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 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 anti-tumor 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 pre-clinical models of GBM.

Impact of phosphoproteomics in the translation of kinase‐targeted therapies

“…Treatment of cells derived from these tumors with the Wee1 inhibitor MK‐1775 increased DNA damage and lead to apoptotic cell death. Similar results were observed in patient‐derived xenografts treated with this kinase inhibitor . Another study identified a hyperphosphorylation of the kinase SRPK2 and its downstream targets in head and neck squamous cell carcinoma cell lines when compared with an untransformed oral cell line.…”

Overexpression of PKMYT1 Facilitates Tumor Development and Is Correlated with Poor Prognosis in Clear Cell Renal Cell Carcinoma

“…PKMYT1 serves as a negative modulator of the cell cycle, preventing cells from transforming from G2 to the mitosis phase through 2 pathways [ 15 ]. One is binding of PKMYT1 localized to the cytoplasm to the cDC2/cyclin B complex, which restrains the complex from entering the nucleus [ 16 ]. In addition, PKMYT1 suppresses cDC2 activity by phosphorylating the Thr14/Thr15 residue on cDC2 [ 17 – 20 ].…”

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

“…AZD1775 is also used in many studies as a probe to interrogate WEE1 biology as it has been described to display high target selectivity, although details on kinome-wide target inhibition have not been reported. We and others have previously observed that AZD1775 does not exhibit anticancer activity just in combination with radiation or chemotherapy − but also as a single agent in various tumor types. − 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. , 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. − In addition, a recent medicinal chemistry study reported superior antiproliferative single agent activity of AZD1775 compared to other similarly potent WEE1 inhibitors . We hypothesized that these differences could be the result of differential cellular target profiles.…”