RESUMEN
Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Factor de Transcripción AP-1 , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Humanos , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genética , Factor de Transcripción AP-1/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Línea Celular Tumoral , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Perfilación de la Expresión GénicaRESUMEN
Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.
RESUMEN
Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.
RESUMEN
Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors have complicated the identification of the mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of the RRM1 protein results in the upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos, and downregulation of c-Myc. Notably, overexpression of c-Jun and c-Fos in Ewing sarcoma cells is sufficient to inhibit cell growth and downregulate the expression of the c-Myc oncogene. We also identified that the upregulation of AP-1 is mediated, in part, by SLFN11, which is a replication stress response protein that is expressed at high levels in Ewing sarcoma. In addition, small-molecule inhibitors of RNR, including gemcitabine, and histone deacetylase inhibitors, which reduce the level of the RRM1 protein, also activate AP-1 signaling and downregulate the level of c-Myc in Ewing sarcoma. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR. Significance: RNR is the rate-limiting enzyme in the synthesis of deoxyribonucleotides. Although RNR is the target of multiple chemotherapy drugs, polypharmacology and off-target effects have complicated the identification of the precise mechanism of action of these drugs. In this work, using a knockout-rescue approach, we identified that inhibition of RNR upregulates AP-1 signaling and downregulates the level of c-Myc in Ewing sarcoma tumors.
Asunto(s)
Traumatismos Craneocerebrales , Tumores Neuroectodérmicos Periféricos Primitivos , Ribonucleótido Reductasas , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/tratamiento farmacológico , Factor de Transcripción AP-1/genética , Transducción de Señal/genética , Proteínas Proto-Oncogénicas c-fos/genética , Replicación del ADN/genética , Proteínas NuclearesRESUMEN
The DNA methyltransferase inhibitor decitabine has classically been used to reactivate silenced genes and as a pretreatment for anticancer therapies. In a variation of this idea, this study explores the concept of adding low-dose decitabine (DAC) following administration of chemotherapy to bolster therapeutic efficacy. We find that addition of DAC following treatment with the chemotherapy agent gemcitabine improves survival and slows tumor growth in a mouse model of high-grade sarcoma. Unlike prior studies in epithelial tumor models, DAC did not induce a robust antitumor T cell response in sarcoma. Furthermore, DAC synergizes with gemcitabine independently of the immune system. Mechanistic analyses demonstrate that the combination therapy induces biphasic cell cycle arrest and apoptosis. Therapeutic efficacy was sequence dependent, with gemcitabine priming cells for treatment with DAC through inhibition of ribonucleotide reductase. This study identifies an apparently unique application of DAC to augment the cytotoxic effects of conventional chemotherapy in an immune-independent manner. The concepts explored in this study represent a promising paradigm for cancer treatment by augmenting chemotherapy through addition of DAC to increase tolerability and improve patient response. These findings have widespread implications for the treatment of sarcomas and other aggressive malignancies.
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Apoptosis , Sarcoma , Ratones , Animales , Decitabina/farmacología , Línea Celular Tumoral , Puntos de Control del Ciclo Celular , Sarcoma/tratamiento farmacológicoRESUMEN
Background: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted along with NF1, a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of MPNSTs is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown. Methods: Using CRISPR-Cas9 targeting of Nf1 + Cdkn2a or Nf1 + Tp53 in the mouse sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Terminal tumors were evaluated by western blot, qRT-PCR, and immunohistochemistry. Results: Mice lacking Rabl6 displayed slower tumor progression and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in Rabl6-null, Nf1 + Cdkn2a lesions whereas loss of RABL6A caused upregulation of the CDK inhibitor, p27, in all tumors. Paradoxically, both models displayed elevated Myc protein and Ki67 staining in terminal tumors lacking RABL6A. In Nf1 + p53 tumors, cellular atypia and polyploidy were evident and increased by RABL6A loss. Conclusions: These findings demonstrate that RABL6A is required for optimal progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for unwanted alterations, including increased Myc, cellular atypia, and polyploidy, that ultimately promote a hyper-proliferative tumor phenotype akin to drug-resistant lesions.
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Sarcomas are difficult to treat and the therapy, even when effective, is associated with long-term and life-threatening side effects. In addition, the treatment regimens for many sarcomas, including Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma, are relatively unchanged over the past two decades, indicating a critical lack of progress. Although differentiation-based therapies are used for the treatment of some cancers, the application of this approach to sarcomas has proven challenging. Here, using a CRISPR-mediated gene knockout approach, we show that Inhibitor of DNA Binding 2 (ID2) is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo in Ewing sarcoma tumors. We also identified that homoharringtonine, which is an inhibitor of protein translation and FDA-approved for the treatment of leukemia, decreases the level of the ID2 protein and significantly reduces tumor growth and prolongs mouse survival in an Ewing sarcoma xenograft model. Furthermore, in addition to targeting ID2, homoharringtonine also reduces the protein levels of ID1 and ID3, which are additional members of the ID family of proteins with well-described roles in tumorigenesis, in multiple types of cancer. Overall, these results provide insight into developmental regulation in Ewing sarcoma tumors and identify a novel, therapeutic approach to target the ID family of proteins using an FDA-approved drug.
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Proteína 2 Inhibidora de la Diferenciación , Sarcoma de Ewing , Animales , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Genes del Desarrollo , Homoharringtonina , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Ratones , Proteínas/genética , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismoRESUMEN
Differentiation blockade is a hallmark of acute myeloid leukemia (AML). A strategy to overcome such a blockade is a promising approach against the disease. The lack of understanding of the underlying mechanisms hampers development of such strategies. Dysregulated ribonucleotide reductase (RNR) is considered a druggable target in proliferative cancers susceptible to deoxynucleoside triphosphate (dNTP) depletion. Herein, we report an unanticipated discovery that hyperactivating RNR enables differentiation and decreases leukemia cell growth. We integrate pharmacogenomics and metabolomics analyses to identify that pharmacologically (eg, nelarabine) or genetically upregulating RNR subunit M2 (RRM2) creates a dNTP pool imbalance and overcomes differentiation arrest. Moreover, R-loop-mediated DNA replication stress signaling is responsible for RRM2 activation by nelarabine treatment. Further aggravating dNTP imbalance by depleting the dNTP hydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) enhances ablation of leukemia stem cells by RRM2 hyperactivation. Mechanistically, excessive activation of extracellular signal-regulated kinase (ERK) signaling downstream of the imbalance contributes to cellular outcomes of RNR hyperactivation. A CRISPR screen identifies a synthetic lethal interaction between loss of DUSP6, an ERK-negative regulator, and nelarabine treatment. These data demonstrate that dNTP homeostasis governs leukemia maintenance, and a combination of DUSP inhibition and nelarabine represents a therapeutic strategy.
Asunto(s)
Leucemia Mieloide Aguda , Ribonucleótido Reductasas , Replicación del ADN , Homeostasis , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Polifosfatos , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismoRESUMEN
Hallux valgus (HV) is a common anatomical deformity leading to pain and difficulty with footwear and mobility. Bilateral HV deformity is much more common than unilateral although it remains unclear whether the severity of deformity is equal between feet. The objective was to investigate the severity and symmetry of HV in patients with bilateral symptomatic deformity presenting for surgery. Weight-bearing radiographs of patients presenting with symptomatic bilateral HV were reviewed. The hallux valgus angle (HVA) and intermetatarsal angle (IMA) were measured and classified as mild, moderate, or severe. Left-to-right comparison was undertaken to assess whether the degree of deformity was similar for each foot. The relationship between age, HVA, and IMA was also assessed. Between July 2014 and June 2020, 322 ft (161 patients with bilateral deformity) underwent corrective HV surgery. Of those, 6.8%, 64.6%, and 28.4% were classified as mild, moderate, and severe, respectively on the left side, and on the right 6.2%, 67.7%, and 26.1% were classified as mild, moderate, and severe respectively. There was no statistically significant difference between feet for either IMA (p = 0.06) or HVA (p = 0.85). There was a moderate correlation (R = 0.41, p ≤ 0.001) between HVA and IMA. There was only a 'weak' or 'very weak' correlation between age and HVA or IMA. Patients presenting for surgery with symptomatic bilateral HV have symmetrical moderate radiographic deformity at the time they present for consideration of surgical intervention.
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Hallux Valgus , Huesos Metatarsianos , Pie , Hallux Valgus/diagnóstico por imagen , Hallux Valgus/cirugía , Humanos , Huesos Metatarsianos/cirugía , Radiografía , Estudios Retrospectivos , Resultado del Tratamiento , Soporte de PesoAsunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazinas/uso terapéutico , Pirazoles/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Humanos , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Pirazoles/farmacología , GemcitabinaRESUMEN
BACKGROUND: There is interest in hallux valgus deformity correction using internal fixation with the minimally invasive chevron and Akin osteotomies (MICA) technique. The objective of this study was to assess the correction measured on postoperative radiographs and clinical outcomes, using validated outcome measures, at 2 years following third-generation MICA. METHODS: This is a prospective single-surgeon case series of 333 consecutive feet that underwent MICA surgery between July 2014 and April 2018. The primary clinical outcome measures included the Manchester-Oxford Foot Questionnaire (MOXFQ), EuroQol-5 Dimensions-5 Level (EQ-5D-5L) Index, EuroQol-visual analogue scale (EQ-VAS), and a VAS for pain (VAS-pain). Secondary outcome measures included radiographic parameters and complication rates. RESULTS: Preoperative and 2-year postoperative patient-reported outcome measures (PROMs) were collected for 292 feet (87.7%). At a minimum 2-year follow-up, the MOXFQ scores (mean ± standard deviation [SD]) had improved in each domain-i.e., reduced from 44.5 ± 21.0 preoperatively to 9.4 ± 15.8 postoperatively for pain (p < 0.001), from 38.7 ± 23.4 to 6.5 ± 14.6 for walking and standing (p < 0.001), and from 48.0 ± 22.3 to 6.6 ± 13.5 for social interaction (p < 0.001). The VAS-pain score improved from 31.4 ± 22.7 preoperatively to 8.4 ± 16.4 at the 2-year follow-up (p < 0.001), the 1-2 intermetatarsal angle was reduced from 15.3° ± 3.6° preoperatively to 5.7° ± 3.2° at the 2-year follow-up (p < 0.001), and the hallux valgus angle was reduced from 32.9° ± 10.2° to 8.7° ± 5.2° (p < 0.001). CONCLUSIONS: The third-generation MICA provided significant improvement in clinical outcome measures at the 2-year follow-up and can be successfully used for correction of a range of hallux valgus deformities with a low rate of symptomatic recurrence. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.
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Hallux Valgus/cirugía , Osteotomía/métodos , Adulto , Anciano , Anciano de 80 o más Años , Tornillos Óseos/efectos adversos , Femenino , Estudios de Seguimiento , Hallux Valgus/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Procedimientos Quirúrgicos Mínimamente Invasivos/estadística & datos numéricos , Osteotomía/estadística & datos numéricos , Evaluación de Resultado en la Atención de Salud , Dimensión del Dolor/métodos , Complicaciones Posoperatorias/etiología , Estudios Prospectivos , Análisis de Regresión , Sesgo de Selección , Posición de Pie , Encuestas y Cuestionarios , Factores de Tiempo , Caminata , Adulto JovenRESUMEN
Precision medicine relies on a detailed molecular understanding of disease pathogenesis. Here, we consider urgently needed therapeutic options for malignant peripheral nerve sheath tumors (MPNSTs) based on emerging insights into druggable pathway alterations found to drive this deadly cancer. Recent observations demonstrate an essential role for an oncogenic GTPase, RABL6A, in promoting MPNST progression through hyperactivation of cyclin-dependent kinases (CDKs) and inactivation of the retinoblastoma (RB1) tumor suppressor. Monotherapies with CDK4/6 inhibitors have shown limited efficacy and durability in pre-clinical studies of MPNSTs and in clinical studies of other tumors. Therefore, we discuss the rationale and clinical benefits of inhibiting multiple RABL6A effectors, particularly CDK4/6 and MEK kinases, in targeted combination therapies suitable for MPNSTs and other Ras-driven malignancies.
RESUMEN
Ribonucleotide reductase (RNR), which is a heterodimeric tetramer composed of RRM1 and RRM2 subunits, is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and essential for both DNA replication and the repair of DNA damage. The activity of RNR is coordinated with the cell cycle and regulated by fluctuations in the level of the RRM2 subunit. Multiple cancer types, including Ewing sarcoma tumors, are sensitive to inhibitors of RNR or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. Here, we show that the expression of the RRM2 protein is dependent on active protein synthesis and that 4E-BP1, a repressor of cap-dependent protein translation, specifically regulates the level of the RRM2 protein. Furthermore, inhibition of mTORC1/2, but not mTORC1, activates 4E-BP1, inhibits protein synthesis, and reduces the level of the RRM2 protein in multiple sarcoma cell lines. This effect of mTORC1/2 inhibitors on protein synthesis and RRM2 levels was rescued in cell lines with the CRISPR/Cas9-mediated knockout of 4E-BP1. In addition, the inducible expression of a mutant 4E-BP1 protein that cannot be phosphorylated by mTOR blocked protein synthesis and inhibited the growth of Ewing sarcoma cells in vitro and in vivo in a xenograft. Overall, these results provide insight into the multifaceted regulation of RRM2 protein levels and identify a regulatory link between protein translation and DNA replication.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Sarcoma de Ewing/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Humanos , Células Jurkat , Células K562 , Ribonucleósido Difosfato Reductasa/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: The treatment of Ewing sarcoma, an aggressive bone and soft tissue sarcoma, is associated with suboptimal outcomes and significant side-effects. Consequently, there is an urgent need to identify novel therapies that will improve outcomes for children and adults with Ewing sarcoma tumors while also decreasing treatment-related toxicities. METHODS: We analyzed data from the PRISM drug repurposing screen, which tested the activity of 4518 drugs across 578 cancer cell lines, to identify drugs that selectively inhibit the growth of Ewing sarcoma cell lines. We then tested the effects of a top hit from the screen on cell proliferation, cell cycle progression, and activation of the DNA damage pathway using Ewing sarcoma cell lines. We also used a CRISPR/Cas9 gene knockout approach to investigate the role of Schlafen 11 (SLFN11), a restriction factor for DNA replication stress that is overexpressed in Ewing sarcoma tumors, in mediating the sensitivity of Ewing sarcoma cells to the drug. RESULTS: We found that eltrombopag, an FDA-approved thrombopoietin-receptor agonist (TPO-RA) that is currently being evaluated as a treatment for chemotherapy-induced thrombocytopenia, inhibits the growth of Ewing sarcoma cell lines in vitro in proliferation and colony formation assays. However, from a mechanistic standpoint, the thrombopoietin receptor is not expressed in Ewing sarcoma cells and we show that eltrombopag impairs DNA replication and causes DNA damage in Ewing sarcoma cells by chelating iron, a known "off-target" effect of the drug. We also found that the sensitivity of Ewing sarcoma cells to eltrombopag is mediated, in part, by SLFN11, which regulates the cellular response to DNA replication stress. CONCLUSIONS: Ewing sarcoma cell lines are sensitive to eltrombopag and this drug could improve outcomes for patients with Ewing sarcoma tumors by both targeting the tumor, via chelation of iron and inhibition of DNA replication, and reducing chemotherapy-induced thrombocytopenia, via stimulation of the thrombopoietin receptor.
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Benzoatos/uso terapéutico , Replicación del ADN/genética , Hidrazinas/uso terapéutico , Quelantes del Hierro/uso terapéutico , Pirazoles/uso terapéutico , Sarcoma de Ewing/tratamiento farmacológico , Benzoatos/farmacología , Proliferación Celular , Humanos , Hidrazinas/farmacología , Quelantes del Hierro/farmacología , Pirazoles/farmacologíaRESUMEN
Sarcomas represent one of the most challenging tumor types to treat due to their diverse nature and our incomplete understanding of their underlying biology. Recent work suggests cyclin-dependent kinase (CDK) pathway activation is a powerful driver of sarcomagenesis. CDK proteins participate in numerous cellular processes required for normal cell function, but their dysregulation is a hallmark of many pathologies including cancer. The contributions and significance of aberrant CDK activity to sarcoma development, however, is only partly understood. Here, we describe what is known about CDK-related alterations in the most common subtypes of sarcoma and highlight areas that warrant further investigation. As disruptions in CDK pathways appear in most, if not all, subtypes of sarcoma, we discuss the history and value of pharmacologically targeting CDKs to combat these tumors. The goals of this review are to (1) assess the prevalence and importance of CDK pathway alterations in sarcomas, (2) highlight the gap in knowledge for certain CDKs in these tumors, and (3) provide insight into studies focused on CDK inhibition for sarcoma treatment. Overall, growing evidence demonstrates a crucial role for activated CDKs in sarcoma development and as important targets for sarcoma therapy.
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Quinasas Ciclina-Dependientes/metabolismo , Sarcoma/etiología , Sarcoma/metabolismo , Factores de Edad , Animales , Antineoplásicos/farmacología , Biomarcadores , Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Susceptibilidad a Enfermedades , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Sarcoma/tratamiento farmacológico , Sarcoma/patología , Transducción de Señal , Transcripción GenéticaRESUMEN
Inhibition of ribonucleotide reductase (RNR), the rate-limiting enzyme in the synthesis of deoxyribonucleotides, causes DNA replication stress and activates the ataxia telangiectasia and rad3-related protein (ATR)-checkpoint kinase 1 (CHK1) pathway. Notably, a number of different cancers, including Ewing sarcoma tumors, are sensitive to the combination of RNR and ATR-CHK1 inhibitors. However, multiple, overlapping mechanisms are reported to underlie the toxicity of ATR-CHK1 inhibitors, both as single agents and in combination with RNR inhibitors, toward cancer cells. Here, we identified a feedback loop in Ewing sarcoma cells in which inhibition of the ATR-CHK1 pathway depletes RRM2, the small subunit of RNR, and exacerbates the DNA replication stress and DNA damage caused by RNR inhibitors. Mechanistically, we identified that the inhibition of ATR-CHK1 activates CDK2, which targets RRM2 for degradation via the proteasome. Similarly, activation of CDK2 by inhibition or knockdown of the WEE1 kinase also depletes RRM2 and causes DNA damage and apoptosis. Moreover, we show that the concurrent inhibition of ATR and WEE1 has a synergistic effect in Ewing sarcoma cells. Overall, our results provide novel insight into the response to DNA replication stress, as well as a rationale for targeting the ATR, CHK1, and WEE1 pathways, in Ewing sarcoma tumors. IMPLICATIONS: Targeting the ATR, CHK1, and WEE1 kinases in Ewing sarcoma cells activates CDK2 and increases DNA replication stress by promoting the proteasome-mediated degradation of RRM2.
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Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Daño del ADN , Inhibidores Enzimáticos/farmacología , Ribonucleósido Difosfato Reductasa/metabolismo , Sarcoma de Ewing/tratamiento farmacológico , Apoptosis/fisiología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Reparación del ADN , Células HEK293 , Humanos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinonas/farmacología , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , TransfecciónRESUMEN
The clinical potential of pharmacologic ascorbate (P-AscH-; intravenous delivery achieving mmol/L concentrations in blood) as an adjuvant in cancer therapy is being reevaluated. At mmol/L concentrations, P-AscH- is thought to exhibit anticancer activity via generation of a flux of H2O2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer to examine the effects of P-AscH- on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H2O2 produced by P-AscH- induces DNA damage. In response to this DNA damage, we observed that PARP1 is hyperactivated. Using our unique absolute quantitation, we found that P-AscH- mediated the overactivation of PARP1, which results in consumption of NAD+, and subsequently depletion of ATP leading to mitotic cell death. We have also found that Chk1 plays a major role in the maintenance of genomic integrity following treatment with P-AscH-. Hyperactivation of PARP1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 analyzer, we demonstrated that the severe decrease in ATP after challenging with P-AscH- is because of increased demand, not changes in the rate of production. Genetic deletion and pharmacologic inhibition of PARP1 preserved both NAD+ and ATP; however, the toxicity of P-AscH- remained. These data indicate that disruption of bioenergetics is a secondary factor in the toxicity of P-AscH-; damage to DNA appears to be the primary factor. IMPLICATIONS: Efforts to leverage P-AscH- in cancer therapy should first focus on DNA damage.
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Ácido Ascórbico/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Daño del ADN , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Transfección , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The treatment of Ewing sarcoma has changed very little in the past two decades and novel treatment approaches are needed. We recently identified that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), the rate-limiting enzyme in the synthesis of deoxyribonucleotides. We subsequently found that the inhibition of checkpoint kinase 1 (CHK1) increases the sensitivity of Ewing sarcoma cells to inhibitors of RNR, such as gemcitabine. However, Ewing sarcoma cells exhibit high levels of the CHK1 protein, which may represent an adaptive response to elevated levels of endogenous DNA replication stress. Consequently, we began this work with the aim of determining the impact of CHK1 levels on drug sensitivity, as well as identifying the mechanisms and pathways that regulate CHK1 levels in Ewing sarcoma cells. In this report, we show that the high levels of the CHK1 protein in Ewing sarcoma cells limit the efficacy of CHK1 inhibitors. However, inhibition of mTORC1/2 activates the translational repressor 4E-BP1, reduces protein synthesis, and decreases levels of the CHK1 protein in Ewing sarcoma cells. Similarly, we identified that the CHK1 inhibitor prexasertib also activates 4E-BP1, inhibits protein synthesis, and reduces CHK1 protein levels in Ewing sarcoma cells. Moreover, the combination of prexasertib and gemcitabine was synergistic in vitro, caused tumor regression in vivo, and significantly prolonged mouse survival in a Ewing sarcoma xenograft experiment. Overall, our results provide insight into Ewing sarcoma biology and support further investigation of the CHK1 pathway as a therapeutic target in Ewing sarcoma tumors.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Biosíntesis de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Sarcoma de Ewing/enzimología , Sarcoma de Ewing/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Sinergismo Farmacológico , Humanos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Pirazinas/farmacología , Pirazoles/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Background/Aims Identifying predictors of recruitment success in clinical trials, particularly prior to study launch, could contribute to higher study completion rates and improved scientific return on investment. This article evaluates the performance of clinical trials funded by the National Heart, Lung, and Blood Institute that began recruitment before and after implementation of National Heart, Lung, and Blood Institute's 2009 Accrual Policy and identifies study-related factors that predict recruitment success. Methods A retrospective analysis of National Heart, Lung, and Blood Institute's cardiovascular clinical trials with initial funding from 1996 to 2012 was performed to assess recruitment success. Success was defined as ≥100% enrollment of the proposed sample size within the duration initially proposed by investigators. Trials were assigned to categories (pre-policy vs post-policy) based on whether the first patient was enrolled before or after the 2009 Accrual Policy implementation. Potential determinants of successful recruitment were evaluated using multivariable logistic regression. Results Of 167 trials analyzed, 26.3% met the definition of success. Twenty-four trials (14.4%) were terminated early and 15 (62.5%) for insufficient recruitment. Trials failed due to <100% enrollment (22.8%), longer duration (19.8%), or both (31.1%). Trials testing behavioral interventions, those conducted within a National Heart, Lung, and Blood Institute-funded network, and those with normal controls were predictive of success. The proportion of successful clinical trials increased from 23% in the pre-policy era to 30% post-policy, although the difference was not statistically significant ( p = 0.29). Conclusion Enrollment success rates for National Heart, Lung, and Blood Institute's clinical trials are concerning. The 2009 National Heart, Lung, and Blood Institute Accrual Policy did not significantly improve trial success. Clinical trials testing behavioral interventions, those conducted within networks, and those with normal controls were predictive of recruitment success. Components of networks may provide model practices to help other trials attain success, including close attention to oversight activities such as recruitment plans, real-time enrollment monitoring, corrective action plans to address shortfalls, and close sponsor-investigator collaborations.
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Ensayos Clínicos como Asunto/estadística & datos numéricos , Selección de Paciente , Terminación Anticipada de los Ensayos Clínicos/estadística & datos numéricos , Humanos , National Heart, Lung, and Blood Institute (U.S.) , Estudios Retrospectivos , Tamaño de la Muestra , Estados UnidosRESUMEN
The limited delivery of chemotherapy agents to cancer cells and the nonspecific action of these agents are significant challenges in oncology. We have previously developed a customizable drug delivery and activation system in which a nucleic acid functionalized gold nanoparticle (Au-NP) delivers a drug that is selectively activated within a cancer cell by the presence of an mRNA unique to the cancer cell. The amount of drug released from sequestration to the Au-NP is determined by both the presence and the abundance of the cancer cell specific mRNA in a cell. We have now developed this technology for the potent, but difficult to deliver, topoisomerase I inhibitor SN-38. Herein, we demonstrate both the efficient delivery and selective release of SN-38 from gold nanoparticles in Ewing sarcoma cells with resulting efficacy in vitro and in vivo. These results provide further preclinical validation for this novel cancer therapy and may be extendable to other cancers that exhibit sensitivity to topoisomerase I inhibitors.