mk-1775 and Pancreatic-Neoplasms

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.. Thirty-four patients with locally advanced pancreatic cancer were enrolled with the intention to receive four 21-day cycles of gemcitabine (1,000 mg/m. 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.. 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.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Proteins; Chemoradiotherapy; Deoxycytidine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Gemcitabine; Humans; Male; Middle Aged; Pancreatic Neoplasms; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones; Survival Rate

Topics: Adenocarcinoma; Chemoradiotherapy; Combined Modality Therapy; Dose-Response Relationship, Drug; Humans; Pancreatic Neoplasms; Progression-Free Survival; Pyrazoles; Pyrimidinones

Autophagy and cell-cycle checkpoints act in concert to confer cellular radioresistance. We investigated the functional interaction between radiation-induced autophagy and G2 checkpoint activation in highly radioresistant human pancreatic ductal adenocarcinoma (PDAC) cells.. Four human PDAC cell lines (MIA PaCa-2, KP-4, Panc-1, and SUIT-2) were analyzed. These cells were first irradiated using x-rays, and their cell cycle status, autophagy, and cell cycle checkpoint marker expression and ATP production levels were evaluated. Autophagic flux assays and siRNA knockdown were used to evaluate autophagy activity. Double thymidine block experiments were performed to synchronize the cells. Two inhibitors (MK-1775 and SCH 900776) were used to attenuate G2 checkpoint activation. Cell survival assays and animal experiments were performed to evaluate the radiosensitizing effects of the G2 checkpoint inhibitors.. Autophagy and G2/M accumulation were synchronously induced in human PDAC cells with an activated G2 checkpoint at 12 hours after x-ray irradiation of 6 Gy. Radiation-induced autophagy produced the ATP levels required for cell survival. Double thymidine block experiments revealed that no autophagy occurred in cells that were solely in G2 phase. MK-1775 or SCH 900776 exposure attenuated not only G2 checkpoint activation but also postirradiation autophagy, indicating the dependence of radiation-induced autophagy on an activated G2 checkpoint. The inhibitors demonstrated a higher radiosensitizing effect in the PDAC cells than the autophagy inhibitor chloroquine. MK-1775 in combination with x-rays significantly suppressed the tumor growth of MIA PaCa-2 xenografts compared with other treatment groups, including radiation or drug exposure alone, to enhance the radiosensitivity of PDAC cells in vivo.. Biological crosstalk exists between the G2 checkpoint activation and radiation-induced autophagy processes that are believed to independently contribute to the radioresistance of human PDAC cells. These findings have important implications for the development of future radiation therapy strategies for PDAC.

Topics: Adenosine Triphosphate; Animals; Autophagy; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Female; G2 Phase Cell Cycle Checkpoints; Humans; Mice; Mice, Inbred BALB C; Pancreatic Neoplasms; Pyrazoles; Pyrimidinones; Quinolines; Radiation Tolerance; Thiazoles

Topics: Cell Cycle Proteins; Deoxycytidine; Gemcitabine; Humans; Nuclear Proteins; Pancreatic Neoplasms; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones

According to results from a phase I/II trial, the investigational WEE1 inhibitor adavosertib combined with gemcitabine and radiation is well tolerated and may benefit patients with locally advanced pancreatic adenocarcinoma by increasing both systemic and local disease control.

Topics: Adenocarcinoma; Cell Cycle Proteins; Deoxycytidine; Gemcitabine; Humans; Nuclear Proteins; Pancreatic Neoplasms; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones

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 (R

Topics: Analysis of Variance; Biomarkers, Tumor; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Checkpoint Kinase 1; Deoxycytidine; DNA Replication; Gemcitabine; Histones; Humans; Nucleosides; Pancreatic Neoplasms; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Roscovitine; Staining and Labeling

The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker γH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genome-scale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin A2; Cyclin-Dependent Kinase 2; DNA Damage; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitosis; Nuclear Proteins; Pancreatic Neoplasms; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Xenograft Model Antitumor Assays

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, in part, because of the lack of effective targeted therapeutic options. MK-1775 (also known as AZD1775), a mitotic inhibitor, has been demonstrated to enhance the anti-tumor effects of DNA damaging agents such as gemcitabine. We evaluated the efficacy of MK-1775 alone or in combination with DNA damaging agents (MMC or oxaliplatin) in PDA cell lines that are either DNA repair proficient (DDR-P) or deficient (DDR-D). PDA cell lines PL11, Hs 766T and Capan-1 harboring naturally selected mutations in DNA repair genes FANCC, FANCG and BRCA2 respectively, were less sensitive to MK-1775 as compared to two out of four representative DDR-P (MIA PaCa2 and PANC-1) cell lines. Accordingly, DDR-P cells exhibit reduced sensitivity to MK-1775 upon siRNA silencing of DNA repair genes, BRCA2 or FANCD2, compared to control cells. Only DDR-P cells showed increased apoptosis as a result of early mitotic entry and catastrophe compared to DDR-D cells. Taken together with other recently published reports, our results add another level of evidence that the efficacy of WEE1 inhibition is influenced by the DNA repair status of a cell and may also be dependent on the tumor type and model evaluated.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; Cell Line, Tumor; DNA Damage; DNA Repair; DNA Repair Enzymes; Drug Resistance, Neoplasm; Drug Synergism; Humans; Inhibitory Concentration 50; Mitomycin; Mitosis; Mutagens; Mutation; Nuclear Proteins; Organoplatinum Compounds; Oxaliplatin; Pancreatic Neoplasms; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones

Pancreatic cancer remains a clinical challenge, thus new therapies are urgently needed. The selective Wee1 inhibitor MK-1775 has demonstrated promising results when combined with DNA damaging agents, and more recently with CHK1 inhibitors in various malignancies. We have previously demonstrated that treatment with the pan-histone deacetylase inhibitor panobinostat (LBH589) can cause down-regulation of CHK1. Accordingly, we investigated using panobinostat to down-regulate CHK1 in combination with MK-1775 to enhance cell death in preclinical pancreatic cancer models. We demonstrate that MK-1775 treatment results in increased H2AX phosphorylation, indicating increased DNA double-strand breaks, and activation of CHK1, which are both dependent on CDK activity. Combination of MK-1775 and panobinostat resulted in synergistic antitumor activity in six pancreatic cancer cell lines. Finally, our in vivo study using a pancreatic xenograft model reveals promising cooperative antitumor activity between MK-1775 and panobinostat. Our study provides compelling evidence that the combination of MK-1775 and panobinostat has antitumor activity in preclinical models of pancreatic cancer and supports the clinical development of panobinostat in combination with MK-1775 for the treatment of this deadly disease.

Topics: Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; DNA Damage; Drug Interactions; Drug Synergism; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Indoles; Mice; Mice, Inbred BALB C; Mice, Nude; Nuclear Proteins; Pancreatic Neoplasms; Panobinostat; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts.. Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry.. MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts.. These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; Cell Line, Tumor; Deoxycytidine; Disease Progression; Drug Synergism; Female; Gemcitabine; Genes, p53; Humans; Mice; Mice, Nude; Mutation; Neoplasms; Nuclear Proteins; Pancreatic Neoplasms; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Tumor Burden; Xenograft Model Antitumor Assays