Wee1 Gene Editing    

The WEE1 kinase family consists of three serine/threonine kinases, which have conserved molecular structures and are encoded by the following genes: WEE1 (WEE1 G2 Checkpoint Kinase), WEE1B (WEE2 oocyte meiosis inhibiting kinase), and PKMYT1 (membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase). In eukaryotic somatic cells, WEE1 and PKMYT1 play a key role in cell cycle regulation and, especially in the entry into mitosis. As part of the DNA damage response (DDR) pathways, their role as regulators is crucial during normal cell cycle progression and in response to DNA damages.

The Function of WEE1 Kinase

Wee1 is a protein kinase that regulates the G2 checkpoint and prevents DNA damage from entering mitosis. The cell cycle is a highly controlled process. Following its synthesis in the cytoplasm, Wee1 is shuttled into the nucleus by the phosphorylated chaperone protein heat-shock protein 90α (Hsp90α) where it is released from Hsp90α to function on its downstream substrates. In human, activation of AKT kinase through growth factors promotes G2-M cell cycle progression by direct phosphorylation of Wee1 protein on the S642 residue at late S to G2 phase, which binds to 14-3-3θ, leading to cytoplasmic localization of Wee1 kinase associated with inactivation of Wee1 kinase against Cdk1.

Figure 1. Wee1 regulates the G2 checkpoint and prevents entry into mitosis in response to DNA damage. (Geenen J J J, Schellens J H M., 2017)

Wee1 has emerged as a key player in regulating histone synthesis by phosphorylating histone H2B at Y37 in the nucleosomes found upstream of the histone gene cluster to inhibit histone transcription in late S phase and inhibit anaphase onset. Wee1 kinase, as a mitotic gatekeeper and an epigenetic modifier, provides a direct link between epigenetic modulation and cell-cycle progression. Loss of expression or inhibition of Wee1 kinase eliminates H2B Y37 phosphorylation with a concomitant increase in histone transcription by reversing the process to recruit nuclear protein mapped to the ATM locus (NPAT, p220) and RNA polymerase II. Therefore, Wee1 kinase acts as a hub for regulating chromatin integrity by blocking replication initiation, G2-M transition, and histone transcription to allow cells to maintain DNA replication fidelity and chromatin integrity. The anaphase-promoting complex/cyclosome (APC/C), a multi-subunit member of the RING finger family of ubiquitin ligase, mediates Wee1 degradation to allow mitotic progression, because Wee1 delays this progression to maintain genomic integrity.

WEE1 as A Target for Anticancer Therapy

WEE1 is highly expressed in several cancer types, including breast cancers, cervical cancers, hepatocellular carcinoma, lung cancers, squamous cell carcinoma, diffuse intrinsic pontine glioma (DIPG), leukemia, glioblastoma, melanoma, medulloblastoma, and ovarian cancers. High expression of WEE1 has been reported in some cancers in response to increased replication stress, and has been associated with tumor progression and poor rates of disease-free survival. However, in NSCLC the lack of WEE1 expression is associated with a poor prognosis. These studies show that cancers that lack WEE1 expression are susceptible to genetic aberrations and may have increased sensitivity to DNA-damaging agents, and treatment with these agents may induce the expression of WEE1. In contrast, those that express WEE1 may be more reliant on an intact G2–M checkpoint for survival and mitosis. Thus, inhibition of WEE1 kinase activity may sensitize cancers dependent on a functional G2-M checkpoint to DNA-damaging therapy.

Some targeted compounds showed an inhibitory activity on WEE1 and PKMYT1 kinases and their efficacy was proven in many tumor types. The pyridopyrimidine, PD0166285, was the first small-molecule Wee1 inhibitor to be reported. PD0166285 has been used to detect the effect of WEE1 inhibition in cervical cancer, lung cancer, melanoma, colon cancer, ovarian cancer, and hepatocellular carcinoma cell lines, and in glioblastoma cell lines and xenografts, but its use as a WEE1 inhibitor is limited due to its non-selective action at low concentrations. Adavosertib (AZD-1775), a pyrazol-pyrimidine derivate, is a potent and ATP-competitive specific small molecule inhibitor of the Wee1 kinase. A large number of preclinical studies evaluated its efficacy in single agent and in combinatory methods. Regarding the mechanism of action, adavosertib induces S and/or G2/M cell cycle checkpoints override, depending on the type of cancer, when used in monotherapy. Cell cycle perturbation is related to a progressive accumulation of DNA damages and by the induction of apoptosis. In the combined treatment strategy, adavosertib can enhance the cytotoxicity of chemo/radiotherapy agents, by inducing cell cycle checkpoint override, inhibition of DNA damage repair, and induction of apoptosis.

WEE1 Gene Editing Services

CRISPR/Cas9 Platform^CB at Creative Biogene is dedicated to offering comprehensive CRISPR/Cas9 gene editing services and products for academic research, biotech research and pharmaceutical drug discovery. With deep gene editing knowledge and extensive experience in experimental operation and data processing, we help you effectively control WEE1 genes knockout/knockin/point mutation in cells or animals via CRISPR/Cas9 technology.

Service	Details	Alternative cell lines or animal species
WEE1 Gene Editing Cell Line Generation	gRNA design and synthesis Transfect the cell lines you're interested Select the high expression cells and sort monoclonal cell Validate the knockout/knockin/point mutation of WEE1 by PCR and sequencing Provide cryogenically preserved vials of stable cells and final reports	HEK239T, Hela, HepG2, U87, Ba/F3, CHO, MDA-MB-453, MDA-MB-231NIH3T3, T47D, Neuro2a, MCF7, RKO, K562, RAW264.7, etc.
WEE1 Gene Editing Animal Model Generation	WEE1 gene conventional knockout animals WEE1 gene conditional knockout animals WEE1 point mutation animals WEE1 knockin animals	Mouse, rat, rabbit, zebrafish, C. elegans, etc.

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