RESUMEN
Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of â¼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.
Asunto(s)
Dihidropiridinas , Rabdomiosarcoma , Humanos , Niño , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/química , Relación Estructura-Actividad , Antihipertensivos/farmacología , Canales de Calcio Tipo L/metabolismo , Rabdomiosarcoma/tratamiento farmacológico , Dihidropiridinas/farmacologíaRESUMEN
Over the past 25 years, chemotherapy regimens for osteosarcoma have failed to improve the 65-70% long-term survival rate. Radiation therapy is generally ineffective except for palliative care. We here investigated whether osteosarcoma can be sensitized to radiation therapy targeting specific molecules in osteosarcoma. Large-scale RNA sequencing analysis in osteosarcoma tissues and cell lines revealed that FGFR1 is the most frequently expressed receptor tyrosine kinase in osteosarcoma. Nuclear FGFR1 (nFGFR1) was observed by subcellular localization assays. The functional studies using a FGFR1IIIb antibody or small molecule FGFR1 inhibitors showed that nFGFR1, but not membrane-bound FGFR1, induces G2 cell-cycle checkpoint adaptation, cell survival and polyploidy following irradiation in osteosarcoma cells. Further, the activation of nFGFR1 induces Histone H3 phosphorylation at Ser 10 and c-jun/c-fos expression to contribute cell survival rendering radiation resistance. Furthermore, an in vivo mouse study revealed that radiation resistance can be reversed by the inhibition of nFGFR1. Our findings provide insights into the potential role of nFGFR1 to radiation resistance. Thus, we propose nFGFR1 could be a potential therapeutic target or a biomarker to determine which patients might benefit from radiation therapy.
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Neoplasias Óseas , Osteosarcoma , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/radioterapia , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , Humanos , Ratones , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Osteosarcoma/radioterapia , Fosforilación , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
We show that both supplemental and ambient magnetic fields modulate myogenesis. A lone 10 min exposure of myoblasts to 1.5 mT amplitude supplemental pulsed magnetic fields (PEMFs) accentuated in vitro myogenesis by stimulating transient receptor potential (TRP)-C1-mediated calcium entry and downstream nuclear factor of activated T cells (NFAT)-transcriptional and P300/CBP-associated factor (PCAF)-epigenetic cascades, whereas depriving myoblasts of ambient magnetic fields slowed myogenesis, reduced TRPC1 expression, and silenced NFAT-transcriptional and PCAF-epigenetic cascades. The expression levels of peroxisome proliferator-activated receptor γ coactivator 1α, the master regulator of mitochondriogenesis, was also enhanced by brief PEMF exposure. Accordingly, mitochondriogenesis and respiratory capacity were both enhanced with PEMF exposure, paralleling TRPC1 expression and pharmacological sensitivity. Clustered regularly interspaced short palindromic repeats-Cas9 knockdown of TRPC1 precluded proliferative and mitochondrial responses to supplemental PEMFs, whereas small interfering RNA gene silencing of TRPM7 did not, coinciding with data that magnetoreception did not coincide with the expression or function of other TRP channels. The aminoglycoside antibiotics antagonized and down-regulated TRPC1 expression and, when applied concomitantly with PEMF exposure, attenuated PEMF-stimulated calcium entry, mitochondrial respiration, proliferation, differentiation, and epigenetic directive in myoblasts, elucidating why the developmental potential of magnetic fields may have previously escaped detection. Mitochondrial-based survival adaptations were also activated upon PEMF stimulation. Magnetism thus deploys an authentic myogenic directive that relies on an interplay between mitochondria and TRPC1 to reach fruition.-Yap, J. L. Y., Tai, Y. K., Fröhlich, J., Fong, C. H. H., Yin, J. N., Foo, Z. L., Ramanan, S., Beyer, C., Toh, S. J., Casarosa, M., Bharathy, N., Kala, M. P., Egli, M., Taneja, R., Lee, C. N., Franco-Obregón, A. Ambient and supplemental magnetic fields promote myogenesis via a TRPC1-mitochondrial axis: evidence of a magnetic mitohormetic mechanism.
Asunto(s)
Campos Magnéticos , Mitocondrias Musculares/metabolismo , Desarrollo de Músculos , Mioblastos Esqueléticos/metabolismo , Transducción de Señal , Canales Catiónicos TRPC/metabolismo , Animales , Línea Celular , Ratones , Mitocondrias Musculares/genética , Mioblastos Esqueléticos/citología , Canales Catiónicos TRPC/genéticaRESUMEN
Differentiation of skeletal muscle cells, like most other cell types, requires a permanent exit from the cell cycle. The epigenetic programming underlying these distinct cellular states is not fully understood. In this study, we provide evidence that the lysine methyltransferase G9a functions as a central axis to regulate proliferation and differentiation of skeletal muscle cells. Transcriptome analysis of G9a knockdown cells revealed deregulation of many cell cycle regulatory genes. We demonstrate that G9a enhances cellular proliferation by two distinct mechanisms. G9a blocks cell cycle exit via methylation-dependent transcriptional repression of the MyoD target genes p21(Cip/Waf1) and Rb1. In addition, it activates E2F1-target genes in a methyltransferase activity-independent manner. We show that G9a is present in the E2F1/PCAF complex, and enhances PCAF occupancy and histone acetylation marks at E2F1-target promoters. Interestingly, G9a preferentially associates with E2F1 at the G1/S phase and with MyoD at the G2/M phase. Our results provide evidence that G9a functions both as a co-activator and a co-repressor to enhance cellular proliferation and inhibit myogenic differentiation.
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Ciclo Celular , Diferenciación Celular , N-Metiltransferasa de Histona-Lisina/metabolismo , Desarrollo de Músculos , Animales , Ciclo Celular/genética , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/genética , Factor de Transcripción E2F1/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones Endogámicos C57BL , Desarrollo de Músculos/genética , Proteína MioD/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Alveolar rhabdomyosarcoma (ARMS) is an aggressive paediatric cancer of skeletal muscle with poor prognosis. A PAX3-FOXO1 fusion protein acts as a driver of malignancy in ARMS by disrupting tightly coupled but mutually exclusive pathways of proliferation and differentiation. While PAX3-FOXO1 is an attractive therapeutic target, no current treatments are designed to block its oncogenic activity. The present work shows that the histone acetyltransferase P/CAF (KAT2B) is overexpressed in primary tumours from ARMS patients. Interestingly, in fusion-positive ARMS cell lines, P/CAF acetylates and stabilizes PAX3-FOXO1 rather than MyoD, a master regulator of muscle differentiation. Silencing P/CAF, or pharmacological inhibition of its acetyltransferase activity, down-regulates PAX3-FOXO1 levels concomitant with reduced proliferation and tumour burden in xenograft mouse models. Our studies identify a P/CAF-PAX3-FOXO1 signalling node that promotes oncogenesis and may contribute to MyoD dysfunction in ARMS. This work exemplifies the therapeutic potential of targeting chromatin-modifying enzymes to inhibit fusion oncoproteins that are a frequent event in sarcomas. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Procesamiento Proteico-Postraduccional , Rabdomiosarcoma Alveolar/genética , Factores de Transcripción p300-CBP/metabolismo , Animales , Carcinogénesis/patología , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Epigenómica , Silenciador del Gen , Xenoinjertos , Ratones , Ratones Desnudos , Músculos/patología , Proteína MioD/genética , Proteína MioD/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Rabdomiosarcoma Alveolar/patología , Transducción de Señal , Factores de Transcripción p300-CBP/genéticaRESUMEN
Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.
Asunto(s)
Diferenciación Celular , N-Metiltransferasa de Histona-Lisina/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/enzimología , Proteína MioD/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Lisina/metabolismo , Metilación , Ratones , Datos de Secuencia Molecular , Desarrollo de Músculos , Proteína MioD/química , Unión ProteicaRESUMEN
Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. Formation of skeletal muscle involves a series of steps in which cells are committed towards the myogenic lineage, undergo expansion to give rise to myoblasts that differentiate into multinucleated myotubes, and mature to form adult muscle fibers. The commitment, proliferation, and differentiation of progenitor cells involve both genetic and epigenetic changes that culminate in alterations in gene expression. Members of the Myogenic regulatory factor (MRF), as well as the Myocyte Enhancer Factor (MEF2) families control distinct steps of skeletal muscle proliferation and differentiation. In addition, -growing evidence indicates that chromatin modifying enzymes and remodeling complexes epigenetically reprogram muscle promoters at various stages that preclude or promote MRF and MEF2 activites. Among these, histone deacetylases (HDACs), histone acetyltransferases (HATs), histone methyltransferases (HMTs) and SWI/SNF complexes alter chromatin structure through post-translational modifications to impact MRF and MEF2 activities. With such new and emerging knowledge, we are beginning to develop a true molecular understanding of the mechanisms by which skeletal muscle development and differentiation is regulated. Elucidation of the mechanisms by which epigenetic regulators control myogenesis will likely provide a new foundation for the development of novel therapeutic drugs for muscle dystrophies, ageing-related regeneration defects that occur due to altered proliferation and differentiation, and other malignancies.
Asunto(s)
Diferenciación Celular/genética , Epigénesis Genética , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Mioblastos Esqueléticos/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/genética , Proliferación Celular , Ensamble y Desensamble de Cromatina , Epigénesis Genética/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Distrofias Musculares/tratamiento farmacológico , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/fisiopatología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma for which subsets of patients have longstanding unmet clinical needs. For example, children with alveolar rhabdomyosarcoma and metastases at diagnosis will experience only 8% disease-free 5-year survival for nonlocalized unresectable recurrent disease. Hence, development of novel therapeutic strategies is urgently needed to improve outcomes. The Plexin-Semaphorin pathway is largely unexplored for sarcoma research. However, emerging interest in the Plexin-Semaphorin signaling axis in pediatric sarcomas has led to phase I cooperative group dose-finding clinical trials, now completed (NCT03320330). In this study, we specifically investigated the protein expression of transmembrane receptor Plexin-B2 and its cognate SEMA4C ligands in clinical RMS tumors and cell models. By RNA interferences, we assessed the role of Plexin-B2 in cell growth and cell migration ability in selected alveolar and embryonal RMS cell model systems. Our results affirmed expression of Plexin-B2 across human samples, while also dissecting expression of the different protein subunits of Plexin-B2 along with the assessment of preferred Semaphorin ligands of Plexin-B2. Plexin-B2 knockdown had positive or negative effects on cell growth, which varied by cell model system. Migration assayed after Plexin-B2 knockdown revealed selective cell line specific migration inhibition, which was independent of Plexin-B2 expression level. Overall, these findings are suggestive of context-specific and possibly patient-specific (stochastic) role of Plexin-B2 and SEMA4 ligands in RMS.
RESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.
Asunto(s)
Neoplasias , Rabdomiosarcoma Alveolar , Rabdomiosarcoma Embrionario , Biología , Niño , ADN Helicasas/genética , Humanos , Proteínas Nucleares/genética , Rabdomiosarcoma Alveolar/genética , Factores de Transcripción/genéticaRESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma occurring in children and carries a dismal prognosis when metastatic disease is detected. Our previous work has suggested the cytokine receptor IL4Rα may play a role in contributing to metastasis in the alveolar subtype of rhabdomyosarcoma (aRMS), and thus could present a therapeutic target. The IL4 signaling axis has been characterized in various adult cancers as well; however, pediatric trials often follow similar adult trials and the role of the IL4Rα receptor has not been explored in the context of a mediator of metastasis in adult disease. Here, we demonstrate that the impact of IL4Rα blockade in an orthotopic allograft model of aRMS is not mediated by a macrophage response. We further examine the effect of IL4 blockade in adult colon, breast, and prostate cancers and find that inhibition of IL4Rα signaling modulates in vitro cell viability of HCT-116 colon carcinoma cells; however, this finding did not translate to an autocrine-related in vivo difference in tumor burden or lung metastasis. Our results suggest that if humanized IL4 mouse host strains are not available (or not ideal due to the need for immunosuppressing the host innate immune response for xenograft systems), then genetically-engineered mice and mouse allograft studies may be the best indicator of therapeutic targeting efficacy.
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Macrófagos/metabolismo , Receptores de Interleucina-4/antagonistas & inhibidores , Rabdomiosarcoma/genética , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Metástasis de la Neoplasia , Rabdomiosarcoma/patologíaRESUMEN
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy, and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades-underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment. To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and four human sarcomas to analyze expression of seven different collagens, fibrillins, and collagen-modifying proteins, with cross-correlation to RNA deep sequencing. We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1, and PLOD2 in human RMS relative to normal skeletal muscle. These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins, and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.
RESUMEN
Aurora kinases are Ser/Thr-directed protein kinases which play pivotal roles in mitosis. Recent evidences highlight the importance of these kinases in multiple biological events including skeletal muscle differentiation. Our earlier study identified the transcription factor POU6F1 (or mPOU) as a novel Aurora kinase (Aurk) A substrate. Here, we report that Aurora kinase A phosphorylates mPOU at Ser197 and inhibit its DNA-binding ability. Delving into mPOU physiology, we find that the phospho-mimic (S197D) mPOU mutant exhibits enhancement, while the wild type or the phospho-deficient mutant shows retardation in C2C12 myoblast differentiation. Interestingly, POU6F1 depletion phenocopies S197D-mPOU overexpression in the differentiation context. Collectively, our results signify mPOU as a negative regulator of skeletal muscle differentiation and strengthen the importance of AurkA in skeletal myogenesis.
Asunto(s)
Aurora Quinasa A/metabolismo , Diferenciación Celular , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Factores del Dominio POU/metabolismo , Células HEK293 , Humanos , Mutación , Factores del Dominio POU/genética , FosforilaciónRESUMEN
Dysregulated activity of the transcription factors of the nuclear factor κb (NF-κB) family has been implicated in numerous cancer types, inflammatory diseases, autoimmune disease, and other disorders. As such, selective NF-κB pathway inhibition is an attractive target to researchers for preclinical and clinical drug development. A plethora of commercially and clinically available inhibitors claim to be NF-κB specific; however, such claims of specificity are rarely quantitative or benchmarked, making the biomedical literature difficult to contextualize. This imprecision is worsened because some NF-κB reporter systems have low signal-to-noise ratios. Herein, we use a robust, defined, commercially available reporter system to benchmark NF-κB agonists and antagonists for the field. We also functionally characterize a RELA fusion-positive ependymoma cell culture with validated NF-κB inhibitor compounds.
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FN-kappa B/agonistas , FN-kappa B/antagonistas & inhibidores , Benchmarking , Fusión Celular , Línea Celular Tumoral , Supervivencia Celular , Ependimoma/patología , Células HEK293 , Humanos , Técnicas In Vitro , Reproducibilidad de los Resultados , Relación Señal-RuidoRESUMEN
BACKGROUND: α-Dystroglycan is the highly glycosylated component of the dystrophin-glycoprotein complex (DGC) that binds with high-affinity to extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains via a unique heteropolysaccharide [-GlcA-beta1,3-Xyl-alpha1,3-]n called matriglycan. Changes in expression of components of the DGC or in the O-glycosylation of α-dystroglycan result in muscular dystrophy but are also observed in certain cancers. In mice, the loss of either of two DGC proteins, dystrophin or α-sarcoglycan, is associated with a high incidence of rhabdomyosarcoma (RMS). In addition, glycosylation of α-dystroglycan is aberrant in a small cohort of human patients with RMS. Since both the glycosylation of α-dystroglycan and its function as an ECM receptor require over 18 post-translational processing enzymes, we hypothesized that understanding its role in the pathogenesis of RMS requires a complete analysis of the expression of dystroglycan-modifying enzymes and the characterization of α-dystroglycan glycosylation in the context of RMS. METHODS: A series of cell lines and biopsy samples from human and mouse RMS were analyzed for the glycosylation status of α-dystroglycan and for expression of the genes encoding the responsible enzymes, in particular those required for the addition of matriglycan. Furthermore, the glycosyltransferase LARGE1 was ectopically expressed in RMS cells to determine its effects on matriglycan modifications and the ability of α-dystroglycan to function as a laminin receptor. RESULTS: Immunohistochemistry and immunoblotting of a collection of primary RMS tumors show that although α-dystroglycan is consistently expressed and glycosylated in these tumors, α-dystroglycan lacks matriglycan and the ability to bind laminin. Similarly, in a series of cell lines derived from human and mouse RMS, α-dystroglycan lacks matriglycan modification and the ability to bind laminin. RNAseq data from RMS cell lines was analyzed for expression of the genes known to be involved in α-dystroglycan glycosylation, which revealed that, for most cell lines, the lack of matriglycan can be attributed to the downregulation of the dystroglycan-modifying enzyme LARGE1. Ectopic expression of LARGE1 in these cell cultures restored matriglycan to levels comparable to those in muscle and restored high-affinity laminin binding to α-dystroglycan. CONCLUSIONS: Collectively, our findings demonstrate that a lack of matriglycan on α-dystroglycan is a common feature in RMS due to the downregulation of LARGE1, and that ectopic expression of LARGE1 can restore matriglycan modifications and the ability of α-dystroglycan to function as an ECM receptor.
Asunto(s)
Distroglicanos/metabolismo , Laminina/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Rabdomiosarcoma/metabolismo , Animales , Línea Celular Tumoral , Glicosilación , Humanos , Ratones , N-Acetilglucosaminiltransferasas/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rabdomiosarcoma/genética , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Embrionario/genética , Rabdomiosarcoma Embrionario/metabolismoRESUMEN
Relapsed and metastatic hepatoblastoma represents an unmet clinical need with limited chemotherapy treatment options. In a chemical screen, we identified volasertib as an agent with in vitro activity, inhibiting hepatoblastoma cell growth while sparing normal hepatocytes. Volasertib targets PLK1 and prevents the progression of mitosis, resulting in eventual cell death. PLK1 is overexpressed in hepatoblastoma biopsies relative to normal liver tissue. As a potential therapeutic strategy, we tested the combination of volasertib and the relapse-related hepatoblastoma chemotherapeutic irinotecan. We found both in vitro and in vivo efficacy of this combination, which may merit further preclinical investigation and exploration for a clinical trial concept.
RESUMEN
BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric cancer population. Survival among metastatic RMS patients has remained dismal yet unimproved for years. We previously identified the class I-specific histone deacetylase inhibitor, entinostat (ENT), as a pharmacological agent that transcriptionally suppresses the PAX3:FOXO1 tumor-initiating fusion gene found in alveolar rhabdomyosarcoma (aRMS), and we further investigated the mechanism by which ENT suppresses PAX3:FOXO1 oncogene and demonstrated the preclinical efficacy of ENT in RMS orthotopic allograft and patient-derived xenograft (PDX) models. In this study, we investigated whether ENT also has antitumor activity in fusion-negative eRMS orthotopic allografts and PDX models either as a single agent or in combination with vincristine (VCR). METHODS: We tested the efficacy of ENT and VCR as single agents and in combination in orthotopic allograft and PDX mouse models of eRMS. We then performed CRISPR screening to identify which HDAC among the class I HDACs is responsible for tumor growth inhibition in eRMS. To analyze whether ENT treatment as a single agent or in combination with VCR induces myogenic differentiation, we performed hematoxylin and eosin (H&E) staining in tumors. RESULTS: ENT in combination with the chemotherapy VCR has synergistic antitumor activity in a subset of fusion-negative eRMS in orthotopic "allografts," although PDX mouse models were too hypersensitive to the VCR dose used to detect synergy. Mechanistic studies involving CRISPR suggest that HDAC3 inhibition is the primary mechanism of cell-autonomous cytoreduction in eRMS. Following cytoreduction in vivo, residual tumor cells in the allograft models treated with chemotherapy undergo a dramatic, entinostat-induced (70-100%) conversion to non-proliferative rhabdomyoblasts. CONCLUSION: Our results suggest that the targeting class I HDACs may provide a therapeutic benefit for selected patients with eRMS. ENT's preclinical in vivo efficacy makes ENT a rational drug candidate in a phase II clinical trial for eRMS.
Asunto(s)
Benzamidas/uso terapéutico , Inhibidores de Histona Desacetilasas/uso terapéutico , Piridinas/uso terapéutico , Rabdomiosarcoma Embrionario/tratamiento farmacológico , Adolescente , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Benzamidas/administración & dosificación , Sistemas CRISPR-Cas , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Niño , Preescolar , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/genética , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Piridinas/administración & dosificación , RNA-Seq , Rabdomiosarcoma Alveolar/tratamiento farmacológico , Rabdomiosarcoma Alveolar/enzimología , Rabdomiosarcoma Alveolar/patología , Rabdomiosarcoma Embrionario/enzimología , Rabdomiosarcoma Embrionario/patología , Carga Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Vincristina/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I-specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3-SMARCA4-miR-27a-PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.
Asunto(s)
ADN Helicasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , MicroARNs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Rabdomiosarcoma Alveolar/metabolismo , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Biología Computacional , Resistencia a Antineoplásicos , Epigénesis Genética , Femenino , Transferencia Resonante de Energía de Fluorescencia , Proteína Forkhead Box O1/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Ratones , Trasplante de Neoplasias , Factor de Transcripción PAX3/metabolismo , Piridinas/farmacología , Análisis de Secuencia de ARN , Vincristina/farmacologíaRESUMEN
Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3ß is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3ß inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models in vivo. In this study, we tested the irreversible GSK3ß-inhibitor, tideglusib for in vivo efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3ß inhibitors may not be a viable treatment for aRMS or eRMS.
RESUMEN
In this study, we demonstrate that the lysine methyltransferase G9a inhibits sarcomere organization through regulation of the MEF2C-HDAC5 regulatory axis. Sarcomeres are essential for muscle contractile function. Presently, skeletal muscle disease and dysfunction at the sarcomere level has been associated with mutations of sarcomere proteins. This study provides evidence that G9a represses expression of several sarcomere genes and its over-expression disrupts sarcomere integrity of skeletal muscle cells. G9a inhibits MEF2C transcriptional activity that is essential for expression of sarcomere genes. Through protein interaction assays, we demonstrate that G9a interacts with MEF2C and its co-repressor HDAC5. In the presence of G9a, calcium signaling-dependent phosphorylation and export of HDAC5 to the cytoplasm is blocked which likely results in enhanced MEF2C-HDAC5 association. Activation of calcium signaling or expression of constitutively active CaMK rescues G9a-mediated repression of HDAC5 shuttling as well as sarcomere gene expression. Our results demonstrate a novel epigenetic control of sarcomere assembly and identifies new therapeutic avenues to treat skeletal and cardiac myopathies arising from compromised muscle function.
RESUMEN
Lysine methylation of histone and non-histone substrates by the methyltransferase G9a is mostly associated with transcriptional repression. Recent studies, however, have highlighted its role as an activator of gene expression through mechanisms that are independent of its methyltransferase activity. Here we review the growing repertoire of molecular mechanisms and substrates through which G9a regulates gene expression. We also discuss emerging evidence for its wide-ranging functions in development, pluripotency, cellular differentiation and cell cycle regulation that underscore the complexity of its functions. The deregulated expression of G9a in cancers and other human pathologies suggests that it may be a viable therapeutic target in various diseases.