mk-1775 and Brain-Neoplasms

A rapid, sensitive, and robust aqueous normal-phase chromatography method coupled with tandem mass spectrometry was developed and validated for the quantitation of AZD1775, a Wee-1 inhibitor, in human plasma and brain tumor tissue. Sample preparation involved simple protein precipitation with acetonitrile. Chromatographic separation was achieved on ethylene bridged hybrid stationary phases (i.e., Waters XBridge Amide column) under an isocratic elution with the mobile phase consisting of acetonitrile/ammonium formate in water (10mM, pH 3.0) (85:15,v/v) at a flow rate of 0.8mL/min for 5min. The lower limit of quantitation (LLOQ) was 0.2ng/mL of AZD1775 in plasma and tissue homogenate. The calibration curve was linear over AZD1775 concentration range of 0.2-1000ng/mL in plasma and tissue homogenate. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method (<15%). The method was successfully applied to assess the penetration of AZD1775 across the blood-brain tumor barrier, as assessed by the unbound brain-to-plasma ratio, in patients with glioblastoma.

Topics: Brain; Brain Neoplasms; Cell Cycle Proteins; Chromatography, High Pressure Liquid; Glioblastoma; Humans; Limit of Detection; Nuclear Proteins; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Tandem Mass Spectrometry

The strategy of using biologically targeted therapeutics for cancer has yet to translate into effective treatment of gliomas. The neuro-oncology community is beginning to recognize that phase 0 studies should be performed to account for the impact of the blood-brain barrier on the ability of a therapeutic to reach its target(s).

Topics: Brain Neoplasms; Glioblastoma; Humans; Pyrazoles; Pyrimidines; Pyrimidinones

Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR.

Topics: Animals; Biomarkers, Tumor; Brain Neoplasms; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mass Spectrometry; Mice; Molecular Targeted Therapy; Nuclear Proteins; Phosphorylation; Protein-Tyrosine Kinases; Proteomics; Pyrazoles; Pyrimidines; Pyrimidinones; Xenograft Model Antitumor Assays

We investigated the efficacy of the Wee1 inhibitor MK-1775 in combination with radiation for the treatment of pediatric high-grade gliomas (HGGs), including diffuse intrinsic pontine gliomas (DIPGs).. Gene expression analysis was performed for 38 primary pediatric gliomas (3 grade I, 10 grade II, 11 grade III, 14 grade IV) and 8 normal brain samples using the Agilent 4 × 44 K array. Clonogenic survival assays were carried out in pediatric and adult HGG cell lines (n = 6) to assess radiosensitizing effects of MK-1775. DNA repair capacity was evaluated by measuring protein levels of γ-H2AX, a marker of double strand DNA breaks. In vivo activity of MK-1775 with radiation was assessed in 2 distinct orthotopic engraftment models of pediatric HGG, including 1 derived from a genetically engineered mouse carrying a BRAF(V600E) mutation, and 1 xenograft model in which tumor cells were derived from a patient's DIPG.. Wee1 is overexpressed in pediatric HGGs, with increasing expression positively correlated with malignancy (P = .007 for grade III + IV vs I + II) and markedly high expression in DIPG. Combination treatment of MK-1775 and radiation reduced clonogenic survival and increased expression of γ-H2AX to a greater extent than achieved by radiation alone. Finally, combined MK-1775 and radiation conferred greater survival benefit to mice bearing engrafted, orthotopic HGG and DIPG tumors, compared with treatment with radiation alone (BRAF(V600E) model P = .0061 and DIPG brainstem model P = .0163).. Our results highlight MK-1775 as a promising new therapeutic agent for use in combination with radiation for the treatment of pediatric HGGs, including DIPG.

Topics: Animals; Brain Neoplasms; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; Chemoradiotherapy; Glioblastoma; Humans; Mice; Mice, Transgenic; Nuclear Proteins; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones

Topics: Animals; Brain Neoplasms; Cell Cycle Proteins; Glioblastoma; Humans; Nuclear Proteins; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones

The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G(2) checkpoint arrest and modulate radiosensitivity in glioblastoma cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G(2) checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G(2) checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells.

Topics: Animals; Brain Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; G2 Phase Cell Cycle Checkpoints; Glioblastoma; Humans; Mice; Mice, Nude; Molecular Targeted Therapy; Nuclear Proteins; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Radiation Tolerance; Radiation-Sensitizing Agents; Up-Regulation; Xenograft Model Antitumor Assays