Wee1 Inhibitor Research Articles

Impaired DNA double-strand break repair and effective radiosensitization of HPV-negative HNSCC cell lines through combined inhibition of PARP and Wee1.

In head and neck squamous cell carcinoma (HNSCC), tumors negative for Human Papillomavirus (HPV) remain a difficult to treat entity and the morbidity of current multimodal treatment is high. Radiotherapy in combination with molecular targeting could represent suitable, less toxic treatment options especially for cisplatin ineligible patients. Therefore, we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity in radioresistant HPV-negative HNSCC cells. Three radioresistant HPV-negative cell lines (HSC4, SAS, UT-SCC-60a) were treated with olaparib, adavosertib and ionizing irradiation. The impact on cell cycle, G2 arrest and replication stress was assessed through flow cytometry after DAPI, phospho-histone H3 and γH2AX staining. Long term cell survival after treatment was determined through colony formation assay and DNA double-strand break (DSB) levels were assessed through quantification of nuclear 53BP1 foci in cell lines and patient-derived HPV±tumor slice cultures. Wee1 and dual targeting induced replication stress but failed to effectively inhibit radiation-induced G2 cell cycle arrest. Single as well as combined inhibition increased radiation sensitivity and residual DSB levels, with the largest effects induced through dual targeting. Dual targeting also enhanced residual DSB levels in patient-derived slice cultures from HPV-negative but not HPV+HNSCC (5/7 vs. 1/6). We conclude that the combined inhibition of PARP and Wee1 results in enhanced residual DNA damage levels after irradiation and effectively sensitizes radioresistant HPV-negative HNSCC cells. Ex vivo tumor slice cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach.

A phase I study of the Wee1 kinase inhibitor adavosertib (AZD1775) in combination with chemoradiation in cervical, upper vaginal, and uterine cancers

ObjectiveWee1 kinase is a crucial regulator of the G2/M checkpoint which prevents entry of damaged DNA into mitosis. Adavosertib (AZD1775), a selective inhibitor of Wee1, induces G2 escape and increases...

WEE1 and PARP-1 play critical roles in myelodysplastic syndrome and acute myeloid leukemia treatment

BackgroundMyelodysplastic syndrome (MDS) is a clonal bone marrow disorder defined by cytopenia and is associated with an increased risk of transformation to acute myeloid leukemia (AML). The outcome of MDS is poor, so alternative therapeutic approaches are needed to improve survival. The inhibition of the DNA damage response pathway, including poly (ADP-ribose) polymerase-1 (PARP-1), has been approved to treat several cancers. In addition, WEE1, a nuclear kinase, is overexpressed in many cancers. Therefore, a WEE1 inhibitor combined with a PARP-1 inhibitor could inhibit the proliferation of MDS and AML.MethodsWe analyzed whether WEE1 was regulated in the progression of MDS and AML. We also evaluated the efficacy of MK-1775 (WEE1 inhibitor) and talazoparib (PARP-1 inhibitor).ResultsPARP-1 expression was higher in the AML cells than in the MDS cells. However, WEE1 expression remained unchanged. MK-1775 or talazoparib alone inhibited MDS and AML cells after 72 h, and cellular cytotoxicity and caspase 3/7 activity were increased. The combined use of MK-1775 and talazoparib produced superior efficacy than either drug alone and SKM-1 colony formation was reduced. Significant cell populations in the sub-G1 phase were found in the cell-cycle analyses. Additionally, γ-H2AX expression and caspase 3 activity were increased. The combined treatment also changed the mitochondrial membrane potential.ConclusionsThe combination of a WEE1 inhibitor and PARP-1 inhibitor had enhanced efficacy and is proposed as a new therapeutic option for patients with MDS or AML. Our findings have clinical implications for a potential novel therapeutic strategy for MDS and AML patients.

Synthetic lethal interaction between WEE1 and PKMYT1 is a target for multiple low-dose treatment of high-grade serous ovarian carcinoma.

Ovarian cancer is driven by genetic alterations that necessitate protective DNA damage and replication stress responses through cell cycle control and genome maintenance. This creates specific vulnerabilities that may be exploited therapeutically. WEE1 kinase is a key cell cycle control kinase, and it has emerged as a promising cancer therapy target. However, adverse effects have limited its clinical progress, especially when tested in combination with chemotherapies. A strong genetic interaction between WEE1 and PKMYT1 led us to hypothesize that a multiple low-dose approach utilizing joint WEE1 and PKMYT1 inhibition would allow exploitation of the synthetic lethality. We found that the combination of WEE1 and PKMYT1 inhibition exhibited synergistic effects in eradicating ovarian cancer cells and organoid models at a low dose. The WEE1 and PKMYT1 inhibition synergistically promoted CDK activation. Furthermore, the combined treatment exacerbated DNA replication stress and replication catastrophe, leading to increase of the genomic instability and inflammatory STAT1 signalling activation. These findings suggest a new multiple low-dose approach to harness the potency of WEE1 inhibition through the synthetic lethal interaction with PKMYT1 that may contribute to the development of new treatments for ovarian cancer.

A Phase Ib Study Assessing the Safety, Tolerability, and Efficacy of the First-in-Class Wee1 Inhibitor Adavosertib (AZD1775) as Monotherapy in Patients with Advanced Solid Tumors.

Adavosertib (AZD1775) is a first-in-class, selective, small-molecule inhibitor of Wee1. The safety, tolerability, pharmacokinetics, and efficacy of adavosertib monotherapy were evaluated in patients with various solid-tumor types and molecular profiles. Eligible patients had the following: confirmed diagnosis of ovarian cancer (OC), triple-negative breast cancer (TNBC), or small-cell lung cancer (SCLC); previous treatment for metastatic/recurrent disease; and measurable disease. Patients were grouped into six matched cohorts based on tumor type and presence/absence of biomarkers and received oral adavosertib 175 mg twice a day on days 1-3 and 8-10 of a 21-day treatment cycle. Eighty patients received treatment in the expansion phase; median total treatment duration was 2.4 months. The most common treatment-related adverse events (AEs) were diarrhea (56.3%), nausea (42.5%), fatigue (36.3%), vomiting (18.8%), and decreased appetite (12.5%). Treatment-related grade ≥3 AEs and serious AEs were reported in 32.5% and 10.0% of patients, respectively. AEs led to dose interruptions in 22.5%, reductions in 11.3%, and discontinuations in 16.3% of patients. One patient died following serious AEs of deep vein thrombosis (treatment related) and respiratory failure (not treatment related). Objective response rate, disease control rate, and progression-free survival were as follows: 6.3%, 68.8%, 4.5 months (OC BRCA wild type); 3.3%, 76.7%, 3.9 months (OC BRCA mutation); 0%, 69.2%, 3.1 months (TNBC biomarker [CCNE1/MYC/MYCL1/MYCN] non-amplified [NA]); 0%, 50%, 2 months (TNBC biomarker amplified); 8.3%, 33.3%, 1.3 months (SCLC biomarker NA); and 0%, 33.3%, 1.2 months (SCLC biomarker amplified). Adavosertib monotherapy was tolerated and demonstrated some antitumor activity in patients with advanced solid tumors. ClinicalTrials.gov identifier NCT02482311; registered June 2015.

Combined Wee1 and EGFR inhibition reveals synergistic antitumor effect in esophageal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is one of the most common amplified and overexpressed oncogenes in esophageal squamous cell carcinoma (ESCC), while the clinical efficacy of EGFR targeted therapy in ESCC is dismal. Here, we evaluated the efficacy of dual blockage using monoclonal antibody against EGFR (Nimotuzumab) and an Wee1 inhibitor (AZD1775) in ESCC. We found that the mRNA and protein expression of EGFR and Wee1 were positively correlated in ESCC. Nimotuzumab-AZD1775 co-treatment inhibited tumor growth in PDX models with different drug susceptibility. Transcriptome sequencing and mass spectrometry analysis indicated that higher sensitive models showed enrichment of the PI3K/Akt or MAPK signaling pathway in Nimotuzumab-AZD1775 group compared with control group. In vitro experiments showed that the combination further inhibit PI3K/Akt and MAPK pathways compared to their monotherapy as indicated by downregulation of pAKT, pS6, pMEK, pErk and p-p38 MAPK. Furthermore, AZD1775 potentiated Nimotuzumab's antitumor effect through inducing apoptosis. Meanwhile, the bioinformatics analysis suggests the POLR2A might be candidate molecule of EGFR/Wee1 downstream. In conclusion, our work uncovers that EGFR-mAb Nimotuzumab combined with Wee1 inhibitor AZD1775 elicited potentiated anticancer activity against ESCC cell line and PDXs partially through PI3K/Akt and MAPK pathways blockade. These pre-clinical data raise the promising that ESCC patients may benefit from dual target EGFR and Wee1.

Synthetic and Medicinal Chemistry Approaches Toward WEE1 Kinase Inhibitors and Its Degraders.

WEE1 is a checkpoint kinase critical for mitotic events, especially in cell maturation and DNA repair. Most cancer cells' progression and survival are linked with elevated levels of WEE1 kinase. Thus, WEE1 kinase has become a new promising druggable target. A few classes of WEE1 inhibitors are designed by rationale or structure-based techniques and optimization approaches to identify selective acting anticancer agents. The discovery of the WEE1 inhibitor AZD1775 further emphasized WEE1 as a promising anticancer target. Therefore, the current review provides a comprehensive data on medicinal chemistry, synthetic approaches, optimization methods, and the interaction profile of WEE1 kinase inhibitors. In addition, WEE1 PROTAC degraders and their synthetic procedures, including a list of noncoding RNAs necessary for regulation of WEE1, are also highlighted. From the standpoint of medicinal chemistry, the contents of this compilation serve as an exemplar for the further design, synthesis, and optimization of promising WEE1-targeted anticancer agents.

A phase 1/2 dose escalation study of the BCL-2 inhibitor ZN-d5 and the WEE1 inhibitor azenosertib (ZN-c3) in patients (Pts) with acute myeloid leukemia (AML).

TPS7077 Background: B-cell lymphoma 2 (BCL-2) is an anti-apoptotic protein and pathway inhibition combined with chemotherapy and/or targeted therapeutics has led to significant clinical benefit in pts with AML. Disruption of cell cycle regulation may complement BCL-2 inhibition as many malignant cells are dependent on proteins that regulate cell cycle progression. The cell cycle checkpoint protein, WEE1, is highly expressed in genomically unstable malignancies and inhibition of WEE1 induces tumor cell apoptosis. It has been previously reported that the combination of ZN-d5 (an oral, selective BCL-2 inhibitor) and azenosertib (an oral, highly potent WEE1 inhibitor) synergistically enhance killing of AML cells both in vitro and in vivo, as well as in TP53-mutated models (Izadi, AACR 2022). Based on this strong pre-clinical rationale, a Phase 1/2 study was designed to evaluate the novel combination of ZN-d5 and azenosertib in pts with relapsed/refractory (R/R) AML. Methods: This phase 1/2 open-label study (ZN-d5-004C, NCT05682170) is determining the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), pharmacokinetics (PK), and clinical activity of ZN-d5 + azenosertib in pts with AML. The phase 1 dose-escalation stage is based on a Bayesian Optimal Interval design. Phase 2 is an open-label expansion to be conducted if supported by safety and efficacy data from the dose-escalation stage. Prior to initiating dose-escalation for the ZN-d5 + azenosertib combination, an azenosertib monotherapy cohort is being enrolled, as it has not been previously administered to pts with hematologic malignancies. For the dose-escalation azenosertib monotherapy (n≈15) and ZN-d5 combination (n≈40) cohorts, pts must have relapsed/refractory (R/R) disease after ≥1 line of AML therapy that may include the BCL-2 inhibitor, venetoclax. In phase 1, an azenosertib monotherapy cohort will be opened prior to investigation of ZN-d5 + azenosertib in combination. In phase 2, ZN-d5 + azenosertib will be given at the RP2D. Alternative dosing schedules may also be investigated based on emerging clinical data. The primary endpoints are adverse events and dose-limiting toxicities. Secondary endpoints include the rate and duration of remission according to the European LeukemiaNet 2017 criteria, including complete remission (CR), CR with incomplete hematologic recovery (CRi), morphologic leukemia-free state (MLFS), partial remission (PR), stable disease, progressive disease, relapse after remission (including molecular relapse), remission rate (CR + CRi), overall response rate (CR + CRi + MLFS + PR), and rate of and time to relapse. Pts will remain on study until disease progression, failure to respond, remission if additional therapy is clinically indicated, withdrawal of consent, loss to follow-up, or death. Clinical trial information: NCT05682170 .

Overexpressed FOXM1 collaborates with MMB to increase WEE1 inhibitor sensitivity in NSCLC.

Non-small cell lung cancer (NSCLC) is a common lung tumor with high mortality. The complex formed by MYB-MuvB complex (MMB) and forkhead box M1 (FOXM1) (MMB-FOXM1) plays a vital role in cell cycle progression to affect the progression of diseases. The role of the FOXM1-MMB complex in Wee1-like protein kinase (WEE1) inhibitor sensitivity in NSCLC keeps unclear. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to measure the mRNA levels of FOXM1, LIN54, Replication Protein A (RPA), gammaH2AX (γH2AX) and Cyclin B (CCNB). The western blot was performed to examine the corresponding protein expressions. The Cell Counting Kit-8 (CCK-8) assay was performed to test cell survival. It was demonstrated that after AZD-1775 treatment, the decrease in cell survival mediated by FOXM1 overexpression (P<0.001) could be reversed by LIN54 knockdown (P<0.01) and that cell survival in the control group did not differ obviously from that in the pcDNA3.1-FOXM1+siLIN54 group, indicating that the FOXM1-MMB complex was necessary for WEE1 inhibitor sensitivity. Moreover, the mRNA and protein expression levels of RPA and γH2AX were increased after AZD-1775 treatment and FOXM1 overexpression (P<0.01), suggesting that FOXM1 upregulation enhanced DNA replication stress and DNA damage. Finally, we found that the increases in the mRNA and protein expression levels of CCNB mediated by FOXM1 (P<0.01) could be rescued by silencing LIN54 (P<0.001) and that CCNB expression in the control group did not differ obviously from that in the pcDNA3.1-FOXM1+siLIN54 group. These findings revealed that the FOXM1-MMB complex activated G2/M checkpoints. In our work, it was discovered that FOXM1 overexpression increased DNA replication stress, which increased DNA replication and pressure on the WEE1 checkpoint. On the other hand, FOXM1 can enhance CCNB expression, increase the threshold content of the CCNB/CDK1 complex, facilitate mitosis, and promote WEE1 dephosphorylation. Under these two conditions, sensitivity to the WEE1 inhibitor AZD-1775 is increased, which leads to the accumulation of DNA damage and drives the activation of apoptosis. Overexpressed FOXM1 collaborates with MMB to increase WEE1 inhibitor sensitivity in NSCLC. This discovery might highlight the regulatory function of FOXM1/MMB in the treatment of NSCLC patients.

Correlation of cyclin E1 expression and clinical outcomes in a phase 1b dose-escalation study of azenosertib (ZN-c3), a WEE1 inhibitor, in combination with chemotherapy (CT) in patients (pts) with platinum-resistant or refractory (R/R) epithelial ovarian, peritoneal, or fallopian tube cancer (EOC).

5513 Background: Azenosertib (ZN-c3) is a novel, selective, and orally bioavailable WEE1 inhibitor demonstrating single-agent antitumor activity. Azenosertib may inhibit CT-induced DNA damage repair and provide benefit in pts with platinum R/R EOC. Cyclin E1 amplification/overexpression is present in ≥35% of metastatic ovarian cancer. High expression/amplification of Cyclin E1 is a poor prognostic marker and predictive of lack of response to platinum-based CT. Ovarian models overexpressing Cyclin E1 have exquisite sensitivity to azenosertib in vitro and in vivo; forced Cyclin E1 overexpression sensitizes cell lines with low endogenous Cyclin E1 to azenosertib. Methods: This open-label, multicenter study (NCT04516447) assessed azenosertib + paclitaxel (PAC), carboplatin (Carbo), gemcitabine (GEM), or pegylated liposomal doxorubicin (PLD) in pts with metastatic high-grade serous EOC after ≤2 lines of CT including platinum CT. Primary endpoint is safety and identification of RP2D of each combination. Secondary endpoints include clinical activity. Azenosertib was given continuously or intermittently QD in 21 or 28D cycles until PD or unacceptable toxicity. Results: 103 pts were enrolled; at data cut-off, 94 were efficacy evaluable. 26.6% had a partial response (PR) and median progression free survival (PFS) = 9.03 mo (95%CI: 5.52-11.01). Azenosertib + PAC demonstrated the highest ORR [9/18 (50%)], followed by Carbo [9/27 (33.3%)]; ORR for azenosertib + PLD or + GEM was 14.3% (5/35, 2/14 respectively). 80 pts had available Cyclin E1 expression data by IHC; higher Cyclin E1 (H-score &gt;50) correlated with higher ORR and longer PFS (ORR = 31.3% vs 7.7%; PFS = 10.35 vs 3.25 mo, HR=0.3; Table). Frequent Grade ≥3 TEAEs (%) were neutropenia (44.4), thrombocytopenia (30.3), anemia (12.1), leukopenia (11.1), fatigue (10.1), diarrhea (6.1), nausea (5.1), and vomiting (5.1). Conclusions: Azenosertib + CT is well tolerated and has encouraging clinical activity, with durable responses in pts with platinum R/R EOC. Pts with Cyclin E1 overexpressing tumors, a subgroup with suboptimal benefits from CT, demonstrated significant improvements in ORR and PFS vs pts with tumors having low expression. These data support a planned trial of azenosertib + CT vs CT alone in Cyclin E1 overexpressing platinum R/R EOC. Clinical trial information: NCT04516447 . [Table: see text]

Debio 0123-101: A phase 1 trial of Debio 0123 in combination with carboplatin in advanced solid tumors—Safety, pharmacokinetic, and preliminary antitumor activity data.

3012 Background: Cell cycle faults are typical in cancer, with tumor cells depending on G2 checkpoints to avoid excess DNA damage. WEE1, a DNA damage-activated kinase, governs G2 by phosphorylating cyclin-dependent kinase 1 (CDK1) to arrest the cell cycle and permit DNA repair. Via CDK1 inhibition, WEE1 also blocks replication initiation, limiting oncogene-induced replication stress. WEE1 inhibition, with accumulating DNA damage, can induce mitotic catastrophe and apoptosis. Debio 0123, an orally available, ATP-competitive WEE1 inhibitor, is being studied as monotherapy and in combination with carboplatin (CP) in patients (pts) with solid tumors. Here, we report safety, pharmacokinetic (PK), pharmacodynamic, and antitumor activity data for the combination. Methods: Debio 0123-101 (NCT03968653) is a two-arm dose-escalation study in pts with advanced solid tumors that recurred or progressed after receiving platinum-containing therapy, where no standard therapy of proven benefit is available. In Arm A, Debio 0123 was given as monotherapy in cycle (C) 1 (as a single dose on day [D] -3 and once daily over D1–3) and in combination with CP from C2 until end of treatment (EOT). In Arm B, Debio 0123 was given in combination with CP on D1–3 and D8–10, from C1 to EOT. CP was given on D1 of each 21-day cycle in both arms. Skin biopsies were taken at baseline and C1D3 to assess target engagement (TE). Results: Arm A data (cut-off 31.12.2022) is shown. Overall, 38 pts were treated (78.9% female, mean age 58.5 years), with two pts ongoing. Using a continual reassessment method-guided dose escalation, tested Debio 0123 doses ranged from 30–520 mg. The maximum tolerated dose was declared at 520 mg. At this dose level, 2/6 pts experienced a dose-limiting toxicity. The treatment was deemed well tolerated, with a manageable safety profile in line with that expected for CP monotherapy. Most Debio 0123-related toxicities were grade 1/2. Preliminary PK data indicate that Debio 0123 plasma levels rise proportionally with dose. TE was seen from the 150 mg dose, becoming more pronounced with increasing dose levels. Confirmed partial responses (PR) occurred in 5/30 evaluable pts; 4/12 pts with platinum-resistant ovarian cancer achieved a PR (one ongoing). Conclusions: Debio 0123, combined with CP, has a manageable safety profile and is well tolerated up to 520 mg; this combination led to observable antitumor activity in pts with platinum-resistant cancer. Further study of Debio 0123 as a therapeutic agent is warranted. Clinical trial information: NCT03968653 . [Table: see text]

A phase 2 study of the WEE1 inhibitor AZD1775 in SETD2-deficient advanced solid tumor malignancies.

3104 Background: AZD1775 is an inhibitor of the WEE1 kinase, leading to loss of genome integrity. Pathogenic SETD2 mutations lead to genome-wide loss of histone H3K36 trimethylation (H3K36me3) and hypersensitivity to WEE1 inhibition with a synthetic lethal effect. AZD1775 was studied in 2 cohorts of patients: A) Patients (pts) with locally advanced/metastatic solid tumor malignancy other than clear cell renal cell carcinoma (ccRCC) and B) pts with locally advanced/metastatic ccRCC. Methods: Eligible pts had a locally advanced/metastatic cancer with progression on ≥ 1 prior systemic therapy (including prior tyrosine kinase inhibitor or checkpoint inhibitor for cohort B), and a pathogenic SETD2 mutation by CLIA-certified next generation sequencing panel. Pts received AZD1775 at a starting dose of 300 mg PO daily on days 1-5, 8-12 of each 21-day cycle. The primary objective was to determine the objective response rate (ORR) by RECIST 1.1 criteria in each cohort. Planned sample size was 9 pts in each cohort in stage 1 of a Simon two-stage design. Secondary objectives were to determine the clinical benefit rate (CBR) (defined as partial response or stable disease [SD] &gt; 4 months), duration of response (DOR), and safety. Correlative assays include the evaluation of H3K36me3 mark by immunohistochemistry (IHC) in archival tumor tissue. Results: Between 05/2019 and 10/2021, 18 pts whose tumor harbored a pathogenic mutation in SETD2 were enrolled (9/cohort). The median age was 60 years (range 45 – 74), ECOG PS 0 in 8 (44.4%). One patient remains on treatment; 17 have discontinued (disease progression, n = 9; adverse event, n = 4; patient withdrawal, n = 2; patient death, n = 1). The median duration of treatment was 1.89 months (range 0.75 – 13.5). There were no confirmed objective responses and therefore enrollment was halted following accrual to stage 1. Tumor regression of any magnitude was observed in 5/17 (29%) evaluable pts. Stable disease was the best overall response in 10/17 (59%) evaluable pts, including 3 pts with SD for &gt; 4 months. One patient with RCC remains on treatment with SD for 14+ months duration. The most common adverse events of any grade were nausea (59%), anemia (41%), diarrhea (41%), and neutropenia (41%). Twelve pts (67%) experienced a Grade ≥ 3 adverse event. Evaluation of H3K36me3 using IHC as an exploratory biomarker is underway. Conclusions: AZD1775 given as monotherapy failed to achieve an objective response in SETD2-altered RCC and other solid tumor malignancies. However, a subset of pts derived clinical benefit as manifested by tumor regressions and/or durable stable disease. Evaluation of AZD1775 in combination with other DNA damage-inducing agents that cause replication stress may be warranted, potentially with the integration of the H3K36me3 mark as a biomarker for patient selection. Clinical trial information: NCT03284385 .

Inhibiting WEE1 Augments the Antitumor Efficacy of Cisplatin in Urothelial Carcinoma by Enhancing the DNA Damage Process.

Urothelial carcinoma (UC) is characterized by a high incidence of TP53 mutation, and overcoming resistance to cisplatin-based chemotherapy in UC is a major concern. Wee1 is a G2/M phase regulator that controls the DNA damage response to chemotherapy in TP53-mutant cancers. The combination of Wee1 blockade with cisplatin has shown synergistic efficacy in several types of cancers, but little is known regarding UC. The antitumor efficacy of the Wee1 inhibitor (AZD-1775) alone or in combination with cisplatin was evaluated in UC cell lines and a xenograft mouse model. AZD-1775 enhanced the anticancer activity of cisplatin by increasing cellular apoptosis. AZD-1775 inhibited the G2/M checkpoint, improving the sensitivity of mutant TP53 UC cells to cisplatin by enhancing the DNA damage process. We confirmed that AZD-1775 combined with cisplatin reduced tumor volume and proliferation activity and increased the markers of cell apoptosis and DNA damage in the mouse xenograft model. In summary, the Wee1 inhibitor AZD-1775 combined with cisplatin elicited a promising anticancer efficacy in UC, and constitutes an innovative and promising therapeutic strategy.

WEE1 inhibitor adavosertib in combination with carboplatin in advanced TP53 mutated ovarian cancer: A biomarker-enriched phase II study

ObjectiveIn the first part of this phase II study (NCT01164995), the combination of carboplatin and adavosertib (AZD1775) was shown to be safe and effective in patients with TP53 mutated platinum-resistant ovarian cancer (PROC). Here, we present the results of an additional safety and efficacy cohort and explore predictive biomarkers for resistance and response to this combination treatment. MethodsThis is a phase II, open-label, non-randomized study. Patients with TP53 mutated PROC received carboplatin AUC 5 mg/ ml·min intravenously and adavosertib 225 mg BID orally for 2.5 days in a 21-day cycle. The primary objective is to determine the efficacy and safety of carboplatin and adavosertib. Secondary objectives include progression-free survival (PFS), changes in circulating tumor cells (CTC) and exploration of genomic alterations. ResultsThirty-two patients with a median age of 63 years (39–77 years) were enrolled and received treatment. Twenty-nine patients were evaluable for efficacy. Bone marrow toxicity, nausea and vomiting were the most common adverse events. Twelve patients showed partial response (PR) as best response, resulting in an objective ORR of 41% in the evaluable patients (95% CI: 23%–61%). The median PFS was 5.6 months (95% CI: 3.8–10.3). In patients with tumors harboring CCNE1 amplification, treatment efficacy was slightly but not significantly better. ConclusionsAdavosertib 225 mg BID for 2.5 days and carboplatin AUC 5 could be safely combined and showed anti-tumor efficacy in patients with PROC. However, bone marrow toxicity remains a point of concern, since this is the most common reason for dose reductions and dose delays.

A phase Ib study of adavosertib, a selective Wee1 inhibitor, in patients with locally advanced or metastatic solid tumors

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.

Combination of PARP and WEE1 inhibitors invitro: Potential for use in the treatment of SHH medulloblastoma.

Medulloblastoma (MB), grouped as either WNT‑activated, Sonic hedgehog (SHH)‑activated, or non-WNT/non-SHH group 3, accounts for almost 20% of all childhood brain cancers. In spite of current intensive treatments, not all patients are cured and survivors suffer from severe side‑effects. The present study therefore examined the effects of the poly‑ADP‑ribose polymerase (PARP) and WEE1‑like protein kinase (WEE1) inhibitors, BMN673 and MK‑1775, respectively, alone or in combination on four MB cell lines. More specifically, the MB cell lines, DAOY, UW228‑3, MED8A and D425, were tested for their sensitivity to BMN673 and MK‑1775 alone or in combination, using cell viability, cell confluency and cytotoxicity assays. The effects on the cell cycle phases were also examined using FACS analysis. Monotherapy with BMN673 and MK‑1775 exerted dose‑dependent inhibitory effects on the viability of almost all MB cell lines. Notably, when BMN673 and MK‑1775 were used in combination, synergistic effects were noted in the SHH group cell lines (DAOY and UW228‑3), but not in the already WEE1‑sensitive group 3 (MED8A and D425) lines. Moreover, the combination treatment decreased the percentage of cells in the G1 phase and induced the novel distribution of both DAOY and UW228‑3 cells in the S and G2/M phases, with the UW228‑3 cells exhibiting a greater delay. To conclude, MK‑1775 was efficient in all and BMN673 in most cell lines, and their combined use exerted synergistic effects on the SHH, but not the group 3 cell lines. These data suggest that MK‑1775 alone may be of interest for all MB cell lines, and that the combination of PARP/WEE1 inhibitors may provide possible therapeutic opportunities for the therapy of SHH MBs. Their use warrants further investigations in the future.

Discovery of pyrido[4,3-d]pyrimidinone derivatives as novel Wee1 inhibitors

Wee1 has emerged as a potential target in cancer therapy due to its critical role in the regulation of the cell cycle. Here, we describe a series of Wee1 inhibitors with a novel scaffold that are potent inhibitors of this kinase (IC50 = 19–1485 nM). These inhibitors demonstrated robust cytotoxicity in MV-4-11 and T47D cell lines (MV-4-11 IC50 = 660–2690 nM, T47D IC50 = 2670–20,000 nM) and displayed good stability in mouse liver microsomes in vitro. Additionally, compound 34 showed remarkable selectivity (more than 500-fold) over the other 9 kinases. Further mechanistic studies demonstrated that compound 34 displayed measurable effects on downstream biomarkers and induced cancer cell apoptosis and cell cycle arrest in the G0/G1 phase. Taken together, these results show that compound 34, potentially a leading Wee1 inhibitor, warrants further investigation.

Development of effective immunotherapy for triple negative breast cancer by WEE1 kinase inhibition

Abstract Treatment options for triple negative breast cancer (TNBC) are limited because they are usually insensitive to most available hormonal or targeted therapeutic agents owing to their triple negative nature. Exciting clinical results using immune checkpoint inhibitors (ICI) directed against PD-1/PD-L1 have been reported. However, only a subset of patients (~20%) respond to ICI. Therefore, identifying rational combinations is needed to enhance the efficacy of immunotherapy in TNBC. We demonstrated there that inhibition of WEE1, a G2/M cell cycle checkpoint regulator induced by DNA damage, increased expression levels of MHC-I, and pro-inflammatory chemokines (CXCL9 and CXCL10) together with PD-L1 in human and mouse TNBC cells. Consequently, WEE1 inhibition enhanced cytotoxic T-lymphocyte (CTL)-mediated killing of tumor cells, and CTL infiltration into the tumor, making tumors susceptible to anti-PD-1 mAb immune checkpoint blockade in mouse TNBC models in a CD8+ T cell–dependent manner. Mechanistically, WEE1 inhibition triggered the cellular damage stress and interferon response through the double-stranded DNA/RNA (dsDNA/RNA) by activating the TLR9/IRF7 pathway. Furthermore, combining WEE1 inhibition and TLR9 agonist CpG DNA resulted in tumor regression, facilitating an effective antitumor T cell response. Our findings have rapid translational implications for the exploration of WEE1 inhibitors alongside agents that stimulate CD8+ T cell activity, including ICI and TLR9 agonists, which are both clinically available to treat TNBC patients. We acknowledge the funding support from NCI R01CA222963 and DoD W81XWH-19-1-0106.

Treatment for ovarian clear cell carcinoma with combined inhibition of WEE1 and ATR

BackgroundStandard platinum-based therapy for ovarian cancer is inefficient against ovarian clear cell carcinoma (OCCC). OCCC is a distinct subtype of epithelial ovarian cancer. OCCC constitutes 25% of ovarian cancers in East Asia (Japan, Korea, China, Singapore) and 6–10% in Europe and North America. The cancer is characterized by frequent inactivation of ARID1A and 10% of cases of endometriosis progression to OCCC. The aim of this study was to identify drugs that are either FDA-approved or in clinical trials for the treatment of OCCC.ResultsHigh throughput screening of 166 compounds that are either FDA-approved, in clinical trials or are in pre-clinical studies identified several cytotoxic compounds against OCCC. ARID1A knockdown cells were more sensitive to inhibitors of either mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738) or MDM2 (RG7388) compared to control cells. Also, compounds targeting BH3 domain (AZD4320) and SRC (AZD0530) displayed preferential cytotoxicity against ARID1A mutant cell lines. In addition, WEE1 inhibitor (AZD1775) showed broad cytotoxicity toward OCCC cell lines, irrespective of ARID1A status.ConclusionsIn a selection of 166 compounds we showed that inhibitors of ATR and WEE1 were cytotoxic against a panel of OCCC cell lines. These two drugs are already in other clinical trials, making them ideal candidates for treatment of OCCC.

Abstract CT087: Phase I/II study of the WEE1 inhibitor adavosertib in combination with carboplatin in children with advanced malignancies: arm C of the AcSé-ESMART trial

Abstract Background: AcSé-ESMART is a proof-of-concept, phase I/II platform trial designed to explore targeted agents in a molecularly enriched pediatric population. WEE1 plays a role in DNA repair and cell cycle control and is overexpressed in pediatric cancers. Adavosertib combinations resulted in enhanced antitumor activity compared to single agent in neuroblastoma, rhabdomyosarcoma, medulloblastoma and high-grade glioma in vivo models. The efficacy and safety of the adavosertib-carboplatin combination has been established in adults with focus on TP53 mutated ovarian cancer. Arm C of AcSé-ESMART applied this regimen to children with advanced malignancies enriched for alterations in TP53, DNA repair/replication stress and cell cycle control. Methods: Adavosertib was administered orally, twice daily on Days 1 to 3 and carboplatin intravenously on Day 1 of a 21-day cycle. Dose finding used the continuous reassessment method starting at adavosertib 100 mg/m2/dose and carboplatin AUC 5. Pharmacokinetic (PK) and retrospective molecular bioinformatic analysis was performed. Results: Twenty patients (median age: 14.0 years, range 3.4-23.5) were included, 18 received a total of 69 cycles. Seven dose-limiting toxicities (DLTs) were observed leading to two de-escalations to adavosertib 75 mg/m2/dose and carboplatin AUC 4. All patients with DLT had thrombocytopenia grade 3/4 requiring transfusions for &amp;gt;7 days and/or neutropenia grade 4 for &amp;gt;7 days. Main overall treatment-related toxicities were hematologic and gastrointestinal. Based on the identified DLT risk, no recommended Phase 2 dose was defined. PK analysis demonstrated equivalent adavosertib exposure in children to that in adults and both doses (75 and 100 mg/m2) achieved the cell kill target. Two patients with neuroblastoma achieved partial response (PR), one with medulloblastoma unconfirmed PR, and five had stable disease (SD) &amp;gt;4 cycles. Patients with PR/SD &amp;gt;4 cycles were considered as clinical benefit (CB) for retrospective molecular analysis. There was no correlation between TP53 genomic alteration alone and response. However, 7 of 8 patients with CB but none of the 10 patients without CB had 1 to 3 genomic alterations in the DNA repair (BRCA2 mutation, 11q loss containing ATM, MRE11A, CHEK1), cell cycle control/replication stress (CCNE1 amplification, RB1 mutation/loss, SETD2 mutation/loss) and RAS pathway (KRAS mutation and amplification, NF1 loss, PTPN11 mutation) in their tumor. Conclusions: Adavosertib combined with carboplatin exhibited significant hematologic toxicity. Activity signals and identified potential molecular biomarkers suggest further combination studies with less hematotoxic DNA damaging therapy in molecularly enriched pediatric cancers. Citation Format: Susanne A. Gatz, Anne C. Harttrampf, Caroline Brard, Francisco J. Bautista, Nicolas André, Samuel Abbou, Jonathan Rubino, Windy Rondof, Marc Deloger, Marc Rübsam, Daniel Hübschmann, Lynley V. Marshall, Souad Nebchi, Isabelle Aerts, Estelle Thebaud, Emilie De Carli, Anne-Sophie Defachelles, Xavier Paoletti, Robert Godin, Kowser Miah, Peter G. Mortimer, Gilles Vassal, Birgit Geoerger. Phase I/II study of the WEE1 inhibitor adavosertib in combination with carboplatin in children with advanced malignancies: arm C of the AcSé-ESMART trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT087.