CHK1 plays a critical role in the anti-leukemic activity of the wee1 inhibitor MK-1775 in acute myeloid leukemia cells

Wenxiu Qi,J Timothy Caldwell,Holly Edwards,Hai Lin,Jeffrey W Taub,Yubin Ge,Chunhuai Li,Chengzhi Xie,Yue Wang

doi:10.1186/s13045-014-0053-9

Abstract

BackgroundAcute myeloid leukemia (AML) remains a difficult disease to treat and requires new therapies to improve treatment outcome. Wee1 inhibitors have been used to prevent activation of the G2 cell cycle checkpoint, thus enhancing the antitumor activity of DNA damaging agents. In this study, we investigated MK-1775 in AML cell lines and diagnostic blast samples to identify sensitive subtypes as well as possible mechanisms of resistance.MethodsIn vitro MK-1775 cytotoxicities of AML cell lines and diagnostic blasts were measured using MTT assays. The effects of MK-1775 on cell cycle progression and related proteins were determined by propidium iodide (PI) staining and flow cytometry analysis and Western blotting. Drug-induced apoptosis was determined using annexin V/PI staining and flow cytometry analysis.ResultsWe found that newly diagnosed and relapsed patient samples were equally sensitive to MK-1775. In addition, patient samples harboring t(15;17) translocation were significantly more sensitive to MK-1775 than non-t(15;17) samples. MK-1775 induced apoptosis in both AML cell lines and diagnostic blast samples, accompanied by decreased phosphorylation of CDK1 and CDK2 on Tyr-15 and increased DNA double-strand breaks (DSBs). Time-course experiments, using AML cell lines, revealed a time-dependent increase in DNA DSBs, activation of CHK1 and subsequent apoptosis following MK-1775 treatment, which could be attenuated by a CDK1/2 inhibitor, Roscovitine. Simultaneous inhibition of CHK1 and Wee1 resulted in synergistic anti-leukemic activity in both AML cell lines and primary patient samples ex vivo.ConclusionsOur study provides compelling evidence that CHK1 plays a critical role in the anti-leukemic activity of MK-1775 and highlights a possible mechanism of resistance to MK-1775. In addition, our study strongly supports the use of MK-1775 to treat both newly diagnosed and relapsed AML, especially cases with t(15;17) translocation, and supports the development of combination therapies with CHK1 inhibitors.

Highlights

Acute myeloid leukemia (AML) is a challenging disease to treat, with overall survival rates of 65% for children and 25% for adults [1,2]
Our study suggests that MK-1775 treatment induces DNA damage which activates CHK1, CHK1 phosphorylates CDC25, inhibiting the dephosphorylation of CDK1/ 2, countering the effects of MK-1775
MK-1775 induces DNA damage and apoptosis in AML cells To investigate the effects of MK-1775 in AML cells, first we determined the protein levels of Wee1, p-CDK1, CDK1, p-CDK2, CDK2 and MYT1 in six AML cell lines

Summary

Introduction

Acute myeloid leukemia (AML) is a challenging disease to treat, with overall survival rates of 65% for children and 25% for adults [1,2]. One mechanism of chemotherapy resistance is the induction of G2/M cell cycle arrest, which allows cells to repair and survive DNA damage [3]. Wee kinase is a cell cycle checkpoint protein whose primary function is inhibitory phosphorylation of CDK1 and CDK2 on Tyr, preventing progression through G2/M and S phase, respectively [4,5]. Upon sensing DNA damage or replication stress ATR phosphorylates CHK1, activating it and eventually leading to S and G2 cell cycle arrest, allowing for DNA repair [8,9]. Acute myeloid leukemia (AML) remains a difficult disease to treat and requires new therapies to improve treatment outcome. We investigated MK-1775 in AML cell lines and diagnostic blast samples to identify sensitive subtypes as well as possible mechanisms of resistance

Methods

Results

Conclusion