RESUMO
The protein kinase checkpoint kinase 1 (Chk1) has been implicated as a key regulator of cell cycle progression and DNA repair, and inhibitors of Chk1 (e.g., UCN-01 and EXEL-9844) potentiate the cytotoxic actions of chemotherapeutic drugs in tumor cells. We have examined the ability of PD-321852, a small-molecule Chk1 inhibitor, to potentiate gemcitabine-induced clonogenic death in a panel of pancreatic cancer cell lines and evaluated the relationship between endpoints associated with Chk1 inhibition and chemosensitization. Gemcitabine chemosensitization by minimally toxic concentrations of PD-321852 ranged from minimal (<3-fold change in survival) in Panc1 cells to >30-fold in MiaPaCa2 cells. PD-321852 inhibited Chk1 in all cell lines as evidenced by stabilization of Cdc25A; in combination with gemcitabine, a synergistic loss of Chk1 protein was observed in the more sensitized cell lines. Gemcitabine chemosensitization, however, did not correlate with abrogation of the S-M or G2-M checkpoint; PD-321852 did not induce premature mitotic entry in gemcitabine-treated BxPC3 or M-Panc96 cells, which were sensitized to gemcitabine 6.2- and 4.6-fold, respectively. In the more sensitized cells lines, PD-321852 not only inhibited gemcitabine-induced Rad51 focus formation and the recovery from gemcitabine-induced replication stress, as evidenced by persistence of gamma-H2AX, but also depleted these cells of Rad51 protein. Our data suggest the inhibition of this Chk1-mediated Rad51 response to gemcitabine-induced replication stress is an important factor in determining gemcitabine chemosensitization by Chk1 inhibition in pancreatic cancer cells.
Assuntos
Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Biocatálise , Carbazóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Dano ao DNA , Desoxicitidina/farmacologia , Humanos , Neoplasias Pancreáticas/genética , Fosforilação/efeitos dos fármacos , GencitabinaRESUMO
In the last three decades, gemcitabine has progressed from the status of a laboratory cytotoxic drug to a standard clinical chemotherapeutic agent and a potent radiation sensitizer. In an effort to improve the efficacy of gemcitabine, additional chemotherapeutic agents have been combined with gemcitabine (both with and without radiation) but with toxicity proving to be a major limitation. Therefore, the integration of molecularly targeted agents, which potentially produce less toxicity than standard chemotherapy, with gemcitabine radiation is a promising strategy for improving chemoradiation. Two of the most promising targets, described in this review, for improving the efficacy of gemcitabine radiation are epidermal growth factor receptor and checkpoint kinase 1.
Assuntos
Desoxicitidina/análogos & derivados , Receptores ErbB/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Quinases , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quinase 1 do Ponto de Checagem , Terapia Combinada , Desoxicitidina/administração & dosagem , Sistemas de Liberação de Medicamentos , Humanos , Modelos Biológicos , Radiossensibilizantes/administração & dosagem , GencitabinaRESUMO
PURPOSE: Gemcitabine-radiotherapy is a standard treatment for locally advanced pancreatic cancer. Clinical data have shown that gemcitabine plus erlotinib is superior to gemcitabine alone for advanced pancreatic cancer. Therefore, we investigated the effects of the combination of epidermal growth factor receptor inhibitors with gemcitabine and radiation on a pancreatic cancer model. EXPERIMENTAL DESIGN: EGFR signaling was analyzed by measuring phosphorylated EGFR (pEGFR(Y845, (Y1173)) and AKT (pAKT(S473)) protein levels in pancreatic cancer cell lines and tumors. The effects of scheduling on gemcitabine-mediated cytotoxicity and radiosensitization combined with erlotinib were determined by clonogenic survival. In vivo, the effects of cetuximab or erlotinib in combination with gemcitabine-radiation on the growth of BxPC-3 tumor xenografts were measured. RESULTS: We found in vitro that gemcitabine induced phosphorylation of EGFR at Y845 and Y1173 that was blocked by erlotinib. Treatment of BxPC-3 cells with gemcitabine before erlotinib enhanced gemcitabine-mediated cytotoxicity without abrogating radiosensitization. In vivo, cetuximab or erlotinib in combination with gemcitabine-radiation inhibited growth compared with gemcitabine-radiation (time to tumor doubling: gemcitabine + radiation, 19 +/- 3 days; cetuximab + gemcitabine + radiation, 30 +/- 3 days; P < 0.05, erlotinib + gemcitabine + radiation 28 +/- 3 days; P < 0.1). Cetuximab or erlotinib in combination with gemcitabine-radiation resulted in significant inhibition of pEGFR(Y1173) and pAKT(S473) early in treatment, and pEGFR(Y845), pEGFR(Y1173), and pAKT(S473) by the end of treatment. This study shows a novel difference pEGFR(Y845) and pEGFR(Y1173) in response to EGFR inhibition. CONCLUSIONS: These results show that the EGFR inhibitors cetuximab and erlotinib increase the efficacy of gemcitabine-radiation. This work supports the integration of EGFR inhibitors with gemcitabine-radiation in clinical trials for pancreatic cancer.
Assuntos
Adenocarcinoma/terapia , Receptores ErbB/antagonistas & inibidores , Neoplasias Pancreáticas/terapia , Radiossensibilizantes/administração & dosagem , Radioterapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular Tumoral , Cetuximab , Terapia Combinada , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Cloridrato de Erlotinib , Humanos , Immunoblotting , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Quinazolinas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/radioterapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Proteínas de Ciclo Celular/antagonistas & inibidores , Quimiorradioterapia , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/análogos & derivados , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Pirimidinonas/administração & dosagem , Pirimidinonas/efeitos adversos , Taxa de Sobrevida , GencitabinaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Replicação do DNA/fisiologia , Desoxicitidina/análogos & derivados , Histonas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas/farmacologia , Análise de Variância , Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Humanos , Nucleosídeos/farmacologia , Neoplasias Pancreáticas , Inibidores de Proteínas Quinases/farmacologia , Roscovitina/farmacologia , Coloração e Rotulagem , GencitabinaRESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Replicação do DNA/efeitos dos fármacos , Neoplasias Pulmonares/terapia , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Radiossensibilizantes/administração & dosagem , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/genética , Camundongos , Mutação , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas , Radiossensibilizantes/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The deoxycytidine analogue 2',2'-difluoro-2'-deoxycytidine (gemcitabine) is a potent radiation sensitizer in a variety of solid tumors and tumor cell lines. Previous studies have shown that radiosensitization by gemcitabine is accompanied by simultaneous depletion of dATP pools (through ribonucleotide reductase inhibition) and accumulation in the S-phase of the cell cycle. Because of the importance of cell cycle redistribution in gemcitabine-mediated radiosensitization, we investigated the role of checkpoint kinase (Chk) 1 and Chk2 in gemcitabine-induced cell cycle arrest. We hypothesized that gemcitabine might induce Chk1 or Chk2 signal transduction pathways that mediate S-phase arrest. We found that radiosensitizing concentrations of gemcitabine induced accumulation of phosphorylated Chk1 and Chk2 and down-regulation of Cdc25A in BxPC-3 (10 nmol/L), Panc-1 (100 nmol/L), A549 (30 nmol/L), RKO (30 nmol/L), and SW620 (30 nmol/L) cells. Depletion of Chk1 from Panc-1 cells prevented the down-regulation of Cdc25A in response to gemcitabine. Furthermore, Chk1 depletion permitted Panc-1 and SW620 cells treated with gemcitabine to enter mitosis despite incomplete DNA synthesis. However, depletion of neither Chk1 nor Chk2 abrogated the inhibition of DNA synthesis in response to gemcitabine. These results provide evidence that Chk1 negatively regulates entry into mitosis in response to gemcitabine. Furthermore, these data imply that Chk1 acts to coordinate the cell cycle with DNA synthesis, thus preventing premature mitotic entry in gemcitabine-treated cells.
Assuntos
Desoxicitidina/análogos & derivados , Mitose/efeitos dos fármacos , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Radiossensibilizantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/biossíntese , Desoxicitidina/farmacologia , Humanos , Mitose/fisiologia , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Transfecção , Fosfatases cdc25/metabolismo , GencitabinaRESUMO
The ability to inappropriately progress through S phase during drug treatment is a key determinant of tumor cell sensitivity to thymidylate synthase inhibitors such as 5-fluoro-2'-deoxyuridine (FdUrd). Previous studies suggest that SW620 cells, which are relatively resistant to FdUrd, have an intact early S-phase checkpoint that protects against FdUrd-induced DNA damage and cytotoxicity and that this checkpoint is defective in the relatively sensitive HT29 cells, which continue to progress through S phase during drug treatment. To test this hypothesis, we examined the expression and activation of known S-phase checkpoint mediators in FdUrd-treated SW620 and HT29 cells. FdUrd induced degradation of cdc25A in SW620, but not HT29 cells, in a manner that correlated with the previously described drug-induced S-phase arrest. This difference, however, could not be attributed to differences in either chk1 activation, which was similar in both cell lines, or chk2 activation, which only occurred in HT29 cells and correlated with uracil misincorporation/misrepair-induced DNA double-stranded breaks. These observations suggest that although FdUrd-induced S-phase arrest and associated cdc25A degradation are impaired in HT29 cells, signaling by ATM/ATR is intact upstream of chk1 and chk2. Finally, FdUrd induced premature mitotic entry, a phenomenon associated with deregulated cdc25A expression, in HT29 but not SW620 cells. Blocking cdc25A expression in HT29 cells with small interfering RNA attenuated FdUrd-induced premature mitotic entry, suggesting that progression of HT29 cells through S phase during drug treatment results in part from the inability of these cells to degrade cdc25A in response to FdUrd-induced DNA damage.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Floxuridina/farmacologia , Fosfatases cdc25/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Dano ao DNA/fisiologia , Células HT29 , Humanos , Mitose/efeitos dos fármacos , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/efeitos dos fármacos , Fase S/fisiologia , Timidilato Sintase/antagonistas & inibidoresRESUMO
Previous work has shown that in humans the dose-limiting toxicity for fluorodeoxyuridine [2-fluoro-5'-deoxyuridine (FdUrd)] when administered by hepatic arterial infusion is biliary sclerosis. The current study was undertaken to attempt to modify this toxicity in a canine model that has been demonstrated to closely mimic the clinical situation. Unlike previous studies using this model, in which animals were sacrificed after extensive fibrosis had already occurred, the current experiments were designed so that observations of pathology were made at an earlier time, when the initial inflammatory injury underlying the fibrotic process was still taking place. Implantable pumps were used to deliver FdUrd into the hepatic artery of animals at a rate of 0.3 mg/kg/day in the presence or absence of 10 mg/week dexamethasone or 100 mg/day of celecoxib for 35 days, at which time the animals were beginning to show signs of toxicity. After evaluation for radiological evidence of biliary obstruction, the animals were sacrificed and portions of their livers were processed for examination of microscopic pathology and 2-bromo-5'deoxyuridine labeling index. Dexamethasone treatment protected the animals from biliary sclerosis determined radiologically, further validating this model as being representative of the response in humans. Similarly the Cox-2 inhibitor, celecoxib, appeared to provide protection against radiological changes of biliary stricture, although possibly to a lesser degree than the resultant from dexamethasone. In addition, FdUrd treatment caused elevation of the DNA 2-bromo-5'deoxyuridine labeling index above control levels in biliary epithelial cells. Dexamethasone and celecoxib each significantly attenuated the FdUrd-induced elevation of DNA labeling index in biliary epithelium. These findings demonstrate the usefulness of this canine model for studying the mechanisms of drug-induced biliary sclerosis and reinforce the hypothesis that blocking inflammation may retard the progression of injury that eventually leads to fibrosis. This study suggests that clinical testing of celecoxib as a preventive for hepatic arterial-FdUrd induced biliary damage could prove valuable.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Dexametasona/uso terapêutico , Floxuridina/toxicidade , Vesícula Biliar/patologia , Sulfonamidas/uso terapêutico , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Celecoxib , Cães , Floxuridina/administração & dosagem , Vesícula Biliar/efeitos dos fármacos , Artéria Hepática , Humanos , Infusões Intra-Arteriais , Testes de Função Hepática , Modelos Animais , PirazóisRESUMO
PURPOSE: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). METHODS AND MATERIALS: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. RESULTS: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. CONCLUSIONS: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.
Assuntos
Carcinoma Hepatocelular/radioterapia , Proteínas de Ciclo Celular/antagonistas & inibidores , Genes p53 , Neoplasias Hepáticas/radioterapia , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Radiossensibilizantes/farmacologia , Proteína Quinase CDC2/análise , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Dano ao DNA , Replicação do DNA , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células Hep G2 , Histonas/análise , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Pirimidinonas , Estresse FisiológicoRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Tiofenos/farmacologia , Ureia/análogos & derivados , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Dano ao DNA , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Mitose , Nocodazol/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Ureia/farmacologia , GencitabinaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Proteínas Nucleares/antagonistas & inibidores , Neoplasias Pancreáticas/terapia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Radiossensibilizantes/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimiorradioterapia , Dano ao DNA/efeitos dos fármacos , Desoxicitidina/farmacologia , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia/efeitos adversos , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Ensaios Clínicos como Assunto , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Humanos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Tolerância a Radiação , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Modelos Animais de Doenças , Feminino , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Radiação , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Carga Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Proteína Quinase CDC2/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Reparo de DNA por Recombinação/efeitos dos fármacos , Fosfatases cdc25/metabolismo , Animais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Neoplasias Pancreáticas/radioterapia , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Interferência de RNA , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Radiossensibilizantes/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quimiorradioterapia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Inibidores de Proteínas Quinases/administração & dosagem , Radiossensibilizantes/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Quinase 1 do Ponto de Checagem , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
PURPOSE: The standard approach of using tumor doubling time to assess growth delay may not accurately represent tumor response, especially if the growth rates are not constant. Therefore, we developed a method to compare the antitumor activities of different treatments in xenograft experiments that uses the entire growth curve to estimate nonconstant growth rates. EXPERIMENTAL DESIGN: A Bayesian hierarchical changepoint (BHC) method was used to model logarithmically transformed tumor volumes (TV). Each tumor was assumed to have a growth profile, represented by a prenadir regression rate, a regression period, a nadir volume, and a postnadir regrowth rate. Confidence intervals were calculated to compare these features between different treatments. We used data from a study assessing the effects of radiation, gemcitabine, and a Chk1/2 inhibitor on MiaPaCa-2 xenografts. RESULTS: We found that the BHC model provided a good fit to the data and more descriptive features than the tumor doubling approach. This model detected significant tumor regression in the AZD7762 + 1 Gy and GEM + 1 Gy that was not detected when comparing the tumor doubling times. The BHC model also provided evidence that the growth inhibition resulted from a direct tumor effect rather than an indirect effect on the tumor bed, as evidenced by dramatic tumor regression in response to effective treatments and similar postnadir regrowth rates across all treatment groups. CONCLUSIONS: Compared with the tumor doubling time approach, the BHC model utilizes all data, providing more descriptive features that address mechanisms underlying tumor growth inhibition and maximize the biological information obtained from tumor xenografts studies.
Assuntos
Proliferação de Células , Modelos Biológicos , Modelos Estatísticos , Neoplasias/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Teorema de Bayes , Intervalos de Confiança , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológico , Tiofenos/uso terapêutico , Ureia/análogos & derivados , Ureia/uso terapêutico , GencitabinaRESUMO
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.
Assuntos
Reparo do DNA/fisiologia , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas Quinases/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Proteína Supressora de Tumor p53/genética , Análise de Variância , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Reparo do DNA/efeitos dos fármacos , Combinação de Medicamentos , Citometria de Fluxo , Histonas/metabolismo , Humanos , Immunoblotting , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mutação/genética , Neoplasias Pancreáticas/radioterapia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Tiofenos/farmacologia , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
PURPOSE: Chk1 inhibitors, such as AZD7762, are in clinical development in combination with cytotoxic agents for the treatment of solid tumors, including pancreatic cancers. To maximize the likelihood of their clinical success, it is essential to optimize drug scheduling as well as pharmacodynamic biomarkers in preclinical models. EXPERIMENTAL DESIGN: We tested multiple schedules of administration of gemcitabine and AZD7762 on the survival of pancreatic cancer cells. Potential pharmacodynamic biomarkers including pChk1, pChk2, pHistone H3, and caspase-3 were evaluated in vitro, followed by assessment of promising candidate biomarkers in vivo. We then went on to determine the contributions of PP2A and DNA damage to the mechanism(s) of induction of the identified biomarker, pS345 Chk1. RESULTS: AZD7762 given during and after or after gemcitabine administration produced maximum chemosensitization. In vivo, AZD7762 significantly inhibited the growth of pancreatic tumor xenografts in response to gemcitabine. Of the biomarkers assessed, pS345 Chk1 was most consistently increased in response to gemcitabine and AZD7762 in tumors and normal tissues (hair follicles). pS345 Chk1 induction in response to gemcitabine and AZD7762 occurred in the presence of PP2A inhibition and in association with elevated γH2AX, suggesting that DNA damage is an underlying mechanism. CONCLUSIONS: AZD7762 sensitizes pancreatic cancer cells and tumors to gemcitabine in association with induction of pS345 Chk1. Together these data support the clinical investigation of AZD7762 with gemcitabine in pancreatic cancer under a dosing schedule in which gemcitabine is administered concurrent with or before AZD7762 and in conjunction with skin biopsies to measure pS345 Chk1.