Wee1 promotes cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1

Abstract

Background The treatment of chronic myeloid leukemia (CML) is facing the dilemma of tyrosine kinase inhibitors (TKIs) resistance and disease recurrence. The dysfunctional DNA damage repair mechanism plays an essential role not only in the initiation and progression of hematological malignancies but also links to the development of TKI resistance. Deciphering the abnormally regulated DNA damage repair and proteins involved brings new insights into the therapy of leukemias. As a G2/M phase checkpoint kinase and a DNA damage repair checkpoint kinase engaged in the DNA damage response (DDR), along with an oncogenic driver present in various cancers, the particular involvement of Wee1 in DNA damage is far from clear. Deciphering its function and targeting it via modulating DNA repair pathways is important for improving our understanding of cancer treatment. Methods Wee1 expression was assessed in cell lines using RT-qPCR and western blot, and Wee1 knockdown efficacy was validated using RT-qPCR, western blot, and immunofluorescence. Wee1 function was investigated by CCK-8, colony formation, and flow cytometry assay in vitro. Wee1 role in DNA repair and its interactions with other proteins were then studied using western blot, immunofluorescence, and double plasmid-repair studies. Finally, the CCK-8 and flow cytometry assay was utilized to investigate Wee1 and imatinib’s synergistic effect, and a CML mouse model was constructed to study Wee1’s role in carcinogenesis in vivo. Results Wee1 was reported to respond quickly to DDR in an ATM-γH2AX-MDC1-dependent way upon DNA double-strand breaks (DSBs) occurrence, and it regulated homologous recombination by stimulating the recruitment of critical proteins RAD51/BRCA1 upon DSB sites. Wee1 was also revealed to be abnormally upregulated in CML cells. Further suppression of Wee1 not only causes cell cycle arrest and inhibits the proliferation of cancer cells but also enhances CML cell sensitivity to Imatinib in vitro and in vivo, possibly through an excessive accumulation of overall DSBs. Conclusion Wee1 is extensively involved in the DRR signaling and DSB repair pathway. Inhibiting abnormally elevated Wee1 benefits CML therapy in both IM-resistant and IM-sensitive cells. Our data demonstrated that Wee1 participated in promoting cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1. In the fight against CML, Wee1’s dysregulation in the DNA damage repair mechanism of CML pathogenesis makes it a viable therapeutic target in clinical applications.

Cellular senescence in cancer: molecular mechanisms and therapeutic targets

“…The phosphorylation of H2A.X, commonly known as γH2A.X, is widely detected in cells undergoing diverse modes of senescence. It is an important sign of DDR activation and necessary for the formation of repair complexes at DSBs 83–85 . Members of the phosphatidylinositol 3‐kinase‐related kinase family (PI3KK), such as ATM–Chk2 and ATR–Chk1 kinases, frequently phosphorylate H2A.X, but may not be the only ones to do so 86 .…”

“…It is an important sign of DDR activation and necessary for the formation of repair complexes at DSBs. 83 , 84 , 85 Members of the phosphatidylinositol 3‐kinase‐related kinase family (PI3KK), such as ATM–Chk2 and ATR–Chk1 kinases, frequently phosphorylate H2A.X, but may not be the only ones to do so. 86 ATM is generally activated by the binding of the MRE11–RAD50–NBS1 complex to DNA breaks.…”

Cyclin E1/CDK2 Activation Defines a Key Vulnerability to WEE1 Kinase Inhibition in Gynecological Cancers

“…It has also been reported that WEE1 can directly regulate Mus81-Eme1 endonuclease activity at stalled replication forks to prevent formation of DSBs (26)(27)(28). As a crucial component of the G2/M checkpoint, WEE1 prevents cells from entering mitosis in response to cellular DNA damage through inhibitory phosphorylation of CDK1 (27,29). In normal cells, DNA damage activates cell cycle checkpoints that arrest cells at G1 and G2 phase to repair DNA, however G1/S checkpoint defects often exist in cancer cells, preventing adequate repair.…”

NDRG3 regulates imatinib resistance by promoting β‑catenin accumulation in the nucleus in chronic myelogenous leukemia

“…This inhibits the autophosphorylation of BCR-ABL, prevents its activation and blocks its downstream signaling (5). However, IM drug resistance and intolerance remain an issue in certain individuals, contributing to recurrence after treatment discontinuation (6). The N-Myc downstream-regulated gene (NDRG) family, which has four members, is functionally involved in multiple biological behaviors and can be used as a biomarker for various types of diseases, including prostate cancer, nervous system diseases and liver damage (7)(8)(9).…”