RESUMEN
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.
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Diterpenos , Compuestos Epoxi , Meduloblastoma , Fenantrenos , Proteínas Proto-Oncogénicas c-myc , Ensayos Antitumor por Modelo de Xenoinjerto , Fenantrenos/farmacología , Diterpenos/farmacología , Compuestos Epoxi/farmacología , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/patología , Meduloblastoma/metabolismo , Animales , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Línea Celular Tumoral , Profármacos/farmacología , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , OrganofosfatosRESUMEN
Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM.
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Neoplasias Encefálicas , Proteínas que Contienen Bromodominio , Proliferación Celular , Resistencia a Antineoplásicos , Glioblastoma , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Proteómica/métodos , Proteínas/metabolismo , Proteínas/antagonistas & inhibidoresRESUMEN
Deep learning is a machine learning technique to model high-level abstractions in data by utilizing a graph composed of multiple processing layers that experience various linear and non-linear transformations. This technique has been shown to perform well for applications in drug discovery, utilizing structural features of small molecules to predict activity. Here, we report a large-scale study to predict the activity of small molecules across the human kinome-a major family of drug targets, particularly in anti-cancer agents. While small-molecule kinase inhibitors exhibit impressive clinical efficacy in several different diseases, resistance often arises through adaptive kinome reprogramming or subpopulation diversity. Polypharmacology and combination therapies offer potential therapeutic strategies for patients with resistant diseases. Their development would benefit from a more comprehensive and dense knowledge of small-molecule inhibition across the human kinome. Leveraging over 650,000 bioactivity annotations for more than 300,000 small molecules, we evaluated multiple machine learning methods to predict the small-molecule inhibition of 342 kinases across the human kinome. Our results demonstrated that multi-task deep neural networks outperformed classical single-task methods, offering the potential for conducting large-scale virtual screening, predicting activity profiles, and bridging the gaps in the available data.
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Aprendizaje Profundo , Humanos , Fosfotransferasas , Descubrimiento de Drogas/métodos , Polifarmacología , Aprendizaje AutomáticoRESUMEN
Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.
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Histonas , Factores de Transcripción , Animales , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Senescencia Celular/genética , Células Madre Hematopoyéticas/metabolismo , Diferenciación Celular , HematopoyesisRESUMEN
Neurobiological disorders are highly prevalent medical conditions that contribute to significant morbidity and mortality. Single-cell RNA sequencing (scRNA-seq) is a technique that measures gene expression in individual cells. In this review, we survey scRNA-seq studies of tissues from patients suffering from neurobiological disease. This includes postmortem human brains and organoids derived from peripheral cells. We highlight a range of conditions, including epilepsy, cognitive disorders, substance use disorders, and mood disorders. These findings provide new insights into neurobiological disease in multiple ways, including discovering novel cell types or subtypes involved in disease, proposing new pathophysiological mechanisms, uncovering novel drug targets, or identifying potential biomarkers. We discuss the quality of these findings and suggest potential future directions and areas open for additional research, including studies of non-cortical brain regions and additional conditions such as anxiety disorders, mood disorders, and sleeping disorders. We argue that additional scRNA-seq of tissues from patients suffering from neurobiological disease could advance our understanding and treatment of these conditions.
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Trastornos de Ansiedad , Trastornos del Humor , Humanos , Trastornos del Humor/genética , Encéfalo , Biomarcadores , Análisis de Secuencia de ARN/métodosRESUMEN
The Hedgehog signaling pathway functions in both embryonic development and adult tissue homeostasis. Importantly, its aberrant activation is also implicated in the progression of multiple types of cancer, including basal cell carcinoma and medulloblastoma. GLI transcription factors function as the ultimate effectors of the Hedgehog signaling pathway. Their activity is regulated by this signaling cascade via their mRNA expression, protein stability, subcellular localization, and ultimately their transcriptional activity. Further, GLI proteins are also regulated by a variety of non-canonical mechanisms in addition to the canonical Hedgehog pathway. Recently, with an increased understanding of epigenetic gene regulation, novel transcriptional regulators have been identified that interact with GLI proteins in multi-protein complexes to regulate GLI transcriptional activity. Such complexes have added another layer of complexity to the regulation of GLI proteins. Here, we summarize recent work on the regulation of GLI transcriptional activity by these novel protein complexes and describe their relevance to cancer, as such GLI regulators represent alternative and innovative druggable targets in GLI-dependent cancers.
RESUMEN
Dysregulation of Sonic hedgehog (SHH) signaling drives the growth of distinct cancer subtypes, including medulloblastoma (MB). Such cancers have been treated in the clinic with a number of clinically relevant SHH inhibitors, the majority of which target the upstream SHH regulator, Smoothened (SMO). Despite considerable efficacy, many of these patients develop resistance to these drugs, primarily due to mutations in SMO. Therefore, it is essential to identify druggable, signaling components downstream of SMO to target in SMO inhibitor resistant cancers. We utilized an integrated functional genomics approach to identify epigenetic regulators of SHH signaling and identified a novel complex of Ubiquitin-like with PHD and RING finger domains 1 (UHRF1), DNA methyltransferase 1 (DNMT1), and GLI proteins. We show that this complex is distinct from previously described UHRF1/DNMT1 complexes, suggesting that it works in concert to regulate GLI activity in SHH driven tumors. Importantly, we show that UHRF1/DNMT1/GLI complex stability is targeted by a repurposed FDA-approved therapy, with a subsequent reduction in the growth of SHH-dependent MB ex vivo and in vivo. IMPLICATIONS: This work describes a novel, druggable UHRF1/DNMT1/GLI complex that regulates SHH-dependent tumor growth, and highlights an FDA-approved drug capable of disrupting this complex to attenuate tumor growth.
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Neoplasias Cerebelosas , Meduloblastoma , Humanos , Proteínas Hedgehog/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Transducción de Señal/genética , Neoplasias Cerebelosas/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Spinal cord injuries (SCI) often lead to multiple neurological deficits as a result from the initial trauma and also the secondary damage that follows. Despite abundant preclinical data proposing anti-inflammatory therapies to minimize secondary injury and improve functional recovery, the field still lacks an effective neuroprotective treatment. Epigenetic proteins, such as bromodomain and extraterminal domain (BET) proteins, are emerging as new targets to regulate inflammation. More importantly, pharmacological inhibition of BET proteins suppresses pro-inflammatory gene transcription after SCI. In this study, we tested the therapeutic potential of inhibiting BET proteins after SCI with clinically relevant compounds, and investigated the role of the BET protein BRD4 in macrophages during progression of SCI pathology. Systemic inhibition of BET proteins with I-BET762 significantly reduced lesion size 8 weeks after a contusion injury in rats. However, we observed no histological or locomotor improvements after SCI when we deleted Brd4 in macrophages through the use of myeloid-specific Brd4 knockout mice or after macrophage-targeted pharmacological BET inhibition. Taken together, our data indicate that systemic I-BET762 treatment is neuroprotective, and the histopathological improvement observed is likely to be a result of effects on non-macrophage targets. Expanding our understanding on the role of BET proteins after SCI is necessary to identify novel therapeutic targets that can effectively promote repair after SCI.
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Neuroprotección , Traumatismos de la Médula Espinal , Animales , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratas , Recuperación de la Función/fisiología , Roedores , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
High rates of recurrence and treatment resistance in the most common malignant adult brain cancer, glioblastoma (GBM), suggest that monotherapies are not sufficiently effective. Combination therapies are increasingly pursued, but the possibility of adverse drug-drug interactions may preclude clinical implementation. Developing single molecules with multiple targets is a feasible alternative strategy to identify effective and tolerable pharmacotherapies for GBM. Here, we report the development of a novel, first-in-class, dual aurora and lim kinase inhibitor termed F114. Aurora kinases and lim kinases are involved in neoplastic cell division and cell motility, respectively. Due to the importance of these cellular functions, inhibitors of aurora kinases and lim kinases are being pursued separately as anti-cancer therapies. Using in vitro and ex vivo models of GBM, we found that F114 inhibits GBM proliferation and invasion. These results establish F114 as a promising new scaffold for dual aurora/lim kinase inhibitors that may be used in future drug development efforts for GBM, and potentially other cancers.
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Antineoplásicos/farmacología , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa B/antagonistas & inhibidores , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Quinasas Lim/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Aurora Quinasa A/metabolismo , Aurora Quinasa B/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Quinasas Lim/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Poor prognosis of glioblastoma patients and the extensive heterogeneity of glioblastoma at both the molecular and cellular level necessitates developing novel individualized treatment modalities via genomics-driven approaches. METHODS: This study leverages numerous pharmacogenomic and tissue databases to examine drug repositioning for glioblastoma. RNA-seq of glioblastoma tumor samples from The Cancer Genome Atlas (TCGA, n = 117) were compared to "normal" frontal lobe samples from Genotype-Tissue Expression Portal (GTEX, n = 120) to find differentially expressed genes (DEGs). Using compound gene expression data and drug activity data from the Library of Integrated Network-Based Cellular Signatures (LINCS, n = 66,512 compounds) CCLE (71 glioma cell lines), and Chemical European Molecular Biology Laboratory (ChEMBL) platforms, we employed a summarized reversal gene expression metric (sRGES) to "reverse" the resultant disease signature for GBM and its subtypes. A multiparametric strategy was employed to stratify compounds capable of blood-brain barrier penetrance with a favorable pharmacokinetic profile (CNS-MPO). RESULTS: Significant correlations were identified between sRGES and drug efficacy in GBM cell lines in both ChEMBL(r = 0.37, P < .001) and Cancer Therapeutic Response Portal (CTRP) databases (r = 0.35, P < 0.001). Our multiparametric algorithm identified two classes of drugs with highest sRGES and CNS-MPO: HDAC inhibitors (vorinostat and entinostat) and topoisomerase inhibitors suitable for drug repurposing. CONCLUSIONS: Our studies suggest that reversal of glioblastoma disease signature correlates with drug potency for various GBM subtypes. This multiparametric approach may set the foundation for an early-phase personalized -omics clinical trial for glioblastoma by effectively identifying drugs that are capable of reversing the disease signature and have favorable pharmacokinetic and safety profiles.
RESUMEN
Bromodomain and extraterminal domain (BET) proteins have emerged as therapeutic targets in multiple cancers, including the most common primary adult brain tumor glioblastoma (GBM). Although several BET inhibitors have entered clinical trials, few are brain penetrant. We have generated UM-002, a novel brain penetrant BET inhibitor that reduces GBM cell proliferation in vitro and in a human cerebral brain organoid model. Since UM-002 is more potent than other BET inhibitors, it could potentially be developed for GBM treatment. Furthermore, UM-002 treatment reduces the expression of cell-cycle related genes in vivo and reduces the expression of invasion related genes within the non-proliferative cells present in tumors as measured by single cell RNA-sequencing. These studies suggest that BET inhibition alters the transcriptional landscape of GBM tumors, which has implications for designing combination therapies. Importantly, they also provide an integrated dataset that combines in vitro and ex vivo studies with in vivo single-cell RNA-sequencing to characterize a novel BET inhibitor in GBM.
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Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Perfilación de la Expresión Génica/métodos , Glioblastoma/tratamiento farmacológico , Piridinas/administración & dosificación , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias Encefálicas/genética , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Glioblastoma/genética , Humanos , Masculino , Ratones , Estructura Molecular , Invasividad Neoplásica , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptido Hidrolasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Caseína Quinasa Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero , Evolución Molecular , Células HEK293 , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Lenalidomida/química , Lenalidomida/farmacología , Ratones , Organoides , Péptido Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Glioblastoma (GBM) is a devastating adult brain cancer with high rates of recurrence and treatment resistance. Cellular heterogeneity and extensive invasion of surrounding brain tissues are characteristic features of GBM that contribute to its intractability. Current GBM model systems do not recapitulate some of the complex features of GBM and have not produced sufficiently-effective treatments. This has cast doubt on the effectiveness of current GBM models and drug discovery paradigms. In search of alternative pre-clinical GBM models, various 3D organoid-based GBM model systems have been developed using human cells. The scalability of these systems and potential to more accurately model characteristic features of GBM, provide promising new avenues for pre-clinical GBM research and drug discovery efforts. Here, we review the current suite of organoid-GBM models, their individual strengths and weaknesses, and discuss their future applications with an emphasis on compound screening.
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The lack of effective treatments for most neurological diseases has prompted the search for novel therapeutic options. Interestingly, neuroinflammation is emerging as a common feature to target in most CNS pathologies. Recent studies suggest that targeted delivery of small molecules to reduce neuroinflammation can be beneficial. However, suboptimal drug delivery to the CNS is a major barrier to modulate inflammation because neurotherapeutic compounds are currently being delivered systemically without spatial or temporal control. Emerging nanomaterial technologies are providing promising and superior tools to effectively access neuropathological tissue in a controlled manner. Here we highlight recent advances in nanomaterial technologies for drug delivery to the CNS. We propose that state-of-the-art nanoparticle drug delivery platforms can significantly impact local CNS bioavailability of pharmacological compounds and treat neurological diseases.
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Hair cells (HCs) play crucial roles in perceiving sound, acceleration, and fluid motion. The tonotopic architecture of the sensory epithelium recognizes mechanical stimuli and convert them into electrical signals. The expression and regulation of the genes in the inner ear is very important to keep the sensory organ functional. Our study is the first to investigate the role of the epigenetic reader Brd4 in the mouse inner ear. We demonstrate that HC specific deletion of Brd4 in vivo in the mouse inner ear is sufficient to cause profound hearing loss (HL), degeneration of stereocilia, nerve fibers and HC loss postnatally in mouse; suggesting an important role in hearing function and maintenance.
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Clinical studies have shown that treating many primary brain tumors is challenging due in part to the lack of safe and effective compounds that cross the blood brain barrier (BBB) (Tan et al., 2018). However, if we were to imagine that we have ideal BBB penetrant compounds that target brain tumor cells selectively, recent studies suggest that those compounds may still not be effective due to the heterogenous nature of the tumors. In other words, there are many subsets of cells within a brain tumor, and compounds that target all those different populations are needed. This is a considerable challenge. Targeting of the cell-of-origin of these brain tumors is equally important. And yet another impediment we face is that brain tumor cells-of-origin may be protean and are able to differentiate into other cell types to drive recurrence. Therefore, an ideal BBB-penetrant compound targeting a cell-of-origin in a brain tumor may be ineffective due to the cell's ability to differentiate into another resistant cell type. One possible means of combating the plastic nature of these cells is targeting epigenetic pathways used by the cells to differentiate into other cell types along with standard treatment regimens. We summarize here some of the epigenetic pathways that have been shown to be active in three different primary brain tumors, glioblastoma (GBM), medulloblastoma (MB), and diffuse intrinsic pontine glioma (DIPG). We also compare recent single-cell RNA sequencing analyses of these tumors in order to identify common epigenetic pathways to treat the respective cells-of-origin for these tumors. Lastly, we discuss possible combination therapies that may be generalizable for treating these and other brain tumors using multi-omics approaches. While our focus on these three tumor types is not exhaustive and certainly other brain tumors can have similar mechanisms, there has been significant recent evidence linking epigenetics, plasticity, and intratumor heterogeneity in these tumors.
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Neoplasias Encefálicas/patología , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Neoplasias Encefálicas/genética , Diferenciación Celular , HumanosRESUMEN
Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ)-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications.
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Ataxia Cerebelosa/genética , Corteza Cerebelosa/crecimiento & desarrollo , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Quinasa Idelta de la Caseína , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Ataxia Cerebelosa/patología , Corteza Cerebelosa/citología , Corteza Cerebelosa/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Ratones , Ratones Noqueados , Neuronas/fisiología , Proteínas Nucleares/genética , Fosforilación/fisiología , Cultivo Primario de Células , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
BACKGROUND: Prenatal exposure to arsenic has been linked to a range of adverse health conditions in later life. Such fetal origins of disease are frequently the result of environmental effects on the epigenome, leading to long-term alterations in gene expression. Several studies have demonstrated effects of prenatal arsenic exposure on DNA methylation; however the impact of arsenic on the generation and decoding of post-translational histone modifications (PTHMs) is less well characterized, and has not been studied in the context of prenatal human exposures. METHODS: In the current study, we examined the effect of exposure to low-to-moderate levels of arsenic in a US birth cohort, on the expression of 138 genes encoding key epigenetic regulators in the fetal portion of the placenta. Our candidate genes included readers, writers and erasers of PTHMs, and chromatin remodelers. RESULTS: Arsenic exposure was associated with the expression of 27 of the 138 epigenetic genes analyzed. When the cohort was stratified by fetal sex, arsenic exposure was associated with the expression of 40 genes in male fetal placenta, and only 3 non-overlapping genes in female fetal placenta. In particular, we identified an inverse relationship between arsenic exposure and expression of the gene encoding the histone methyltransferase, PRDM6 (p < 0.001). Mutation of PRDM6 has been linked to the congenital heart defect, patent ductus arteriosus. CONCLUSIONS: Our findings suggest that prenatal arsenic exposure may have sex-specific effects on the fetal epigenome, which could plausibly contribute to its subsequent health impacts.
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Arsénico/orina , Contaminantes Ambientales/orina , Epigénesis Genética , Placenta/metabolismo , Caracteres Sexuales , Transcriptoma , Adulto , Femenino , Humanos , Recién Nacido , Masculino , Intercambio Materno-Fetal , Embarazo , Segundo Trimestre del Embarazo/orinaRESUMEN
PURPOSE: Although most children with medulloblastoma are cured of their disease, Sonic Hedgehog (SHH) subgroup medulloblastoma driven by TRP53 mutations is essentially lethal. Casein kinase 1α (CK1α) phosphorylates and destabilizes GLI transcription factors, thereby inhibiting the key effectors of SHH signaling. We therefore tested a second-generation CK1α activator against TRP53-mutant, MYCN-amplified medulloblastoma. EXPERIMENTAL DESIGN: The ability of this CK1α activator to block SHH signaling was determined in vitro using GLI reporter cells, granular precursor primary cultures, and PATCHED1 (PTCH1)-mutant sphere cultures. While in vivo efficacy was tested using 2 different medulloblastoma mouse models: PTCH1 and ND2:SMOA1. Finally, the clinical relevance of CK1α activators was demonstrated using a TRP53-mutant, MYCN-amplified patient-derived xenograft. RESULTS: SSTC3 inhibited SHH activity in vitro, acting downstream of the vismodegib target SMOOTHENED (SMO), and reduced the viability of sphere cultures derived from SHH medulloblastoma. SSTC3 accumulated in the brain, inhibited growth of SHH medulloblastoma tumors, and blocked metastases in a genetically engineered vismodegib-resistant mouse model of SHH medulloblastoma. Importantly, SSTC3 attenuated growth and metastasis of orthotopic patient-derived TRP53-mutant, MYCN-amplified, SHH subgroup medulloblastoma xenografts, increasing overall survival. CONCLUSIONS: Using a newly described small-molecule, SSTC3, we show that CK1a activators could address a significant unmet clinical need for patients with SMO inhibitor-resistant medulloblastoma, including those harboring mutations in TRP53.
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Benzoatos/farmacología , Caseína Quinasa Ialfa/genética , Meduloblastoma/tratamiento farmacológico , Receptor Smoothened/genética , Anilidas/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Xenoinjertos , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Metástasis de la Neoplasia , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Glioblastoma (GBM) is the most common primary adult brain tumor. Despite extensive efforts, the median survival for GBM patients is approximately 14 months. GBM therapy could benefit greatly from patient-specific targeted therapies that maximize treatment efficacy. Here we report a platform termed SynergySeq to identify drug combinations for the treatment of GBM by integrating information from The Cancer Genome Atlas (TCGA) and the Library of Integrated Network-Based Cellular Signatures (LINCS). We identify differentially expressed genes in GBM samples and devise a consensus gene expression signature for each compound using LINCS L1000 transcriptional profiling data. The SynergySeq platform computes disease discordance and drug concordance to identify combinations of FDA-approved drugs that induce a synergistic response in GBM. Collectively, our studies demonstrate that combining disease-specific gene expression signatures with LINCS small molecule perturbagen-response signatures can identify preclinical combinations for GBM, which can potentially be tested in humans.