility of hindbrain lineages to medulloblastoma

DEAD-Box Helicase 3 X-Linked (DDX3X) is frequently mutated in the Wingless (WNT) and Sonic hedghog (SHH) subtypes of medulloblastoma—the commonest malignant childhood brain tumor, but whether DDX3X functions as a medulloblastoma oncogene or tumor suppressor gene is not known. Here, we show that Ddx3x regulates hindbrain patterning and development by controlling Hox gene expression and cell stress signaling. In mice predisposed to Wnt- or Shh medulloblastoma, Ddx3x sensed oncogenic stress and suppressed tumor formation. WNT and SHH medulloblastomas normally arise only in the lower and upper rhombic lips, respectively. Deletion of Ddx3x removed this lineage restriction, enabling both medulloblastoma subtypes to arise in either germinal zone. Thus, DDX3X is a medulloblastoma tumor suppressor that regulates hindbrain development and restricts the competence of cell lineages to form medulloblastoma subtypes

The G-protein Alpha Subunit Gsα Is A Tumor Suppressor In Sonic Hedgehog-driven Medulloblastoma

Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G-protein Gsα, as a potent tumor suppressor gene that defines a subset of aggressive Sonic Hedgehog (Shh)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically-distinct progenitors is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gsα is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh-signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation of a Gsα effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas mutants. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gsα that acts as a molecular link across Shh-group medulloblastomas of disparate cellular and anatomical origins, illuminating G-protein modulation as a potential therapeutic avenue

Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma

Human induced pluripotent stem cell engineering establishes a humanized mouse platform for pediatric low-grade glioma modeling

A major obstacle to identifying improved treatments for pediatric low-grade brain tumors (gliomas) is the inability to reproducibly generate human xenografts. To surmount this barrier, we leveraged human induced pluripotent stem cell (hiPSC) engineering to generate low-grade gliomas (LGGs) harboring the two most common pediatric pilocytic astrocytoma-associated molecular alterations, NF1 loss and KIAA1549:BRAF fusion. Herein, we identified that hiPSC-derived neuroglial progenitor populations (neural progenitors, glial restricted progenitors and oligodendrocyte progenitors), but not terminally differentiated astrocytes, give rise to tumors retaining LGG histologic features for at least 6 months in vivo. Additionally, we demonstrated that hiPSC-LGG xenograft formation requires the absence of CD4 T cell-mediated induction of astrocytic Cxcl10 expression. Genetic Cxcl10 ablation is both necessary and sufficient for human LGG xenograft development, which additionally enables the successful long-term growth of patient-derived pediatric LGGs in vivo. Lastly, MEK inhibitor (PD0325901) treatment increased hiPSC-LGG cell apoptosis and reduced proliferation both in vitro and in vivo. Collectively, this study establishes a tractable experimental humanized platform to elucidate the pathogenesis of and potential therapeutic opportunities for childhood brain tumors

Automatic Segmentation of Glottal Space from Video Images Based on Mathematical Morphology and the hough Transform

Vocal disorders directly arise from the physical shape of the vocal cords. Videostroboscopic imaging provides doctors with valuable information about the physical shape of the vocal cords and about the way these cords move. Segmentation of the glottal space is necessary in order to characterize morphological disorders of vocal folds. One of the main problems with the methods presented is their low level of accuracy. To solve this problem, an automatic method based on Mathematical Morphology edge detection and the Hough transformation is presented in this article to extract the glottal space from the videostroboscopic images presented. Our method compared with the histogram and active contours methods and the findings showed that our proposed method yields better results.DOI:http://dx.doi.org/10.11591/ijece.v2i2.21

Consensus Paper: Cerebellar Development

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum

Spatial proteomics finds CD155 and Endophilin-A1 as mediators of growth and invasion in medulloblastoma

The composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the fast-endophilin-mediated endocytosis effector endophilin-A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells

Spatiotemporal expansion of primary progenitor zones in the developing human cerebellum

We present histological and molecular analyses of the developing human cerebellum from 30 days after conception to 9 months after birth. Differences in developmental patterns between humans and mice include spatiotemporal expansion of both ventricular and rhombic lip primary progenitor zones to include subventricular zones containing basal progenitors. The human rhombic lip persists longer through cerebellar development than in the mouse and undergoes morphological changes to form a progenitor pool in the posterior lobule, which is not seen in other organisms, not even in the nonhuman primate the macaque. Disruptions in human rhombic lip development are associated with posterior cerebellar vermis hypoplasia and Dandy-Walker malformation. The presence of these species-specific neural progenitor populations refines our insight into human cerebellar developmental disorders

Biological mechanisms of disease relapse in childhood medulloblastoma

PhD ThesisOver 30% of patients diagnosed with a medulloblastoma experience disease recurrence. Relapse is almost universally fatal, only infants who receive delayed radiotherapy at disease recurrence typically survive long term. Consequently relapse is the single leading cause of mortality disease-wide. Improved understanding of medulloblastoma at diagnosis has led to the identification of four distinct molecular subgroups with differing biology and outcome. These comprise of medulloblastomas associated with WNT and SHH pathway disruption (MBWNT and MBSHH respectively), and Group 3 and Group 4 tumours (MBGroup3 and MBGroup4). In contrast, very little is understood about the disease at recurrence, and at present there are only two published studies interrogating the biology of relapsed medulloblastoma. However, improved understanding of the biology at relapse is critical to improving treatment. Events at disease relapse could be explored as therapeutic targets or, if predictive of disease recurrence, provide an opportunity to escalate upfront therapy with the aim of preventing relapse. This study compiled a cohort of medulloblastoma tumours sampled at relapse (n=29), paired with their diagnostic counterparts. All clinicopathological and molecular features, with an established relationship to disease prognosis at diagnosis, were interrogated in this paired relapse cohort. With the exception of molecular subgroup, all features investigated displayed evidence of alteration and predominantly acquisition at recurrence. Most strikingly, the emergence of combined p53-MYC defects was commonly observed at relapse and these features were associated with locally aggressive, rapidly progressive disease following relapse. Through collaborative work, this discovery was explored further, with the development of a novel GTML/Trp53 KI/KI mouse model which faithfully recapitulated the clinicopathological and molecular features of the p53-MYC human tumours, and demonstrated the dependency of tumourigenesis and maintenance on this genetic interaction. Moreover, therapeutic inhibition of Aurora A kinase using MLN8237 in these mouse tumours led to degradation of MYCN, tumour reduction and prolonged survival. v A novel genome-wide DNA methylation analysis was next undertaken in the paired relapse cohort, focusing on MBGroup4 tumours, to interrogate maintained and acquired DNA methylation events between diagnosis and relapse, which may play a role in tumour development. Individual CpG sites on the Infinium DNA methylation 450K array were assessed for changes in their DNA methylation status between diagnosis and relapse. Fifteen candidate genes demonstrated tumour-specific methylation states that emerged at relapse and correlated with gene expression. The T-box and Homeobox gene families accounted for 8/15 (53%) candidates identified. Both these families are reportedly important for tumour development in other cancers. In addition, several studies suggest that epigenetic mechanisms, such as DNA methylation, play a regulatory role in their gene expression. Finally, a large cohort of medulloblastoma tumours (n=206), sampled at diagnosis, from patients who are known to go on and recur, was assembled to investigate any subgroup-specific patterns and timings of relapse. MBWNT rarely relapsed, whereas MBSHH frequently relapsed at both local and distant sites, but were the tumour subgroup most readily salvaged by radiotherapy in patients who were not treated with craniospinal irradiation (CSI) at diagnosis (8/12, 67%). Both MBGroup3 and MBGroup4 were widely metastatic at recurrence (34/41 (83%) and 52/61 (85%)) but contrastingly MBGroup3 relapsed quickly (p=0.0022), whereas MBGroup4 relapsed more slowly (p=0.0008). In patients who did not receive upfront CSI, MYC amplification at diagnosis was associated with rapid disease progression after relapse (p=0.0003). No diagnostic feature was significantly associated with time to death following relapse in the cohort of patients who received upfront CSI. This finding was supported by data from the paired relapse cohort where, in patients who received upfront CSI, it was the biological features of the tumour at relapse and not diagnosis, which were associated with disease course. In summary, this study has discovered emergent combined p53-MYC defects at medulloblastoma relapse which are associated with disease behaviour, identified potentially epigenetically regulated candidate genes in relapsed MBGroup4 tumours, and shown that the patterns of disease relapse are associated with radiotherapy and molecular subgroup. Together these findings demonstrate that medulloblastoma tumour biology is significantly different at relapse and that the timings and location of vi disease recurrence should be considered in the context of molecular subgroup and treatment. Biopsy at disease recurrence is now essential to validate and expand on these novel findings, interrogate all molecular subgroups at disease recurrence, and translate these discoveries into improved outcomes for the patients suffering from this devastating diagnosis.Action Medical Researc

Long-term speech and language deficits and associated neural correlates in survivors of paediatric posterior fossa tumours

Background: The present research is the first multi-modal study of language deficits in long-term PFT survivors to date. In addition to a detailed neuropsychological assessment of language, this thesis investigated neural correlates of language processing by employing functional (fMRI) and diffusion (DTI) magnetic resonance imaging techniques. Method: Twenty-one PFT survivors, aged 16-21, and twenty-two matched healthy volunteers completed a series of neuropsychological assessments, task-based language fMRI study (targeting semantic retrieval and speech articulation), and a DTI study. The patients’ clinical profiles and oro-motor functioning were also analysed. Results: Patients demonstrated significantly poorer performance in semantic content, expressive and receptive skills, verbal memory, reading and writing, visuo-motor coordination and non-verbal intelligence, with elevated internal variability of the linguistic profiles, when compared to controls. Semantically-related language skills contributed more to the between-group differences than non-verbal cognitive skills. A significant association between language and non-verbal cognitive abilities in both patients and controls was unconfounded by the age and disease. On fMRI, patients lacked metabolic response in the pre-central and post-central gyri during semantic retrieval. Broca’s, Wernike’s and Geschwind’s areas responded similarly in both hemispheres, with no significant differences between the groups. In all participants Language Content Index predicted the BOLD response on the border of the left lateral occipital cortex and angular gyrus. Expressive Language Index predicted BOLD response in the right frontal pole, paracingulate gyrus, superior frontal gyrus, and middle frontal gyrus. In healthy controls, articulation of speech was associated with activation in the Crus I and Crus II of the right cerebellar hemisphere. Semantic load triggered activation in the Crus VI and VIIb of the vermis, as well as right lobules V and VI of the cerebellum. DTI revealed a global decrease in the fractional anisotropy and increase in the diffusivity scalars in patients, compared to controls, but not different between those patients that received and did not receive radiotherapy. Patients also demonstrated significant reduction in FA index in the bilateral arcuate fasciculus and increased diffusivity in the bilateral SCP. The FA index in the segments of the left-hemispheric cortico-spinal tract, anterior thalamic radiation, superior longitudinal fasciculus and inferior fronto-occipital fasciculus, positively predicted Language Content Index score in patients. Clinical profiles analysis indicated that younger age at diagnosis, radiotherapy treatment and longer duration of mutism (if present) were associated with the poorest language outcomes. Patients with the longest recovery time demonstrated the best manual dexterity abilities. 38% of the patients that met the criteria for a diagnosable language disorder also had reduced oro-motor functioning and reduced FA within left arcuate fasciculus, compared to the remaining patients. Conclusions: Despite the fact that PFTs do not directly impact cortical language-associated areas, patients, particularly those treated with radiotherapy at a younger age, demonstrate deficits in all aspects of language processing. Semantic processing difficulties in PFT survivors are underpinned by the diminished cortical metabolic response during associated task performance, and microstructural changes in the left-hemispheric white matter. Tumours affecting the right cerebellar hemisphere may further predispose patients to developing difficulties in accessing language semantics