Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma

Abstract

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 G 2 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. Ó2016 AACR.

Wee1 Inhibitor AZD1775 Effectively Inhibits the Malignant Phenotypes of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo

“…Increasing evidence showed that AZD1775 can induce significant apoptosis in various cancers including sarcomas (Kreahling et al, 2012), glioblastoma (Lescarbeau et al, 2016), lung cancer (Chen et al, 2017), and gastric cancer (Kim et al, 2016). In agreement with these findings, in our study, we observed that AZD1775 treatment indeed led to an increase in cell apoptosis in ESCC cells, as demonstrated by a dose- and time-dependent increase in annexin-V-binding cells and upregulation of cleaved PARP and caspase-3.…”

“…In the presence of cellular DNA damage, Wee1 phosphorylates CDK1 at Y15 residue, thus preventing the Cyclin B-CDK1 complex from driving cells into mitosis, leading to cell cycle arrest in G 2 phase, allowing time for DNA repair (Russell and Nurse, 1987; Harris et al, 2014; Matheson et al, 2016; Geenen and Schellens, 2017). High expression of Wee1 has been found in several cancer types including acute leukemia (Weisberg et al, 2015), rhabdomyosarcoma (Stewart et al, 2018), glioblastoma (Lescarbeau et al, 2016), lung cancer (Hai et al, 2017; Richer et al, 2017), and head and neck squamous cell carcinoma (Tanaka et al, 2015). Importantly, high expression of Wee1 has been associated with tumor metastasis and poor prognosis (Ge et al, 2017; Yuan et al, 2018).…”

Dual Targeting of WEE1 and PLK1 by AZD1775 Elicits Single Agent Cellular Anticancer Activity

“…We and others have previously observed that AZD1775 does not just exhibit anticancer activity in combination with radiation or chemotherapy, 5–8 but also as a single agent in various tumor types. 9–12 Such single agent activity has been confirmed in early phase clinical trials and appears to be accompanied by a better safety profile than when AZD1775 is administered in combination with cytotoxic chemotherapy drugs or CHK1 kinase inhibitors. 13, 14 However, the underlying mechanism for this is not fully understood considering that single agent AZD1775 activity was found to be independent of TP53 mutational status.…”

Phosphoproteomics of Retinoblastoma: A Pilot Study Identifies Aberrant Kinases

“…Phosphoproteomic approaches have identified targets for early screening and therapy in hepatitis C virus (HCV)-mediated hepatocellular carcinoma [ 13 ]. Phosphoproteomics has been applied to understanding disease progression and therapy in breast, lung, and brain cancers [ 14 , 15 , 16 ].…”