Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Abstract

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. 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 MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.

The Efficacy of the Wee1 Inhibitor MK-1775 Combined with Temozolomide Is Limited by Heterogeneous Distribution across the Blood–Brain Barrier in Glioblastoma

“…Endonuclease-mediated cleavage of chicken-foot DNA structures associated with stalled replication forks then leads to replication associated DNA double-strand breaks (25). Consistent with this model, MK-1775 monotherapy is associated with induction of pan-nuclear γH2AX staining in this current report and several other studies, and a similar phenotype is seen with Wee1 siRNA (7, 20–22). Interestingly, Myc overexpression is associated increased replicative stress (26, 27), and the highly sensitive GBM22 xenograft line from this study harbors high level Myc amplification (unpublished data).…”

“…Consistent with the presence of replicative stress in this model, GBM22 cells are highly aneuploid and exhibited marked basal elevation in γH2AX staining that was further accentuated with MK-1775 treatment. In a neurosphere assay, which provides results similar to a clonogenic assay and is generally more sensitive than the CyQuant assay for assessment of cytotoxicity, GBM22 cells (IC 50 of 12 nM; Figure 1C) were exquisitely sensitive to single agent MK-1775 as compared to previously published studies in sarcoma and neuroblastoma with IC 50 s ranging from 100 to over 1000 nM (7, 9, 22). In a transgenic model of neuroblastomas, homozygous expression of the MYCN oncogene was associated with an almost 3 fold-reduction in IC 50 (62 nM) as compared to heterozygous expression of MYCN (161 nM) (9).…”

“…Previous studies have suggested that MK-1775 can induce DNA damage (7, 20–22), and consistent with the differential sensitivity observed, treatment of GBM6 cells with 300 nM MK-1775 had a modest impact on the total number of cells staining positive for γH2AX (Figure 2A and B; 3.8 ± 1.1% DMSO control vs. 20.5 ± 6.5% MK-1775 treated; p=0.04) and had no significant impact on cells exhibiting pan-nuclear γH2AX staining (0% untreated vs. 2.6 ± 2.2% treated; p=0.22). While pan-nuclear staining was uncommon in GBM6 cells, the majority of GBM22 cells with γH2AX staining exhibited pan-nuclear staining, and the fraction of cells with pan-nuclear staining significantly increased after MK-1775 treatment (72.2 ± 4.5%) as compared to controls (30.2 ± 6.1%; p=0.0007).…”

Wee-1 Kinase Inhibition Overcomes Cisplatin Resistance Associated with High-RiskTP53Mutations in Head and Neck Cancer through Mitotic Arrest Followed by Senescence

“…It has been shown that unscheduled mitosis results in micro-nuclei formation and apoptosis in tumor cells treated with gemcitabine and MK-1775 (32, 33). Therefore, we tested whether sensitization of the isogenic PCI-13 cells to cisplatin by addition of MK-1775 led to apoptosis induction.…”

Synergistic anti-leukemic interactions between panobinostat and MK-1775 in acute myeloid leukemia ex vivo

“…Therefore, inhibition of checkpoint kinases in combination with DNA damaging agents may become a therapeutic strategy for the treatment of various malignancies. [15][16][17][18] Histone deacetylase (HDAC) inhibitors (HDACIs) have demonstrated anticancer activity in numerous malignancies including AML. [19][20][21][22][23][24][25][26] They have been demonstrated to induce cell cycle arrest, differentiation, and apoptosis in cancer cells, but less so in normal cells.…”