Inhibition of WEE1 Induces Cell Death of Both Bortezomib- and Lenalidomide-Resistant Multiple Myeloma Cells: A Novel Therapeutic Approach Targeting Cell-Cycle Checkpoint Kinase
Authors: Tabayashi Takayuki , Tanaka Yuka , Takahashi Yasuyuki , Kimura Yuta , Tomikawa Tatsuki , Sagawa Morihiko , Anan-Nemoto Tomoe , Watanabe Reiko , Tokuhira Michihide , Kizaki Masahiro 
Published: October 2019

Abstract
Multiple myeloma (MM) is a hematological malignancy that derives from the proliferation of unregulated plasma cells. Dramatic improvement in the clinical outcomes of both newly diagnosed and relapsed/refractory patients with MM has been achieved using many clinical approaches, including use of high-dose chemotherapy followed by hematopoietic stem cell transplantation, and new drugs, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors. However, most patients eventually relapse and develop drug resistance. Moreover, the prognosis of patients with bortezomib (BTZ) and/or lenalidomide (LEN)-resistant MM (key drugs in the treatment of MM) is very poor. Therefore, novel therapeutic approaches to overcome BTZ and LEN resistance are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 catalyzes inhibitory phosphorylation of both cyclin-dependent kinase1 and 2 (CDK1 and CDK2), leading to CDK1- and CDK2-induced cell cycle arrest at the G1, S, or G2-M phases. This cell-cycle arrest, in turn, allows for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells harboring unrepaired damaged DNA from mitotic lethality. Furthermore, recent research has shown that knockdown of WEE1 leads to DNA double-strand breaks specifically in S-phase cells undergoing DNA replication, and that WEE1 is most active in the S-phase, suggesting that WEE1 is involved in DNA synthesis. Overexpression of WEE1 has been observed in many types of cancers, including hepatic cancer, breast cancer, glioblastoma and gastric cancer, and high expression of WEE1 has been shown to correlate with poor prognosis. In addition, research has shown that inhibition of checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response, potentiates the cytotoxicity of chemotherapy on p53-deficient MM cells, which are regarded as chemotherapy-resistant, suggesting that inhibition of cell-cycle checkpoint kinase is involved in re-sensitization of refractory MM cells to anticancer drugs. These data suggest that WEE1 might be an attractive target for novel therapeutic agents against this incurable hematological malignancy. MK-1775 is a potent and highly-selective small-molecule inhibitor of WEE1. In the present study, we investigated the role of WEE1 in MM as a potential therapeutic target using MK-1775. MTSassays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including the intrinsically LEN-resistant cell line, RPMI-8226, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. Furthermore, the growth inhibition effect is irrespective of p53 status. To examine the mechanisms behind the growth inhibition effect induced by MK-1775, assays for apoptotic cell death were performed. These assays demonstrated that MK-1775 induces both early and late apoptosis in MM cells. To investigate the molecular mechanisms of MK-1775-induced cell death in MM cells, the expression of various cell death-associated proteins and downstream molecules of WEE1 were examined. Western blotting analysis showed that MK-1775 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by inhibiting both, the expression of the target molecules of Bcl-2 and MCL1, and the cleavage of PARP and Caspase 3. Similarly, there was a substantial inhibition of CDK1 phosphorylation downstream of WEE1. Moreover, an increased expression of histone H2AX was observed following administration of MK-1775, suggesting that MK-1775 results in cytotoxicity by direct DNA damage. Next, we examined the effects of MK-1775 on BTZ-resistant MM cells. Interestingly, MK-1775 inhibited the proliferation of both BTZ-sensitive wild-type MM cells and BTZ-resistant MM cells, suggesting that BTZ resistance can be overcome by targeting WEE1. Furthermore, in combination with BTZ, MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that inhibition of WEE1 might serve as an attractive therapeutic option for patients with both BTZ-resistant and LEN-resistant MM. In conclusion, our data suggest that WEE1 might be a promising molecular target for the treatment of MM.

Disclosures
Tokuhira: Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria.