MK1775, a Selective Wee1 Inhibitor, Shows Single-Agent Antitumor Activity against Sarcoma Cells

Abstract

Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by a selective small molecule inhibitor MK1775 can abrogate G2/M checkpoint resulting in premature mitotic entry and cell death. MK1775 has recently been tested in preclinical and clinical studies of human carcinoma to enhance the cytotoxic effect of DNA damaging agents. However, its role in mesenchymal tumors, especially as a single agent has not been explored. Here we studied the cytotoxic effect of MK1775 in various sarcoma cell lines and patient-derived tumor explants ex vivo. Our data demonstrate that MK1775 treatment at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. In MK1775 treated cells CDC2 activity was enhanced, as determined by decreased inhibitory phosphorylation of tyrosine-15 residue and increased expression of phosphorylated histone H3, a marker of mitotic entry. The cytotoxic effect of Wee1 inhibition on sarcoma cells appears to be independent of p53 status as all sarcoma cell lines with different p53 mutation were highly sensitive to MK1775 treatment. Finally, in patient-derived sarcoma samples we showed that MK1775 as a single agent causes significant apoptotic cell death suggesting that Wee1 inhibition may represent a novel approach in the treatment of sarcomas.

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

“…S1B). Also, Wee1 inhibition resulted in reduced levels of Cdk2 phosphorylation at Tyr-15, and elevated γ-H2AX levels as described (25). MK-1775-induced cell death was visible from 6 h of treatment onwards, as evidenced by PARP cleavage (Fig.…”

“…4). Multinucleation upon Wee1 inhibition was previously noted, although it was suggested that these effects might have resulted from S-phase defects (25,44). However, our data show that the aberrant mitotic exit was not caused indirectly by defective S-phase progression, because addition of Wee1 inhibitor during release from a nocodazole-induced mitotic arrest showed near-complete cytokinesis failure (Fig.…”

“…Inhibition of Wee1 potentiates DNA-damaging chemotherapeutics and radiotherapy and has cytotoxic effects as a single agent (19)(20)(21)(22). The consensus view is that Wee1 inhibition facilitates tumor cell killing through G 2 /M checkpoint inactivation, which would catalyze mitotic catastrophe (23)(24)(25). As expected, Wee1 inhibition is synergistic with DNA-damaging agents, specifically in TP53-mutant cancer cell lines (3).…”

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

“…MK-1775 monotherapy has been previously documented by Kreahling et al (30) where tumor explants treated with 500 nM MK-1775 for 24 hours demonstrated decreased CDK1 phosphorylation and features of cell death. Building on these results, here we have used multiple tumor derived cell lines and a flank PDX model to assess the effectiveness of monotherapy with MK-1775 in in vitro and in vivo models of GBM with a range of characteristics, Table 1.…”

“…At 1 µM MK-1775 treatment, the cell counts were 17.8% (p=1.3E-6) and 15.7% (p=3.6E-6) of the DMSO control treatment for MGPP6 and MGPP7, respectively. This dose was used throughout, similar to a study treating sarcoma cells with MK-1775 as a single agent at 500 nM (30). Treatment with MK-1775 effectively decreased levels of phosphorylation at CDK1 Y15, indicating on-target inhibition of Wee1, Figure 3D and Supplementary Figure S4B.…”