RESUMO
Major progress in recent decades has furthered our clinical and biological understanding of rhabdomyosarcoma (RMS) with improved stratification for treatment based on risk factors. Clinical risk factors alone were used to stratify patients for treatment in the European Pediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS 2005 protocol. The current EpSSG overarching study for children and adults with frontline and relapsed rhabdomyosarcoma (FaR-RMS NCT04625907) includes FOXO1 fusion gene status in place of histology as a risk factor. Additional molecular features of significance have recently been recognized, including the MYOD1L122R gene mutation. Here, we review biological information showing that MYOD1L122R blocks cell differentiation and has a MYC-like activity that enhances tumorigenesis and is linked to an aggressive cellular phenotype. MYOD1L122R mutations can be found together with mutations in other genes, such as PIK3CA, as potentially cooperating events. Using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, ten publications in the clinical literature involving 72 cases were reviewed. MYOD1L122R mutation in RMS can occur in both adults and children and is frequent in sclerosing/spindle cell histology, although it is also significantly reported in a subset of embryonal RMS. MYOD1L122R mutated tumors most frequently arise in the head and neck and extremities and are associated with poor outcome, raising the issue of how to use MYOD1L122R in risk stratification and how to treat these patients most effectively.
RESUMO
Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach.
RESUMO
Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Carcinogênese/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Mutação em Linhagem Germinativa/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação de Sentido Incorreto , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Polimorfismo de Nucleotídeo Único/fisiologia , Prognóstico , Fatores de Risco , Transdução de Sinais/genética , Resultado do TratamentoRESUMO
The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS. SIGNIFICANCE: These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aurora Quinase A/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/tratamento farmacológico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Aurora Quinase A/antagonistas & inibidores , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/metabolismo , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Resistencia a Medicamentos Antineoplásicos/genética , Osteossarcoma/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Conjuntos de Dados como Assunto , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Mutagênese , Mutação , Osteossarcoma/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/metabolismoRESUMO
BACKGROUND: MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01. METHODS: MYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively. RESULTS: MYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed. CONCLUSIONS: An alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/metabolismo , RNA Longo não Codificante/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/fisiopatologia , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/fisiopatologiaRESUMO
Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by expression of SS18-SSX fusions, where treatment options are limited. To identify therapeutically actionable genetic dependencies in SS, we performed a series of parallel, high-throughput small interfering RNA (siRNA) screens and compared genetic dependencies in SS tumor cells with those in >130 non-SS tumor cell lines. This approach revealed a reliance of SS tumor cells upon the DNA damage response serine/threonine protein kinase ATR. Clinical ATR inhibitors (ATRi) elicited a synthetic lethal effect in SS tumor cells and impaired growth of SS patient-derived xenografts. Oncogenic SS18-SSX family fusion genes are known to alter the composition of the BAF chromatin-remodeling complex, causing ejection and degradation of wild-type SS18 and the tumor suppressor SMARCB1. Expression of oncogenic SS18-SSX fusion proteins caused profound ATRi sensitivity and a reduction in SS18 and SMARCB1 protein levels, but an SSX18-SSX1 Δ71-78 fusion containing a C-terminal deletion did not. ATRi sensitivity in SS was characterized by an increase in biomarkers of replication fork stress (increased γH2AX, decreased replication fork speed, and increased R-loops), an apoptotic response, and a dependence upon cyclin E expression. Combinations of cisplatin or PARP inhibitors enhanced the antitumor cell effect of ATRi, suggesting that either single-agent ATRi or combination therapy involving ATRi might be further assessed as candidate approaches for SS treatment. Cancer Res; 77(24); 7014-26. ©2017 AACR.
Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular/métodos , Sarcoma Sinovial/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Interferência de RNA/fisiologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Sarcoma Sinovial/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fusion of genes in tumours can have oncogenic roles in reprogramming cells through overexpression of oncogenes or the production of novel fusion proteins. A fundamental question in cancer biology is what genetic events are critical for initiation and whether these are also required for cancer progression. In recent work published in The Journal of Pathology, dependency on a fusion protein was addressed using a model of alveolar rhabdomyosarcomas - a sarcoma subtype with frequent fusion of PAX3 and FOXO1 genes that is associated with poor outcome. PAX3-FOXO1 encodes a potent transcription factor that together with MYCN alters the transcriptional landscape of cells. Building on previous work, an inducible model in human myoblast cells was used to show that PAX3-FOXO1 and MYCN can initiate rhabdomyosarcoma development but, contrary to current thinking, tumour recurrences occasionally arose independent of the fusion protein. Further work needs to identify the molecular nature of this independence and assess any relevance in human tumours. Such functional approaches are required together with computational modeling of molecular data to unravel spatial and temporal dependencies on specific genetic events. This may support molecular prognostic markers and therapeutic targets. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Fusão Gênica , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Animais , Modelos Animais de Doenças , Proteína Forkhead Box O1/genética , Humanos , Camundongos , Recidiva Local de Neoplasia/genética , Fator de Transcrição PAX3/genéticaRESUMO
The role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transformação Celular Neoplásica/metabolismo , Neoplasias Musculares/metabolismo , Fosfoproteínas/fisiologia , Rabdomiossarcoma Embrionário/metabolismo , Células Satélites de Músculo Esquelético/patologia , Animais , Diferenciação Celular/genética , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Dosagem de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias Musculares/mortalidade , Neoplasias Musculares/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Proteína MyoD , Transplante de Neoplasias , Proteínas Nucleares/metabolismo , Oncogenes , Rabdomiossarcoma Embrionário/mortalidade , Rabdomiossarcoma Embrionário/patologia , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
PURPOSE: To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths. EXPERIMENTAL DESIGN: The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA-mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations. RESULTS: PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP-ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations. CONCLUSIONS: Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.