To maximize the physiological relevance of in vivo brain tumor mouse models designed to study the downstream effects of oncogenic mutations, it is important to express the mutated genes at ...appropriate levels, in relevant cell types, and in the proper developmental context. For recurrent mutations found in the heterozygous state in tumors, expression of the mutation from the endogenous locus is a more physiologically relevant recapitulation of the brain tumor genome. Here, we describe an approach to generate knock-in mice with an inducible mutation recombined into the endogenous locus. In these engineered mice, the mutated allele is designed for expression controlled by the endogenous promoter and regulatory elements after Cre recombinase-mediated deletion of a loxP-STOP-loxP cassette inserted upstream of the translational start site. To preserve the structure of the endogenous locus, mutations or additional elements may need to be inserted at a considerable distance from the loxP-STOP-loxP cassette. We used recombineering to build a construct with two selectable markers and multiple genetic alterations that can be introduced into the endogenous allele in cis with a single ES cell targeting.
From 2014 to 2017, the World Health Organization convened a working group to evaluate influenza disease burden and vaccine efficacy to inform estimates of maternal influenza immunization program ...impact. The group evaluated existing systematic reviews and relevant primary studies, and conducted four new systematic reviews. There was strong evidence that maternal influenza immunization prevented influenza illness in pregnant women and their infants, although data on severe illness prevention were lacking. The limited number of studies reporting influenza incidence in pregnant women and infants under six months had highly variable estimates and underrepresented low- and middle-income countries. The evidence that maternal influenza immunization reduces the risk of adverse birth outcomes was conflicting, and many observational studies were subject to substantial bias. The lack of scientific clarity regarding disease burden or magnitude of vaccine efficacy against severe illness poses challenges for robust estimation of the potential impact of maternal influenza immunization programs.
GenomePaint (https://genomepaint.stjude.cloud/) is an interactive visualization platform for whole-genome, whole-exome, transcriptome, and epigenomic data of tumor samples. Its design captures the ...inter-relatedness between DNA variations and RNA expression, supporting in-depth exploration of both individual cancer genomes and full cohorts. Regulatory non-coding variants can be inspected and analyzed along with coding variants, and their functional impact further explored by examining 3D genome data from cancer cell lines. Further, GenomePaint correlates mutation and expression patterns with patient outcomes, and supports custom data upload. We used GenomePaint to unveil aberrant splicing that disrupts the RING domain of CREBBP, discover cis activation of the MYC oncogene by duplication of the NOTCH1-MYC enhancer in B-lineage acute lymphoblastic leukemia, and explore the inter- and intra-tumor heterogeneity at EGFR in adult glioblastomas. These examples demonstrate that deep multi-omics exploration of individual cancer genomes enabled by GenomePaint can lead to biological insights for follow-up validation.
CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in ...individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3β. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.
Purpose
Ribociclib, an orally bioavailable small-molecule CDK4/6 inhibitor is currently undergoing evaluation to treat pediatric central nervous system (CNS) tumors. However, it is crucial that it ...penetrates the brain and tumor. Thus, the objectives of the present study were to derive a clinically relevant mouse dosage for cerebral microdialysis studies, and to characterize ribociclib CNS penetration in non-tumor bearing mice and in mice bearing DIPGx7 (glioma) cortical allograft tumors.
Methods
A plasma pharmacokinetic study of ribociclib (100 mg/kg, orally) was performed in CD1 nude mice bearing glioma cortical allografts to obtain initial plasma pharmacokinetic parameters and to derive D-optimal plasma sampling time-points for microdialysis studies. Using a cerebral microdialysis technique, the extracellular fluid (ECF) disposition of ribociclib was evaluated after a single oral ribociclib dose (100 mg/kg) in non-tumor bearing mice and in mice bearing glioma cortical allografts. A one-compartment plasma model with absorption and ECF compartments were fit to plasma and ECF concentration–time data using a nonlinear mixed effects modeling approach (NONMEM 7.2).
Results
The mean unbound ribociclib plasma exposure (6812 ng/ml*h) was similar to that observed clinically at recommended dosages in adults. The median ribociclib ECF to plasma partition coefficient (
K
p,uu
) in non-tumor bearing and glioma mice was 0.10 and 0.07, respectively, and was not statistically different (
t
test,
p
= 0.19).
Conclusions
The CNS penetration observed was encouraging enough to move ribociclib forward with preclinical efficacy studies in models of pediatric brain tumors.
The AKT family, comprising three highly homologous kinases, is an essential mediator of the PTEN/PI3K pathway, which is deregulated in many human cancers. A thorough understanding of the specific ...activities of each isoform in normal and disease tissues is lacking. We evaluated the role of each Akt isoform in gliomagenesis by using a model system driven by common glioma abnormalities, loss of function of p53 and Pten, and expression of EGFRvIII. Both Pten deletion and EGFRvIII expression accelerated the proliferation of p53-null primary murine astrocytes. All three Akt isoforms were expressed and phosphorylated in astrocytes, with significantly higher activation in Pten-null cells. Despite substantial compensation in many contexts when individual Akt isoforms were inhibited, isoform-specific effects were also identified. Specifically, loss of Akt1 or Akt2 decreased proliferation of Pten wild-type astrocytes, whereas combined loss of multiple isoforms was needed to inhibit proliferation of Pten-null astrocytes. In addition, Akt3 was required for anchorage-independent growth of transformed astrocytes and human glioma cells, and Akt3 loss inhibited invasion of transformed astrocytes. EGFRvIII expression transformed p53-null astrocytes with or without Pten deletion, causing rapid development of high-grade astrocytoma on intracranial transplantation. Furthermore, tumorigenesis of Pten;p53-null astrocytes expressing EGFRvIII was delayed by Akt1 loss and accelerated by Akt2 loss. Taken together, these results indicate context-dependent roles for individual Akt isoforms and suggest that there may be heterogeneous tumor response to isoform-specific inhibitors.
Oligodendrocytes-the myelin-forming cells of the central nervous system-can be regenerated during adulthood. In adults, new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs), ...but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized, the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1, a protein deacetylase implicated in energy metabolism, increases the production of new OPCs in the adult mouse brain, in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally, generating fully myelinating oligodendrocytes. Remarkably, SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling, in particular PDGF receptor α (PDGFRα). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK-signalling molecules downstream of PDGFRα. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases, such as multiple sclerosis.
Non‐technical summary The sizes of neurons and their synaptic connections are regulated by multiple molecular mechanisms to provide neuronal networks that perform well‐defined functions. Deletion of ...the tumour suppressor phosphatase and tensin homologue (PTEN) during early development leads to a 2‐ to 3‐fold increase in neuronal and synaptic size and abnormalities in synaptic plasticity, the cellular mechanism underlying learning and memory. Whether PTEN deletion affects synaptic plasticity directly or as a consequence of its effect on the neuronal and synaptic size remained unclear. Here we show that deletion of the Pten gene Pten in mice during postnatal development, when the central nervous system is formed, does not affect neuronal or synaptic size but impairs synaptic plasticity. Thus, PTEN affects neuronal structure and synaptic plasticity through independent mechanisms.
The tumour suppressor PTEN is the central negative regulator of the phosphatidylinositol 3‐kinase (PI3K) signalling pathway, which mediates diverse processes in various tissues. In the nervous system, the PI3K pathway modulates proliferation, migration, cellular size, synaptic transmission and plasticity. In humans, neurological abnormalities such as autism, seizures and ataxia are associated with inherited PTEN mutations. In rodents, Pten loss during early development is associated with extensive deficits in neuronal migration and substantial hypertrophy of neurons and synaptic densities; however, whether its effect on synaptic transmission and plasticity is direct or mediated by structural abnormalities remains unknown. Here we analysed neuronal and synaptic structures and function in Pten‐conditional knockout mice in which the gene was deleted from excitatory neurons postnatally. Using two‐photon imaging, Golgi staining, immunohistochemistry, electron microscopy, and electrophysiological tools, we determined that Pten loss does not affect hippocampus development, neuronal or synaptic structures, or basal excitatory synaptic transmission. However, it does cause deficits in both major forms of synaptic plasticity, long‐term potentiation and long‐term depression, of excitatory synaptic transmission. These deficits coincided with impaired spatial memory, as measured in water maze tasks. Deletion of Pdk1, which encodes a positive downstream regulator of the PI3K pathway, rescued Pten‐mediated deficits in synaptic plasticity but not in spatial memory. These results suggest that PTEN independently modulates functional and structural properties of hippocampal neurons and is directly involved in mechanisms of synaptic plasticity.
Diffuse intrinsic pontine glioma (DIPG) is most commonly diagnosed based on imaging criteria, with biopsy often reserved for pontine tumors with imaging features not typical for DIPG (atypical DIPG, ...'aDIPG'). The histopathologic and molecular spectra of the clinical entity aDIPG remain to be studied systematically. In this study, thirty-three patients with newly diagnosed pontine-centered tumors with imaging inconsistent with DIPG for whom a pathologic diagnosis was subsequently obtained were included. Neoplasms were characterized by routine histology, immunohistochemistry, interphase fluorescence in situ hybridization, Sanger and next-generation DNA/RNA sequencing, and genome-wide DNA methylome profiling. Clinicopathologic features and survival outcomes were analyzed and compared to those of a contemporary cohort with imaging features consistent with DIPG (typical DIPG, 'tDIPG'). Blinded retrospective neuroimaging review assessed the consistency of the initial imaging-based diagnosis and correlation with histopathology. WHO grade II-IV infiltrating gliomas were observed in 54.6% of the cases; the remaining were low-grade gliomas/glioneuronal tumors or CNS embryonal tumors. Histone H3 K27M mutation, identified in 36% of the cases, was the major prognostic determinant. H3 K27M-mutant aDIPG and H3 K27M-mutant tDIPG had similar methylome profiles but clustered separately from diffuse midline gliomas of the diencephalon and spinal cord. In the aDIPG cohort, clinicoradiographic features did not differ by H3 status, yet significant differences in clinical and imaging features were observed between aDIPG without H3 K27M mutation and tDIPG. Neuroimaging review revealed discordance between the classification of aDIPG and tDIPG and did not correlate with the histology of glial/glioneuronal tumors or tumor grade. One patient (3.1%) developed persistent neurologic deficits after surgery; there were no surgery-related deaths. Our study demonstrates that surgical sampling of aDIPG is well-tolerated and provides significant diagnostic, therapeutic, and prognostic implications, and that neuroimaging alone is insufficient to distinguish aDIPG from tDIPG. H3 K27M-mutant aDIPG is epigenetically and clinically similar to H3 K27M-mutant tDIPG.
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