RESUMEN
Radiotherapy induces a type I interferon-mediated (T1IFN-mediated) antitumoral immune response that we hypothesized could be potentiated by a first-in-class ataxia telangiectasia mutated (ATM) inhibitor, leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound, AZD0156, on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced antitumoral immune responses and sensitized tumors to anti-PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells, as therapeutic efficacy was blunted in CD8+ T cell-depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN, leading to both innate and subsequent adaptive antitumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.
Asunto(s)
Ataxia Telangiectasia , Interferón Tipo I , Neoplasias Pancreáticas , Piridinas , Quinolonas , Animales , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Neoplasias Pancreáticas/patología , InmunidadRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is a rare but highly lethal pediatric and adolescent tumor located in the pons of the brainstem. DIPGs harbor unique and specific pathological and molecular alterations, such as the hallmark lysine 27-to-methionine (H3K27M) mutation in histone H3, which lead to global changes in the epigenetic landscape and drive tumorigenesis. While fractionated radiotherapy, the current standard of care, improves symptoms and delays tumor progression, DIPGs inevitably recur, and despite extensive efforts chemotherapy-driven radiosensitization strategies have failed to improve survival. Advances in our understanding of the role of epigenetics in the cellular response to radiation-induced DNA damage, however, offer new opportunities to develop combinational therapeutic strategies selective for DIPGs expressing H3K27M. In this review, we provide an overview of preclinical studies that explore potential radiosensitization strategies targeting the unique epigenetic landscape of H3K27M mutant DIPG. We further discuss opportunities to selectively radiosensitize DIPG through strategic inhibition of the radiation-induced DNA damage response. Finally, we discuss the potential for using radiation to induce anti-tumor immune responses that may be potentiated in DIPG by radiosensitizing-therapeutic strategies.
Asunto(s)
Neoplasias del Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Glioma , Adolescente , Humanos , Niño , Glioma Pontino Intrínseco Difuso/genética , Glioma/genética , Recurrencia Local de Neoplasia , Histonas/genética , Mutación , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patologíaRESUMEN
Targeting the DNA damage response in combination with radiation enhances type I interferon (T1IFN)-driven innate immune signaling. It is not understood, however, whether DNA-dependent protein kinase (DNA-PK), the kinase critical for repairing the majority of radiation-induced DNA double-strand breaks in cancer cells, is immunomodulatory. We show that combining radiation with DNA-PK inhibition increases cytosolic double-stranded DNA and tumoral T1IFN signaling in a cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-independent, but an RNA polymerase III (POL III), retinoic acid-inducible gene I (RIG-I), and antiviral-signaling protein (MAVS)-dependent manner. Although DNA-PK inhibition and radiation also promote programmed death-ligand 1 (PD-L1) expression, the use of anti-PD-L1 in combination with radiation and DNA-PK inhibitor potentiates antitumor immunity in pancreatic cancer models. Our findings demonstrate a novel mechanism for the antitumoral immune effects of DNA-PK inhibitor and radiation that leads to increased sensitivity to anti-PD-L1 in poorly immunogenic pancreatic cancers. IMPLICATIONS: Our work nominates a novel therapeutic strategy as well as its cellular mechanisms pertinent for future clinical trials combining M3814, radiation, and anti-PD-L1 antibody in patients with pancreatic cancer.
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Proteína Quinasa Activada por ADN , Neoplasias Pancreáticas , Inhibidores de Proteínas Quinasas , ARN Polimerasa III , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas , Piridazinas , Quinazolinas , Neoplasias PancreáticasRESUMEN
Combination therapies with agents targeting the DNA damage response (DDR) offer an opportunity to selectively enhance the therapeutic index of chemoradiation or eliminate use of chemotherapy altogether. The successful translation of DDR inhibitors to clinical use requires investigating both their direct actions as (chemo)radiosensitizers and their potential to stimulate tumor immunogenicity. Beginning with high-throughput screening using both viability and DNA damage-reporter assays, followed by validation in gold-standard radiation colony-forming assays and in vitro assessment of mechanistic effects on the DDR, we describe proven strategies and methods leading to the clinical development of DDR inhibitors both with radiation alone and in combination with chemoradiation. Beyond these in vitro studies, we discuss the impact of key features of human xenograft and syngeneic mouse models on the relevance of in vivo tumor efficacy studies, particularly with regard to the immunogenic effects of combined therapy with radiation and DDR inhibitors. Finally, we describe recent technological advances in radiation delivery (using the small animal radiation research platform) that allow for conformal, clinically relevant radiation therapy in mouse models. This overall approach is critical to the successful clinical development and ultimate Food and Drug Administration approval of DDR inhibitors as (chemo)radiation sensitizers.
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Daño del ADN , Animales , Reparación del ADN , Laboratorios , Ratones , Neoplasias , Fármacos Sensibilizantes a RadiacionesRESUMEN
The development of molecular targeted drugs with radiation and chemotherapy is critically important for improving the outcomes of patients with hard-to-treat, potentially curable cancers. However, too many preclinical studies have not translated into successful radiation oncology trials. Major contributing factors to this insufficiency include poor reproducibility of preclinical data, inadequate preclinical modeling of intertumoral genomic heterogeneity that influences treatment sensitivity in the clinic, and a reliance on tumor growth delay instead of local control (TCD50) endpoints. There exists an urgent need to overcome these barriers to facilitate successful clinical translation of targeted radiosensitizers. To this end, we have used 3-dimensional (3D) cell culture assays to better model tumor behavior in vivo. Examples of successful prediction of in vivo effects with these 3D assays include radiosensitization of head and neck cancers by inhibiting epidermal growth factor receptor or focal adhesion kinase signaling, and radioresistance associated with oncogenic mutation of KRAS. To address the issue of tumor heterogeneity, we leveraged institutional resources that allow high-throughput 3D screening of radiation combinations with small-molecule inhibitors across genomically characterized cell lines from lung, head and neck, and pancreatic cancers. This high-throughput screen is expected to uncover genomic biomarkers that will inform the successful clinical translation of targeted agents from the National Cancer Institute Cancer Therapy Evaluation Program portfolio and other sources. Screening "hits" need to be subjected to refinement studies that include clonogenic assays, addition of disease-specific chemotherapeutics, target/biomarker validation, and integration of patient-derived tumor models. The chemoradiosensitizing activities of the most promising drugs should be confirmed in TCD50 assays in xenograft models with or without relevant biomarker and using clinically relevant radiation fractionation. We predict that appropriately validated and biomarker-directed targeted therapies will have a higher likelihood than past efforts of being successfully incorporated into the standard management of hard-to-treat tumors.
Asunto(s)
Terapia Molecular Dirigida , Biomarcadores de Tumor , Humanos , Neoplasias , Preparaciones Farmacéuticas , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Reproducibilidad de los ResultadosRESUMEN
Programmed death-ligand 1 (PD-L1) promotes tumor immune evasion by engaging the PD-1 receptor and inhibiting T-cell activity. While the regulation of PD-L1 expression is not fully understood, its expression is associated with tumor mutational burden and response to immune checkpoint therapy. Here, we report that Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) is an important regulator of PD-L1 expression. Using an APOBEC3A inducible expression system as well as siRNA against endogenous APOBEC3A, we found that APOBEC3A regulates PD-L1 mRNA and protein levels as well as PD-L1 cell surface expression in cancer. Mechanistically, APOBEC3A-induced PD-L1 expression was dependent on APOBEC3A catalytic activity as catalytically dead APOBEC3A mutant (E72A) failed to induce PD-L1 expression. Furthermore, APOBEC3A-induced PD-L1 expression was dependent on replication-associated DNA damage and JNK/c-JUN signaling but not interferon signaling. In addition, we confirmed the relevance of these finding in patient tumors as APOBEC3A expression and mutational signature correlated with PD-L1 expression in multiple patient cancer types. These data provide a novel link between APOBEC3A, its DNA mutagenic activity and PD-L1-mediated antitumoral immunity. This work nominates APOBEC3A as a mechanism of immune evasion and a potential biomarker for the therapeutic efficacy of immune checkpoint blockade. IMPLICATIONS: APOBEC3A catalytic activity induces replication-associated DNA damage to promote PD-L1 expression implying that APOBEC3A-driven mutagenesis represents both a mechanism of tumor immune evasion and a therapeutically targetable vulnerability in cancer cells.
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Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/metabolismo , Citidina Desaminasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Neoplasias/patología , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Apoptosis , Antígeno B7-H1/genética , Biomarcadores de Tumor/genética , Proliferación Celular , Citidina Desaminasa/genética , Humanos , Proteína Quinasa 8 Activada por Mitógenos/genética , Neoplasias/genética , Neoplasias/metabolismo , Pronóstico , Proteínas/genética , Proteínas Proto-Oncogénicas c-jun/genética , Células Tumorales CultivadasRESUMEN
PARP inhibitor monotherapy (olaparib) was recently FDA approved for the treatment of BRCA1/2-mutant, homologous recombination (HR) repair-deficient pancreatic cancer. Most pancreatic cancers, however, are HR proficient and thus resistant to PARP inhibitor monotherapy. We tested the hypothesis that combined therapy with radiation and ataxia telangiectasia and Rad3-related (ATR) inhibitor (AZD6738) would extend the therapeutic indication of olaparib to HR-proficient pancreatic cancers. We show that olaparib combined with AZD6738 significantly reduced radiation survival relative to either agent alone, regardless of HR status. Whereas catalytic inhibition of PARP with low concentrations of olaparib radiosensitized HR-deficient models, maximal sensitization in HR-proficient models required concentrations of olaparib that induce formation of PARP1-DNA complexes. Furthermore, CRISPR-Cas9-mediated PARP1 deletion failed to recapitulate the effects of olaparib on radiosensitivity and negated the combinatorial efficacy of olaparib and AZD6738 on radiosensitization, suggesting that PARP1-DNA complexes, rather than PARP catalytic inhibition, were responsible for radiosensitization. Mechanistically, therapeutic concentrations of olaparib in combination with radiation and AZD6738 increased DNA double-strand breaks. DNA fiber combing revealed that high concentrations of olaparib did not stall replication forks but instead accelerated replication fork progression in association with an ATR-mediated replication stress response that was antagonized by AZD6738. Finally, in HR-proficient tumor xenografts, the combination of olaparib, radiation, and AZD6738 significantly delayed tumor growth compared with all other treatments. These findings suggest that PARP1-DNA complexes are required for the therapeutic activity of olaparib combined with radiation and ATR inhibitor in HR-proficient pancreatic cancer and support the clinical development of this combination for tumors intrinsically resistant to PARP inhibitors.
Asunto(s)
Terapia Combinada/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/patología , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias PancreáticasRESUMEN
PURPOSE: AZD1775 (adavosertib) is an inhibitor of the Wee1 kinase. In this study, we built on our preclinical studies to evaluate the safety and efficacy of AZD1775 in combination with gemcitabine and radiation in patients with newly diagnosed locally advanced pancreatic cancer. PATIENTS AND METHODS: Thirty-four patients with locally advanced pancreatic cancer were enrolled with the intention to receive four 21-day cycles of gemcitabine (1,000 mg/m2 days 1 and 8) with AZD1775 (once daily on days 1, 2, 8, and 9). Cycles 2 and 3 were administered concurrently with radiation, and cycles 5 to 8 were optional. AZD1775 was dose escalated using a time-to-event continual reassessment method on the basis of the rate of dose-limiting toxicities within the first 15 weeks of therapy. The primary objective was to determine the maximum tolerated dose of AZD1775 given in conjunction with gemcitabine and radiation. Secondary objectives were to estimate overall and progression-free survival and determine pharmacodynamic activity of AZD1775 in surrogate tissues. RESULTS: The recommended phase II dose of AZD1775 was 150 mg/d. Eight patients (24%) experienced a dose-limiting toxicity, most commonly anorexia, nausea, or fatigue. The median overall survival for all patients was 21.7 months (90% CI, 16.7 to 24.8 months), and the median progression-free survival was 9.4 months (90% CI, 8.0 to 9.9 months). Hair follicle biopsy samples demonstrated evidence of Wee1 inhibition with decreased phosphorylation of cyclin-dependent kinase 1 staining by immunohistochemistry after AZD1775 administration at the recommended phase II dose. CONCLUSION: AZD1775 in combination with gemcitabine and radiation therapy was well tolerated at a dose that produced target engagement in a surrogate tissue. The overall survival is substantially higher than prior results combining gemcitabine with radiation therapy and warrants additional investigation.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Quimioradioterapia , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Desoxicitidina/análogos & derivados , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/administración & dosificación , Pirazoles/efectos adversos , Pirimidinonas/administración & dosificación , Pirimidinonas/efectos adversos , Tasa de Supervivencia , GemcitabinaRESUMEN
Combinatorial strategies are needed to overcome the resistance of pancreatic cancer to immune checkpoint blockade (ICB). DNA damage activates the innate immune response and improves ICB efficacy. Because ATM is an apical kinase in the radiation-induced DNA damage response, we investigated the effects of ATM inhibition and radiation on pancreatic tumor immunogenicity. ATM was inhibited through pharmacologic and genetic strategies in human and murine pancreatic cancer models both in vitro and in vivo. Tumor immunogenicity was evaluated after ATM inhibition alone and in combination with radiation by assessing TBK1 and Type I interferon (T1IFN) signaling as well as tumor growth following PD-L1/PD-1 checkpoint inhibition. Inhibition of ATM increased tumoral T1IFN expression in a cGAS/STING-independent, but TBK1- and SRC-dependent, manner. The combination of ATM inhibition with radiation further enhanced TBK1 activity, T1IFN production, and antigen presentation. Furthermore, ATM silencing increased PD-L1 expression and increased the sensitivity of pancreatic tumors to PD-L1-blocking antibody in association with increased tumoral CD8+ T cells and established immune memory. In patient pancreatic tumors, low ATM expression inversely correlated with PD-L1 expression. Taken together, these results demonstrate that the efficacy of ICB in pancreatic cancer is enhanced by ATM inhibition and further potentiated by radiation as a function of increased tumoral immunogenicity, underscoring the potential of ATM inhibition in combination with ICB and radiation as an efficacious treatment strategy for pancreatic cancer. SIGNIFICANCE: This study demonstrates that ATM inhibition induces a T1IFN-mediated innate immune response in pancreatic cancer that is further enhanced by radiation and leads to increased sensitivity to anti-PD-L1 therapy.See related commentary by Gutiontov and Weichselbaum, p. 3815.
Asunto(s)
Linfocitos T CD8-positivos , Neoplasias Pancreáticas , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Humanos , Inmunoterapia , Interferones , Ratones , Transducción de SeñalRESUMEN
Small molecule inhibitors of the checkpoint proteins CHK1 and WEE1 are currently in clinical development in combination with the antimetabolite gemcitabine. It is unclear, however, if there is a therapeutic advantage to CHK1 vs. WEE1 inhibition for chemosensitization. The goals of this study were to directly compare the relative efficacies of the CHK1 inhibitor MK8776 and the WEE1 inhibitor AZD1775 to sensitize pancreatic cancer cell lines to gemcitabine and to identify pharmacodynamic biomarkers predictive of chemosensitization. Cells treated with gemcitabine and either MK8776 or AZD1775 were first assessed for clonogenic survival. With the exception of the homologous recombination-defective Capan1 cells, which were relatively insensitive to MK8776, we found that these cell lines were similarly sensitized to gemcitabine by CHK1 or WEE1 inhibition. The abilities of either the CDK1/2 inhibitor roscovitine or exogenous nucleosides to prevent MK8776 or AZD1775-mediated chemosensitization, however, were both inhibitor-dependent and variable among cell lines. Given the importance of DNA replication stress to gemcitabine chemosensitization, we next assessed high-intensity, pan-nuclear γH2AX staining as a pharmacodynamic marker for sensitization. In contrast to total γH2AX, aberrant mitotic entry or sub-G1 DNA content, high-intensity γH2AX staining correlated with chemosensitization by either MK8776 or AZD1775 (R2 0.83 - 0.53). In summary, we found that MK8776 and AZD1775 sensitize to gemcitabine with similar efficacy. Furthermore, our results suggest that the effects of CHK1 and WEE1 inhibition on gemcitabine-mediated replication stress best predict chemosensitization and support the use of high-intensity or pan-nuclear γH2AX staining as a marker for therapeutic response.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Replicación del ADN/fisiología , Desoxicitidina/análogos & derivados , Histonas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Pirimidinonas/farmacología , Análisis de Varianza , Biomarcadores de Tumor/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/farmacología , Humanos , Nucleósidos/farmacología , Neoplasias Pancreáticas , Inhibidores de Proteínas Quinasas/farmacología , Roscovitina/farmacología , Coloración y Etiquetado , GemcitabinaRESUMEN
KRAS mutations in non-small cell lung cancer (NSCLC) cause increased levels of DNA damage and replication stress, suggesting that inhibition of the DNA damage response (DDR) is a promising strategy for radiosensitization of NSCLC. This study investigates the ability of a WEE1 inhibitor (AZD1775) and a PARP inhibitor (olaparib) to radiosensitize KRAS-mutant NSCLC cells and tumors. In addition to inhibiting the DDR, these small-molecule inhibitors of WEE1 and PARP induce DNA replication stress via nucleotide exhaustion and PARP trapping, respectively. As monotherapy, AZD1775 or olaparib alone modestly radiosensitized a panel of KRAS-mutant NSCLC lines. The combination of agents, however, significantly increased radiosensitization. Furthermore, AZD1775-mediated radiosensitization was rescued by nucleotide repletion, suggesting a mechanism involving AZD1775-mediated replication stress. In contrast, radiosensitization by the combination of AZD1775 and olaparib was not rescued by nucleosides. Whereas both veliparib, a PARP inhibitor that does not efficiently trap PARP1 to chromatin, and PARP1 depletion radiosensitized NSCLC cells as effectively as olaparib, which does efficiently trap PARP, only olaparib potentiated AZD1775-mediated radiosensitization. Taken together, these mechanistic data demonstrate that although nucleotide depletion is sufficient for radiosensitization by WEE1 inhibition alone, and inhibition of PARP catalytic activity is sufficient for radiosensitization by olaparib alone, PARP1 trapping is required for enhanced radiosensitization by the combination of WEE1 and PARP inhibitors.Implications: This study highlights DNA replication stress caused by nucleotide depletion and PARP1 trapping as an important mechanism of radiosensitization in KRAS-mutant tumors and supports further development of DNA replication as a therapeutic target. Mol Cancer Res; 16(2); 222-32. ©2017 AACR.
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Carcinoma de Pulmón de Células no Pequeñas/terapia , Replicación del ADN/efectos de los fármacos , Neoplasias Pulmonares/terapia , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/genética , Ratones , Mutación , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Pirimidinonas , Fármacos Sensibilizantes a Radiaciones/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In order to determine the relative contribution of checkpoint abrogation and subsequent aberrant mitotic entry to gemcitabine chemosensitization by CHK1 inhibition, we established a model utilizing the CDK inhibitors roscovitine or purvalanol A to re-establish cell cycle arrest and prevent aberrant mitotic entry in pancreatic cancer cells treated with gemcitabine and the CHK inhibitor AZD7762. In this study, we report that the extent of aberrant mitotic entry, as determined by flow cytometry for the mitotic marker phospho-Histone H3 (Ser10), did not reflect the relative sensitivities of pancreatic cancer cell lines to gemcitabine chemosensitization by AZD7762. In addition, re-establishing gemcitabine-induced cell cycle arrest either pharmacologically, with roscovitine or purvalanol A, or genetically, with cyclin B1 siRNA, did not inhibit chemosensitization uniformly across the cell lines. Furthermore, we found that AZD7762 augmented high-intensity γH2AX signaling in gemcitabine-treated cells, suggesting the presence of replication stress when CHK1 is inhibited. Finally, the ability of roscovitine to prevent chemosensitization correlated with its ability to inhibit AZD7762-induced high-intensity γH2AX, but not aberrant pHH3, suggesting that the effects of AZD7762 on DNA replication or repair rather than aberrant mitotic entry determine gemcitabine chemosensitization in pancreatic cancer cells.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Desoxicitidina/análogos & derivados , Tiofenos/farmacología , Urea/análogos & derivados , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Ciclina B1/metabolismo , Daño del ADN , Desoxicitidina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Puntos de Control de la Fase G2 del Ciclo Celular , Humanos , Mitosis , Nocodazol/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Urea/farmacología , GemcitabinaRESUMEN
To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Desoxicitidina/análogos & derivados , Proteínas Nucleares/antagonistas & inhibidores , Neoplasias Pancreáticas/terapia , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Quimioradioterapia , Daño del ADN/efectos de los fármacos , Desoxicitidina/farmacología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Desnudos , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.
RESUMEN
PURPOSE: While the addition of radiation to chemotherapy improves survival in patients with locally advanced pancreatic cancer, more effective therapies are urgently needed. Thus, we investigated the radiosensitizing efficacy of the novel drug combination of Wee1 and PARP1/2 inhibitors (AZD1775 and olaparib, respectively) in pancreatic cancer. EXPERIMENTAL DESIGN: Radiosensitization of AsPC-1 or MiaPaCa-2 human pancreatic cancer cells was assessed by clonogenic survival and tumor growth assays. Mechanistically, the effects of AZD1775, olaparib, and radiation on cell cycle, DNA damage (γH2AX), and homologous recombination repair (HRR) were determined. RESULTS: Treatment of AsPC-1 and MiaPaCa-2 cells with either AZD1775 or olaparib caused modest radiosensitization, whereas treatment with the combination significantly increased radiosensitization. Radiosensitization by the combination of AZD1775 and olaparib was associated with G2 checkpoint abrogation and persistent DNA damage. In addition, AZD1775 inhibited HRR activity and prevented radiation-induced Rad51 focus formation. Finally, in vivo, in MiaPaCa-2-derived xenografts, olaparib did not radiosensitize, whereas AZD1775 produced moderate, yet significant, radiosensitization (P < 0.05). Importantly, the combination of AZD1775 and olaparib produced highly significant radiosensitization (P < 0.0001) evidenced by a 13-day delay in tumor volume doubling (vs. radiation alone) and complete eradication of 20% of tumors. CONCLUSIONS: Taken together, these results demonstrate the efficacy of combined inhibition of Wee1 and PARP inhibitors for radiosensitizing pancreatic cancers and support the model that Wee1 inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization through inhibition of HRR and abrogation of the G2 checkpoint, ultimately resulting in unrepaired, lethal DNA damage and radiosensitization. Clin Cancer Res; 20(19); 5085-96. ©2014 AACR.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tolerancia a Radiación , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Modelos Animales de Enfermedad , Femenino , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Radiación , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Carga Tumoral/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chemoradiation is the standard therapy for the majority of inoperable, locally advanced cancers. Although there is a need to improve chemoradiation efficacy, normal-tissue toxicity limits our ability to give additional chemotherapy or higher doses of radiation. Thus, there is excitement about the addition of molecularly targeted agents, which tend to be less toxic than chemotherapy, to chemoradiation regimens. Unfortunately, initial empiric attempts have not been successful. This review will focus on the evidence that supports rational combinations of targeted agents with chemoradiation, with an emphasis on agents that target the DNA damage response and radiation-induced membrane signaling.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quimioradioterapia/efectos adversos , Terapia Molecular Dirigida/métodos , Neoplasias/terapia , Ensayos Clínicos como Asunto , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiaciónRESUMEN
PURPOSE: The combination of radiation with chemotherapy is the most effective therapy for unresectable pancreatic cancer. To improve upon this regimen, we combined the selective Checkpoint kinase 1 (Chk1) inhibitor MK8776 with gemcitabine-based chemoradiation in preclinical pancreatic cancer models. EXPERIMENTAL DESIGN: We tested the ability of MK8776 to sensitize to gemcitabine-radiation in homologous recombination repair (HRR)-proficient and -deficient pancreatic cancer cells and assessed Rad51 focus formation. In vivo, we investigated the efficacy, tumor cell selectivity, and pharmacodynamic biomarkers of sensitization by MK8776. RESULTS: We found that MK8776 significantly sensitized HRR-proficient (AsPC-1, MiaPaCa-2, BxPC-3) but not -deficient (Capan-1) pancreatic cancer cells to gemcitabine-radiation and inhibited Rad51 focus formation in HRR-proficient cells. In vivo, MiaPaCa-2 xenografts were significantly sensitized to gemcitabine-radiation by MK8776 without significant weight loss or observable toxicity in the small intestine, the dose-limiting organ for chemoradiation therapy in pancreatic cancer. We also assessed pChk1 (S345), a pharmacodynamic biomarker of DNA damage in response to Chk1 inhibition in both tumor and small intestine and found that MK8776 combined with gemcitabine or gemcitabine-radiation produced a significantly greater increase in pChk1 (S345) in tumor relative to small intestine, suggesting greater DNA damage in tumor than in normal tissue. Furthermore, we demonstrated the utility of an ex vivo platform for assessment of pharmacodynamic biomarkers of Chk1 inhibition in pancreatic cancer. CONCLUSIONS: Together, our results suggest that MK8776 selectively sensitizes HRR-proficient pancreatic cancer cells and xenografts to gemcitabine-radiation and support the clinical investigation of MK8776 in combination with gemcitabine-radiation in locally advanced pancreatic cancer.
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Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quimioradioterapia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Inhibidores de Proteínas Quinasas/administración & dosificación , Fármacos Sensibilizantes a Radiaciones/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
PURPOSE: To identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and thus improve survival, we conducted an siRNA library screen in pancreatic cancer cells. We investigated PPP2R1A, a scaffolding subunit of protein phosphatase 2A (PP2A) as a lead radiosensitizing target. EXPERIMENTAL DESIGN: We determined the effect of PP2A inhibition by genetic (PPP2R1A siRNA) and pharmacologic (LB100, a small molecule entering phase I clinical trials) approaches on radiosensitization of Panc-1 and MiaPaCa-2 pancreatic cancer cells both in vitro and in vivo. RESULTS: PPP2R1A depletion by siRNA radiosensitized Panc-1 and MiaPaCa-2 cells, with radiation enhancement ratios of 1.4 (P < 0.05). Likewise, LB100 produced similar radiosensitization in pancreatic cancer cells, but minimal radiosensitization in normal small intestinal cells. Mechanistically, PPP2R1A siRNA or LB100 caused aberrant CDK1 activation, likely resulting from accumulation of the active forms of PLK1 (pPLK1 T210) and CDC25C (pCDC25C T130). Furthermore, LB100 inhibited radiation-induced Rad51 focus formation and homologous recombination repair (HRR), ultimately leading to persistent radiation-induced DNA damage, as reflected by γ-H2AX expression. Finally, we identified CDC25C as a key PP2A substrate involved in LB100-mediated radiosensitization as depletion of CDC25C partially reversed LB100-mediated radiosensitization. In a mouse xenograft model of human pancreatic cancer, LB100 produced significant radiosensitization with minimal weight loss. CONCLUSIONS: Collectively, our data show that PP2A inhibition radiosensitizes pancreatic cancer both in vitro and in vivo via activation of CDC25C/CDK1 and inhibition of HRR, and provide proof-of-concept evidence that PP2A is a promising target for the improvement of local therapy in pancreatic cancer.
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Proteína Quinasa CDC2/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Reparación del ADN por Recombinación/efectos de los fármacos , Fosfatasas cdc25/metabolismo , Animales , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Neoplasias Pancreáticas/radioterapia , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Interferencia de ARN , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Fármacos Sensibilizantes a Radiaciones/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Checkpoint kinase 1 (Chk1) inhibition sensitizes pancreatic cancer cells and tumors to gemcitabine. We hypothesized that Chk1 inhibition would sensitize pancreatic cancer stem cells to gemcitabine. We tested this hypothesis by using two patient-derived xenograft models (designated J and F) and the pancreatic cancer stem cell markers CD24, CD44, and ESA. We determined the percentage of marker-positive cells and their tumor-initiating capacity (by limiting dilution assays) after treatment with gemcitabine and the Chk1 inhibitor, AZD7762. We found that marker-positive cells were significantly reduced by the combination of gemcitabine and AZD7762. In addition, secondary tumor initiation was significantly delayed in response to primary tumor treatment with gemcitabine + AZD7762 compared with control, gemcitabine, or AZD7762 alone. Furthermore, for the same number of stem cells implanted from gemcitabine- versus gemcitabine + AZD7762-treated primary tumors, secondary tumor initiation at 10 weeks was 83% versus 43%, respectively. We also found that pS345 Chk1, which is a measure of DNA damage, was induced in marker-positive cells but not in the marker-negative cells. These data demonstrate that Chk1 inhibition in combination with gemcitabine reduces both the percentage and the tumor-initiating capacity of pancreatic cancer stem cells. Furthermore, the finding that the Chk1-mediated DNA damage response was greater in stem cells than in non-stem cells suggests that Chk1 inhibition may selectively sensitize pancreatic cancer stem cells to gemcitabine, thus making Chk1 a potential therapeutic target for improving pancreatic cancer therapy.
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Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
We have recently shown that inhibition of HRR (homologous recombination repair) by Chk1 (checkpoint kinase 1) inhibition radiosensitizes pancreatic cancer cells and others have demonstrated that Chk1 inhibition selectively sensitizes p53 mutant tumor cells. Furthermore, PARP1 [poly (ADP-ribose) polymerase-1] inhibitors dramatically radiosensitize cells with DNA double strand break repair defects. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 [via olaparib (AZD2281)] would selectively sensitize p53 mutant pancreatic cancer cells to radiation. We also used 2 isogenic p53 cell models to assess the role of p53 status in cancer cells and intestinal epithelial cells to assess overall cancer specificity. DNA damage response and repair were assessed by flow cytometry, γH2AX, and an HRR reporter assay. We found that the combination of AZD7762 and olaparib produced significant radiosensitization in p53 mutant pancreatic cancer cells and in all of the isogenic cancer cell lines. The magnitude of radiosensitization by AZD7762 and olaparib was greater in p53 mutant cells compared with p53 wild type cells. Importantly, normal intestinal epithelial cells were not radiosensitized. The combination of AZD7762 and olaparib caused G 2 checkpoint abrogation, inhibition of HRR, and persistent DNA damage responses. These findings demonstrate that the combination of Chk1 and PARP1 inhibition selectively radiosensitizes p53 mutant pancreatic cancer cells. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRCA1/2 'deficient-like' phenotype in p53 mutant tumor cells, while sparing normal tissue.