WEE1 kinase inhibitor MK-1775 sensitizes oral tongue squamous cell carcinoma cells to radiation irrespective of TP53 status.

OBJECTIVE: Oral tongue squamous cell carcinoma (OTSCC) frequently harbors non-functional p53 and depends on G2/M checkpoint mediated by WEE1. WEE1 suppression has been identified as a promising anti-tumor strategy. This study investigated the capacity of WEE1 kinase inhibitor (MK-1775) and its underlying mechanisms in enhancing radiation responses of OTSCC cells in vitro. MATERIALS AND METHODS: WEE1 kinase expression and its downstream target (CDK1) were investigated in OTSCC versus normal oral tissue. A synergistic combination of MK-1775 with radiation on OTSCC cell lines with different p53 statuses was assessed by viability assay. The radio-sensitizing effects of MK-1775 on apoptosis, cell cycle, DNA damage, and mitotic entry were also determined. RESULTS: Irradiation enhanced CDK1 expression in all tested cell lines, though the effect was far more pronounced in p53 mutated cell lines. MK-1775 exhibited inhibitory effects against the survival of all cell lines and enhanced their response to the radiation. These effects were strongly elicited by induction of apoptosis and lethal mitosis, but less likely by abrogation of radiation-induced G2 arrest. CONCLUSION: These results demonstrate the efficacy of MK-1775 in enhancing the radiation effect on OTSCC in vitro associated with a significant apoptotic death rate, identifying WEE1 inhibitor as a potent radiosensitizer in OTSCC irrespective of p53 mutational status.

Wee1 Kinase: A Potential Target to Overcome Tumor Resistance to Therapy.

During the cell cycle, DNA suffers several lesions that need to be repaired prior to entry into mitosis to preserve genome integrity in daughter cells. Toward this aim, cells have developed complex enzymatic machinery, the so-called DNA damage response (DDR), which is able to repair DNA, temporarily stopping the cell cycle to provide more time to repair, or if the damage is too severe, inducing apoptosis. This DDR mechanism is considered the main source of resistance to DNA-damaging therapeutic treatments in oncology. Recently, cancer stem cells (CSCs), which are a small subset of tumor cells, were identified as tumor-initiating cells. CSCs possess self-renewal potential and persistent tumorigenic capacity, allowing for tumor re-growth and relapse. Compared with cancer cells, CSCs are more resistant to therapeutic treatments. Wee1 is the principal gatekeeper for both G2/M and S-phase checkpoints, where it plays a key role in cell cycle regulation and DNA damage repair. From this perspective, Wee1 inhibition might increase the effectiveness of DNA-damaging treatments, such as radiotherapy, forcing tumor cells and CSCs to enter into mitosis, even with damaged DNA, leading to mitotic catastrophe and subsequent cell death.

The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls.

Galls induced by plant-parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident. We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode-induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1-knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1-knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root-knot nematodes. Together with data on DNA-damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls.

WEE1 inhibition and genomic instability in cancer.

One of the hallmarks of cancer is genomic instability controlled by cell cycle checkpoints. The G1 and G2 checkpoints allow DNA damage responses, whereas the mitotic checkpoint enables correct seggregation of the sister chromosomes to prevent aneuploidy. Cancer cells often lack a functional G1 arrest and rely on G2 arrest for DNA damage responses. WEE1 kinase is an important regulator of the G2 checkpoint and is overexpressed in various cancer types. Inhibition of WEE1 is a promising strategy in cancer therapy in combination with DNA-damaging agents, especially when cancer cells harbor p53 mutations, as it causes mitotic catastrophy when DNA is not repaired during G2 arrest. Cancer cell response to WEE1 inhibition monotherapy has also been demonstrated in various types of cancer, including p53 wild-type cancers. We postulate that chromosomal instability can explain tumor response to WEE1 monotherapy. Therefore, chromosomal instability may need to be taken into account when determining the most effective strategy for the use of WEE1 inhibitors in cancer therapy.

Single-agent Adavosertib Shows Anticancer Effects Against Colorectal Cancer Cells.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer-related deaths worldwide. Adavosertib (AZD1775), a small molecule inhibitor of WEE1 kinase, abrogates G2/M cell cycle arrest and induces double-stranded DNA breaks. According to previous findings, adavosertib, in combination with other DNA-damaging agents, causes premature mitosis and cell death in p53-mutated cancer cells mainly via abrogation of the G2/M cell cycle checkpoint. This study aims to evaluate the inhibition of WEE1 kinase by adavosertib as monotherapy in the TP53-wildtype human CRC cell line HCT116. MATERIALS AND METHODS: In this study, HCT116 cells were treated with different concentrations of adavosertib for 24 to 72 hours. Cell viability was assessed by Water-Soluble Tetrazolium 1 (WST-1) assay and crystal violet assays. Cell migration was evaluated by the wound healing assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The IC50 value of adavosertib for the HCT116 cell line was 0.1310 µM. Adavosertib monotherapy (both 0.125 and 0.250 µM) significantly reduced cell viability, inhibited cell migration and abrogated intra-S phase cell cycle arrest. In addition, 0.250 µM of adavosertib significantly induced apoptosis in HCT116 cells. CONCLUSION: Adavosertib effectively inhibits the TP53-wildtype HCT116 cells via the abrogation of intra-S phase cell cycle arrest. Our findings suggest that adavosertib monotherapy may be a potential targeted therapy for CRC.

Insightful t-SNE guided exploration spotlighting Palbociclib and Ribociclib analogues as novel WEE1 kinase inhibitory candidates.

In the era of targeted therapeutics, protein kinases like WEE1 have become pivotal drug targets, especially for cancer therapy. Utilizing a multi-faceted approach, our study adds fresh insights to this endeavour. We employed the t-SNE algorithm, combined with ECFP4 fingerprints, to analyse the molecular similarity between FDA-approved drugs and known clinical trial inhibitors. Our t-SNE analysis identified the closest clusters to known inhibitors and selected 11 FDA-approved drugs for further study. Using the DrugSpaceX platform, we generated analogues for these 11 FDA-approved drugs. These analogues were refined according to Lipinski's Rule of Five and Synthetic Accessibility scores, yielding 68,640 analogues for additional scrutiny. Among these, derivatives of Palbociclib and Ribociclib stood out as the most promising WEE1 inhibitors, based on docking scores and interaction patterns. Molecular dynamics simulations validated the stability of these protein-ligand interactions, particularly for DE50607359, a top-ranked Palbociclib analogue, which also met most pharmacokinetic parameters within acceptable limits. Our study uncovers new candidates for WEE1 inhibition not previously reported. With our multi-layered computational strategy, we provide a solid foundation for future experimental validation and targeted drug development in cancer therapeutics.Communicated by Ramaswamy H. Sarma.

Employed the t-SNE algorithm and ECFP4 fingerprints to discern molecular similarities between FDA-approved drugs and known clinical trial inhibitors, identifying 11 key drugs.Leveraged the DrugSpaceX platform to generate analogues for these selected FDA-approved drugs, yielding a massive collection of 68,640 refined analogues based on Lipinski's Rule of Five and Synthetic Accessibility scores.Derivatives of Palbociclib and Ribociclib emerged as the most promising WEE1 inhibitors, supported by their docking scores and interaction patterns.Validated protein-ligand interactions through molecular dynamics simulations, spotlighting DE50607359, a superior Palbociclib analogue, meeting critical pharmacokinetic parameters.

Broadening the scope of WEE1 inhibitors: identifying novel drug candidates via computational approaches and drug repurposing.

The protein kinase Wee1 plays a vital role in the G2/M cell cycle checkpoint activation, triggered by double-stranded DNA disruptions. It fulfills this task by phosphorylating and consequently deactivating the cyclin B linked to Cdk1/Cdc2 at the Tyr15 residue, leading to a G2 cell cycle halt and subsequent delay of mitosis post DNA damage. Despite advancements, only the Wee1 inhibitor MK1775 has made it to Phase II clinical trials, presenting a challenge in innovative chemical structure development for small molecule discovery. To navigate this challenge, we employed an e-pharmacophore model of the MK1775-WEE1 complex (PDB ID: 5V5Y), using in silico screening of FDA-approved drugs. We chose six drugs for analog creation, guided by docking scores, key residue interactions, and ligand occupancy. Utilizing the 'DrugSpaceX' database, we generated 2,776 analogues via expert-defined transformations. Our findings identified DE90612 as the top-ranked analogue, followed by DE363106, DE489678, DE395383, DE90548, DE689343, DE395019, and DE538066. These analogues introduced unique structures not found in other databases. A t-SNE structurally diversified distribution map unveiled promising transformations linked to Temozolomide for WEE1 inhibitor development. Simulations of the WEE1-DE90612 complex (a Temozolomide analogue) for 200 nanoseconds demonstrated stability, with DE90612 forging robust bonds with active site residues and sustaining vital contacts at ASN376 and CYS379. These results underscore DE90612's potential inhibitory properties at the WEE1 binding site, warranting additional in vitro and in vivo exploration for its anticancer activity. Our approach outlines a promising pathway for creating diverse WEE1 inhibitors with suitable biological properties for potential oncology therapeutics.Communicated by Ramaswamy H. Sarma.

Cell cycle involvement in cancer therapy; WEE1 kinase, a potential target as therapeutic strategy.

Mitosis is the process of cell division and is regulated by checkpoints in the cell cycle. G1-S, S, and G2-M are the three main checkpoints that prevent initiation of the next phase of the cell cycle phase until previous phase has completed. DNA damage leads to activation of the G2-M checkpoint, which can trigger a downstream DNA damage response (DDR) pathway to induce cell cycle arrest while the damage is repaired. If the DNA damage cannot be repaired, the replication stress response (RSR) pathway finally leads to cell death by apoptosis, in this case called mitotic catastrophe. Many cancer treatments (chemotherapy and radiotherapy) cause DNA damages based on SSBs (single strand breaks) or DSBs (double strand breaks), which cause cell death through mitotic catastrophe. However, damaged cells can activate WEE1 kinase (as a part of the DDR and RSR pathways), which prevents apoptosis and cell death by inducing cell cycle arrest at G2 phase. Therefore, inhibition of WEE1 kinase could sensitize cancer cells to chemotherapeutic drugs. This review focuses on the role of WEE1 kinase (as a biological macromolecule which has a molecular mass of 96 kDa) in the cell cycle, and its interactions with other regulatory pathways. In addition, we discuss the potential of WEE1 inhibition as a new therapeutic approach in the treatment of various cancers, such as melanoma, breast cancer, pancreatic cancer, cervical cancer, etc.

Inhibitors of cell cycle checkpoint target Wee1 kinase - a patent review (2003-2022).

INTRODUCTION: DNA damage repair in most malignancies with mutation of p53 is more dependent on the G2/M checkpoint. Wee1 kinase is a key regulator of the G2/M checkpoint. If Wee1 is inhibited, it results in cells with unrepaired DNA damage entering mitosis prematurely, leading to mitotic catastrophe and subsequent cell death via the apoptotic program. Therefore, inhibition of Wee1 kinase which overexpressed in several cancer cell lines has emerged as a promising therapy for cancer treatment. AREAS COVERED: This review summarizes for the first time the structures of small-molecule inhibitors of Wee1 reported in patents published from 2003 to 2022 and the recent clinical developments. It also provides perspectives on the challenges and the future directions. We used different methods to search different databases (PubMed, Reaxys, clinicaltrials.gov)for the literature we needed. EXPERT OPINION: Although the small-molecule inhibitors of Wee1, Adavosertib, and ZN-C3 have entered the clinical phase II, the clinical toxicity exhibited by Adavosertib remains the subject of greater concern. The use of Wee1 inhibitors as monotherapy or in combination therapy remains the main trend in Wee1 inhibitors at present.

Polo-like kinase-1, Aurora kinase A and WEE1 kinase are promising druggable targets in CML cells displaying BCR::ABL1-independent resistance to tyrosine kinase inhibitors.

In chronic myeloid leukemia (CML), Aurora kinase A and Polo like kinase 1 (PLK1), two serine-threonine kinases involved in the maintenance of genomic stability by preserving a functional G2/M checkpoint, have been implicated in BCR::ABL1-independent resistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate and in leukemic stem cell (LSC) persistence. It can be speculated that the observed deregulated activity of Aurora A and Plk1 enhances DNA damage, promoting the occurrence of additional genomic alterations contributing to TKI resistance and ultimately driving progression from chronic phase to blast crisis (BC). In this study, we propose a new therapeutic strategy based on the combination of Aurora kinase A or PLK1 inhibition with danusertib or volasertib, respectively, and WEE1 inhibition with AZD1775. Danusertib and volasertib used as single drugs induced apoptosis and G2/M-phase arrest, associated with accumulation of phospho-WEE1. Subsequent addition of the WEE1 inhibitor AZD1775 in combination significantly enhanced the induction of apoptotic cell death in TKI-sensitive and -resistant cell lines as compared to both danusertib and volasertib alone and to the simultaneous combination. This schedule indeed induced a significant increase of the DNA double-strand break marker γH2AX, forcing the cells through successive replication cycles ultimately resulting in apoptosis. Finally, combination of danusertib or volasertib+AZD1775 significantly reduced the clonogenic potential of CD34+ CML progenitors from BC patients. Our results may have implications for the development of innovative therapeutic approaches aimed to improve the outcomes of patients with multi-TKI-resistant or BC CML.

Computational simulation studies on the binding selectivity of Wee1 and Checkpoint kinase 1 by molecular dynamics simulation combined with free energy calculations.

Wee1 kinase and Checkpoint kinase 1 (Chk1) kinase, which are well known to be involved in cancer, are promising targets for cancer therapy. Most of developed Wee1 inhibitors can inhibit activity of Chk1 kinase to different degrees as well. The poor selectivity brought side effects and selective inhibitor is needed. However, the selective mechanisms of Wee1 versus Chk1 are not clear. Therefore, the design of selective Wee1 and Chk1 inhibitors would provide a meaningful starting for the development of anticancer drugs with optimal efficacy. In this study, Wee1 inhibitors with different selectivity over Chk1 were chosen to analyze the selectivity mechanism by means of molecular docking, molecular dynamics simulations and binding free energy calculations. Two key residues of Wee1 kinase and two critical residues of Chk1 were mutated to detect their effect on ligand binding into protein. The results indicated that these residues play a pivotal role in the binding interactions of ligands to receptors through hydrogen bond and hydrophobic interaction with inhibitors. This may provide a better understanding of the selective mechanism of Wee1 and Chk1. It would be beneficial to the discovery and optimization of selective Wee1 and Chk1 inhibitors.Communicated by Ramaswamy H. Sarma.

Targeting Wee1 kinase as a therapeutic approach in Hematological Malignancies.

Hematologic malignancies include various diseases that develop from hematopoietic stem cells of bone marrow or lymphatic organs. Currently, conventional DNA-damage-based chemotherapy drugs are approved as standard therapeutic regimens for these malignancies. Although many improvements have been made, patients with relapsed or refractory hematological malignancies have a poor prognosis. Therefore, novel and practical therapeutic approaches are required for the treatment of these diseases. Interestingly several studies have shown that targeting Wee1 kinase in the Hematological malignancies, including AML, ALL, CML, CLL, DLBCL, BL, MCL, etc., can be an effective therapeutic strategy. It plays an essential role in regulating the cell cycle process by abrogating the G2-M cell-cycle checkpoint, which provides time for DNA damage repair before mitotic entry. Consistently, Wee1 overexpression is observed in various Hematological malignancies. Also, in healthy normal cells, repairing DNA damages occurs due to G1-S checkpoint function; however, in the cancer cells, which have an impaired G1-S checkpoint, the damaged DNA repair process depends on the G2-M checkpoint function. Thus, Wee1 inhibition could be a promising target in the presence of DNA damage in order to potentiate multiple therapeutic drugs. This review summarized the potentials and challenges of Wee1 inhibition combined with other therapies as a novel effective therapeutic strategy in Hematological malignancies.

Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells.

Inhibitors of WEE1 and ATR kinases are considered promising for cancer treatment, either as monotherapy or in combination with chemo- or radiotherapy. Here, we addressed whether simultaneous inhibition of WEE1 and ATR might be advantageous. Effects of the WEE1 inhibitor MK1775 and ATR inhibitor VE822 were investigated in U2OS osteosarcoma cells and in four lung cancer cell lines, H460, A549, H1975, and SW900, with different sensitivities to the WEE1 inhibitor. Despite the differences in cytotoxic effects, the WEE1 inhibitor reduced the inhibitory phosphorylation of CDK, leading to increased CDK activity accompanied by ATR activation in all cell lines. However, combining ATR inhibition with WEE1 inhibition could not fully compensate for cell resistance to the WEE1 inhibitor and reduced cell viability to a variable extent. The decreased cell viability upon the combined treatment correlated with a synergistic induction of DNA damage in S-phase in U2OS cells but not in the lung cancer cells. Moreover, less synergy was found between ATR and WEE1 inhibitors upon co-treatment with radiation, suggesting that single inhibitors may be preferable together with radiotherapy. Altogether, our results support that combining WEE1 and ATR inhibitors may be beneficial for cancer treatment in some cases, but also highlight that the effects vary between cancer cell lines.

Targeting Replicative Stress and DNA Repair by Combining PARP and Wee1 Kinase Inhibitors Is Synergistic in Triple Negative Breast Cancers with Cyclin E or BRCA1 Alteration.

The identification of biomarker-driven targeted therapies for patients with triple negative breast cancer (TNBC) remains a major clinical challenge, due to a lack of specific targets. Here, we show that cyclin E, a major regulator of G1 to S transition, is deregulated in TNBC and is associated with mutations in DNA repair genes (e.g., BRCA1/2). Breast cancers with high levels of cyclin E not only have a higher prevalence of BRCA1/2 mutations, but also are associated with the worst outcomes. Using several in vitro and in vivo model systems, we show that TNBCs that harbor either mutations in BRCA1/2 or overexpression of cyclin E are very sensitive to the growth inhibitory effects of AZD-1775 (Wee 1 kinase inhibitor) when used in combination with MK-4837 (PARP inhibitor). Combination treatment of TNBC cell lines with these two agents results in synergistic cell killing due to induction of replicative stress, downregulation of DNA repair and cytokinesis failure that results in increased apoptosis. These findings highlight the potential clinical application of using cyclin E and BRCA mutations as biomarkers to select only those patients with the highest replicative stress properties that may benefit from combination treatment with Wee 1 kinase and PARP inhibitors.

Inhibition of AKT Signaling Alters ßIV Spectrin Distribution at the AIS and Increases Neuronal Excitability.

The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, ßIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing ßIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on ßIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in ßIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.

Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex.

Voltage-gated Na+ (Nav) channels are a primary molecular determinant of the action potential (AP). Despite the canonical role of the pore-forming α subunit in conferring this function, protein-protein interactions (PPI) between the Nav channel α subunit and its auxiliary proteins are necessary to reconstitute the full physiological activity of the channel and to fine-tune neuronal excitability. In the brain, the Nav channel isoforms 1.2 (Nav1.2) and 1.6 (Nav1.6) are enriched, and their activities are differentially regulated by the Nav channel auxiliary protein fibroblast growth factor 14 (FGF14). Despite the known regulation of neuronal Nav channel activity by FGF14, less is known about cellular signaling molecules that might modulate these regulatory effects of FGF14. To that end, and building upon our previous investigations suggesting that neuronal Nav channel activity is regulated by a kinase network involving GSK3, AKT, and Wee1, we interrogate in our current investigation how pharmacological inhibition of Wee1 kinase, a serine/threonine and tyrosine kinase that is a crucial component of the G2-M cell cycle checkpoint, affects the Nav1.2 and Nav1.6 channel macromolecular complexes. Our results show that the highly selective inhibitor of Wee1 kinase, called Wee1 inhibitor II, modulates FGF14:Nav1.2 complex assembly, but does not significantly affect FGF14:Nav1.6 complex assembly. These results are functionally recapitulated, as Wee1 inhibitor II entirely alters FGF14-mediated regulation of the Nav1.2 channel, but displays no effects on the Nav1.6 channel. At the molecular level, these effects of Wee1 inhibitor II on FGF14:Nav1.2 complex assembly and FGF14-mediated regulation of Nav1.2-mediated Na+ currents are shown to be dependent upon the presence of Y158 of FGF14, a residue known to be a prominent site for phosphorylation-mediated regulation of the protein. Overall, our data suggest that pharmacological inhibition of Wee1 confers selective modulatory effects on Nav1.2 channel activity, which has important implications for unraveling cellular signaling pathways that fine-tune neuronal excitability.

Identifying and Overcoming Mechanisms of PARP Inhibitor Resistance in Homologous Recombination Repair-Deficient and Repair-Proficient High Grade Serous Ovarian Cancer Cells.

High grade serous ovarian cancer (HGSOC) is a major cause of female cancer mortality. The approval of poly (ADP-ribose) polymerase (PARP) inhibitors for clinical use has greatly improved treatment options for patients with homologous recombination repair (HRR)-deficient HGSOC, although the development of PARP inhibitor resistance in some patients is revealing limitations to outcome. A proportion of patients with HRR-proficient cancers also benefit from PARP inhibitor therapy. Our aim is to compare mechanisms of resistance to the PARP inhibitor olaparib in these two main molecular categories of HGSOC and investigate a way to overcome resistance that we considered particularly suited to a cancer like HGSOC, where there is a very high incidence of TP53 gene mutation, making HGSOC cells heavily reliant on the G2 checkpoint for repair of DNA damage and survival. We identified alterations in multiple factors involved in resistance to PARP inhibition in both HRR-proficient and -deficient cancers. The most frequent change was a major reduction in levels of poly (ADP-ribose) glycohydrolase (PARG), which would be expected to preserve a residual PARP1-initiated DNA damage response to DNA single-strand breaks. Other changes seen would be expected to boost levels of HRR of DNA double-strand breaks. Growth of all olaparib-resistant clones isolated could be controlled by WEE1 kinase inhibitor AZD1775, which inactivates the G2 checkpoint. Our work suggests that use of the WEE1 kinase inhibitor could be a realistic therapeutic option for patients that develop resistance to olaparib.

WEE1 inhibitor, AZD1775, overcomes trastuzumab resistance by targeting cancer stem-like properties in HER2-positive breast cancer.

Although trastuzumab has greatly improved the outcome of HER2-positive breast cancer, the emergence of resistance hampers its clinical benefits. Trastuzumab resistance is a multi-factorial consequence predominantly due to presence of cancer stem-like cells (CSCs). AZD1775, a potent anti-cancer agent targeting WEE1 kinase to drive tumor cells with DNA damage to premature mitosis, has previously shown high efficacies when targeting different cancers with a well-tolerated cytotoxic profile, but has not been evaluated in trastuzumab-resistant (TrR) breast cancer. We sought to investigate the effect of AZD1775 on cancer stem-like cell (CSC) properties, apoptosis, cell cycle regulation in TrR breast cancer. Our study for the first time demonstrated that AZD1775 induces apoptosis and arrests TrR cells at G2/M phase. More importantly, AZD1775 effectively targeted CSC properties by suppressing MUC1 expression levels. AZD1775 administration also induced apoptosis in our in-house patient-derived tumor cell line at passage 0, implying its significant clinical relevance. These findings highlight the potential clinical application of AZD1775 in overcoming trastuzumab resistance in breast cancer.

WEE1 kinase limits CDK activities to safeguard DNA replication and mitotic entry.

Precise execution of the cell division cycle is vital for all organisms. The Cyclin dependent kinases (CDKs) are the main cell cycle drivers, however, their activities must be precisely fine-tuned to ensure orderly cell cycle progression. A major regulatory axis is guarded by WEE1 kinase, which directly phosphorylates and inhibits CDK1 and CDK2. The role of WEE1 in the G2/M cell-cycle phase has been thoroughly investigated, and it is a focal point of multiple clinical trials targeting a variety of cancers in combination with DNA-damaging chemotherapeutic agents. However, the emerging role of WEE1 in S phase has so far largely been neglected. Here, we review how WEE1 regulates cell-cycle progression highlighting the importance of this kinase for proper S phase. We discuss how its function is modulated throughout different cell-cycle stages and provide an overview of how WEE1 levels are regulated. Furthermore, we outline recent clinical trials targeting WEE1 and elaborate on the mechanisms behind the anticancer efficacy of WEE1 inhibition. Finally, we consider novel biomarkers that may benefit WEE1-inhibition approaches in the clinic.

p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775.

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.