RESUMO
Cerebellar development requires regulated proliferation of cerebellar granule neuron progenitors (CGNPs). Inadequate CGNP proliferation causes cerebellar hypoplasia whereas excessive CGNP proliferation can cause medulloblastoma, the most common malignant pediatric brain tumor. Although sonic hedgehog (SHH) signaling is known to activate CGNP proliferation, the mechanisms downregulating proliferation are less defined. We investigated CGNP regulation by GSK-3, which downregulates proliferation in the forebrain, gut and breast by suppressing mitogenic WNT signaling in mouse. In striking contrast to these systems, we found that co-deleting Gsk3a and Gsk3b blocked CGNP proliferation, causing severe cerebellar hypoplasia. The GSK-3 inhibitor CHIR-98014 similarly downregulated SHH-driven proliferation. Transcriptomic analysis showed activated WNT signaling and upregulated Cdkn1a in Gsk3a/b-deleted CGNPs. Ctnnb co-deletion increased CGNP proliferation and rescued cerebellar hypoproliferation in Gsk3a/b mutants, demonstrating physiological control of CGNPs by GSK-3, mediated through WNT. SHH-driven medulloblastomas similarly required GSK-3, as co-deleting Gsk3a/b blocked tumor growth in medulloblastoma-prone SmoM2 mice. These data show that a GSK-3/WNT axis modulates the developmental proliferation of CGNPs and the pathological growth of SHH-driven medulloblastoma. The requirement for GSK-3 in SHH-driven proliferation suggests that GSK-3 may be targeted for SHH-driven medulloblastoma therapy.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Neurogênese/fisiologia , Aminopiridinas/farmacologia , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Proteínas Hedgehog/genética , Meduloblastoma/genética , Camundongos , Camundongos Mutantes , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Pirimidinas/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
MOTIVATION: Low-dimensional representations of high-dimensional data are routinely employed in biomedical research to visualize, interpret and communicate results from different pipelines. In this article, we propose a novel procedure to directly estimate t-SNE embeddings that are not driven by batch effects. Without correction, interesting structure in the data can be obscured by batch effects. The proposed algorithm can therefore significantly aid visualization of high-dimensional data. RESULTS: The proposed methods are based on linear algebra and constrained optimization, leading to efficient algorithms and fast computation in many high-dimensional settings. Results on artificial single-cell transcription profiling data show that the proposed procedure successfully removes multiple batch effects from t-SNE embeddings, while retaining fundamental information on cell types. When applied to single-cell gene expression data to investigate mouse medulloblastoma, the proposed method successfully removes batches related with mice identifiers and the date of the experiment, while preserving clusters of oligodendrocytes, astrocytes, and endothelial cells and microglia, which are expected to lie in the stroma within or adjacent to the tumours. AVAILABILITY AND IMPLEMENTATION: Source code implementing the proposed approach is available as an R package at https://github.com/emanuelealiverti/BC_tSNE, including a tutorial to reproduce the simulation studies. CONTACT: aliverti@stat.unipd.it.
Assuntos
Células Endoteliais , Software , Algoritmos , Animais , Expressão Gênica , Perfilação da Expressão Gênica , CamundongosRESUMO
We report a nanoparticle formulation of the SHH-pathway inhibitor vismodegib that improves efficacy for medulloblastoma, while reducing toxicity. Limited blood-brain barrier (BBB) penetration and dose-limiting extitle/citraneural toxicities complicate systemic therapies for brain tumors. Vismodegib is FDA-approved for SHH-driven basal cell carcinoma, but implementation for medulloblastoma has been limited by inadequate efficacy and excessive bone toxicity. To address these issues through optimized drug delivery, we formulated vismodegib in polyoxazoline block copolymer micelles (POx-vismo). We then evaluated POx-vismo in transgenic mice that develop SHH-driven medulloblastomas with native vasculature and tumor microenvironment. POx-vismo improved CNS pharmacokinetics and reduced bone toxicity. Mechanistically, the nanoparticle carrier did not enter the CNS, and acted within the vascular compartment to improve drug delivery. Unlike conventional vismodegib, POx-vismo extended survival in medulloblastoma-bearing mice. Our results show the broad potential for non-targeted nanoparticle formulation to improve systemic brain tumor therapy, and specifically to improve vismodegib therapy for SHH-driven cancers.
Assuntos
Anilidas/farmacocinética , Anilidas/uso terapêutico , Sistema Nervoso Central/patologia , Neoplasias Cerebelares/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Meduloblastoma/tratamento farmacológico , Nanopartículas/química , Oxazóis/química , Piridinas/farmacocinética , Piridinas/uso terapêutico , Anilidas/efeitos adversos , Anilidas/farmacologia , Animais , Disponibilidade Biológica , Modelos Animais de Doenças , Portadores de Fármacos/química , Camundongos , Micelas , Tamanho da Partícula , Ligação Proteica , Piridinas/efeitos adversos , Piridinas/farmacologia , Albumina Sérica/metabolismoRESUMO
New targets for brain tumor therapies may be identified by mutations that cause hereditary microcephaly. Brain growth depends on the repeated proliferation of stem and progenitor cells. Microcephaly syndromes result from mutations that specifically impair the ability of brain progenitor or stem cells to proliferate, by inducing either premature differentiation or apoptosis. Brain tumors that derive from brain progenitor or stem cells may share many of the specific requirements of their cells of origin. These tumors may therefore be susceptible to disruptions of the protein products of genes that are mutated in microcephaly. The potential for the products of microcephaly genes to be therapeutic targets in brain tumors are highlighted hereby reviewing research on EG5, KIF14, ASPM, CDK6, and ATR. Treatments that disrupt these proteins may open new avenues for brain tumor therapy that have increased efficacy and decreased toxicity.
Assuntos
Neoplasias Encefálicas/patologia , Microcefalia/patologia , Animais , Encéfalo/patologia , Diferenciação Celular/fisiologia , Glioma/patologia , Humanos , Meduloblastoma/patologia , Mitose/fisiologiaRESUMO
Microcephaly and medulloblastoma may both result from mutations that compromise genomic stability. We report that ATR, which is mutated in the microcephalic disorder Seckel syndrome, sustains cerebellar growth by maintaining chromosomal integrity during postnatal neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation, apoptosis and cerebellar hypoplasia in mice. Co-deletions of either p53 or Bax and Bak prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. ATR-deficient CGNPs had impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to ATR-deficient proliferation was highly p53 dependent and markedly attenuated by p53 co-deletion. Acute ATR inhibition in vivo by nanoparticle-formulated VE-822 reproduced the developmental disruptions seen with Atr deletion. Genetic deletion of Atr blocked tumorigenesis in medulloblastoma-prone SmoM2 mice. Our data show that p53-driven apoptosis and cell cycle arrest - and, in the absence of p53, non-apoptotic cell death - redundantly limit growth in ATR-deficient progenitors. These mechanisms may be exploited for treatment of CGNP-derived medulloblastoma using ATR inhibition.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Cerebelares/genética , Cerebelo/crescimento & desenvolvimento , Instabilidade Cromossômica/genética , Meduloblastoma/genética , Neurogênese/genética , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Cerebelares/patologia , Cerebelo/anormalidades , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Instabilidade Cromossômica/efeitos dos fármacos , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoxazóis/farmacologia , Masculino , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Pirazinas/farmacologiaRESUMO
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.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Neoplasias Cerebelares/fisiopatologia , Cerebelo/crescimento & desenvolvimento , Meduloblastoma/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Animais , Western Blotting , Proteínas de Ligação a Calmodulina/genética , Dano ao DNA/genética , Deleção de Genes , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Mitose/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologiaRESUMO
Commitment to survival or apoptosis within expanding progenitor populations poses distinct risks and benefits to the organism. We investigated whether specialized mechanisms regulate apoptosis in mouse neural progenitors and in the progenitor-derived brain tumor medulloblastoma. Here, we identified constitutive activation of proapoptotic Bax, maintained in check by Bcl-xL, as a mechanism for rapid cell death, common to postnatal neural progenitors and medulloblastoma. We found that tonic activation of Bax in cerebellar progenitors, along with sensitivity to DNA damage, was linked to differentiation state. In cerebellar progenitors, active Bax localized to mitochondria, where it was bound to Bcl-xL. Disruption of Bax:Bcl-xL binding by BH3-mimetic ABT 737 caused rapid apoptosis of cerebellar progenitors and primary murine medulloblastoma cells. Conditional deletion of Mcl-1, in contrast, did not cause death of cerebellar progenitors. Our findings identify a mechanism for the sensitivity of brain progenitors to typical anticancer therapies and reveal that this mechanism persists in medulloblastoma, a malignant brain tumor markedly sensitive to radiation and chemotherapy.
Assuntos
Apoptose/fisiologia , Meduloblastoma/metabolismo , Células-Tronco Neurais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Células-Tronco Neurais/patologia , Ligação Proteica/fisiologia , Fatores de TempoRESUMO
Analysis of single-cell RNA sequencing typically includes the clustering of cells and subsequent determination of the population size of each cluster, relative to the whole. In an experimental setting, two or more conditions are compared to assess changes in cellular composition of the sampled tissue. Cluster populations are frequently normalized to the total number of cells from each replicate in order to facilitate comparisons. After normalization, they become interdependent fractions and therefore cannot be compared using individual t-tests. Here we describe the use of Dirichlet regression to compare changes in cellular composition between two or more conditions when multiple biological replicates (three or more) are sampled under each condition. We provide an example of R code to conduct a similar analysis and interpret the results.
Assuntos
Microcefalia , Análise de Célula Única , Humanos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Análise por Conglomerados , Sequenciamento do ExomaRESUMO
Neural progenitors show a strong tendency to undergo apoptosis in response to DNA damage, and both impaired DNA repair and increased neural progenitor apoptosis are associated with microcephaly. Here we present an immunohistochemistry-based method for assessing DNA damage and apoptosis in the neonatal mouse brain. These methods are suitable for determining in specific experimental conditions the fractions of cells with DNA double-strand breaks, the fractions of cells undergoing apoptosis, or both. While DNA damage in neural progenitors can trigger apoptosis, inappropriate apoptosis may also result from other processes. Simultaneous analysis of DNA damage and apoptosis in mouse models of microcephaly can determine how genetic instability and cell death contribute to the observed phenotype.
Assuntos
Microcefalia , Animais , Camundongos , Dano ao DNA , Encéfalo , Apoptose , Coloração e Rotulagem , ImunofluorescênciaRESUMO
Analyzing sections of neonatal mouse brain using immunohistochemistry can inform microcephaly pathogenesis, but obtaining and staining high-quality sections can be challenging. The neonatal brain shows less structural integrity than the adult brain. As a result, embedding technique must be optimized to allow sections without cracks or other anatomic disruptions. Moreover, paraffin embedding, which maximized tissue preservation, can reduce antigenicity of proteins in the embedded tissues. We describe an optimized embedding technique and antigen recovery technique that allows successful sectioning and immunohistochemical staining.
Assuntos
Encéfalo , Dano ao DNA , Animais , Camundongos , Animais Recém-Nascidos , Inclusão em Parafina , ApoptoseRESUMO
Medulloblastoma (MB) is the most common pediatric brain malignancy and is divided into four molecularly distinct subgroups: WNT, Sonic Hedgehog (SHHp53mut and SHHp53wt), Group 3, and Group 4. Previous reports suggest that SHH MB features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB tumor cells interact with and potentially modify their microenvironment, we performed cytokine array analysis of culture media from freshly isolated MB patient tumor cells, spontaneous SHH MB mouse tumor cells and mouse and human MB cell lines. We found that the SHH MB cells produced elevated levels of IGFBP2 compared to non-SHH MBs. We confirmed these results using ELISA, western blotting, and immunofluorescence staining. IGFBP2 is a pleiotropic member of the IGFBP super-family with secreted and intracellular functions that can modulate tumor cell proliferation, metastasis, and drug resistance, but has been understudied in medulloblastoma. We found that IGFBP2 is required for SHH MB cell proliferation, colony formation, and cell migration, through promoting STAT3 activation and upregulation of epithelial to mesenchymal transition markers; indeed, ectopic STAT3 expression fully compensated for IGFBP2 knockdown in wound healing assays. Taken together, our findings reveal novel roles for IGFBP2 in SHH medulloblastoma growth and metastasis, which is associated with very poor prognosis, and they indicate an IGFBP2-STAT3 axis that could represent a novel therapeutic target in medulloblastoma.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Humanos , Criança , Animais , Camundongos , Meduloblastoma/metabolismo , Proteínas Hedgehog/metabolismo , Transição Epitelial-Mesenquimal , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Cerebelares/metabolismo , Microambiente Tumoral , Fator de Transcrição STAT3/metabolismoRESUMO
We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proliferação de Células , Cerebelo/metabolismo , Diferenciação Celular , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismoRESUMO
We show that inactivating AMPK in a genetic medulloblastoma model depletes tumor stem cells and slows progression. In medulloblastoma, the most common malignant pediatric brain tumor, drug-resistant stem cells co-exist with transit-amplifying cells and terminally differentiated neuronal progeny. Prior studies show that Hk2-dependent glycolysis promotes medulloblastoma progression by suppressing neural differentiation. To determine how the metabolic regulator AMPK affects medulloblastoma growth and differentiation, we inactivated AMPK genetically in medulloblastomas. We bred conditional Prkaa1 and Prkaa2 deletions into medulloblastoma-prone SmoM2 mice and compared SmoM2-driven medulloblastomas with intact or inactivated AMPK. AMPK-inactivation increased event-free survival (EFS) and altered cellular heterogeneity, increasing differentiation and decreasing tumor stem cell populations. Surprisingly, AMPK-inactivation decreased mTORC1 activity and decreased Hk2 expression. Hk2 deletion similarly depleted medulloblastoma stem cells, implicating reduced glycolysis in the AMPK-inactivated phenotype. Our results show that AMPK inactivation disproportionately impairs medulloblastoma stem cell populations typically refractory to conventional therapies.
RESUMO
Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.
RESUMO
The therapeutic potential of CDK4/6 inhibitors for brain tumors has been limited by recurrence. To address recurrence, we tested a nanoparticle formulation of CDK4/6 inhibitor palbociclib (POx-Palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma, alone or in combination with specific agents suggested by our analysis. Nanoparticle encapsulation reduced palbociclib toxicity, enabled parenteral administration, improved CNS pharmacokinetics, and extended mouse survival, but recurrence persisted. scRNA-seq identified up-regulation of glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes in proliferating medulloblastoma cells in POx-Palbo-treated mice, suggesting mTORC1 signaling suppression, subsequently confirmed by decreased 4EBP1 phosphorylation. Combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects and prolonged mouse survival compared to either agent alone, contrasting markedly with other tested drug combinations. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment and identify POx-Palbo + Sapanisertib as effective combinatorial therapy for SHH medulloblastoma.
RESUMO
SRY (sex determining region Y)-box 2 (SOX2)-labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Neoplasias Cerebelares/genética , Criança , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Recidiva Local de Neoplasia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
BACKGROUND: Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models. METHODS: To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups. RESULTS: Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB. CONCLUSIONS: These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Animais , Neoplasias Cerebelares/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/genética , Camundongos , Transcriptoma , Microambiente Tumoral/genéticaRESUMO
The tendency of brain cells to undergo apoptosis in response to exogenous events varies across neural development, with apoptotic threshold dependent on proliferation state. Proliferative neural progenitors show a low threshold for apoptosis, while terminally differentiated neurons are relatively refractory. To define the mechanisms linking proliferation and apoptotic threshold, we examined the effect of conditionally deleting Bcl2l1, the gene that codes the antiapoptotic protein BCL-xL, in cerebellar granule neuron progenitors (CGNPs), and of co-deleting Bcl2l1 homologs, antiapoptotic Mcl-1, or pro-apoptotic Bax. We found that cerebella in conditional Bcl2l1-deleted (Bcl-xLcKO) mice were severely hypoplastic due to the increased apoptosis of CGNPs and their differentiated progeny, the cerebellar granule neurons (CGNs). Apoptosis was highest as Bcl-xLcKO CGNPs exited the cell cycle to initiate differentiation, with proliferating Bcl-xLcKO CGNPs relatively less affected. Despite the overall reduction in cerebellar growth, SHH-dependent proliferation was prolonged in Bcl-xLcKO mice, as more CGNPs remained proliferative in the second postnatal week. Co-deletion of Bax rescued the Bcl-xLcKO phenotype, while co-deletion of Mcl-1 enhanced the phenotype. These findings show that CGNPs require BCL-xL to regulate BAX-dependent apoptosis, and that this role can be partially compensated by MCL-1. Our data further show that BCL-xL expression regulates MCL-1 abundance in CGNPs, and suggest that excessive MCL-1 in Bcl-xLcKO mice prolongs CGNP proliferation by binding SUFU, resulting in increased SHH pathway activation. Accordingly, we propose that BCL-xL and MCL-1 interact with each other and with developmental mechanisms that regulate proliferation, to adjust the apoptotic threshold as CGNPs progress through postnatal neurogenesis to CGNs.
Assuntos
Neoplasias Cerebelares/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Proliferação de Células , Neoplasias Cerebelares/patologia , Humanos , Camundongos , Neurogênese , Transdução de SinaisRESUMO
It is unclear why medulloblastoma patients receiving similar treatments experience different outcomes. Transcriptomic profiling identified subgroups with different prognoses, but in each subgroup, individuals remain at risk of incurable recurrence. To investigate why similar-appearing tumors produce variable outcomes, we analyzed medulloblastomas triggered in transgenic mice by a common driver mutation expressed at different points in brain development. We genetically engineered mice to express oncogenic SmoM2, starting in multipotent glio-neuronal stem cells, or committed neural progenitors. Both groups developed medulloblastomas with similar transcriptomic profiles. We compared medulloblastoma progression, radiosensitivity, and cellular heterogeneity, determined by single-cell transcriptomic analysis (scRNA-seq). Stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing stem-like cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, down-regulated stem-like cells and were curable with radiation. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, with more Ccr2+ macrophages and fewer Igf1+ microglia, indicating that developmental events affected the subsequent tumor microenvironment. Reduced mTORC1 activity in M-Smo tumors suggests that differential Igf1 contributed to differences in phenotype. Developmental events in tumorigenesis that were obscure in transcriptomic profiles thus remained cryptic determinants of tumor composition and outcome. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic/methylomic studies with analyses that resolve cellular heterogeneity.
Assuntos
Linhagem da Célula , Neoplasias Cerebelares/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Meduloblastoma/patologia , Tolerância a Radiação/genética , Células-Tronco/patologia , Transcriptoma/efeitos da radiação , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/radioterapia , Heterogeneidade Genética , Humanos , Meduloblastoma/genética , Meduloblastoma/radioterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Célula Única , Células-Tronco/efeitos da radiação , Microambiente TumoralRESUMO
Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1-dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.