RESUMO
Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.
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Regulação Neoplásica da Expressão Gênica , Glioma , Fator B de Elongação Transcricional Positiva , Humanos , Glioma/radioterapia , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Animais , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Camundongos , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Transcrição Gênica/efeitos da radiação , Apoptose/efeitos da radiação , Apoptose/genética , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Reparo do DNA/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening across MB cell lines, we identified the Mediator-associated kinase CDK8 as the top dependence for MYC-driven MB. Loss of CDK8 markedly reduces MYC expression and impedes MB growth. Mechanistically, we demonstrate that CDK8 depletion suppresses ribosome biogenesis and mRNA translation. CDK8 regulates occupancy of phospho-Polymerase II at specific chromatin loci facilitating an epigenetic alteration that promotes transcriptional regulation of ribosome biogenesis. Additionally, CDK8-mediated phosphorylation of 4EBP1 plays a crucial role in initiating eIF4E-dependent translation. Targeting CDK8 effectively suppresses cancer stem and progenitor cells, characterized by increased ribosome biogenesis activity. We also report the synergistic inhibition of CDK8 and mTOR in vivo and in vitro . Overall, our findings establish a connection between transcription and translation regulation, suggesting a promising therapeutic approach targets multiple points in the protein synthesis network for MYC-driven MB.
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BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.
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Neoplasias Cerebelares , Meduloblastoma , Humanos , Criança , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Linhagem Celular Tumoral , Proteínas Tirosina Fosfatases/genética , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Tirosina , Proteínas Nucleares/genética , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
PURPOSE OF REVIEW: Correlative studies should leverage clinical trial frameworks to conduct biospecimen analyses that provide insight into the bioactivity of the intervention and facilitate iteration toward future trials that further improve patient outcomes. In pediatric cellular immunotherapy trials, correlative studies enable deeper understanding of T cell mobilization, durability of immune activation, patterns of toxicity, and early detection of treatment response. Here, we review the correlative science in adoptive cell therapy (ACT) for childhood central nervous system (CNS) tumors, with a focus on existing chimeric antigen receptor (CAR) and T cell receptor (TCR)-expressing T cell therapies. RECENT FINDINGS: We highlight long-standing and more recently understood challenges for effective alignment of correlative data and offer practical considerations for current and future approaches to multi-omic analysis of serial tumor, serum, and cerebrospinal fluid (CSF) biospecimens. We highlight the preliminary success in collecting serial cytokine and proteomics from patients with CNS tumors on ACT clinical trials.
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Neoplasias do Sistema Nervoso Central , Receptores de Antígenos Quiméricos , Humanos , Criança , Imunoterapia Adotiva , Neoplasias do Sistema Nervoso Central/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos TRESUMO
The most aggressive subtype of medulloblastoma (MB), Group 3, is characterized by MYC amplifications. However, targeting MYC has proven unsuccessful, and there remains a lack of therapeutic targets for treating MB. Studies have shown that the B7 homolog 3 (B7H3) promotes cell proliferation and tumor cell invasion in a variety of cancers. Similarly, it was recently revealed that B7H3 promotes angiogenesis in Group 3 MB and likely facilitates MB metastasis through exosome biogenesis. While therapies targeting B7H3 remain in the early stages of development, targeting upstream regulators of B7H3 expression may be more effective for halting MB progression. Notably, MYC and the enhancer of zeste homolog 2 (EZH2) are known to regulate B7H3 expression, and a previous study by the authors suggested that B7H3 amplifications present in MB are likely the result of EZH2MYC mediated activities. In the present study, it was reported that overexpression of EZH2 is associated with lower overall survival in Group 3 MB patients. It was also revealed that inhibition of EZH2 significantly reduces B7H3 and MYC transcript levels and upregulates miR29a, indicating that EZH2 posttranscriptionally regulates B7H3 expression in Group 3 MB cells. Pharmacological inhibition of EZH2 using EPZ005687 attenuated MB cell viability and reduced the expression of B7H3. Similarly, pharmacological inhibition and knockdown of EZH2 led to the downregulation of MYC, B7H3, and H3K27me3. Further, EZH2 silencing induced apoptosis and reduced colonyforming ability in MB cells, whereas EZH2 inhibition in MYCamplified C17.2 neural stem cells induced G2/M phase arrest while downregulating B7H3 expression. Collectively, the current study positions EZH2 as a viable target for the future development of MB treatments and that targeting EZH2 in combination with B7H3 immunotherapy may be an effective treatment for halting MB progression.
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Neoplasias Cerebelares , Meduloblastoma , Humanos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Proliferação de Células , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismoRESUMO
Background: Atypical teratoid rhabdoid tumors (ATRT) are highly aggressive pediatric brain tumors. The available treatments rely on toxic chemotherapy and radiotherapy, which themselves can cause poor outcomes in young patients. Poly (ADP-ribose) polymerases (PARP), multifunctional enzymes which play an important role in DNA damage repair and genome stability have emerged as a new target in cancer therapy. An FDA-approved drug screen revealed that Rucaparib, a PARP inhibitor, is important for ATRT cell growth. This study aims to investigate the effect of Rucaparib treatment in ATRT. Methods: This study utilized cell viability, colony formation, flow cytometry, western blot, immunofluorescence, and immunohistochemistry assays to investigate Rucaparib's effectiveness in BT16 and MAF737 ATRT cell lines. In vivo, intracranial orthotopic xenograft model of ATRT was used. BT16 cell line was transduced with a luciferase-expressing vector and injected into the cerebellum of athymic nude mice. Animals were treated with Rucaparib by oral gavaging and irradiated with 2 Gy of radiation for 3 consecutive days. Tumor growth was monitored using In Vivo Imaging System. Results: Rucaparib treatment decreased ATRT cell growth, inhibited clonogenic potential of ATRT cells, induced cell cycle arrest and apoptosis, and led to DNA damage accumulation as shown by increased expression of γH2AX. In vivo, Rucaparib treatment decreased tumor growth, sensitized ATRT cells to radiation and significantly increased mice survival. Conclusion: We demonstrated that Rucaparib has potential to be a new therapeutic strategy for ATRT as seen by its ability to decrease ATRT tumor growth both in vitro and in vivo.
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Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.
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Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Maximum safe resection, postoperative craniospinal irradiation, and chemotherapy are the standard of care for MB patients. MB is classified into four subgroups: Shh, Wnt, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis, necessitating alternative therapies. There is increasing interest in targeting epigenetic modifiers for treating pediatric cancers, including MB. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3, as a crucial epigenetic regulator that drives the growth of Group 3 Myc+ MB cells. We demonstrated that SMYD3 directly binds to the cyclin D3 promoter to activate its transcription. Further, SMYD3 depletion significantly reduced MB cell proliferation and led to the downregulation of cyclin D3, cyclin D1, pRBSer795, with concomitant upregulations in RB in vitro. Similar results were obtained following pharmacological inhibition of SMYD3 using BCI-121 ex vivo. SMYD3 knockdown also promoted cyclin D1 ubiquitination, indicating that SMYD3 plays a vital role in stabilizing the cyclin D1 protein. Collectively, our studies demonstrate that SMYD3 drives cell cycle progression in Group 3 Myc+ MB cells and that targeting SMYD3 has the potential to improve clinical outcomes for high-risk patients.
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PURPOSE: Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism. EXPERIMENTAL DESIGN: We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-naïve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models. RESULTS: BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2-BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors. CONCLUSIONS: This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies demonstrate the potential use of the BCL2 inhibitor venetoclax combined with radiotherapy for pediatric DMG.
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Antineoplásicos , Glioma , Animais , Antineoplásicos/farmacologia , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Glioma/genética , Glioma/radioterapia , Humanos , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2 , Radiação Ionizante , Espécies Reativas de Oxigênio , SulfonamidasRESUMO
von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.
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Doenças de von Willebrand , Fator de von Willebrand , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Célula Única , Corpos de Weibel-Palade/metabolismo , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, children usually have a poor survival time. In the present study, the role of TP53/MDM2 interaction in ATRT was investigated. A functional genomic screen identified the TP53/MDM2 axis as a therapeutic target in the central nervous system (CNS) ATRT. Gene expression analysis revealed that all ATRT subgroups expressed high levels of MDM2, which is a negative regulator of TP53. Using cell viability, colony formation and methylcellulose assays it was found that genetic MDM2 inhibition with short hairpin RNA or chemical MDM2 inhibition with small molecule inhibitors, Nutlin3 and idasanutlin (RG7388) decreased the growth of ATRT cell lines. Furthermore, idasanutlin significantly decreased the growth of intracranial orthotopic ATRT brain tumors, as evaluated using T2 MRI, and prolonged survival time relative to control animals. MRI of intracranial tumors showed that diffusion coefficient, an effective marker for successful treatment, significantly increased with idasanutlin treatment showing tumor necrosis/apoptosis. Immunohistochemistry revealed an increased number of caspase3positive cells in the idasanutlin treatment group, confirming the induction of apoptosis in vivo. Using flow cytometry and western blot analysis we show that inhibition of MDM2 enhanced radiation sensitivity in vitro by potentiating DNA damage via the induction of the TP53/Bax/Puma proapoptotic axis. Furthermore, DNA damage was associated with increased mitochondrial reactive oxygen species accumulation. The present study demonstrated that MDM2 expression level was increased in ATRT patient samples and MDM2 inhibition suppressed ATRT cell growth in vitro, and leads to apoptosis in vivo. MDM2 inhibition potentiates DNA damage and sensitizes ATRT cells to radiation. These findings highlight the TP53/MDM2 axis as a rational therapeutic target in CNS ATRT.
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Proteínas Proto-Oncogênicas c-mdm2/efeitos dos fármacos , Tolerância a Radiação/efeitos dos fármacos , Tumor Rabdoide/radioterapia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proliferação de Células/efeitos dos fármacos , Colorado , Humanos , Tolerância a Radiação/genética , Teratoma/radioterapiaRESUMO
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.
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Neoplasias Cerebelares , Meduloblastoma , Animais , Neoplasias Cerebelares/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/genética , Camundongos , Transcriptoma , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. METHODS: Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. RESULTS: PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. CONCLUSIONS: These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.
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Neoplasias Cerebelares , Meduloblastoma , Apoptose , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/patologia , Criança , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Meduloblastoma/radioterapia , Piperazinas , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis , QuinazolinasRESUMO
Radiation-induced high-grade gliomas (RIGs) are an incurable late complication of cranial radiation therapy. We performed DNA methylation profiling, RNA-seq, and DNA sequencing on 32 RIG tumors and an in vitro drug screen in two RIG cell lines. We report that based on DNA methylation, RIGs cluster primarily with the pediatric receptor tyrosine kinase I high-grade glioma subtype. Common copy-number alterations include Chromosome (Ch.) 1p loss/1q gain, and Ch. 13q and Ch. 14q loss; focal alterations include PDGFRA and CDK4 gain and CDKN2A and BCOR loss. Transcriptomically, RIGs comprise a stem-like subgroup with lesser mutation burden and Ch. 1p loss and a pro-inflammatory subgroup with greater mutation burden and depleted DNA repair gene expression. Chromothripsis in several RIG samples is associated with extrachromosomal circular DNA-mediated amplification of PDGFRA and CDK4. Drug screening suggests microtubule inhibitors/stabilizers, DNA-damaging agents, MEK inhibition, and, in the inflammatory subgroup, proteasome inhibitors, as potentially effective therapies.
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Glioma/genética , Glioma/patologia , Radiação , Adolescente , Criança , Estudos de Coortes , Simulação por Computador , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Gradação de Tumores , Transcriptoma/genética , Adulto JovemRESUMO
MYC-driven medulloblastoma is a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. MYC gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in MYC-amplified medulloblastoma, we employ a CRISPR-Cas9 essentiality screen targeting 1,140 genes. We identify CDK7 as a mediator of medulloblastoma tumorigenesis. Using chemical inhibitors and genetic depletion, we observe cessation of tumor growth in xenograft mouse models and increases in apoptosis. The results are attributed to repression of a core set of MYC-driven transcriptional programs mediating DNA repair. CDK7 inhibition alters RNA polymerase II (RNA Pol II) and MYC association at DNA repair genes. Blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage, extending survival and tumor latency in xenograft mouse models. Our studies establish the selective inhibition of MYC-driven medulloblastoma by CDK7 inhibition combined with radiation as a viable therapeutic strategy.
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Neoplasias Cerebelares/radioterapia , Reparo do DNA/genética , Meduloblastoma/radioterapia , Animais , Proliferação de Células , Neoplasias Cerebelares/patologia , Humanos , Meduloblastoma/patologia , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Diffuse midline gliomas (DMGs) are incurable pediatric tumors with extraordinarily limited treatment options. Decades of clinical trials combining conventional chemotherapies with radiation therapy have failed to improve these outcomes, demonstrating the need to identify and validate druggable biologic targets within this disease. NTRK1/2/3 fusions are found in a broad range of pediatric cancers, including high-grade gliomas and a subset of DMGs. Phase 1/2 studies of TRK inhibitors have demonstrated good tolerability, effective CNS penetration, and promising objective responses across all patients with TRK fusion-positive cancers, but their use has not been explored in TRK fusion-positive DMG. Here, we report 3 cases of NTRK fusions co-occurring within H3K27M-positive pontine diffuse midline gliomas. We employ a combination of single-cell and bulk transcriptome sequencing from TRK fusion-positive DMG to describe the phenotypic consequences of this co-occurring alteration. We then use ex vivo short-culture assays to evaluate the potential response to TRK inhibition in this disease. Together, these data highlight the importance of routine molecular characterization of these highly aggressive tumors and identify a small subset of patients that may benefit from currently available targeted therapies.
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Neoplasias do Tronco Encefálico/genética , Glioma/genética , Histona Desmetilases com o Domínio Jumonji/genética , Glicoproteínas de Membrana/genética , Mutação/genética , Receptor trkB/genética , Receptor trkC/genética , Neoplasias do Tronco Encefálico/diagnóstico por imagem , Criança , Pré-Escolar , Evolução Fatal , Feminino , Glioma/diagnóstico por imagem , Humanos , MasculinoRESUMO
Polo-like kinase 1 (PLK1) is highly expressed in group 3 medulloblastoma (MB), and it has been preclinically validated as a cancer therapeutic target in medulloblastoma. Here, we demonstrate that PLK1 inhibition with PCM-075 or BI6727 significantly reduces the growth of MB cells and causes a decrease of c-MYC mRNA and protein levels. We show that MYC activates PLK1 transcription, while the inhibition of PLK1 suppresses MB tumor development and causes a decrease in c-MYC protein level by suppressing FBXW7 auto poly-ubiquitination. FBXW7 physically interacts with PLK1 and c-MYC, facilitating their protein degradation by promoting ubiquitination. These results demonstrate a PLK1-FBXW7-MYC regulatory loop in MYC-driven medulloblastoma. Moreover, FBXW7 is significantly downregulated in group 3 patient samples. The overexpression of FBXW7 induced apoptosis and suppressed proliferation in vitro and in vivo, while constitutive phosphorylation mutation attenuated its tumor suppressor function. Altogether, these findings demonstrated that PLK1 inhibition stabilizes FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing medulloblastoma progression.
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Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor of childhood characterized by histone mutations at lysine 27, which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. Using a combined RNAi and chemical screen targeting epigenomic regulators, we identify the polycomb repressive complex 1 (PRC1) component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy is enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 decreases cell self-renewal and attenuates tumor growth due to induction of senescence. Prolonged BMI1 inhibition induces a senescence-associated secretory phenotype, which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to enhance tumor cell killing in vivo.
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Envelhecimento/genética , Glioma Pontino Intrínseco Difuso/genética , Complexo Repressor Polycomb 1/metabolismo , Astrocitoma/genética , Neoplasias do Tronco Encefálico/tratamento farmacológico , Neoplasias do Tronco Encefálico/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Criança , Pré-Escolar , Cromatina/genética , Glioma Pontino Intrínseco Difuso/tratamento farmacológico , Glioma Pontino Intrínseco Difuso/metabolismo , Epigenômica , Feminino , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Mutação , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genéticaRESUMO
Ependymoma (EPN) is a brain tumor commonly presenting in childhood that remains fatal in most children. Intra-tumoral cellular heterogeneity in bulk-tumor samples significantly confounds our understanding of EPN biology, impeding development of effective therapy. We, therefore, use single-cell RNA sequencing, histology, and deconvolution to catalog cellular heterogeneity of the major childhood EPN subgroups. Analysis of PFA subgroup EPN reveals evidence of an undifferentiated progenitor subpopulation that either differentiates into subpopulations with ependymal cell characteristics or transitions into a mesenchymal subpopulation. Histological analysis reveals that progenitor and mesenchymal subpopulations co-localize in peri-necrotic zones. In conflict with current classification paradigms, relative PFA subpopulation proportions are shown to determine bulk-tumor-assigned subgroups. We provide an interactive online resource that facilitates exploration of the EPN single-cell dataset. This atlas of EPN cellular heterogeneity increases understanding of EPN biology.
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Ependimoma/genética , Células Neoplásicas Circulantes/metabolismo , Análise de Célula Única/métodos , Criança , HumanosRESUMO
BACKGROUND: Hundreds of systemic chemotherapy trials in diffuse intrinsic pontine glioma (DIPG) have not improved survival, potentially due to lack of intratumoral penetration, which has not previously been assessed in humans. METHODS: We used gemcitabine as a model agent to assess DIPG intratumoral pharmacokinetics (PK) using mass spectrometry. RESULTS: In a phase 0 clinical trial of i.v. gemcitabine prior to biopsy in children newly diagnosed with DIPG by MRI, mean concentration in 4 biopsy cores in patient 1 (H3K27M diffuse midline glioma) was 7.65 µM. These compare favorably to levels for patient 2 (mean 3.85 µM, found to have an H3K27-wildtype low-grade glioma on histology), and from a similar study in adult glioblastoma (adjusted mean 3.48 µM). In orthotopic patient-derived xenograft (PDX) models of DIPG and H3K27M-wildtype pediatric glioblastoma, gemcitabine levels and clearance were similar in tumor, pons, and cortex and did not depend on H3K27 mutation status or tumor location. Normalized gemcitabine levels were similar in patient 1 and the DIPG PDX. CONCLUSIONS: These findings, while limited to one agent, provide preliminary evidence for the hypotheses that lack of intratumoral penetration is not why systemic chemotherapy has failed in DIPG, and orthotopic PDX models can adequately model intratumoral PK in human DIPG.