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Abstract Background Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence. Results Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers. Conclusions Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

Purpose: Adavosertib may alter exposure to substrates of the cytochrome P450 (CYP) family of enzymes. This study assessed its effect on the pharmacokinetics of a cocktail of probe substrates for CYP3A (midazolam), CYP2C19 (omeprazole), and CYP1A2 (caffeine). Methods: Period 1: patients with locally advanced or metastatic solid tumors received 'cocktail': caffeine 200 mg, omeprazole 20 mg, and midazolam 2 mg (single dose); period 2: after 7- to 14-day washout, patients received adavosertib 225 mg twice daily on days 1–3 (five doses), with cocktail on day 3. After cocktail alone or in combination with adavosertib administration, 24-h pharmacokinetic sampling occurred for probe substrates and their respective metabolites paraxanthine, 5-hydroxyomeprazole (5-HO), and 1′-hydroxymidazolam (1′-HM). Safety was assessed throughout. Results: Of 33 patients (median age 60.0 years, range 41–83) receiving cocktail, 30 received adavosertib. Adavosertib co-administration increased caffeine, omeprazole, and midazolam exposure by 49%, 80%, and 55% (AUC0–12), respectively; AUC0–t increased by 61%, 98%, and 55%. Maximum plasma drug concentration (Cmax) increased by 4%, 46%, and 39%. Adavosertib co-administration increased 5-HO and 1′-HM exposure by 43% and 54% (AUC0–12) and 49% and 58% (AUC0–t), respectively; paraxanthine exposure was unchanged. Adavosertib co-administration decreased Cmax for paraxanthine and 5–HO by 19% and 7%; Cmax increased by 33% for 1′-HM. After receiving adavosertib, 19 (63%) patients had treatment-related adverse events (six [20%] grade ≥ 3). Conclusion: Adavosertib (225 mg bid) is a weak inhibitor of CYP1A2, CYP2C19, and CYP3A. ClinicalTrials.gov: NCT03333824 [ABSTRACT FROM AUTHOR]

Purpose: Adavosertib is a small-molecule, ATP-competitive inhibitor of Wee1 kinase. Molecularly targeted oncology agents have the potential to increase the risk of cardiovascular events, including prolongation of QT interval and associated cardiac arrhythmias. This study investigated the effect of adavosertib on the QTc interval in patients with advanced solid tumors. Methods: Eligible patients were ≥ 18 years of age with advanced solid tumors for which no standard therapy existed. Patients received adavosertib 225 mg twice daily on days 1–2 at 12-h intervals and once on day 3. Patients underwent digital 12-lead electrocardiogram and pharmacokinetic assessments pre-administration and time-matched assessments during the drug administration period. The relationship between maximum plasma drug concentration (Cmax) and baseline-adjusted corrected QT interval by Fridericia (QTcF) was estimated using a prespecified linear mixed-effects model. Results: Twenty-one patients received adavosertib. Concentration–QT modeling of ΔQTcF and the upper limit of the 90% confidence interval corresponding to the geometric mean of Cmax observed on days 1 and 3 were below the threshold for regulatory concern (not > 10 ms). No significant relationship between ΔQTcF (vs baseline) and adavosertib concentration was identified (P = 0.27). Pharmacokinetics and the adverse event (AE) profile were consistent with previous studies at this dose. Eleven (52.4%) patients experienced 17 treatment-related AEs in total, including diarrhea and nausea (both reported in six [28.6%] patients), vomiting (reported in two [9.5%] patients), anemia, decreased appetite, and constipation (all reported in one [4.8%] patient). Conclusion: Adavosertib does not have a clinically important effect on QTc prolongation. ClinicalTrials.gov: NCT03333824. [ABSTRACT FROM AUTHOR]

Background: Inhibition of the WEE1 kinase by adavosertib (AZD1775) potentiates replicative stress from genomic instability or chemotherapy. This study reports the pediatric solid tumor phase 2 results of the ADVL1312 trial combining irinotecan and adavosertib. Methods: Pediatric patients with recurrent neuroblastoma (part B), medulloblastoma/central nervous system embryonal tumors (part C), or rhabdomyosarcoma (part D) were treated with irinotecan and adavosertib orally for 5 days every 21 days. The combination was considered effective if there were at least three of 20 responses in parts B and D or six of 19 responses in part C. Tumor tissue was analyzed for alternative lengthening of telomeres and ATRX. Patient's prior tumor genomic analyses were provided. Results: The 20 patients with neuroblastoma (part B) had a median of three prior regimens and 95% had a history of prior irinotecan. There were three objective responses (9, 11, and 18 cycles) meeting the protocol defined efficacy end point. Two of the three patients with objective responses had tumors with alternative lengthening of telomeres. One patient with pineoblastoma had a partial response (11 cycles), but parts C and D did not meet the protocol defined efficacy end point. The combination was well tolerated and there were no dose limiting toxicities at cycle 1 or beyond in any parts of ADVL1312 at the recommended phase 2 dose. Conclusion: This is first phase 2 clinical trial of adavosertib in pediatrics and the first with irinotecan. The combination may be of sufficient activity to consider further study of adavosertib in neuroblastoma. This is the first phase 2 clinical trial of the WEE1 inhibitor adavosertib in pediatrics and the first in combination with irinotecan in cancer. The combination met the phase 2 clinical trial efficacy end point in neuroblastoma. [ABSTRACT FROM AUTHOR]

Adavosertib selectively inhibits Wee1, which regulates intra-S and G2/M cell-cycle checkpoints. This study investigated dosing schedules for adavosertib monotherapy, determining the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) in patients with advanced solid tumors. Patients received oral adavosertib qd or bid on a 5/9 schedule (5 days on treatment, 9 days off) in 14-day cycles, or qd on one of two 5/2 schedules (weekly, or for 2 of 3 weeks) in 21-day cycles. Safety, efficacy, and pharmacokinetic analyses were performed. Sixty-two patients (female, 64.5%; median age, 61.5 years; most common primary tumors: lung [24.2%], ovary [21.0%]) received treatment (qd schedules, n = 50; bid schedules, n = 12) for 1.8 months (median). Median time to maximum adavosertib concentration was 2.2–4.1 h; mean half-life was 5–12 h. Adverse events (AEs) caused dose reductions, interruptions and discontinuations in 17 (27.4%), 25 (40.3%) and 4 (6.5%) patients, respectively. Most common grade ≥ 3 AEs were anemia, neutropenia (each n = 9, 14.5%) and diarrhea (n = 8, 12.9%). Seven (11.3%) patients experienced 10 treatment-related serious AEs (pneumonia n = 2 [3.2%], dehydration n = 2 [3.2%], anemia n = 1 [1.6%], febrile neutropenia n = 1 [1.6%], and thrombocytopenia n = 1 [1.6%]). Overall objective response rate was 3.4% (2/58); disease control rate was 48.4% (30/62); median progression-free survival was 2.7 months. MTDs were 125 mg (bid 5/9) and 300 mg (qd 5/9 and 5/2 for 2 of 3 weeks); RP2D was 300 mg (qd 5/2 for 2 of 3 weeks). The safety profile was manageable, acceptable, and generally concordant with the known safety profile. [ABSTRACT FROM AUTHOR]

Targeting the G2/M checkpoint mediator WEE1 has been explored as a novel treatment strategy in ovarian cancer, but mechanisms underlying its efficacy and resistance remains to be understood. Here, it is demonstrated that the WEE1 inhibitor AZD1775 induces endoplasmic reticulum stress and activates the protein kinase RNA‐like ER kinase (PERK) and inositol‐required enzyme 1α (IRE1α) branches of the unfolded protein response (UPR) in TP53 mutant (mtTP53) ovarian cancer models. This is facilitated through NF‐κB mediated senescence‐associated secretory phenotype. Upon AZD1775 treatment, activated PERK promotes apoptotic signaling via C/EBP‐homologous protein (CHOP), while IRE1α‐induced splicing of XBP1 (XBP1s) maintains cell survival by repressing apoptosis. This leads to an encouraging synergistic antitumor effect of combining AZD1775 and an IRE1α inhibitor MKC8866 in multiple cell lines and preclinical models of ovarian cancers. Taken together, the data reveal an important dual role of the UPR signaling network in mtTP53 ovarian cancer models in response to AZD1775 and suggest that inhibition of the IRE1α‐XBP1s pathway may enhance the efficacy of AZD1775 in the clinics. [ABSTRACT FROM AUTHOR]

Abstract Targeting the G2/M checkpoint mediator WEE1 has been explored as a novel treatment strategy in ovarian cancer, but mechanisms underlying its efficacy and resistance remains to be understood. Here, it is demonstrated that the WEE1 inhibitor AZD1775 induces endoplasmic reticulum stress and activates the protein kinase RNA‐like ER kinase (PERK) and inositol‐required enzyme 1α (IRE1α) branches of the unfolded protein response (UPR) in TP53 mutant (mtTP53) ovarian cancer models. This is facilitated through NF‐κB mediated senescence‐associated secretory phenotype. Upon AZD1775 treatment, activated PERK promotes apoptotic signaling via C/EBP‐homologous protein (CHOP), while IRE1α‐induced splicing of XBP1 (XBP1s) maintains cell survival by repressing apoptosis. This leads to an encouraging synergistic antitumor effect of combining AZD1775 and an IRE1α inhibitor MKC8866 in multiple cell lines and preclinical models of ovarian cancers. Taken together, the data reveal an important dual role of the UPR signaling network in mtTP53 ovarian cancer models in response to AZD1775 and suggest that inhibition of the IRE1α‐XBP1s pathway may enhance the efficacy of AZD1775 in the clinics.

Patients with small-cell lung cancer (SCLC) are in dire need of more effective therapeutic options. Frequent disruption of the G1 checkpoint in SCLC cells creates a dependency on the G2/M checkpoint to maintain genomic integrity. Indeed, in pre-clinical models, inhibiting the G2/M checkpoint kinase WEE1 shows promise in inhibiting SCLC growth. However, toxicity and acquired resistance limit the clinical effectiveness of this strategy. Here, using CRISPR-Cas9 knockout screens in vitro and in vivo , we identified multiple factors influencing the response of SCLC cells to the WEE1 kinase inhibitor AZD1775, including the GCN2 kinase and other members of its signaling pathway. Rapid activation of GCN2 upon AZD1775 treatment triggers a stress response in SCLC cells. Pharmacological or genetic activation of the GCN2 pathway enhances cancer cell killing by AZD1775. Thus, activation of the GCN2 pathway represents a promising strategy to increase the efficacy of WEE1 inhibitors in SCLC. [Display omitted] • CRISPR-Cas9 screens identify genetic determinants of response to the WEE1 inhibitor AZD1775 • GCN2 inactivation leads to resistance to AZD1775 treatment in vitro and in vivo • GCN2 gets rapidly activated upon WEE1 inhibition by AZD1775 • Activation of the GCN2 pathway sensitizes cells to AZD1775 treatment Drainas et al. identify GCN2 as a factor influencing small-cell lung cancer (SCLC) response to the WEE1 inhibitor AZD1775. AZD1775 directly activates the GCN2 stress response pathway, increasing its effectiveness in killing SCLC cells. Thus, activating the GCN2 pathway presents a novel opportunity to enhance WEE1 inhibitor efficacy in SCLC. [ABSTRACT FROM AUTHOR]

Purpose: We investigated the role of Wee1 kinase in cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines and determined the efficacy of either Wee1 inhibitor, AZD1775 alone, or in combination with cisplatin, on cisplatin-resistant HNSCC inhibition. Methods: Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell viability and apoptosis were examined by MTS assay and flow cytometry, respectively. Results: Wee1 kinase protein expression levels in five cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin-sensitive partners. Importantly, Wee1 knockdown inhibited cell proliferation and re-sensitized cells to cisplatin treatment. Interestingly, previous studies have also shown that Wee1 inhibitor AZD1775 synergizes with cisplatin to suppress cell proliferation of cisplatin-sensitive HNSCC. We found that AZD1775 inhibited both cisplatin-sensitive and resistant HNSCC with similar IC50 values, which suggested that AZD1775 could overcome cisplatin resistance in cisplatin-resistant HNSCC. Mechanistically, AZD1775 and cisplatin cooperatively induced DNA damage and apoptosis. Conclusion: Wee1 inhibitor, AZD1775, and cisplatin coordinately suppressed proliferation and survival of HNSCC. [ABSTRACT FROM AUTHOR]

Purpose of Review: WEE1 inhibitor has been shown to potential chemotherapy or radiotherapy sensitivity in preclinical models, particularly in p53-mutated or deficient cancer cells although not exclusively. Here, we review the clinical development of WEE1 inhibitor in combination with chemotherapy or radiotherapy with concurrent chemotherapy as well as its combination with different novel agents. Recent Findings: Although several clinical trials have shown that WEE1 inhibitor can be safely combined with different chemotherapy agents as well as radiotherapy with concurrent chemotherapy, its clinical development has been hampered by the higher rate of grade 3 toxicities when added to standard treatments. A few clinical trials had also been conducted to test WEE1 inhibitor using TP53 mutation as a predictive biomarker. However, TP53 mutation has not been shown to be the most reliable predictive biomarker and the benefit of adding WEE1 inhibitor to chemotherapy has been modest, even in TP53 biomarker-driven studies. Summary: There are ongoing clinical trials testing WEE1 inhibitor with novel agents such as ATR and PAPR inhibitors as well as anti-PDL1 immunotherapy, which may better define the role of WEE1 inhibitor in the future if any of the novel treatment combination will show superior anti-tumor efficacy with a good safety profile compared to monotherapy and/or standard treatment. [ABSTRACT FROM AUTHOR]

p53 is among the most frequently mutated tumor suppressor genes given its prevalence in >50% of all human cancers. One critical tumor suppression function of p53 is to regulate transcription of downstream genes and maintain genomic stability by inducing the G1/S checkpoint in response to DNA damage. Tumor cells lacking functional p53 are defective in the G1/S checkpoint and become highly dependent on the G2/M checkpoint to maintain genomic stability and are consequently vulnerable to Wee1 inhibitors, which override the cell cycle G2/M checkpoint and induce cell death through mitotic catastrophe. In addition to the lost tumor suppression function, many mutated p53 (Mutp53) proteins acquire gain-of-function (GOF) activities as oncogenes to promote cancer progression, which manifest through aberrant expression of p53. In cancer cells with GOF Mutp53, statins can induce CHIP-mediated degradation of Mutp53 within the mevalonate pathway by blocking the interaction between mutp53 and DNAJA1. Therefore, targeting critical downstream pathways of Mutp53 provides an alternative strategy for treating cancers expressing Mutp53. In this review, we summarize recent advances with Wee1 inhibitors, statins, and mevalonate pathway inhibitors in cancers with p53 mutations.

Despite the success of antiestrogens in extending overall survival of patients with estrogen receptor positive (ER+) breast tumors, resistance to these therapies is prevalent. ER+ tumors that progress on antiestrogens are treated with antiestrogens and CDK4/6 inhibitors. However, 20% of these tumors never respond to CDK4/6 inhibitors due to intrinsic resistance. Here, we used endocrine sensitive ER+ MCF7 and T47D breast cancer cells to generate long-term estrogen deprived (LTED) endocrine resistant cells that are intrinsically resistant to CDK4/6 inhibitors. Since treatment with antiestrogens arrests cells in the G1 phase of the cell cycle, we hypothesized that a defective G1 checkpoint allows resistant cells to escape this arrest but increases their dependency on G2 checkpoint for DNA repair and growth, and hence, targeting the G2 checkpoint will induce cell death. Indeed, inhibition of WEE1, a crucial G2 checkpoint regulator, with AZD1775 (Adavosertib), significantly decreased cell proliferation and increased G2/M arrest, apoptosis and gamma-H2AX levels (a marker for DNA double stranded breaks) in resistant cells compared with sensitive cells. Thus, targeting WEE1 is a promising anti-cancer therapeutic strategy in standard therapy resistant ER+ breast cancer. [ABSTRACT FROM AUTHOR]

Background: PARP inhibitor (PARPi) monotherapy is a new strategy in BRCA germ-line deficient triple negative breast cancer (TNBC). However, not all patients respond, and the development of resistance limits the use of PARPi monotherapy. Therefore, the development of alternative synthetic lethality strategy, including in sporadic TNBC, is a priority. XRCC1, a key player in base excision repair, single strand break repair, nucleotide excision repair and alternative non-homologous end joining, interacts with PARP1 and coordinates DNA repair. ATR, ATM and Wee1 have essential roles in DNA repair and cell cycle regulation. Methods: Highly selective inhibitors of ATR (AZD6738), ATM (AZ31) and Wee1 (AZD1775) either alone or in combination with olaparib were tested for synthetic lethality in XRCC1 deficient TNBC or HeLa cells. Clinicopathological significance of ATR, ATM or Wee1 co-expression in XRCC1 proficient or deficient tumours was evaluated in a large cohort of 1650 human breast cancers. Results: ATR (AZD6738), ATM (AZ31) or Wee1 (AZD1775) monotherapy was selectively toxic in XRCC1 deficient cells. Selective synergistic toxicity was evident when olaparib was combined with AZD6738, AZ31 or AZD1775. The most potent synergistic interaction was evident with the AZD6738 and olaparib combination therapy. In clinical cohorts, ATR, ATM or Wee1 overexpression in XRCC1 deficient breast cancer was associated with poor outcomes. Conclusion: XRCC1 stratified DNA repair targeted combinatorial approach is feasible and warrants further clinical evaluation in breast cancer. [ABSTRACT FROM AUTHOR]

Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): ‘PTEN low’ BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while ‘PTEN high’ BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.

WEE1 is a cell cycle and DNA damage response kinase that is emerging as a therapeutic target for cancer. AZD1775 is a small molecule inhibitor of WEE1, currently in early phase clinical trials as a single agent and in combination with more conventional anti-neoplastic agents. As resistance to kinase inhibitors is frequent, we sought to identify mechanisms of resistance to WEE1 inhibition in acute leukemia. We found that AZD1775 resistant cell lines are dependent upon increased HDAC activity for their survival, in part due to increased KDM5A activity. In addition, gene expression analyses demonstrate HDAC dependent increase in MYC expression and c-MYC activity in AZD1775 treated resistant cells. Overexpression of c-MYC confers resistance to AZD1775 in cell lines with low baseline expression. Pharmacologic inhibition of BRD4, and thereby c-MYC, partially abrogated resistance to AZD1775. Thus, acquired resistance to WEE1 inhibition may be reversed by HDAC or BRD4 inhibition in leukemia cells.

Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies. [ABSTRACT FROM AUTHOR]

Purpose: MYC-driven medulloblastomas are highly aggressive childhood tumors with dismal outcomes and a lack of new treatment paradigms. We identified that targeting replication stress through WEE1 inhibition to suppress the S-phase replication checkpoint, combined with the attenuation of nucleotide synthesis with gemcitabine, is an effective strategy to induce apoptosis in MYC-driven medulloblastoma that could be rapidly translated into early phase clinical trials in children. Attenuation of replication stress is a key component of MYC-driven oncogenesis. Previous studies revealed a vulnerability in MYC medulloblastoma through WEE1 inhibition. Here, we focused on elucidating combinations of agents to synergize with WEE1 inhibition and drive replication stress toward cell death. Methods: We first analyzed WEE1 expression in patient tissues by immunohistochemistry. Next, we used high-throughput drug screens to identify agents that would synergize with WEE1 inhibition. Synergy was confirmed by in vitro live cell imaging, ex vivo slice culture models, and in vivo studies using orthotopic and flank xenograft models. Results: WEE1 expression was significantly higher in Group 3 and 4 medulloblastoma patients. The WEE1 inhibitor AZD1775 synergized with inhibitors of nucleotide synthesis, including gemcitabine. AZD1775 with gemcitabine suppressed proliferation and induced apoptosis. Ex vivo modeling demonstrated efficacy in Group 3 medulloblastoma patients, and in vivo modeling confirmed that combining AZD1775 and gemcitabine effectively suppressed tumor growth. Conclusion: Our results identified a potent new synergistic treatment combination for MYC-driven medulloblastoma that warrants exploration in early phase clinical trials. [ABSTRACT FROM AUTHOR]

WEE1 is a cell cycle and DNA damage response kinase that is emerging as a therapeutic target for cancer. AZD1775 is a small molecule inhibitor of WEE1, currently in early phase clinical trials as a single agent and in combination with more conventional anti-neoplastic agents. As resistance to kinase inhibitors is frequent, we sought to identify mechanisms of resistance to WEE1 inhibition in acute leukemia. We found that AZD1775 resistant cell lines are dependent upon increased HDAC activity for their survival, in part due to increased KDM5A activity. In addition, gene expression analyses demonstrate HDAC dependent increase in MYC expression and c-MYC activity in AZD1775 treated resistant cells. Overexpression of c-MYC confers resistance to AZD1775 in cell lines with low baseline expression. Pharmacologic inhibition of BRD4, and thereby c-MYC, partially abrogated resistance to AZD1775. Thus, acquired resistance to WEE1 inhibition may be reversed by HDAC or BRD4 inhibition in leukemia cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tumor cells activate the G2/M cell cycle checkpoint in response to ionizing radiation (IR) and effector immune cell-derived granzyme B to facilitate repair and survival. Wee1 kinase inhibition reverses the ability of tumor cells to pause at G2/M. Here, we hypothesized that AZD1775, a small molecule inhibitor of Wee1 kinase, could sensitize tumor cells to IR and T-lymphocyte killing and improve responses to combination IR and programmed death (PD)-axis immune checkpoint blockade (ICB). Multiple models of head and neck carcinoma, lung carcinoma and melanoma were used in vitro and in vivo to explore this hypothesis. AZD1775 reversed G2/M cell cycle checkpoint activation following IR, inducing cell death. Combination IR and AZD1775 induced accumulation of DNA damage in M-phase cells and was rescued with nucleoside supplementation, indicating mitotic catastrophe. Combination treatment enhanced control of syngeneic MOC1 tumors in vivo, and on-target effects of systemic AZD1775 could be localized with targeted IR. Combination treatment enhanced granzyme B-dependent T-lymphocyte killing through reversal of additive G2/M cell cycle block induced by IR and granzyme B. Combination IR and AZ1775-enhanced CD8+ cell-dependent MOC1 tumor growth control and rate of complete rejection of established tumors in the setting of PD-axis ICB. Functional assays demonstrated increased tumor antigen-specific immune responses in sorted T-lymphocytes. The combination of IR and AZD1775 not only lead to enhanced tumor-specific cytotoxicity, it also enhanced susceptibility to T-lymphocyte killing and responses to PD-axis ICB. These data provide the pre-clinical rationale for the combination of these therapies in the clinical trial setting.

Esophageal squamous cell carcinoma (ESCC) is a common malignant diagnosed cancer with increasing incidence rate and few treatment options. As a specific small-molecule inhibitor of the Wee1 tyrosine kinase, AZD1775 has previously shown potent antitumor effect on multiple types of cancer in various preclinical studies and clinical trials. However, the expression of Wee1 and the role of AZD1775 in ESCC remain unclear. In the present study, we found that the expression of Wee1 was much higher in ESCC cell lines and clinical samples than that of the corresponding controls. In addition, we demonstrated that AZD1775 exhibited strong inhibitory effect against Wee1 kinase in both tested ESCC cells at nanomolar concentrations. Moreover, AZD1775 effectively suppressed ESCC cell growth and triggered apoptosis via the mitochondrial-dependent signaling pathway. AZD1775 also diminished cell migration and invasion as well as the expression of MMP-2 and MMP-9. Interestingly, knockdown of Wee1 displayed a similar inhibitory effect of AZD1775 on ESCC cells. In addition, there was a synergism between AZD1775 and 5-fluorouracil or cisplatin in inducing cell death. More importantly, the in vivo experiments also demonstrated that AZD1775 potently inhibited ESCC cell growth and metastasis. In summary, our data suggest that the Wee1 inhibitor AZD1775 may be a potential therapeutic agent and warrants a clinical trial for patients with ESCC, even those with metastasis.

Esophageal squamous cell carcinoma (ESCC) is a common malignant diagnosed cancer with increasing incidence rate and few treatment options. As a specific small-molecule inhibitor of the Wee1 tyrosine kinase, AZD1775 has previously shown potent antitumor effect on multiple types of cancer in various preclinical studies and clinical trials. However, the expression of Wee1 and the role of AZD1775 in ESCC remain unclear. In the present study, we found that the expression of Wee1 was much higher in ESCC cell lines and clinical samples than that of the corresponding controls. In addition, we demonstrated that AZD1775 exhibited strong inhibitory effect against Wee1 kinase in both tested ESCC cells at nanomolar concentrations. Moreover, AZD1775 effectively suppressed ESCC cell growth and triggered apoptosis via the mitochondrial-dependent signaling pathway. AZD1775 also diminished cell migration and invasion as well as the expression of MMP-2 and MMP-9. Interestingly, knockdown of Wee1 displayed a similar inhibitory effect of AZD1775 on ESCC cells. In addition, there was a synergism between AZD1775 and 5-fluorouracil or cisplatin in inducing cell death. More importantly, the in vivo experiments also demonstrated that AZD1775 potently inhibited ESCC cell growth and metastasis. In summary, our data suggest that the Wee1 inhibitor AZD1775 may be a potential therapeutic agent and warrants a clinical trial for patients with ESCC, even those with metastasis. [ABSTRACT FROM AUTHOR]

Malignant mesothelioma (MM) is a very aggressive asbestos-related cancer, for which no therapy proves to be effective. We have recently shown that the oncolytic adenovirus dl922-947 had antitumor effects in MM cell lines and murine xenografts. Previous studies demonstrated that dl922-947-induced host cell cycle checkpoint deregulation and consequent DNA lesions associated with the virus efficacy. However, the cellular DNA damage response (DDR) can counteract this virus action. Therefore, we assessed whether AZD1775, an inhibitor of the G2/M DNA damage checkpoint kinase WEE1, could enhance MM cell sensitivity to dl922-947. Through cell viability assays, we found that AZD1775 synergized with dl922-947 selectively in MM cell lines and increased dl922-947-induced cell death, which showed hallmarks of apoptosis (annexinV-positivity, caspase-dependency, BCL-XL decrease, chromatin condensation). Predictably, dl922-947 and/or AZD1775 activated the DDR, as indicated by increased levels of three main DDR players: phosphorylated histone H2AX (γ-H2AX), phospho-replication protein A (RPA)32, phospho-checkpoint kinase 1 (CHK1). Dl922-947 also increased inactive Tyr-15-phosphorylated cyclin-dependent kinase 1 (CDK1), a key WEE1 substrate, which is indicative of G2/M checkpoint activation. This increase in phospho-CDK1 was effectively suppressed by AZD1775, thus suggesting that this compound could, indeed, abrogate the dl922-947-induced DNA damage checkpoint in MM cells. Overall, our data suggest that the dl922-947-AZD1775 combination could be a feasible strategy against MM.

The main reasons for failure of cancer chemotherapy are intrinsic and acquired drug resistance. The Hippo pathway effector Yes‐associated protein (YAP) is associated with resistance to both cytotoxic and molecular targeted drugs. Several lines of evidence indicate that YAP activates transcriptional programmes to promote cell cycle progression and DNA damage responses. Therefore, we hypothesised that YAP is involved in the sensitivity of cancer cells to small‐molecule agents targeting cell cycle‐related proteins. Here, we report that the inactivation of YAP sensitises the OVCAR‐8 ovarian cancer cell line to AZD1775, a small‐molecule WEE1 kinase inhibitor. The accumulation of DNA damage and mitotic failures induced by AZD1775‐based therapy were further enhanced by YAP depletion. YAP depletion reduced the expression of the Fanconi anaemia (FA) pathway components required for DNA repair and their transcriptional regulator E2F1. These results suggest that YAP activates the DNA damage response pathway, exemplified by the FA pathway and E2F1. Furthermore, we aimed to apply this finding to combination chemotherapy against ovarian cancers. The regimen containing dasatinib, which inhibits the nuclear localisation of YAP, improved the response to AZD1775‐based therapy in the OVCAR‐8 ovarian cancer cell line. We propose that dasatinib acts as a chemosensitiser for a subset of molecular targeted drugs, including AZD1775, by targeting YAP. [ABSTRACT FROM AUTHOR]

Introduction Wee1 is an important kinase involved in the G2 cell cycle checkpoint and frequently upregulated in intracranial neoplasms such as glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG). Two small molecules are available that target Wee1, AZD1775 and PD0166285, and clinical trials with AZD1775 have already been started. Since GBM and DIPG are highly invasive brain tumors, they are at least to some extent protected by the blood-brain barrier (BBB) and its ATP-binding cassette (ABC) efflux transporters. Methods We have here conducted a comprehensive set of in vitro and in vivo experiments to determine to what extent two dominant efflux transporters in the BBB, P-gp (ABCB1) and BCRP (ABCG2), exhibit affinity towards AZD1775 and PD0166285 and restrict their brain penetration. Results Using these studies, we demonstrate that AZD1775 is efficiently transported by both P-gp and BCRP, whereas PD0166285 is only a substrate of P-gp. Nonetheless, the brain penetration of both compounds was severely restricted in vivo, as indicated by a 5-fold (PD0166285) and 25-fold (AZD1775) lower brain-plasma ratio in wild type mice compared to Abcb1a/b;Abcg2−/− mice. Conclusion The brain penetration of these Wee1 inhibitors is severely limited by ABC transporters, which may compromise their clinical efficacy against intracranial neoplasms such as DIPG and GBM. [ABSTRACT FROM AUTHOR]

Small molecule inhibitors of the checkpoint proteins CHK1 and WEE1 are currently in clinical development in combination with the antimetabolite gemcitabine. It is unclear, however, if there is a therapeutic advantage to CHK1 vs. WEE1 inhibition for chemosensitization. The goals of this study were to directly compare the relative efficacies of the CHK1 inhibitor MK8776 and the WEE1 inhibitor AZD1775 to sensitize pancreatic cancer cell lines to gemcitabine and to identify pharmacodynamic biomarkers predictive of chemosensitization. Cells treated with gemcitabine and either MK8776 or AZD1775 were first assessed for clonogenic survival. With the exception of the homologous recombination-defective Capan1 cells, which were relatively insensitive to MK8776, we found that these cell lines were similarly sensitized to gemcitabine by CHK1 or WEE1 inhibition. The abilities of either the CDK1/2 inhibitor roscovitine or exogenous nucleosides to prevent MK8776 or AZD1775-mediated chemosensitization, however, were both inhibitor-dependent and variable among cell lines. Given the importance of DNA replication stress to gemcitabine chemosensitization, we next assessed high-intensity, pan-nuclear γH2AX staining as a pharmacodynamic marker for sensitization. In contrast to total γH2AX, aberrant mitotic entry or sub-G1 DNA content, high-intensity γH2AX staining correlated with chemosensitization by either MK8776 or AZD1775 (R2 0.83 - 0.53). In summary, we found that MK8776 and AZD1775 sensitize to gemcitabine with similar efficacy. Furthermore, our results suggest that the effects of CHK1 and WEE1 inhibition on gemcitabine-mediated replication stress best predict chemosensitization and support the use of high-intensity or pan-nuclear γH2AX staining as a marker for therapeutic response. [ABSTRACT FROM AUTHOR]

Inhibition of WEE1 is emerging as a promising chemosensitization strategy in many cancers including acute leukemia. Our lab and others have demonstrated that a small-molecule inhibitor of WEE1, AZD1775, sensitizes acute leukemia cells to cytarabine; however, a mechanism of combinatorial activity has remained elusive. Thus, we sought to determine the relative contribution of WEE1 targets CDK1 and CDK2 to the combinatorial activity of AZD1775 and cytarabine. To accomplish this, we expressed “WEE1 resistant” CDK1 (CDK1-AF) and CDK2 (CDK2-AF) constructs in a T-ALL cell line. Expression of CDK1/2-AF together, but neither alone, enhanced the anti-proliferative effects, DNA damage and apoptosis induced by cytarabine. Furthermore, pharmacologic inhibition of CDK1 alone or CDK1 and CDK2 together reduced the combinatorial activity of AZD1775 and cytarabine. Thus, increased activity of both CDK1 and CDK2 in response to WEE1 inhibition is necessary for the combinatorial activity of AZD1775 and cytarabine. This suggests the role of WEE1 in cells with accumulated DNA damage extends beyond regulation of CDK1 and the G2/M checkpoint and highlights the importance of WEE1 in mediating progression through the cell cycle. [ABSTRACT FROM AUTHOR]

A rapid, sensitive, and robust aqueous normal-phase chromatography method coupled with tandem mass spectrometry was developed and validated for the quantitation of AZD1775, a Wee-1 inhibitor, in human plasma and brain tumor tissue. Sample preparation involved simple protein precipitation with acetonitrile. Chromatographic separation was achieved on ethylene bridged hybrid stationary phases ( i.e. , Waters XBridge Amide column) under an isocratic elution with the mobile phase consisting of acetonitrile/ammonium formate in water (10 mM, pH 3.0) (85:15,v/v) at a flow rate of 0.8 mL/min for 5 min. The lower limit of quantitation (LLOQ) was 0.2 ng/mL of AZD1775 in plasma and tissue homogenate. The calibration curve was linear over AZD1775 concentration range of 0.2–1000 ng/mL in plasma and tissue homogenate. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method (<15%). The method was successfully applied to assess the penetration of AZD1775 across the blood-brain tumor barrier, as assessed by the unbound brain-to-plasma ratio, in patients with glioblastoma. [ABSTRACT FROM AUTHOR]

Purpose: Systemic therapy has improved rhabdomyosarcoma event-free and overall survival; however, approximately 40 % of patients will have progressive or recurrent disease which is difficult to cure and remains a considerable challenge. Minimal progress has been made in improving outcomes for metastatic or relapsed RMS due to a lack of effective therapeutic agents. Targeted therapies are likely to be incorporated into regimens which rely on conventional cytotoxic chemotherapy. A system to evaluate novel combinations of interest is needed.Methods: In this study, we explored 8 agents, 5 that are routinely used or similar to agents used in the clinical management of RMS and 3 biologically targeted agents with novel mechanisms of action, the Wee1 inhibitor AZD1775, the tyrosine kinase inhibitor cabozantinib, and the proteasome inhibitor bortezomib. All were tested individually at clinically achievable concentrations for activity in 4 RMS cell lines and then for potential synergy in two-drug combinations.Results: We found single-agent activity in five of the agents (or their active metabolites) that constitute the standard of care in RMS and for AZD1775 with mean IC50 values of 207 ng/ml, well below clinically achievable levels. In addition, the combination of individual cytotoxic chemotherapeutics currently used for RMS demonstrated largely synergistic activity with higher, but clinically achievable concentrations of AZD1775 in our assays.Conclusions: Prioritization of chemotherapeutics in RMS is possible using an in vitro system that can define novel drug combinations worthy of future investigation. AZD1775 exhibits single-agent activity, as well as synergy with conventional cytotoxic chemotherapy, and is a novel targeted agent that warrants further study in RMS. [ABSTRACT FROM AUTHOR]

Summary Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts. [ABSTRACT FROM AUTHOR]

Ewing sarcoma is an aggressive childhood cancer for which treatment options remain limited and toxic. There is an urgent need for the identification of novel therapeutic strategies. Our group has recently shown that Ewing cells rely on the S-phase kinase CDC7 (DDK) to maintain replication rates and cell viability and that DDK inhibition causes an increase in the phosphorylation of CDK1 and a significant delay in mitotic entry. Here, we expand on our previous findings and show that DDK inhibitor-induced mitotic entry delay is dependent upon WEE1 kinase. Specifically, WEE1 phosphorylates CDK1 and prevents mitotic entry upon DDK inhibition due to the presence of under-replicated DNA, potentially limiting the cytotoxic effects of DDK inhibition. To overcome this, we combined the inhibition of DDK with the inhibition of WEE1 and found that this results in elevated levels of premature mitotic entry, mitotic catastrophe, and apoptosis. Importantly, we have found that DDK and WEE1 inhibitors display a synergistic relationship with regards to reducing cell viability of Ewing sarcoma cells. Interestingly, the cytotoxic nature of this combination can be suppressed by the inhibition of CDK1 or microtubule polymerization, indicating that mitotic progression is required to elicit the cytotoxic effects. This is the first study to display the potential of utilizing the combined inhibition of DDK and WEE1 for the treatment of cancer. We believe this will offer a potential therapeutic strategy for the treatment of Ewing sarcoma as well as other tumor types that display sensitivity to DDK inhibitors., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest.

Ovarian cancer is the most lethal gynecological malignancy. Recurrence and chemoresistance are tough challenges leading to poor prognosis. HJURP is a molecular chaperone of CENP-A, which is associated with aggressive progression in multiple tumors. However, the function of HJURP in ovarian cancer has not been elucidated. In our study, we found HJURP was over-expressed in ovarian cancer and high expression of HJURP was correlated to unfavorable prognosis. HJURP knockdown could inhibit proliferation, metastasis and induce G0/G1 stagnation of ovarian cancer cells. Besides, next-generation sequencing (NGS) unveiled that WEE1 was down-regulated by silencing HJURP. Further mechanistic research revealed that HJURP regulated WEE1 through MYC, and luciferase assay indicated that MYC was a transcription factor of WEE1. Additionally, we investigated that silencing HJURP increased sensitivity of ovarian cancer cells to cisplatin via MYC/WEE1 axis, and HJURP participated in DNA repair of cisplatin-induced damage. More interestingly, silencing HJURP could enhance sensitivity of ovarian cancer cells to AZD1775 and improve the synergistic effect of cisplatin plus AZD1775 combined therapy. Collectively, our data displays that HJURP promotes tumor progression and chemoresistance of ovarian cancer, and HJURP has potential to be a novel therapeutic target in the combined treatment with cisplatin and AZD1775 in ovarian cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Antiapoptotic and proapoptotic BCL-2 protein family members regulate mitochondrial apoptotic pathway. Small molecule inhibitors of antiapoptotic BCL-2 proteins including BCL-2-specific inhibitor ABT-199 (Venetoclax) are in clinical development. However, the efficiency of ABT-199 as a single agent in solid tumors is limited. We performed a high-throughput RNAi kinome screen targeting 691 kinases to identify potentially targetable kinases to enhance ABT-199 response in breast cancer cells. Our studies identified Wee1 as the primary target kinase to overcome resistance to ABT-199. Depletion of Wee1 by siRNA-mediated knockdown or inhibition of Wee1 by the small molecule Wee1 inhibitor AZD1775 sensitized SKBR3, MDA-MB-468, T47D and CAMA-1 breast cancer cells to ABT-199 along with decreased MCL1. BH3-only proteins PUMA and BIM functionally contribute to apoptosis signaling following co-targeting BCL-2 and Wee1. Suppression of Wee1 function increased mitochondrial cell death priming. Furthermore, we found that Wee1 inhibition altered MCL1 phosphorylation and protein stability, which led to HUWE1-mediated MCL1 degradation. Our findings suggest that Wee1 inhibition can overcome resistance to ABT-199 and provide a rationale for further translational investigation of BCL-2 inhibitor/Wee1 inhibitor combination in breast cancer. [ABSTRACT FROM AUTHOR]