RESUMO
The primary cilium, a signaling organelle projecting from the surface of a cell, controls cellular physiology and behavior. The presence or absence of primary cilia is a distinctive feature of a given tumor type; however, whether and how the primary cilium contributes to tumorigenesis are unknown for most tumors. Medulloblastoma (MB) is a common pediatric brain cancer comprising four groups: SHH, WNT, group 3 (G3), and group 4 (G4). From 111 cases of MB, we show that primary cilia are abundant in SHH and WNT MBs but rare in G3 and G4 MBs. Using WNT and G3 MB mouse models, we show that primary cilia promote WNT MB by facilitating translation of mRNA encoding ß-catenin, a major oncoprotein driving WNT MB, whereas cilium loss promotes G3 MB by disrupting cell cycle control and destabilizing the genome. Our findings reveal tumor type-specific ciliary functions and underlying molecular mechanisms. Moreover, we expand the function of primary cilia to translation control and reveal a molecular mechanism by which cilia regulate cell cycle progression, thereby providing new frameworks for studying cilium function in normal and pathologic conditions.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Neoplasias Cerebelares/genética , Cílios/genética , Humanos , Meduloblastoma/genética , CamundongosRESUMO
PURPOSE: We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy. METHODS: We used a gene expression dataset to identify PLK1 as a second target in MB cells and validated the relevance of PLK1 in MB. We measured cell metabolic activity, viability, and cycle progression in MB cells after treatment with PLK1-specific inhibitors (PLK1i). Chou-Talalay synergy calculations were used to determine the nature of class I HDACi entinostat and PLK1i interaction which was validated. Finally, the clinical potential of the combination was assessed in the in vivo experiment. RESULTS: MYC-amplified tumor cells are highly sensitive towards treatment with ATP-competitive PLK1i as a monotherapy. Entinostat and PLK1i in combination act synergistically in MYC-driven MB cells, exerting cytotoxic effects at clinically relevant concentrations. The downstream effect is exerted via MYC-related pathways, pointing out the potential of MYC amplification as a clinically feasible predictive biomarker for patient selection. While entinostat significantly extended survival of mice implanted with orthotopic MYC-amplified MB PDX, there was no evidence of the improvement of survival when treating the animals with the combination. CONCLUSION: The combination of entinostat and PLK1i showed synergistic interaction in vitro, but not in vivo. Therefore, further screening of blood-brain barrier penetrating PLK1i is warranted to determine the true potential of the combination as no on-target activity was observed after PLK1i volasertib treatment in vivo.
Assuntos
Antineoplásicos , Neoplasias Cerebelares , Meduloblastoma , Camundongos , Animais , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Meduloblastoma/tratamento farmacológico , Meduloblastoma/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias Cerebelares/tratamento farmacológico , Linhagem Celular TumoralRESUMO
PURPOSE: Tumor infiltration by immunosuppressive myeloid cells or tumor-associated macrophages (TAMs) contributes to tumor progression and metastasis. In contrast to their adult counterparts, higher TAM signatures do not correlate with aggressive tumor behavior in pediatric brain tumors. While prominent TAM infiltrates exist before and after radiation, the degree to which irradiated macrophages and microglia support progression or leptomeningeal metastasis remains unclear. Patients with medulloblastoma often present with distant metastases and tumor recurrence is largely incurable, making them prime candidates for the study of novel approaches to prevent neuroaxis dissemination and recurrence. METHODS: Macrophage depletion was achieved using CSF-1 receptor inhibitors (CSF-1Ri), BLZ945 and AFS98, with or without whole brain radiation in a variety of medulloblastoma models, including patient-derived xenografts bearing Group 3 medulloblastoma and a transgenic Sonic Hedgehog (Ptch1+/-, Trp53-/-) medulloblastoma model. RESULTS: Effective reduction of microglia, TAM, and spinal cord macrophage with CSF-1Ri resulted in negligible effects on the rate of local and spinal recurrences or survival following radiation. Results were comparable between medulloblastoma subgroups. While notably few tumor-infiltrating lymphocytes (TILs) were detected, average numbers of CD3+ TILs and FoxP3+ Tregs did not differ between groups following treatment and tumor aggressiveness by Ki67 proliferation index was unaltered. CONCLUSION: In the absence of other microenvironmental influences, medulloblastoma-educated macrophages do not operate as tumor-supportive cells or promote leptomeningeal recurrence in these models. Our data add to a growing body of literature describing a distinct immunophenotype amid the medulloblastoma microenvironment and highlight the importance of appropriate pediatric modeling prior to clinical translation.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Transdução de Sinais , Criança , Proteínas Hedgehog , Humanos , Fator Estimulador de Colônias de Macrófagos , Macrófagos , Receptores Proteína Tirosina Quinases , Receptor de Fator Estimulador de Colônias de Macrófagos , Microambiente TumoralRESUMO
Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3â pM; BRD4 DC50 =0.87â nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Piperidonas/química , Ubiquitina-Proteína Ligases/química , Humanos , Hidrólise , ProteóliseRESUMO
Pediatric brain tumors are the leading cause of cancer-related death in children. Patient-derived orthotopic xenografts (PDOX) of childhood brain tumors have recently emerged as a biologically faithful vehicle for testing novel and more effective therapies. Herein, we provide the histopathological and molecular analysis of 37 novel PDOX models generated from pediatric brain tumor patients treated at St. Jude Children's Research Hospital. Using a combination of histopathology, whole-genome and whole-exome sequencing, RNA-sequencing, and DNA methylation arrays, we demonstrate the overall fidelity and inter-tumoral molecular heterogeneity of pediatric brain tumor PDOX models. These models represent frequent as well as rare childhood brain tumor entities, including medulloblastoma, ependymoma, atypical teratoid rhabdoid tumor, and embryonal tumor with multi-layer rosettes. PDOX models will be valuable platforms for evaluating novel therapies and conducting pre-clinical trials to accelerate progress in the treatment of brain tumors in children. All described PDOX models and associated datasets can be explored using an interactive web-based portal and will be made freely available to the research community upon request.
Assuntos
Neoplasias Encefálicas , Modelos Animais de Doenças , Xenoenxertos , Animais , Criança , Humanos , CamundongosRESUMO
Choroid plexus carcinomas (CPCs) are highly malignant brain tumours predominantly found in children and associated to poor prognosis. Improved therapy for these cancers would benefit from the generation of animal models. Here we have created a novel mouse CPC model by expressing a stabilised form of c-Myc (MycT58A) and inactivating Trp53 in the choroid plexus of newborn mice. This induced aberrant proliferation of choroid plexus epithelial cells, leading to aggressive tumour development and death within 150 days. Choroid plexus tumours occurred with a complete penetrance in all brain ventricles, with prevalence in the lateral and fourth ventricles. Histological and cellular analysis indicated that these tumours were CPCs resembling their human counterparts. Comparison of gene expression profiles of CPCs and non-neoplastic tissues revealed profound alterations in cell cycle regulation and DNA damage responses, suggesting that dysregulation of cell division and DNA checkpoint pathways may represent key vulnerabilities. This novel animal model of CPC provides an invaluable tool to elucidate the mechanism of CPC formation and to develop successful therapies against this devastating paediatric cancer.
Assuntos
Carcinoma/genética , Carcinoma/patologia , Neoplasias do Plexo Corióideo/genética , Neoplasias do Plexo Corióideo/patologia , Plexo Corióideo/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Proliferação de Células , Dano ao DNA , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Mutação , TranscriptomaRESUMO
Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.
Assuntos
Ependimoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Infratentoriais/genética , Mutação/genética , Proteínas Oncogênicas/genética , Metilação de DNA , Ependimoma/classificação , Ependimoma/patologia , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Histonas/genética , Humanos , Neoplasias Infratentoriais/classificação , Neoplasias Infratentoriais/patologia , Masculino , TransfecçãoRESUMO
Epigenetics is the process by which gene expression is regulated by events other than alterations of the genome. This includes DNA methylation, histone modifications, chromatin remodeling, microRNAs, and long non-coding RNAs. Methylation of DNA, chromatin remodeling, and histone modifications regulate the chromatin and access of transcription factors to DNA and in turn gene transcription. Alteration of chromatin is now recognized to be deregulated in many cancers. Medulloblastoma is an embryonal tumor of the cerebellum and the most common malignant brain tumor in children, that occurs only rarely in adults. Medulloblastoma is characterized by four major molecularly and histopathologically distinct groups, wingless (WNT), sonic hedgehog (SHH), group 3 (G3), and group 4 (G4), that, except for WNT, are each now subdivided in several subgroups. Gene expression array, next-generation sequencing, and methylation profiling of several hundred primary tumors by several consortia and independent groups revealed that medulloblastomas harbor a paucity of mutations most of which occur in epigenetic regulators, genetic alterations in oncogenes and tumor suppressors, in addition to copy number alterations and chromosome gains and losses. Remarkably, some tumors have no reported mutations, suggesting that some genes required for oncogenesis might be regulated by epigenetic mechanisms which are still to be uncovered and validated. This review will highlight several epigenetic regulators focusing mainly on histone modifiers identified in medulloblastoma.
Assuntos
Neoplasias Cerebelares/genética , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Meduloblastoma/genética , Neoplasias Cerebelares/metabolismo , Humanos , Meduloblastoma/metabolismo , Transdução de SinaisRESUMO
Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC50 values comparable to BET inhibitor (BETi) clinical candidates.
Assuntos
Proteínas/antagonistas & inibidores , Quinolinas/farmacologia , Termodinâmica , Água/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas/metabolismo , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.
Assuntos
Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/genética , Meduloblastoma/classificação , Meduloblastoma/genética , Mutação/genética , Animais , Antígenos CD , Proteína de Ligação a CREB/genética , Caderinas/genética , Proteínas Cdh1 , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Linhagem da Célula , Neoplasias Cerebelares/patologia , Criança , Classe I de Fosfatidilinositol 3-Quinases , RNA Helicases DEAD-box/genética , Variações do Número de Cópias de DNA , DNA Helicases/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Genoma Humano/genética , Genômica , Proteínas Hedgehog/metabolismo , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Meduloblastoma/patologia , Metilação , Camundongos , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/genética , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.
Assuntos
Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/genética , Genoma Humano/genética , Variação Estrutural do Genoma/genética , Meduloblastoma/classificação , Meduloblastoma/genética , Proteínas de Transporte/genética , Neoplasias Cerebelares/metabolismo , Criança , Variações do Número de Cópias de DNA/genética , Duplicação Gênica/genética , Genes myc/genética , Genômica , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/metabolismo , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Fusão Oncogênica/genética , Proteínas/genética , RNA Longo não Codificante , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Translocação Genética/genéticaRESUMO
Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation.
Assuntos
Proteínas Culina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Conformação Proteica , Dobramento de Proteína , Ubiquitina-Proteína Ligases/química , Ubiquitinas/químicaRESUMO
Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2'-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Tetra-Hidrouridina/administração & dosagem , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/sangue , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapêutico , Relação Dose-Resposta a Droga , Epigênese Genética/efeitos dos fármacos , Camundongos , Camundongos Nus , Tetra-Hidrouridina/sangue , Tetra-Hidrouridina/farmacocinética , Tetra-Hidrouridina/uso terapêuticoRESUMO
Medulloblastoma encompasses a collection of clinically and molecularly diverse tumour subtypes that together comprise the most common malignant childhood brain tumour. These tumours are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) after aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT subtype) arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumours infiltrate the dorsal brainstem, whereas SHH-subtype tumours are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem which included aberrantly proliferating Zic1(+) precursor cells. These lesions persisted in all mutant adult mice; moreover, in 15% of cases in which Tp53 was concurrently deleted, they progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence, to our knowledge, that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH- and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.
Assuntos
Tronco Encefálico/patologia , Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mutação , beta Catenina/genéticaRESUMO
Background: Medulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. Methods: To determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Children's Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. Results: Our RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. Conclusions: These results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key Points: Several Type I MAGE CTAs are expressed in >60% of group 3 MBs. Type I MAGEs affect MB cell proliferation and apoptosis. MAGEs are potential biomarkers and therapeutic targets for group 3 MBs. Importance of the Study: This study is the first comprehensive analysis of all Type I MAGE CTAs ( MAGEA , -B , and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options.
RESUMO
Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.
Assuntos
Proteína p300 Associada a E1A , Redes Reguladoras de Genes , Meduloblastoma , Humanos , Meduloblastoma/genética , Meduloblastoma/tratamento farmacológico , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteína p300 Associada a E1A/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/antagonistas & inibidores , Linhagem Celular Tumoral , Redes Reguladoras de Genes/efeitos dos fármacos , Animais , Domínios Proteicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Antineoplásicos/farmacologiaRESUMO
Most children with medulloblastoma (MB) achieve remission, but some face very aggressive metastatic tumors. Their dismal outcome highlights the critical need to advance therapeutic approaches that benefit such high-risk patients. Minnelide, a clinically relevant analog of the natural product triptolide, has oncostatic activity in both preclinical and early clinical settings. Despite its efficacy and tolerable toxicity, this compound has not been evaluated in MB. Utilizing a bioinformatic data set that integrates cellular drug response data with gene expression, we predicted that Group 3 (G3) MB, which has a poor 5-year survival, would be sensitive to triptolide/Minnelide. We subsequently showed that both triptolide and Minnelide attenuate the viability of G3 MB cells ex vivo. Transcriptomic analyses identified MYC signaling, a pathologically relevant driver of G3 MB, as a downstream target of this class of drugs. We validated this MYC dependency in G3 MB cells and showed that triptolide exerts its efficacy by reducing both MYC transcription and MYC protein stability. Importantly, Minnelide acted on MYC to reduce tumor growth and leptomeningeal spread, which resulted in improved survival of G3 MB animal models. Moreover, Minnelide improved the efficacy of adjuvant chemotherapy, further highlighting its potential for the treatment of MYC-driven G3 MB.
Assuntos
Diterpenos , Compostos de Epóxi , Meduloblastoma , Fenantrenos , Proteínas Proto-Oncogênicas c-myc , Ensaios Antitumorais Modelo de Xenoenxerto , Fenantrenos/farmacologia , Diterpenos/farmacologia , Compostos de Epóxi/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular Tumoral , Pró-Fármacos/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , OrganofosfatosRESUMO
Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in â¼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for >2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Coristoma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Núcleo Vestibular Lateral/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Cóclea/citologia , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Núcleo Vestibular Lateral/citologia , Núcleo Vestibular Lateral/crescimento & desenvolvimentoRESUMO
Traditionally, well-defined three-dimensional structure has been thought to be essential for protein function. However, myriad biological functions are performed by highly dynamic, intrinsically disordered proteins (IDPs). IDPs often fold upon binding their biological targets and frequently show 'binding diversity' by targeting multiple ligands. We sought to understand the physical basis of IDP binding diversity and report here that the cyclin-dependent kinase (Cdk) inhibitor p21(Cip1) adaptively binds to and inhibits the various Cdk-cyclin complexes that regulate eukaryotic cell division. Using results from NMR spectroscopy and biochemical and cellular assays, we show that structural adaptability of a helical subdomain within p21, termed LH, enables two other subdomains, D1 and D2, to specifically bind conserved surface features of the cyclin and Cdk subunits, respectively, within otherwise structurally distinct Cdk-cyclin complexes. Adaptive folding upon binding is likely to mediate the diverse biological functions of the thousands of IDPs present in eukaryotes.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Sequência de Aminoácidos , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21/química , Inibidor de Quinase Dependente de Ciclina p21/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclinas/química , Ciclinas/genética , Eucariotos/citologia , Eucariotos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Selective targeting of N-Myc-driven Sonic hedgehog (SHH) medulloblastoma has been a challenge for many years and, despite decades of research, few targeted therapy opportunities exist. Recently, Kuzuoglu-Ozturk et al. characterized the translatome of N-Myc-driven medulloblastoma as a promising therapeutic target. The study showed that N-Myc controls a subset of members of the protein folding machinery that could be inhibited pharmacologically and validated a subset of Hsp70 functions as required for medulloblastoma progression in vitro and in vivo.