Wee1 Kinase Research Articles

Deubiquitinase-targeting chimeras for targeted protein stabilization.

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.

Cancer cells typically heavily rely on the G2/M checkpoint to survive endogenous and exogenous DNA damage, such as genotoxic stress due to genome instability or radiation and chemotherapy. The key regulator of the G2/M checkpoint, the cyclin-dependent kinase 1 (CDK1), is tightly controlled, including by its phosphorylation state. This posttranslational modification, which is determined by the opposing activities of the phosphatase cdc25 and the kinase Wee1, allows for a more rapid response to cellular stress than via the synthesis or degradation of modulatory interacting proteins, such as p21 or cyclin B. Reducing Wee1 activity results in ectopic activation of CDK1 activity and drives premature entry into mitosis with unrepaired or under-replicated DNA and causing mitotic catastrophe. Here, we review efforts to use small molecule inhibitors of Wee1 for therapeutic purposes, including strategies to combine Wee1 inhibition with genotoxic agents, such as radiation therapy or drugs inducing replication stress, or inhibitors of pathways that show synthetic lethality with Wee1. Furthermore, it become increasingly clear that Wee1 inhibition can also modulate therapeutic immune responses. We will discuss the mechanisms underlying combination treatments identifying both cell intrinsic and systemic anti-tumor activities.

Exposure to Low UV-B Dose Induces DNA Double-Strand Breaks Mediated Onset of Endoreduplication in Vigna radiata (L.) R. Wilczek Seedlings.

Multiple lines of evidence indicate that solar UV-B light acts as an important environmental signal in plants, regulating various cellular and metabolic activities, gene expression, growth and development. Here, we show that low levels of UV-B (4.0 kJ m-2) significantly influence plant response during early seedling development in the tropical legume crop Vigna radiata (L.) R. Wilczek. Exposure to low doses of UV-B showed relatively less growth inhibition yet remarkably enhanced lateral root formation in seedlings. Both low and high (8.0 kJ m-2) doses of UV-B treatment induced DNA double-strand breaks and activated the SOG1-related ATM-ATR-mediated DNA damage response pathway. These effects led to G2-M-phase arrest with a compromised expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1 and CYCB1;1, respectively. However, along with these effects, imbibitional exposure of seeds to a low UV-B dose resulted in enhanced accumulation of FZR1/CCS52A, E2Fa and WEE1 kinase and prominent induction of endoreduplication in 7-day-old seedlings. Low dose of UV-B mediated phenotypical responses, while the onset of endoreduplication appeared to be regulated at least in part via UV-B induced reactive oxygen species accumulation. Transcriptome analyses further revealed a network of co-regulated genes associated with DNA repair, cell cycle regulation and oxidative stress response pathways that are activated upon exposure to low doses of UV-B.

Identifying and overcoming a mechanism of resistance to WEE1 kinase inhibitor AZD1775 in high grade serous ovarian cancer cells

Objective: As a result of TP53 gene mutation high grade serous ovarian cancer (HGSOC) is dependent on the G2 checkpoint for the repair of DNA damage and survival. The key role of WEE1 kinase at this checkpoint makes inhibition of WEE1 kinase in combination with DNA damaging agents an attractive therapeutic strategy for HGSOC. Our aim was to characterise resistance mechanisms to WEE1 inhibitor AZD1775 and identify ways to overcome resistance ready for use in the clinic. Methods: AZD1775-resistant HGSOC cell clones were isolated and western blotting, cell cycle analysis, growth assays, RNA-Seq and gene expression analysis were used to characterise resistance mechanisms and investigate a way to overcome resistance. Results: A resistance mechanism previously reported in small cell lung cancer did not operate in HGSOC. Instead, resistance resulted from different cell cycle control pathway changes that slow AZD1775-induced cell cycle progression and reduce accumulation of replication associated DNA damage. One major change was reduced levels of CDK1, the substrate for WEE1 kinase inhibition; another was increased levels of PKMYT1, which can also inhibit CDK1. Increased expression of TGFβ signalling to slow cell cycle progression occurred in resistant clones. A TGFβR1 inhibitor overcame resistance in a clone with the highest TGFβR1 receptor expression. Conclusions: Although overexpression of the membrane glycoprotein MDR1 is a common mechanism of drug resistance, it was not involved in our HGSOC cells. Instead AZD1775 resistance resulted from cell cycle control pathway changes that combine to slow AZD1775-induced cell cycle progression and so reduce accumulation of replication-associated DNA damage.

Increased Susceptibility to Doxorubicin-Induced Cell Death in Acute Lymphocytic Leukemia Cells by Inhibiting Serine/Threonine WEE1 Kinase Expression

Increased Susceptibility to Doxorubicin-Induced Cell Death in Acute Lymphocytic Leukemia Cells by Inhibiting Serine/Threonine WEE1 Kinase Expression

WEE1 kinase protects the stability of stalled DNA replication forks by limiting CDK2 activity.

Cellular feedback systems ensure genome maintenance during DNA replication. When replication forks stall, newly replicated DNA is protected by pathways that limit excessive DNA nuclease attacks. Here we show that WEE1 activity guards against nascent DNA degradation at stalled forks. Furthermore, we identify WEE1-dependent suppression of cyclin-dependent kinase 2 (CDK2) as a major activity counteracting fork degradation. We establish DNA2 as the nuclease responsible for excessive fork degradation in WEE1-inhibited cells. In addition, WEE1 appears to be unique among CDK activity suppressors in S phase because neither CHK1 nor p21 promote fork protection as WEE1 does. Our results identify a key role of WEE1 in protecting stalled forks, which is separate from its established role in safeguarding DNA replication initiation. Our findings highlight how WEE1 inhibition evokes massive genome challenges during DNA replication, and this knowledge may improve therapeutic strategies to specifically eradicate cancer cells that frequently harbor elevated DNA replication stress.

Wee1 kinase inhibitor adavosertib with radiation in newly diagnosed diffuse intrinsic pontine glioma: A Children's Oncology Group phase I consortium study.

BackgroundChildren with diffuse intrinsic pontine gliomas (DIPG) have a dismal prognosis. Adavosertib (AZD1775) is an orally available, blood-brain barrier penetrant, Wee1 kinase inhibitor. Preclinical efficacy against DIPG is heightened by radiation induced replication stress.MethodsUsing a rolling six design, 7 adavosertib dose levels (DLs) (50 mg/m2 alternating weeks, 50 mg/m2 alternating with weeks of every other day, 50 mg/m2, then 95, 130, 160, 200 mg/m2) were assessed. Adavosertib was only given on days of cranial radiation therapy (CRT).The duration of CRT (54 Gy over 30 fractions; 6 weeks) constituted the dose limiting toxicity (DLT) period. Endpoints included tolerability, pharmacokinetics, overall survival (OS) and peripheral blood γH2AX levels as a marker of DNA damage.ResultsA total of 46 eligible patients with newly diagnosed DIPG [median (range) age 6 (3–21) years; 52% female] were enrolled. The recommend phase 2 dose (RP2D) of adavosertib was 200 mg/m2/d during days of CRT. Dose limiting toxicity included ALT elevation (n = 1, DL4) and neutropenia (n = 1, DL7). The mean Tmax, T1/2 and Clp on Day 1 were 2 h, 4.4 h, and 45.2 L/hr/m2, respectively. Modest accumulation of adavosertib was observed comparing day 5 versus day 1 AUC0-8h (accumulation ratio = 1.6). OS was 11.1 months (95% CI: 9.4, 12.5) and did not differ from historical control.ConclusionAdavosertib in combination with CRT is well tolerated in children with newly diagnosed DIPG, however, compared to historical controls, did not improve OS. These results can inform future trial design in children with high-risk cancer.

Structural conservation of WEE1 and its role in cell cycle regulation in plants

The WEE1 kinase is ubiquitous in plant development and negatively regulates the cell cycle through phosphorylations. However, analogies with the control of the human cell cycle by tyrosine- (Tyr-) phosphorylation of cyclin-dependent kinases (CDKs) are sometimes questioned. In this in silico study, we assessed the structural conservation of the WEE1 protein in the plant kingdom with a particular focus on agronomically valuable plants, the legume crops. We analyzed the phylogenetic distribution of amino-acid sequences among a large number of plants by Bayesian analysis that highlighted the general conservation of WEE1 proteins. A detailed sequence analysis confirmed the catalytic potential of WEE1 proteins in plants. However, some substitutions of an arginine and a glutamate at the entrance of the catalytic pocket, illustrated by 3D structure predictions, challenged the specificity of this protein toward the substrate and Tyr-phosphorylation compared to the human WEE1. The structural differences, which could be responsible for the loss of specificity between human and plants, are highlighted and suggest the involvement of plant WEE1 in more cell regulation processes.

Cell cycle checkpoint control in response to DNA damage by environmental stresses.

Being sessile organisms, plants are ubiquitously exposed to stresses that can affect the DNA replication process or cause DNA damage. To cope with these problems, plants utilize DNA damage response (DDR) pathways, consisting of both highly conserved and plant-specific elements. As a part of this DDR, cell cycle checkpoint control mechanisms either pause the cell cycle, to allow DNA repair, or lead cells into differentiation or programmed cell death, to prevent the transmission of DNA errors in the organism through mitosis or to its offspring via meiosis. The two major DDR cell cycle checkpoints control either the replication process or the G2/M transition. The latter is largely overseen by the plant-specific SOG1 transcription factor, which drives the activity of cyclin-dependent kinase inhibitors and MYB3R proteins, which are rate limiting for the G2/M transition. By contrast, the replication checkpoint is controlled by different players, including the conserved kinase WEE1 and likely the transcriptional repressor RBR1. These checkpoint mechanisms are called upon during developmental processes, in retrograde signaling pathways, and in response to biotic and abiotic stresses, including metal toxicity, cold, salinity, and phosphate deficiency. Additionally, the recent expansion of research from Arabidopsis to other model plants has revealed species-specific aspects of the DDR. Overall, it is becoming evidently clear that the DNA damage checkpoint mechanisms represent an important aspect of the adaptation of plants to a changing environment, hence gaining more knowledge about this topic might be helpful to increase the resilience of plants to climate change.

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.

ATR/CHK1/WEE1 Dependency in SRSF2-Mutated MDS/AML

Background: Mutations in splicing factor gene SRSF2 are recurrent drivers in 5-15 % of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A key property of SRSF2 mutations is that they occur early in the pathogenesis of MDS and are therefore present in all tumor cells in a patient. This property makes targetable vulnerabilities caused by SRSF2 mutations exceptionally important as they provide a way to inhibit the whole tumor. We previously demonstrated that splicing factor mutations induce R-loop-dependent activation of ATR, rendering cells sensitive to ATR inhibition. R-loops are transcription intermediates consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. Accumulation of aberrant R-loops induces the ATR kinase, which activates the G2-M cell cycle checkpoint via CHK1- and WEE1-mediated signaling. In normal cells, activation of the G2-M checkpoint halts the cell cycle until R-loops have been resolved. When the ATR pathway is inhibited, checkpoint activation does not occur, causing cells with unresolved R-loops to proceed to mitosis, resulting in DNA damage and cell death. We, therefore, sought to assess primary human MDS/AML samples for sensitivity to perturbation of the ATR/CHK1/WEE1 pathway and identify mechanisms of resistance.Methods: Sensitivity of 147 AML patient samples to 515 oncology drugs was tested ex vivo. Bone marrow mononuclear cells were incubated with 5 concentrations of each drug for 72 h followed by measurement of cell viability by CTG assay. Somatic mutations were identified by exome sequencing of matched leukemic bone marrow and skin biopsy samples.Isogenic K562 cell line clones carrying SRSF2 P95H/L/R mutations were generated using CRISPR/Cas9 editing. The presence of SRSF2 mutation was confirmed by CRISPR-sequencing and expression by whole transcriptome RNA-sequencing. Drug sensitivity of the K562 clones with and without SRSF2 mutation was determined by incubating cells with 16 concentrations of prexasertib, SRA-737, adavosertib, or BAY-1895344, followed by determination of cell viability by the MTS assay.Results: Analysis of ex vivo drug sensitivities in AML patient samples identified vulnerability to CHK1 and WEE1 inhibition in SRSF2-mutated AML: SRSF2 mutation is associated with sensitivity to the CHK1 inhibitors prexasertib (p = 0.006) (Fig 1 A and B) and PF-00477736 (p = 0.002) and the WEE1 inhibitor adavosertib (p = 0.003).To establish whether the isogenic SRSF2-mutant K562 cell line models recapitulate known downstream aberrations associated with SRSF2 mutations in patients, we analyzed gene expression and splicing. SRSF2 contains an RNA binding domain with affinity to CCNG or GGNG exonic splicing enhancer sequences. Similar to what has been observed in patients, the K562 clones with SRSF2 mutation show reduced use of GGNG sequence motifs at skipped exons. These results demonstrate that isogenic K562 clones recapitulate known alterations caused by mutant SRSF2.To determine whether SRSF2 mutations induce sensitivity to inhibition of ATR, CHK1, and WEE1, we tested 10 isogenic SRSF2 mutant and 4 wild-type K562 clones. Cells with SRSF2 mutation show increased sensitivity to ATR/CHK1/WEE1 inhibition (Fig 1C). We found no significant difference in drug sensitivity between clones carrying SRSF2 P95H/L/R substitutions. Clones with higher SRSF2 mutant allele dosage are more sensitive (Fig 1D).We identified a subset of SRSF2 mutated AML samples that were resistant to CHK1 and WEE1 inhibition. All resistant AML have co-occurring RUNX1 mutations (Fig 1B). In AML, RUNX1 mutations are associated with therapy resistance, suggesting that these mutations contribute to drug resistance. To test whether RUNX1 mutations induce resistance to ATR/CHK1/WEE1 inhibition in SRSF2-mutant leukemia, we introduced RUNX1 loss-of-function mutations in isogenic K562 carrying SRSF2 mutations. Candidate resistance factors identified by ATAC and RNA-sequencing will be validated in functional assays.Conclusions: Our results indicate that SRSF2-mutated leukemia harbor a vulnerability to the inhibition of ATR, CHK1, and WEE1 kinases. Cell line models indicate that sensitivity is similar across mutant alleles and dependent on allelic copy number. Several ATR/CHK1 and WEE1 inhibitors are in development, and our results suggest that these compounds could be effective treatments for SRSF2-mutated MDS and AML. [Display omitted] DisclosuresGraubert: astrazeneca: Research Funding; Janssen: Research Funding; Calico: Research Funding.

Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

Targeting MUS81 promotes the anticancer effect of WEE1 inhibitor and immune checkpoint blocking combination therapy via activating cGAS/STING signaling in gastric cancer cells

BackgroundIdentification of genomic biomarkers to predict the anticancer effects of indicated drugs is considered a promising strategy for the development of precision medicine. DNA endonuclease MUS81 plays a pivotal role in various biological processes during malignant diseases, mainly in DNA damage repair and replication fork stability. Our previous study reported that MUS81 was highly expressed and linked to tumor metastasis in gastric cancer; however, its therapeutic value has not been fully elucidated.MethodsBioinformatics analysis was used to define MUS81-related differential genes, which were further validated in clinical tissue samples. Gain or loss of function MUS81 cell models were constructed to elucidate the effect and mechanism of MUS81 on WEE1 expression. Moreover, the antitumor effect of targeting MUS81 combined with WEE1 inhibitors was verified using in vivo and in vitro assays. Thereafter, the cGAS/STING pathway was evaluated, and the therapeutic value of MUS81 for immunotherapy of gastric cancer was determined.ResultsIn this study, MUS81 negatively correlated with the expression of cell cycle checkpoint kinase WEE1. Furthermore, we identified that MUS81 regulated the ubiquitination of WEE1 via E-3 ligase β-TRCP in an enzymatic manner. In addition, MUS81 inhibition could sensitize the anticancer effect of the WEE1 inhibitor MK1775 in gastric cancer in vitro and in vivo. Interestingly, when MUS81 was targeted, it increased the accumulation of cytosolic DNA induced by MK1775 treatment and activated the DNA sensor STING-mediated innate immunity in the gastric cancer cells. Thus, the WEE1 inhibitor MK1775 specifically enhanced the anticancer effect of immune checkpoint blockade therapy in MUS81 deficient gastric cancer cells.ConclusionsOur data provide rational evidence that targeting MUS81 could elevate the expression of WEE1 by regulating its ubiquitination and could activate the innate immune response, thereby enhancing the anticancer efficacy of WEE1 inhibitor and immune checkpoint blockade combination therapy in gastric cancer cells.

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.

Transcription factor KLF2 enhances the sensitivity of breast cancer cells to cisplatin by suppressing kinase WEE1

ABSTRACT Cisplatin is an effective chemotherapeutic agent in facilitating the inhibition of proliferation, migration, and invasion in cancerous cells. However, the detailed mechanism of the regulation by cisplatin of human breast cancer cells is still unclear. This study aimed to investigate the mechanism of kruppel-like factor 2 (KLF2) transcription factor in cisplatin therapy for breast cancer. RT-qPCR was performed to quantify the expression of KLF2 and WEE1 in clinical tissue samples from breast cancer patients and in MDA-MB-231 cells. ChIP assay and dual-luciferase reporter assay were used to analyze the potential-binding sites of KLF2 and WEE1 promoter. Gain- or loss-of-function approaches were used to manipulate KLF2 and WEE1 in cisplatin-treated MDA-MB-231 cells, and the mechanism of KLF2 in breast cancer was evaluated both via CCK-8 assay, flow cytometry, Transwell assay, and Western blot. Further validation of the KLF2 was performed on nude mouse models. Breast cancer tissues and cells showed a relative decline of KLF2 expression and abundant WEE1 expression. Cisplatin inhibited the proliferation, migration, and invasion of MDA-MB-231 cells. Overexpression of KLF2 enhanced the inhibitory effect of cisplatin on the malignant characteristics of MDA-MB-231 cells in vitro. KLF2 targeted WEE1 and negatively regulated its expression, thus enhancing the sensitivity to cisplatin of breast cancer cells as well as tumor-bearing mice. Overall, these results suggest that KLF2 can potentially inhibit WEE1 expression and sensitize breast cancer cells to cisplatin, thus presenting a promising adjunct treatment.

382O Inhibition of WEE1 is effective in TP53 and RAS mutant metastatic colorectal cancer (mCRC): A randomised phase II trial (FOCUS4-C) comparing adavosertib (AZD1775) with active monitoring

Outcomes in RAS-mutant metastatic colorectal cancer (mCRC) remain poor and patients have limited therapeutic options. Adavosertib is the first small molecule inhibitor of WEE1 kinase. We hypothesised that aberrations in DNA replication seen in mCRC with both RAS and TP53 mutations would sensitise tumours to WEE1 inhibition.

Targeted inhibition of the WEE1 kinase as a novel therapeutic strategy in neuroendocrine neoplasms

Neuroendocrine neoplasms (NENs) represent a rare and heterogeneous group of malignancies, sharing features of both neural and endocrine cells. NENs G3 appear as a highly aggressive subset with a poor prognosis and limited therapeutic options. The small-molecule inhibitor of the WEE1 tyrosine kinase, adavosertib (AZD1775), has previously demonstrated potent anti-tumor effects on various types of cancer in preclinical and clinical studies. However, the role of adavosertib in NENs G3 had remained elusive. We evaluated the effects of adavosertib on pancreatic (BON-1, QGP-1) and bronchopulmonary (NCI-H720) neuroendocrine tumor cell lines applying 2D and 3D spheroid models. We newly demonstrated that adavosertib is sufficient to reduce cell viability and proliferation in neuroendocrine cell lines with features of high-grade NENs. As underlying mechanisms, we identified adavosertib-mediated DNA double-strand breaks and a G2/M cell cycle checkpoint abrogation leading into mitotic catastrophe and cancer cell apoptosis. Silencing of WEE1 via siRNA transfection resulted in a phenotype similar to adavosertib treatment. Together, inhibition of the WEE1 tyrosine kinase applying adavosertib on NENs G3 outlines a promising novel therapeutic strategy.

A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU.

During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the polα-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase.

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

Simple SummaryCancer cells often show elevated replication stress and loss of cell cycle checkpoints. The ataxia telangiectasia and Rad3-related (ATR) and WEE1 kinases play roles in protecting cancer cells from high replication stress and in regulating the remaining cell cycle checkpoints. Inhibitors of ATR or WEE1 therefore have the potential to selectively kill cancer cells and are currently being tested in clinical trials. However, more studies are needed to understand how these inhibitors work in various types of cancer and to find the most effective ways of using them. Here, we have explored whether simultaneous treatment with ATR and WEE1 inhibitors is a promising approach. Effects were investigated in cell lines from osteosarcoma and lung cancer. We expect our results to be of importance for future treatment strategies with these inhibitors.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.

Selected Articles from This Issue

Adavosertib (AZD1775) is a Wee1 kinase inhibitor that abrogates G1/S and G2/M cell cycle arrest, leading to replication stress and, ultimately, tumor cell death. Takebe and colleagues performed a clinical trial of once-daily adavosertib in patients with solid tumors. Adavosertib was well tolerated, and plasma exposures were similar to a twice-daily regimen. Six patients (14%) showed a partial response. Elevated tumor CCNE1 mRNA expression was identified as a potential biomarker for response, particularly in ovarian and endometrial cancers. These results suggest adavosertib monotherap. may hold promise in patients with advanced solid tumors when combined with biomarker selection using CCNE1 expression as a precision medicine approach to induce synthetic lethality.Checkpoint inhibitors demonstrated clinically meaningful antitumor activity and a manageable safety profile in chemotherapy-refractory gastroesophageal cancer (GEC). Lei and colleagues performed an exploratory analysis of the CheckMate 032 GEC cohort comparing the clinical utility of PD-L1, assessed using percentage of tumor cell (%TC) versus combined positive score (CPS), to predict nivolumab ± ipilimumab treatment efficacy. The analysis demonstrated that increased objective response and overall survival were observed with CPS compared with %TCs at higher cut-offs. Additionally, the study showed that multiple inflammatory gene expression signatures, including the four-gene signature of CD274, CD8A, LAG3, and STAT1, were associated with response, warranting further investigation.Maximal safe resection is an important first step to the successful management of glioma patients. Intraoperative fluorescent labeling is effective in maximizing the extent of tumor resection while minimizing the risk of excessive resection of the non-tumorous surrounding brain; however, the currently used probe lacks sufficient sensitivity and is not able to be readministered. Kitagawa and colleagues established a novel fluorescent labeling system for rapid intraoperative detection of glioblastoma using a topical, proline-arginine hydroxymethyl rhodamine green-based probe (PR-HMRG). This probe successfully distinguished tumor from peritumoral tissues in animal studies due to Calpain-1 protease activity. Further work will be needed to determine this probe's clinical applicability.Combining immune checkpoint blockade (ICB) with radiation therapy is considered a promising strategy, but only a subset of patients responds to treatment. Chen and colleagues assessed changes in immune infiltration after chemoradiotherapy (CCRT) in patients with cervical cancer. CCRT led to increased CD8+ T-cell infiltration and interferon induction, but only in a subset of patients. This heterogeneity in antitumor immunity and interferon response validates the observation that combination CCRT and ICB will only be of clinical benefit to certain patients. Additional studies are needed to identify biomarkers for patients who will benefit from CCRT.