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
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
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
Diffuse intrinsic pontine glioma (DIPG) is a universally fatal childhood cancer of the brain. Despite the introduction of conventional chemotherapy and radiotherapy, improvements in survival have been marginal and long-term survivorship is uncommon. Thus, new targets for therapeutics are critically needed. Early phase clinical trials exploring molecularly-targeted therapies against the epidermal growth factor receptor (EGFR) and novel immunotherapies targeting interleukin receptor-13α2 (IL-13Rα2) have demonstrated activity in this disease. To identify additional therapeutic markers for cell surface receptors, we performed exome sequencing (16 new samples, 22 previously published samples, total 38 with 26 matched normal DNA samples), RNA deep sequencing (17 new samples, 11 previously published samples, total 28 with 18 matched normal RNA samples), and immunohistochemistry (17 DIPG tissue samples) to examine the expression of the interleukin-4 (IL-4) signaling axis components (IL-4, interleukin 13 (IL-13), and their respective receptors IL-4Rα, IL-13Rα1, and IL-13Rα2). In addition, we correlated cytokine and receptor expression with expression of the oncogenes EGFR and c-MET. In DIPG tissues, transcript-level analysis found significant expression of IL-4, IL-13, and IL-13Rα1/2, with strong differential expression of IL-13Rα1/2 in tumor versus normal brain. At the protein level, immunohistochemical studies revealed high content of IL-4 and IL-13Rα1/2 but notably low expression of IL-13. Additionally, a strong positive correlation was observed between c-Met and IL-4Rα. The genomic and transcriptional landscape across all samples was also summarized. These data create a foundation for the design of potential new immunotherapies targeting IL-13 cell surface receptors in DIPG.
Asunto(s)
Neoplasias del Tronco Encefálico/tratamiento farmacológico , Glioma/tratamiento farmacológico , Receptores de Interleucina-13/efectos de los fármacos , Neoplasias del Tronco Encefálico/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Humanos , Interleucina-4/metabolismo , Mutación Puntual , Receptores de Interleucina-13/genética , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/metabolismo , Análisis de Secuencia de ADNRESUMEN
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
Risk or presence of metastasis in medulloblastoma causes substantial treatment-related morbidity and overall mortality. Through the comparison of cytokines and growth factors in the cerebrospinal fluid (CSF) of metastatic medulloblastoma patients with factors also in conditioned media of metastatic MYC amplified medulloblastoma or leptomeningeal cells, we were led to explore the bioactivity of IGF1 in medulloblastoma by elevated CSF levels of IGF1, IGF-sequestering IGFBP3, IGFBP3-cleaving proteases (MMP and tPA), and protease modulators (TIMP1 and PAI-1). IGF1 led not only to receptor phosphorylation but also accelerated migration/adhesion in MYC amplified medulloblastoma cells in the context of appropriate matrix or meningothelial cells. Clinical correlation suggests a peri-/sub-meningothelial source of IGF-liberating proteases that could facilitate leptomeningeal metastasis. In parallel, studies of key factors responsible for cell autonomous growth in MYC amplified medulloblastoma prioritized IGF1R inhibitors. Together, our studies identify IGF1R as a high value target for clinical trials in high risk medulloblastoma.