RESUMO
Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.
Assuntos
Genômica , Meduloblastoma/genética , Meduloblastoma/patologia , Análise de Célula Única , Transcriptoma , Adolescente , Adulto , Animais , Linhagem da Célula , Cerebelo/metabolismo , Cerebelo/patologia , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Lactente , Meduloblastoma/classificação , Camundongos , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is classified into four primary molecular subgroups: wingless (WNT), sonic hedgehog (SHH), Group 3 (G3), and Group 4 (G4), and further genomic and proteomic subtypes have been reported. Subgroup heterogeneity and few actionable mutations have hindered the development of targeted therapies, especially for G3 MB, which has a particularly poor prognosis. To identify novel therapeutic targets for MB, we performed mass spectrometry-based deep expression proteomics and phosphoproteomics in 20 orthotopic patient-derived xenograft (PDX) models of MB comprising SHH, G3, and G4 subgroups. We found that the proteomic profiles of MB PDX tumors are closely aligned with those of primary human MB tumors illustrating the utility of PDX models. SHH PDXs were enriched for NFκB and p38 MAPK signaling, while G3 PDXs were characterized by MYC activity. Additionally, we found a significant association between actinomycin D sensitivity and increased abundance of MYC and MYC target genes. Our results highlight several candidate pathways that may serve as targets for new MB therapies. Mass spectrometry data are available via ProteomeXchange with identifier PXD035070.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Neoplasias Encefálicas/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Criança , Modelos Animais de Doenças , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/uso terapêutico , Xenoenxertos , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , ProteômicaRESUMO
The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3ß implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.
Assuntos
Adenosina Desaminase/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Adenosina Desaminase/genética , Processamento Alternativo , Animais , Crise Blástica/etiologia , Crise Blástica/genética , Crise Blástica/metabolismo , Crise Blástica/patologia , Transformação Celular Neoplásica , Progressão da Doença , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Mediadores da Inflamação/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide de Fase Crônica/genética , Leucemia Mieloide de Fase Crônica/metabolismo , Leucemia Mieloide de Fase Crônica/patologia , Camundongos , Edição de RNA , Proteínas de Ligação a RNA , Transcriptoma , Transplante Heterólogo , Ensaio Tumoral de Célula-TroncoRESUMO
Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities.
Assuntos
Neoplasias , Engenharia Tecidual , Sistemas CRISPR-Cas/genética , Detecção Precoce de Câncer , Genoma , Humanos , Neoplasias/genéticaRESUMO
Relapse is the leading cause of death in patients with medulloblastoma, the most common malignant pediatric brain tumor. A better understanding of the mechanisms underlying recurrence could lead to more effective therapies for targeting tumor relapses. Here, we observed that SOX9, a transcription factor and stem cell/glial fate marker, is limited to rare, quiescent cells in high-risk medulloblastoma with MYC amplification. In paired primary-recurrent patient samples, SOX9-positive cells accumulated in medulloblastoma relapses. SOX9 expression anti-correlated with MYC expression in murine and human medulloblastoma cells. However, SOX9-positive cells were plastic and could give rise to a MYC high state. To follow relapse at the single-cell level, an inducible dual Tet model of medulloblastoma was developed, in which MYC expression was redirected in vivo from treatment-sensitive bulk cells to dormant SOX9-positive cells using doxycycline treatment. SOX9 was essential for relapse initiation and depended on suppression of MYC activity to promote therapy resistance, epithelial-mesenchymal transition, and immune escape. p53 and DNA repair pathways were downregulated in recurrent tumors, whereas MGMT was upregulated. Recurrent tumor cells were found to be sensitive to treatment with an MGMT inhibitor and doxorubicin. These findings suggest that recurrence-specific targeting coupled with DNA repair inhibition comprises a potential therapeutic strategy in patients affected by medulloblastoma relapse. SIGNIFICANCE: SOX9 facilitates therapy escape and recurrence in medulloblastoma via temporal inhibition of MYC/MYCN genes, revealing a strategy to specifically target SOX9-positive cells to prevent tumor relapse.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Humanos , Camundongos , Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. SIGNIFICANCE: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Medicina de Precisão/métodos , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Criança , Dactinomicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Meduloblastoma/genética , Camundongos Endogâmicos NOD , Mutação , Polimorfismo de Nucleotídeo Único , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-ß receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.
Assuntos
Neoplasias Cerebelares/imunologia , Meduloblastoma/imunologia , Evasão Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Proteína Supressora de Tumor p53/imunologia , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Drugs that modify the epigenome are powerful tools for treating cancer, but these drugs often have pleiotropic effects, and identifying patients who will benefit from them remains a major clinical challenge. Here we show that medulloblastomas driven by the transcription factor Gfi1 are exquisitely dependent on the enzyme lysine demethylase 1 (Kdm1a/Lsd1). We demonstrate that Lsd1 physically associates with Gfi1, and that these proteins cooperate to inhibit genes involved in neuronal commitment and differentiation. We also show that Lsd1 is essential for Gfi1-mediated transformation: Gfi1 proteins that cannot recruit Lsd1 are unable to drive tumorigenesis, and genetic ablation of Lsd1 markedly impairs tumor growth in vivo. Finally, pharmacological inhibitors of Lsd1 potently inhibit growth of Gfi1-driven tumors. These studies provide important insight into the mechanisms by which Gfi1 contributes to tumorigenesis, and identify Lsd1 inhibitors as promising therapeutic agents for Gfi1-driven medulloblastoma.
Assuntos
Carcinogênese/efeitos dos fármacos , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Meduloblastoma/patologia , Fatores de Transcrição/metabolismo , Animais , Antibióticos Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/terapia , Proteínas de Ligação a DNA/genética , Doxorrubicina/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Histona Desmetilases/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Células NIH 3T3 , Transplante de Neoplasias , Vírus Oncogênicos , Retroviridae , Fatores de Transcrição/genéticaRESUMO
A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.
Assuntos
Caseína Quinase II/metabolismo , Neoplasias Cerebelares/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Fosfoproteínas/metabolismo , Proteômica/métodos , Transdução de Sinais , Anilidas/farmacologia , Animais , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Linhagem Celular Tumoral , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Estimativa de Kaplan-Meier , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Células NIH 3T3 , Naftiridinas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Fenazinas , Fosfoproteínas/genética , Piridinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Cerebelares/genética , Meduloblastoma/genética , Proteômica/métodos , Linhagem Celular Tumoral , Neoplasias Cerebelares/patologia , Humanos , Espectrometria de Massas , Meduloblastoma/patologia , Fatores de RiscoRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor in children, where one-third of patients succumb to their disease. This SnapShot describes the classification of MB subgroups, historically by histopathology and currently based on genomic information. Genomics-based classification has identified four major subgroups and provides greater opportunity for developing targeted therapies more successful than current conventional therapy.