Wee1 Inhibitor MK-1775 Research Articles

Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Chemotherapy is crucial in NSCLC treatment, and targeting Wee1 kinase, a key regulator of the G2/M cell cycle checkpoint, may enhance the efficacy of cytotoxic agents. This study investigates the potential of the Wee1 inhibitor MK-1775 in combination with gemcitabine and pemetrexed to enhance cytotoxicity in NSCLC cell lines. MethodsHuman NSCLC cell lines H1975, HCC827, A549, and H460 were treated with MK-1775 and chemotherapeutic agents, both alone and in combination. Growth inhibitory effects were assessed using the CCK8 assay. Apoptotic markers were evaluated via Western blotting, and cell cycle distribution was analyzed using FACS. In vivo efficacy was assessed using xenograft mouse models with H1975 and H460 cells, monitoring tumor growth and treatment toxicity. ResultsMK-1775 combined with gemcitabine or pemetrexed significantly decreased cell survival rates and IC50 values in A549 and HCC827 cell lines. Increased levels of phosphorylated cdc2, γ-H2AX, and PARP indicated enhanced apoptosis. Cell cycle analysis revealed G2/M phase arrest in p53-mutant HCC827 and H1975 cells treated with MK-1775 and gemcitabine. In xenograft models, the combination significantly inhibited tumor growth without significant toxicity. ConclusionsMK-1775 enhances the cytotoxic effects of gemcitabine and pemetrexed in NSCLC cell lines and effectively inhibits tumor growth in vivo. These findings suggest that Wee1 inhibition by MK-1775, combined with chemotherapy, represents a promising therapeutic strategy for NSCLC treatment.

Identification of DNA methylation-regulated WEE1 with potential implications in prognosis and immunotherapy for low-grade glioma.

WEE1 is a critical kinase in the DNA damage response pathway and has been shown to be effective in treating serous uterine cancer. However, its role in gliomas, specifically low-grade glioma (LGG), remains unclear. The impact of DNA methylation on WEE1 expression and its correlation with the immune landscape in gliomas also need further investigation. This study used data from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and Gene Expression Omnibus (GEO) and utilized various bioinformatics tools to analyze gene expression, survival, gene correlation, immune score, immune infiltration, genomic alterations, tumor mutation burden, microsatellite instability, clinical characteristics of glioma patients, WEE1 DNA methylation, prognostic analysis, single-cell gene expression distribution in glioma tissue samples, and immunotherapy response prediction based on WEE1 expression. WEE1 was upregulated in LGG and glioblastoma (GBM), but it had a more significant prognostic impact in LGG compared to other cancers. High WEE1 expression was associated with poorer prognosis in LGG, particularly when combined with wild-type IDH. The WEE1 inhibitor MK-1775 effectively inhibited the proliferation and migration of LGG cell lines, which were more sensitive to WEE1 inhibition. DNA methylation negatively regulated WEE1, and high DNA hypermethylation of WEE1 was associated with better prognosis in LGG than in GBM. Combining WEE1 inhibition and DNA methyltransferase inhibition showed a synergistic effect. Additionally, downregulation of WEE1 had favorable predictive value in immunotherapy response. Co-expression network analysis identified key genes involved in WEE1-mediated regulation of immune landscape, differentiation, and metastasis in LGG. Our study shows that WEE1 is a promising indicator for targeted therapy and prognosis evaluation. Notably, significant differences were observed in the role of WEE1 between LGG and GBM. Further investigation into WEE1 inhibition, either in combination with DNA methyltransferase inhibition or immunotherapy, is warranted in the context of LGG.

Abstract 4744: Overcoming MK1775 resistance in upper gastrointestinal cancers with combined WEE1 and CHK1 inhibition

Abstract Background: Upper gastrointestinal cancers (UGC), encompassing esophageal and gastric cancers, rank among the top causes of cancer-related deaths globally, with esophageal cancer being the sixth and gastric cancer the fourth leading cause. Esophageal adenocarcinoma (EAC), predominant in the western world, shares histopathological and molecular characteristics with gastric cancer (GC). Despite these similarities, treatment challenges persist, exemplified by the limited efficacy of the WEE1 inhibitor MK1775 in clinical trials due to drug resistance. Our study investigates novel mechanisms underlying this resistance in UGC. Methods and results: RNA sequencing and immunohistochemistry staining demonstrated notable overexpression of WEE1 in over two hundred human EAC and GC samples compared to normal tissues. Concurrently, Western blot analysis revealed a positive correlation between WEE1 and CHK1 overexpression across 16 UGC cell lines. To categorize UGC cells based on their sensitivity to MK1775, we conducted ATP-glo cell viability assays, determining their half-maximal inhibitory concentration (IC50) values. Utilizing nuclear-cytosol separation, our data intriguingly uncovered, for the first time, that in MK1775-resistant cells, MK1775 treatment induces the abnormal translocation of phosphorylated CHK1 (p-CHK1) from the nucleus to the cytoplasm, a shift not observed in MK1775-sensitive cells. Further analysis through Western blot suggested that the cytoplasmic presence of p-CHK1 might trigger the activation of oncogenic STAT3 following MK1775 treatment. This activation appears to stimulate the expression of genes that promote cancer cell survival and drug resistance by enhancing cell survival and inhibiting apoptosis. Notably, combining the WEE1 inhibitor with the CHK1 inhibitor CHIR124 effectively inhibited CHK1 kinase activity and abrogated STAT3 phosphorylation, DNA binding, and transcriptional activity in UGC cells. Our findings strongly indicate that a combined therapy of WEE1 and CHK1 inhibitors could be a promising strategy to overcome MK1775 resistance in UGC cells. Our ongoing research will employ human-derived UGC organoids, patient-derived xenografts (PDXs), and human tissue samples to further validate these findings. Conclusions: Our study underscores the potential of combining WEE1 and CHK1 inhibitors to effectively combat MK1775 resistance in UGC, offering a promising direction for improved treatment strategies and patient outcomes in these challenging malignancies. Citation Format: Krishnapriya Thangaretnam, MD Obaidul Islam, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, EL-Rifai Wael, Zheng Chen. Overcoming MK1775 resistance in upper gastrointestinal cancers with combined WEE1 and CHK1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4744.

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

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

Abstract 375: Smoking induces Wee1 expression through miRNA deregulation, promoting docetaxel resistance in esophageal adenocarcinoma

Abstract Esophageal Adenocarcinoma (EAC) is the most common subtype of Esophageal Cancer in Western countries, and its incidence rate has increased over the past few decades. The overall five-year survival rate of EAC is less than 20%. Smoking is a major risk factor for EAC development. WEE1 kinase plays a crucial role in the cell cycle by regulating the G2/M checkpoint, providing a time frame for the cells to repair DNA damage. Thus, targeting WEE1 using an inhibitor could potentiate the effect of chemotherapeutic drugs. This study investigated how smoking induces WEE1 protein expression, promoting docetaxel resistance in EAC. IHC staining for WEE1 in the normal esophagus and EAC tissue sections revealed significant over-expression of WEE1 protein in EAC. Nicotine, Nicotine derived Nitrosamine Ketone (NNK), and 2% cigarette smoke extract (CSE) treatment induced WEE1 expression with a concomitant increase in CDC2 phosphorylation (p-CDC2, Y15), a well-established read-out of WEE1 activity in EAC cells. In addition, we found that smoking upregulates WEE1 expression by decreasing miR-195-5p expression levels in EAC. Based on the differential gene expression signatures in EAC cell lines FLO1 and OE33 following WEE1 knockdown, drug and small molecule induced gene expression signatures analysis, L1000 fireworks display (L1000FWD), predicted that docetaxel is one of the best drug candidates which can synergize with the WEE1 inhibitor MK1775. Smoking-induced docetaxel resistance in EAC cells measured by cell viability assay. Down-regulating WEE1 expression with siRNA or pharmacological inhibition using MK1775 significantly sensitized EAC cells to docetaxel treatment, as evidenced by a remarkable decrease in the IC50 value of docetaxel. Inhibition of WEE1 combined with docetaxel can be considered an ideal therapeutic strategy due to its superior anti-tumor efficacy compared to the standard single-agent treatment. In conclusion, our data provide a firm rationale for the clinical combination of docetaxel with MK1775 in EAC patients. Citation Format: Krishnapriya Thangaretnam, Islam MD Obaidul, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, Zheng Chen. Smoking induces Wee1 expression through miRNA deregulation, promoting docetaxel resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 375.

Abstract 6187: The combination of Trifluridine/Tipiracil and a WEE1 inhibitor is an effective and tolerable candidate strategy against ESCC

Abstract Background: Recently we reported that trifluridine (FTD)/Tipiracil (TPI) is tolerable for unresectable Esophageal squamous cell carcinoma (ESCC) patients although the anti-tumor effect was modest (Mori Y, et al. Esophagus 2022). Therefore, we aimed at developing a combination therapy with FTD/TPI with another small molecule to achieve better efficacy against ESCC. CHK1 inhibitor was considered to be the candidate, because combination treatment with FTD/TPI and prexasertib showed potent antitumor effects in p53-mutant ESCC cells through the synthetic lethality (Ohashi S, et al. Mol Cancer Ther 2020); however, CHK1 inhibitors are not clinically available for further clinical development. Here we explored the concept of synthetic lethality with CHK1 perturbation to the whole ATR-CHK1-WEE1 pathway, especially to the downward WEE1 which directly targets a cell cycle regulator CDK1. The aim of this study is to elucidate the efficacy of the combination of a WEE1 inhibitor (WEE1i) MK1775 with FTD/TPI in ESCC. Methods: ESCC cells (TE-8 and TE-11) are used for in vitro assay, with compounds including FTD, MK1775 (WEE1i). Mitosis assay (flowcytometry with phospho-Histone H3 [p-hH3] antibody), cell viability assay (WST-1 and clonogenic assays), cytotoxicity assay (CytoTox-Glo assay), and western blotting (double-strand DNA break [γ-H2AX], DDR activity [phospho-CHK1], and CDK1 activity [phospho-Tyr15-CDK1]) were performed. Antitumor effects and tolerability were observed in vivo with TE-8 xenograft model with nu/nu nude mice. Results: FTD induced activation of CHK1 and inhibition of CDK1 sequentially in ESCC cells. FTD also decreased the proportion of p-hH3 positive cells in mitosis assay. Combination treatment with WEE1i and FTD had activated CDK1, increased p-hH3 positive cells, and induced γ-H2AX. The WST-1 cell viability assay showed significant sensitizing effect of WEE1i to FTD in ESCC cells. The cytotoxicity assay also revealed the significant increase of dead cells by the combination treatment (p = 0.003). Furthermore, we confirmed the combination treatment significantly suppressed xenografted tumor growth (-86%) without major adverse events in vivo (two-way ANOVA and Tukey post-hoc analyses: FTD/TPI vs. control, P < 0.05; MK1775 vs. control, P < 0.05, without significant interaction between the FTD/TPI treatment and MK1775 treatment). Conclusion: FTD/TPI and WEE1i combination showed potent cytotoxicity, and is considered as a candidate treatment strategy against ESCC. Citation Format: Hoang Trang Nguyen Vu, Osamu Kikuchi, Tomoki Saito, Yukie Nakai, Tomomi Ida, Yuki Kondo, Shigeki Kataoka, Yosuke Mitani, Shinya Ohashi, Manabu Muto. The combination of Trifluridine/Tipiracil and a WEE1 inhibitor is an effective and tolerable candidate strategy against ESCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6187.

Abstract LB542: Overexpression of the short isoform of Brd4 in ovarian carcinoma leads to highly aggressive tumor phenotype

Abstract High-grade serous ovarian carcinoma (HGSOC) is one of the deadliest types of cancer in the US. Occurrence of sporadic HGSOC is associated with high genomic instability, which causes amplification and deletion of several different genes. BRD4 is one of the most frequently amplified genes in ovarian carcinoma patients. BRD4 amplification leads to overexpression of BRD4 proteins including long and short BRD4 isoforms (BRD4-L and BRD4-S, respectively), and is associated with poor patient prognosis. Recently, it has been shown that the short isoform BRD4-S contributes to oncogenic properties of several tumor types; however, little is known about its role in ovarian cancer. Therefore, in order to interrogate the function of different BRD4 isoforms in HGSOC, we generated ovarian cancer cell lines stably overexpressing BRD4 isoforms (BRD4-L and BRD-S). Our data revealed that overexpression of BRD4-S in ovarian carcinoma cell lines induces a highly aggressive tumor phenotype in vitro and in vivo. Ovcar4-BRD4-S cells showed significantly higher cell proliferation rate and increased anchorage-independent growth when compared with parental cell lines. In addition, Ovcar4-BRD4-S presents a high level of DNA damage reflected by increased pH2AX foci. We also observed a disruption of cell cycle in Ovcar4-BRD4-S cells associated with a higher number of cells arrested in G2/M phase and increased population of polyploid cells. In vivo experiments demonstrated that BRD4-S expressing tumors grow significantly faster than parental tumors. BRD4-S overexpressing tumors are also very resistant to DNA-damage agents including standard of care platinum drugs. Finally, our preliminary drug screening demonstrated that Ovcar4-BRD4-S cells are highly sensitive to Wee1 inhibitor MK-1775. Furthermore, the combination of MK-1775 with paclitaxel synergistically increased the antitumor effect. In summary, our studies indicate that amplification of the BRD4 gene in HGSOC patients might lead to an increase in splicing of BRD4-S transcript, which in consequence promotes highly aggressive and platinum-resistant tumors. Precision therapies targeting BRD4 amplified tumors, such as Wee1 inhibitors in combination with paclitaxel hold great promise to improve ovarian cancer treatment and reduce chemotherapy resistance, which is a critical problem for eradication of this aggressive tumor. Citation Format: Ana Luiza Drumond-Bock, Luyao Wang, Lin Wang, Magdalena Cybula, Maria Rostworowska, Magdalena Bieniasz. Overexpression of the short isoform of Brd4 in ovarian carcinoma leads to highly aggressive tumor phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB542.

An isogenic cell line panel for sequence-based screening of targeted anticancer drugs

SummaryWe describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

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.

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.

Targeting WEE1 Kinase after G2/M Checkpoint Arrest Induces Apoptosis in Chronic Myeloid Leukemia Progenitors

BackgroundThe integrity of signaling pathways involved in cell cycle arrest, chromatin remodeling and DNA repair are critical for maintaining the fidelity of replicated DNA. Normal cells repair damaged DNA during G1 arrest; in contrast, leukemic cells often have a deficient G1/S checkpoint and depend on a functional G2/M checkpoint for DNA repair. Aurora Kinase A (AKA) is a key component of centrosome cycle and polar spindle assembly required for regulated progression from G2 to M and throughout M phase. It is frequently overexpressed in cancers, where it correlates with a poor prognosis. Notably, AKA overexpression is associated with genomic instability, one major trait of chronic myeloid leukemia (CML). Our hypothesis is that AKA, together with Polo like kinase 1 (Plk1), cooperates with the constitutive TK activity of the BCR-ABL1 fusion protein by increasing DNA damage, promoting the occurrence of additional genomic alterations and driving TKIs resistance and disease progression to blast crisis.AimsIn this study, the effects of AKA and Plk1 inhibition were evaluated in K562 cells sensitive (K562-S) and resistant (K562-R) to imatinib.MethodsProtein expression and activation was assessed by Western Blotting (WB). Danusertib and Volasertib were used to investigate the effects of AKA and Plk1 inhibition in K562-S and K562-R cells. Both cell lines were incubated with increasing concentrations of Danusertib and Volasertib (0.5-1 µM) or control medium for 24, 48, 72 and 96 hours to perform growth curve experiments and to identify the best sub-lethal drug doses in liquid medium. The percentage of apoptotic cells was quantified by annexin V/propidium iodide staining and flow cytometry. WB experiments were performed to evaluate AKA and Plk1 expression and activation before and after treatment with Danusertib and Volasertib alone or in combination with the Wee1 inhibitor MK1775. Further experiments were performed using polyclonal antibodies against cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 to confirm apoptosis activation. PARP cleavage and γH2AX expression and activation were also tested. The effects of Danusertib and Volasertib on cell cycle progression were evaluated by flow cytometry in K562-S and K562-R cells after incubation in medium containing 0.5 µM of each drugs for 24 h.ResultsBoth Danusertib and Volasertib (0.5 µM for 24h) were effective in cell growth inhibition in K562-S and K562-R by inducing cells to undergo cell cycle arrest and apoptosis. Moreover, they caused a dose- and time-dependent reduction of the G0/G1 cell fraction and an increase of the G2/M fraction. Cell cycle arrest was associated with increased levels of phospho (p)-Chk1 and p-Chk2, p-cyclin B1, p-cdc2 and p-Wee1. Using a Wee1 inhibitor (MK1775) after 24h treatment with Danusertib and Volasertib 0.5 µM, when cells were arrested in G2 phase and Wee1 was overexpressed and hyper-activated, resulted in a synergistic inhibition of cell viability in both K562-S and -R (Figure 1). MK1775 combined with either Danusertib or Volasertib caused a time-dependent increase of annexin-V-positive cells by activating the mitochondrial apoptotic pathway as reflected by an increment of Bax expression and induction of the cleavage of caspase-3, -8 and -9 and PARP. Moreover, both drug combinations induced a significant increase of the DNA double-strand break marker γH2AX, suggesting that Wee1 inhibition promotes mitosis and propagates genomic instability by forcing the cells through successive replication cycles, ultimately resulting in apoptosis from mitotic catastrophe.ConclusionsOur results provide support for MK1775 to be used in synergistic combination therapies to improve the outcomes of patients with CML in blast crisis or resistant to multiple lines of TKI therapy.Supported by ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. [Display omitted] DisclosuresCastagnetti:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rosti:Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Martinelli:Johnson&Johnson: Consultancy; Roche: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Soverini:Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

Downregulation of Cell Cycle and Checkpoint Genes by Class I HDAC Inhibitors Limits Synergism with G2/M Checkpoint Inhibitor MK-1775 in Bladder Cancer Cells.

Since genes encoding epigenetic regulators are often mutated or deregulated in urothelial carcinoma (UC), they represent promising therapeutic targets. Specifically, inhibition of Class-I histone deacetylase (HDAC) isoenzymes induces cell death in UC cell lines (UCC) and, in contrast to other cancer types, cell cycle arrest in G2/M. Here, we investigated whether mutations in cell cycle genes contribute to G2/M rather than G1 arrest, identified the precise point of arrest and clarified the function of individual HDAC Class-I isoenzymes. Database analyses of UC tissues and cell lines revealed mutations in G1/S, but not G2/M checkpoint regulators. Using class I-specific HDAC inhibitors (HDACi) with different isoenzyme specificity (Romidepsin, Entinostat, RGFP966), cell cycle arrest was shown to occur at the G2/M transition and to depend on inhibition of HDAC1/2 rather than HDAC3. Since HDAC1/2 inhibition caused cell-type-specific downregulation of genes encoding G2/M regulators, the WEE1 inhibitor MK-1775 could not overcome G2/M checkpoint arrest and therefore did not synergize with Romidepsin inhibiting HDAC1/2. Instead, since DNA damage was induced by inhibition of HDAC1/2, but not of HDAC3, combinations between inhibitors of HDAC1/2 and of DNA repair should be attempted.

An Isogenic Cell Line Panel for Sequence-Based Screening of Targeted Anti-Cancer Drugs

We describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

Abstract 328: DNA damage inhibitors with topotecan in a complex spheroid model in PDM and established cell lines

Abstract Many solid tumor treatment regimens include combination chemotherapy. Over thirty-five human tumor cell lines, some recently developed and some well-established, were grown as complex spheroids, tumor cells mixed with HUVEC and MSC, to better model human solid tumors. The complex spheroids were established for 3 days, then the drug(s) and/or investigational agent(s) were added. After 7 days, the experiment was terminated with CellTiterGlo 3D. Cell viability was determined relative to a vehicle treated control and IC50s were calculated from concentration response curves. The response to topotecan was determined over a concentration range achievable in patients alone and in combination with nedisertib (M3814) or VX984, DNA PK inhibitors, M6620 or BAY-1895344, ATR inhibitors, MK-1775, a Wee-1 inhibitor, AZD-1390, an ATM inhibitor or olaparib or talazoparib, PARP inhibitors. The clinical Cmax of topotecan is 0.15 uM and the topotecan concentration range tested was 0.001 to 0.3 uM. Topotecan IC50's spanned more than 2 logs from 0.003 uM to >>0.3 uM. The ATRi inhibitors were tested over the concentration range from 0.01 to 10 uM. The M6620 IC50's covered 2 logs from 0.015 to >>1 uM and the BAY-189-5344 IC50 range was similar. Combinations of topotecan and M6620 produced greater than additive cytotoxicity in Rh28 and Rh30 rhabdomyosarcoma and 328373-195-R-J1 squamous cell carcinoma with >1 log increase in cell killing. BAY-1895344 in combination with topotecan was greater than additive in the COR L88 SCLC line and the 287954-098-R-J1 Ewing sarcoma line. The combination of topotecan and M6620 resulted in approximately additive cell killing in 929823-356-R-J2 squamous carcinoma, and NCI-H196 and NCI-H1618 SCLC with 4-6-fold increase in cell killing. Topotecan along with the ATM inhibitor AZD-1390, produced greater than additive cell killing in the 287954-098-R-J1 Ewing sarcoma line at the lower concentrations of topotecan (0.003 and 0.01 uM) tested. Primarily additive effects of the ATM inhibitor and topotecan were seen in most other cell lines tested. The combination of topotecan and the Wee-1 inhibitor MK1775 was tested in complex spheroids of the same cell lines. Only in the NCI-H196 SCLC line did topotecan and MK1775 produce greater than additive cell killing. There was a 1.5- to 4-fold increase in cell killing with topotecan and MK1775 in the RH28 and Rh30 rhabdomyosarcomas, 929823-356-R-J2 and 328373-195-R-J1 squamous cell carcinomas and the NCI-H1618 SCLC which was additive to sub-additive. The combination of topotecan with the DNA PK inhibitor M3814 produced little or no increase in cell killing (1.2- to 2-fold) compared with topotecan alone in complex spheroids. These findings may provide guidance regarding selection of agent combinations worthy of further investigation. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Beverly A. Teicher, Austin Doyle, John Wright, Charles Kunos, Thomas Silvers, Rene Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Nathan Coussens, Joel Morris, James Doroshow. DNA damage inhibitors with topotecan in a complex spheroid model in PDM and established cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 328.

The Apparent Requirement for Protein Synthesis during G2 Phase Is due to Checkpoint Activation

SUMMARYProtein synthesis inhibitors (e.g., cycloheximide) block mitotic entry, suggesting that cell cycle progression requires protein synthesis until right before mitosis. However, cycloheximide is also known to activate p38 mitogen-activated protein kinase (MAPK), which can delay mitotic entry through a G2/M checkpoint. Here, we ask whether checkpoint activation or a requirement for protein synthesis is responsible for the cycloheximide effect. We find that p38 inhibitors prevent cycloheximide-treated cells from arresting in G2 phase and that G2 duration is normal in approximately half of these cells. The Wee1 inhibitor MK-1775 and Wee1/Myt1 inhibitor PD0166285 also prevent cycloheximide from blocking mitotic entry, raising the possibility that Wee1 and/or Myt1 mediate the cycloheximide-induced G2 arrest. Thus, protein synthesis during G2 phase is not required for mitotic entry, at least when the p38 checkpoint pathway is abrogated. However, M phase progression is delayed in cycloheximide-plus-kinase-inhibitor-treated cells, emphasizing the different requirements of protein synthesis for timely entry and completion of mitosis.

Abstract 15: Imputing a more targeted therapy for triple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) is a clinically defined subset of breast cancers that are inherently difficult to treat since, unlike the other breast cancer subtypes, they are defined by their lack of a distinct molecular target. Previously our lab developed a computational framework to impute drug response in patients that accurately mirrored the observed patient response. In short, this methodology involves building unsupervised linear regression models between cell line baseline gene expression values and the respective drug sensitivity metric (EC50 or AUC), which allows us to generate imputed sensitivity scores for cell line or patient tumor samples for which we have gene expression data. For this study, we applied this methodology to prospectively identify agents that are predicted to be more effective specifically in triple-negative breast cancer. We began by expanding the drug imputation training dataset to the Broad’s Cell Therapeutics Response Portal, which contains 545 drugs and over 1,000 cancer cell lines (CCLs) and imputed sensitivity in The Cancer Genome Atlas’s (TCGA’s) breast cancer cohort. Our imputation data indicated that TNBC tumors were expected to be comparatively sensitive to cell cycle pathway inhibitors. Among these compounds, the Wee1 inhibitor MK1775 (aka adavosertib and AZD1775) was identified for having the most significant predicted effect in TNBC patients compared to the receptor-positive patients. We were able to validate the accuracy of the MK1775 imputation model using traditional cross-validation approaches as well as in independent cell line dataset and showed that the measured and our predicted response to Wee1 inhibition strongly correlated. Additionally, we were able to uses our predicted MK1775 response scores to recapitulate the significant association between MK1775 response and p53 mutation status. We then looked to validate the efficacy of MK1775’s efficacy in TNBC cell line and xenograft experiments. We observed strong inhibition of cancer growth by MK1775 alone but also identified a combination effect between MK1775 and the TNBC standard-of-care agent paclitaxel as assayed by both in vitro viability experiments as well as in vivo tumor growth assays. We believe these results indicate that this methodology could generally be applied as a hypothesis-generating tool for identifying targeted agents for other disease subtypes as well as supporting the use of MK1775, especially in combination with paclitaxel, for TNBC. Citation Format: Robert F. Gruener, Geoffrey L. Greene, R. Stephanie Huang. Imputing a more targeted therapy for triple-negative breast cancers [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 15.

The Wee1 kinase inhibitor MK1775 suppresses cell growth, attenuates stemness and synergises with bortezomib in multiple myeloma.

Multiple myeloma stem-like cells (MMSCs) are responsible for initiation and relapse, though novel treatment paradigms that effectively eradicate MMSCs are yet to be developed. Selective inhibition of the cell cycle regulatory kinase Wee1 by MK1775 is being explored as a potential anti-cancer therapeutic. We report that higher expression of Wee1 is correlated with poor survival in multiple myeloma (MM). The MM models and patient-derived CD138+ plasma cells are particularly sensitive to the growth-inhibitory effects of the Wee1 inhibitor MK1775. MK1775 induces Mus81-Eme1 endonuclease-mediated DNA damage in S-phase cell cycle that results in a blockade of replication and then apoptosis. Furthermore, MK1775 strongly suppresses the features of stemness in vitro, in vivo and in primary CD138+ cells by decreasing ALDH1+ cell fraction and the expression of ALDH1. In addition, co-treatment of MK1775 with bortezomib is synergistic in vitro and in vivo. Bortezomib, although it enhances ALDH1+ cells, when combined with MK1775 abrogates this stimulatory effect on stemness. Considering MM as an invariably incurable malignancy due to the presence of heterogenic myeloma stem-like cells, our study presents inhibition of Wee1 as a promising targeted therapy for MM and provides a compelling rationale to further investigate the activity of MK1775 against myeloma in clinical settings.

Abstract 3163: The role of early response genes (ERG’s) as a biomarker of response to Wee1 targeted therapies

Abstract Introduction: WEE1 kinase is a key component in maintaining the G2/M cell cycle checkpoint for pre-mitotic DNA repair, and is overexpressed in several cancer types. Novel therapeutics are currently being developed to target WEE1 kinase in cancer, however, to date no predictive biomarkers have been approved to aid patient stratification and clinical trial design. To address this, we employed a siRNA screening to identify tumour suppressor genes (TSGs) whose loss mediates sensitivity to WEE1 inhibition. Experimental procedures: U2OS cells were reverse transfected with a customised siRNA library containing 3 independent siRNAs targeting 178 tumour suppressor genes and 24 hours later treated with either DMSO control or MK-1775 (Wee1 Kinase Inhibitor). Cell viability was measured using a cell titer-glo luminescent Cell Viability Assay 72 hours post-treatment. Hits were selected based on robust z-score analysis. Those genes with 2 or more targeted siRNAs demonstrating a robust z-score of ±1 median absolute deviation (MAD) were taken forward for validation studies. Sensitive hits were selected on a z-score of <-1 and resistant hits were selected on a z-score of >1. siRNA knockdown of WEE1 was performed in multiple human cancer cell lines and confirmed by western blotting and RT-q-PCR. Basal expression levels of phosphorylated WEE1, total WEE1, FOS and JUNB were assessed by western blotting. Results: Consistent with previously published findings, the siRNA screen demonstrated that loss of BRCA2 conferred increased sensitivity to WEE1 inhibition (Aarts et al. 2015). The siRNA screen also identified an additional 12 TSGs whose loss mediated sensitivity and 14 TSGs whose loss mediated resistance to WEE1 kinase inhibition. Interestingly, we found that loss of two early response genes, FOS and JUNB conferred resistance to WEE1 inhibition. FOS and JUNB interact to form the AP1 heterodimer, and previous published work has demonstrated the presence of an AP1 binding motif on the WEE1 promoter (Kawasaki et al. 2003). Using publically available gene expression data (TCGA) we have shown a significant correlation between expression of WEE1 with FOS and JUNB in multiple cancer types. Conclusions: Using a TSG siRNA screen, we have identified that loss of JUNB and FOS confers resistance to the WEE1 inhibitor MK1775. Future studies will investigate the mechanisms by which the loss of these genes affects response to WEE1 inhibition, and will also investigate the utility of these genes as predictive biomarkers for response to WEE1 inhibition in clinical samples, thereby aiding patient stratification. Citation Format: Victoria L. Dunne, Niamh McGivern, Kienan I. Savage, Nuala McCabe, Richard Kennedy. The role of early response genes (ERG’s) as a biomarker of response to Wee1 targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3163.

P21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775

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

Effect of WEE1 inhibitor MK-1775 on self-renewal of gallbladder cancer stem cells

Effect of WEE1 inhibitor MK-1775 on self-renewal of gallbladder cancer stem cells