Wee1 Kinase Research Articles

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

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

Anti-Tumor Effects of Wee1 Kinase Inhibitor with Radiotherapy in Human Cervical Cancer

Although the concurrent use of a chemotherapeutic agent and radiotherapy improves survival in patients with locally advanced or recurrent cervical cancer, severe side effects related to chemotherapy are frequent and may result in a low quality of life for the patients. In this study, we investigated the effects of a combination of Wee1 inhibitor (AZD1775) and irradiation in cervical cancer. In vitro effects of AZD1775 with irradiation in human cervical cancer cells were assessed by clonogenic survival and apoptosis assays. The effects on DNA damage response signaling and the cell cycle were also explored. Tumor growth delay was evaluated to investigate the in vivo effects of AZD1775 with irradiation in cervical cancer mouse models, including xenografts and patient-derived xenografts (PDXs). The co-treatment of AZD1775 and irradiation significantly decreased clonogenic survival and increased apoptosis in cervical cancer cells. These effects were associated with G2 checkpoint abrogation which resulted in persistent DNA damage. Both in the xenografts and the PDXs, the co-treatment significantly decreased tumor growth compared tothe irradiation alone (p < 0.05). These results demonstrate that the Wee1 inhibitor (AZD1775) can be considered as a potential alternative as a radiosensitizer in cervical cancer instead of a chemotherapeutic agent such as cisplatin.

Synergism Through WEE1 and CHK1 Inhibition in Acute Lymphoblastic Leukemia.

Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. Methods: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. Results: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. Conclusion: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.

A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1.

To enter into mitosis, cells must shut off the cell cycle inhibitor Wee1. SAD family protein kinases regulate Wee1 signaling in yeast and humans. In Schizosaccharomyces pombe, two SAD kinases (Cdr1/Nim1 and Cdr2) act as upstream inhibitors of Wee1. Previous studies found that S. pombe Cdr1/Nim1 directly phosphorylates and inhibits Wee1 in vitro, but different results were obtained for budding yeast and human SAD kinases. Without a full understanding of Cdr1 action on Wee1, it has been difficult to assess the in vivo relevance and conservation of this mechanism. Here, we show that both Cdr1 and Cdr2 promote Wee1 phosphorylation in cells, but only Cdr1 inhibits Wee1 kinase activity. Inhibition occurs when Cdr1 phosphorylates a cluster of serine residues linking α-helices G and H of the Wee1 kinase domain. This region is highly divergent among different Wee1 proteins, consistent with distinct regulatory mechanisms. A wee(4A) mutant that impairs phosphorylation by Cdr1 delays mitotic entry and causes elongated cells. By disrupting and retargeting Cdr1 localization, we show that Cdr1 inhibition of Wee1 occurs in cells at cortical nodes formed by Cdr2. On the basis of our results, we propose a two-step model for inhibition of Wee1 by Cdr1 and Cdr2 at nodes.

P1.03-25 Synergistic Lethality of Ferroptosis Inducer-Disulfiram/Copper Complex and WEE1 Kinase Inhibitor for RAS/TP53 Mutant NSCLC

P1.03-25 Synergistic Lethality of Ferroptosis Inducer-Disulfiram/Copper Complex and WEE1 Kinase Inhibitor for RAS/TP53 Mutant NSCLC

Combining ERBB family and MET inhibitors is an effective therapeutic strategy in cutaneous malignant melanoma independent of BRAF/NRAS mutation status

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common. A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway). IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival. In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs. We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs. The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model. The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status. Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo. Importantly, this combination therapy had minimal therapy-related toxicity in mice. Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination. Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells. In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.

Defining and targeting wild-type BRCA high-grade serous ovarian cancer: DNA repair and cell cycle checkpoints

ABSTRACTIntroduction: Molecular analyzes including molecular descriptor/phenotype interactions have led to better characterization of epithelial ovarian cancer patients, including a definition of a BRCA wild-type (BRCAwt) phenotype. Understanding how and when to use agents targeted against dependent BRCAwt pathways or other molecular events at disease progression is an important translational and therapeutic direction in ovarian cancer research.Areas covered: In this overview, we provide definitions and descriptions of a BRCAwt genotype and phenotype. We discuss novel investigational drugs that hold promise for the treatment of BRCAwt ovarian cancer, including inhibitors of poly(ADP-ribose) polymerase, ribonucleotide reductase, DNA protein kinase-catalytic subunit, ataxia-telangiectasia-mutated kinase (ATM), ataxia-telangiectasia mutated and Rad3-related kinase (ATR), CHK 1/2, cyclin kinases, glutaminase-1, WEE1 kinase, as well as tumor microenvironment and angiogenesis inhibitors. This article explores the known and the emerging areas of clinical research on patients with BRCAwt ovarian cancer.Expert opinion: Discovery of molecular changes tied to annotated disease information, along with an expanding array of pathway targets and targeted therapeutic agents, creates optimism and opportunity for women with ovarian cancer. Using precision oncology approaches, clinical researchers are, and will be, poised to select more effective treatments for ovarian cancer patients.

WEE1 kinase inhibitor shows promise.

WEE1 kinase inhibitor shows promise.

Wee1 Inhibitor AZD1775 Effectively Inhibits the Malignant Phenotypes of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo.

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.

Ensemble docking-based virtual screening toward identifying inhibitors against Wee1 kinase.

Aim:Wee1 kinase plays a key role in the arrest of G2/M checkpoint that prevents mitotic entry in response to DNA damage. This work is to discover potent Wee1 inhibitors which can be considered valuable. Materials & Methods: Herein, Ensemble docking using multiple crystal structures was considered an effective strategy in the virtual screening. The performance of 17 scoring functions obtained from different docking software was evaluated for molecular docking. Results: Two novel compounds B1 and A2 were identified as Wee1 inhibitors with IC50 values of 10.23±0.505 and 8.72±0.323μM, respectively. Further cell viability assay demonstrated that the two active compounds exhibited good anticancer activities. Conclusion: This provides a meaningful starting point for further structure optimization to discover more potent Wee1 inhibitors.

Enhancing direct cytotoxicity and response to immune checkpoint blockade following ionizing radiation with Wee1 kinase inhibition

ABSTRACTTumor 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.

Differential Activity of ATR and WEE1 Inhibitors in a Highly Sensitive Subpopulation of DLBCL Linked to Replication Stress.

DNA damage checkpoint kinases ATR and WEE1 are among key regulators of DNA damage response pathways protecting cells from replication stress, a hallmark of cancer that has potential to be exploited for therapeutic use. ATR and WEE1 inhibitors are in early clinical trials and success will require greater understanding of both their mechanism of action and biomarkers for patient selection. Here, we report selective antitumor activity of ATR and WEE1 inhibitors in a subset of non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines, characterized by high MYC protein expression and CDKN2A/B deletion. Activity correlated with the induction of replication stress, indicated by increased origin firing and retardation of replication fork progression. However, ATR and WEE1 inhibitors caused different amounts of DNA damage and cell death in distinct phases of the cell cycle, underlying the increased potency observed with WEE1 inhibition. ATR inhibition caused DNA damage to manifest as 53BP1 nuclear bodies in daughter G1 cells leading to G1 arrest, whereas WEE1 inhibition caused DNA damage and arrest in S phase, leading to earlier onset apoptosis. In vivo xenograft DLBCL models confirmed differences in single-agent antitumor activity, but also showed potential for effective ATR inhibitor combinations. Importantly, insights into the different inhibitor mechanisms may guide differentiated clinical development strategies aimed at exploiting specific vulnerabilities of tumor cells while maximizing therapeutic index. Our data therefore highlight clinical development opportunities for both ATR and WEE1 inhibitors in non-GCB DLBCL subtypes that represent an area of unmet clinical need. SIGNIFICANCE: ATR and WEE1 inhibitors demonstrate effective antitumor activity in preclinical models of DLBCL associated with replication stress, but new mechanistic insights and biomarkers of response support a differentiated clinical development strategy.

Enhancement of chemosensitivity by WEE1 inhibition in EGFR-TKIs resistant non-small cell lung cancer

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is the first-line treatment in non-resectable non-small lung cancer (NSCLC) with EGFR mutation. However, EGFR-TIKs resistance would inevitably develop within 9–14 months after treatment. And, chemotherapy is the main treatment for EGFR-TKIs resistant patients. WEE1 kinase, a G2/M checkpoint regulator, was recently considered as a putative biomarker for the platinum-based chemo-response. The aim of this study is to clarify the relationship between WEE1 kinase and chemosensitivity in EGFR-TKIs resistant NSCLC. WEE1 expression was tested in EGFR-TKIs resistant cell lines (H1299, PC9/G2) and patients’ specimens by western blot, qPCR and immunohistochemistry (IHC). In in vitro experiment, WEE1 expression was higher in EGFR-TKIs resistant than EGFR-TKIs sensitive cell lines and was gradually increased following cisplatin or gemcitabine treatment with the enrichment of G2/M cell cycle phase. And, for patients with acquired Icotinib/Gefitinib resistance, 58.4% (7/12) had increased WEE1 expression compared to its initial expression level. In order to explore the impact of WEE1 on chemo-response, WEE1 knockdown was conducted in EGFR-TKIs resistant H1299 and PC9/G2 cells. MTT and colony formation assay showed that the efficacy of cisplatin and gemcitabine was enhanced in the two cell lines after WEE1 knockdown. And, the IC50 value of cisplatin decreased from 8.64 μg/ml to 3.10 μg/ml or 2.38 μg/ml in H1299 and from 3.66 μg/ml to 0.97 μg/ml or 1.18 μg/ml in PC9/G2 after WEE1 knockdown with two specific shRNAs. This study revealed that WEE1 expression was increased after EGFR-TKIs resistance, and WEE1 knockdown could enhance chemosensitivity in EGFR-TKIs resistant NSCLC. It is suggested the combination of WEE1 inhibitor and chemotherapy might improve the clinical outcome of NSCLC patients with acquired EGFR-TKIs resistance.

Phylogenetic Analyses of Proteins Coordinating G2 Size Control in Fission Yeast

Regulation of G2 phase is based on inhibition of MPF (M-phase Promoting Factor) through phosphorylation by Wee1-like kinases. Removal of the inhibiting phosphate group requires Cdc25-like phosphatases. In fission yeast, size control is achieved by monitoring cell length via interactions of Pom1, Nif1, Cdr1 and Cdr2 proteins, regulating MPF via the Wee1 kinase. Here, a search for homologues of these key proteins was performed in the genomes of several model organisms to analyze the evolution of G2 size control. Both the known upstream pathways regulating Wee1 protein (Pom1 → Cdr2, and Nif1 → Cdr1) have been found to be characteristic only in fission yeasts. Mik1, a backup copy of Wee1 kinase probably appeared in the common ancestor of the fission yeasts. The duplication resulting in Wee1A and Wee1B isoforms probably happened in a common ancestor of higher animals, while the Myt1 protein (found only in animals) could be a variant between an ancient serine / threonine kinase and the Wee1 tyrosine kinase. Probably both the ancestors of plants and that of fungi may have lost the myt1 gene. In fission yeasts, Pyp3 is a backup phosphatase of Cdc25, also activating MPF in late G2. Interestingly, we found that the small Ibp1 phosphatase appeared to be a closer homologue of Cdc25, although its function is different. Moreover, Cdc25 homologues identified in plants were found to be more closely related to Ibp1 rather than to Cdc25 of fission yeast. In the Cdc25-like proteins, a novel conserved region was found with the consensus sequence LxxG(Y/F).

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 &amp;lt;-1 and resistant hits were selected on a z-score of &amp;gt;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.

Abstract CT138: NCI-MATCH EAY131 -Z1I: Phase II study of AZD1775, a wee-1 kinase inhibitor, in patients with tumors containing BRCA1 and BRCA2 mutations

Abstract Background: Wee1 is a tyrosine kinase implicated in the inhibitory phosphorylation of CDK1/CDC2-bound cyclin B complex responsible for G2 arrest. Wee 1 kinase inhibition results in premature mitotic entry and double strand DNA breaks in preclinical models. AZD1775 is a selective, adenosine-triphosphate (ATP) competitive, small molecule inhibitor of Wee1 kinase that directly inhibits phosphorylation of CDC2 at Tyr15. Partial responses (PR) were observed in patients (pts) with BRCAm+ solid tumors following treatment with AZD1775 in a single agent, Phase I trial (J Clin Oncol. 2015;33(30):3409-15). Clinical benefit in pts with BRCAm+ solid tumors was further evaluated in this trial, conducted as a subprotocol of the NCI-MATCH trial. Study Design: Eligible pts met all criteria for the NCI-MATCH master protocol and provided tissue specimen for central next generation sequencing, which revealed a known pathogenic mutation in BRCA 1 or 2. Pts with ovarian cancer were required to have received prior poly ADP ribose polymerase (PARP) inhibitor treatment. AZD1775 was administered orally, 300 mg once daily, 5 days on and 2 days off, 2 weeks on and 1 week off; cycle length 21 days. Cycles were repeated until disease progression or unacceptable toxicity. Radiologic assessment was performed every 3 cycles. Dose of AZD1775 was held and modified for grade ≥ 3 toxcities; which were required to resolve to ≤ Grade 2 prior to starting next cycle. Results: 33 pts were enrolled; 31 received study treatment. Male:female (11/20), age range 36-79 yrs.; ECOG PS 0/1 5/26; 67% had &amp;gt; 3 prior therapies. Tumor types: breast (9), ovarian/fallopian tube/mixed (5/1/1), pancreas (4), gallbladder (1), GI tract (4), melanoma (1), solitary fibrous tumor (1), mesothelioma (1), salivary gland (1), anaplastic oligodendroglioma (1), lung (1). BRCA 1/2 13/18. Clinical benefit: response rate 3.2% (90% CI 0.15%-14.4%); 6-month PFS rate 19% (90% CI 10%-36%): 1 PR (serous fallopian tube cancer); 4 SD &amp;gt; 6 months (ovarian cancer (3), solitary fibrous tumor (1). Median number of cycles: 2 (range 1- 18).Treatment discontinued for disease progression/adverse event/ patient withdrawal/other 17/6/3/3; 2 pts remain on study (4 and 20 cycles). Prior PARP exposure: Gyn cancer 71%; Breast cancer 22%. Prior platinum: Gyn cancer 100%; breast cancer 56%. Common side effects: myelosuppression, fatigue, nausea, vomiting, diarrhea. Conclusions: Single agent AZD1775, at the dose and schedule evaluated, was well tolerated. Modest single agent activity was observed in heavily pretreated patients with BRCA 1/2 mutated solid tumors. Evaluation of AZD1775 in combination with PARP inhibitors and/or chemotherapy may be warranted in less heavily pretreated patients with BRCA 1/2 mutated solid tumors. Citation Format: Shivaani Kummar, Shuli Li, Kim Reiss, James M. Ford, Edith P. Mitchell, James A. Zwiebel, Naoko Takebe, Robert J. Gray, Lisa M. McShane, Larry V. Rubinstein, David Patton, Paul Mickey Williams, Stanley R. Hamilton, Adam Brufsky, Barbara A. Conley, Carlos L. Arteaga, Lyndsay N. Harris, Peter J. O'Dwyer, Alice P. Chen, Keith T. Flaherty. NCI-MATCH EAY131 -Z1I: Phase II study of AZD1775, a wee-1 kinase inhibitor, in patients with tumors containing BRCA1 and BRCA2 mutations [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 CT138.

Abstract 3503: Biomarkers for inhibitors of the replication stress response proteins WEE1 and ATR in triple negative breast cancer

Abstract Replication stress (RS) is a hallmark of cancer and has the potential to be exploited by selective DNA Damage Response inhibitors. During replication stress, the DNA polymerase is uncoupled from the replisome helicase activity resulting in extended regions of single strand DNA that lead to the initiation of a replication stress response (RSR). Two key regulators of the RSR are the ATR and WEE1 kinases. Here, we aimed to identify patient selection biomarkers for WEE1 and ATR inhibitors (WEE1i, ATRi) from a cohort of 37 patient-derived tumor xenografts (PDX) from ovarian or triple negative breast cancer, 20 of which harbored BRCA1/BRCA2deleterious mutations. The antitumor response of the WEE1i AZD1775 and the ATRi AZD6837 was evaluated, and PDX tumors were characterized by exome sequencing, metabolomics, western blot and immunohistochemistry. AZD1775 treatment response in PDXs, measured as tumor regressions (≤-30% change in tumor volume), was observed in 28% of models tested. Exome sequencing identified an association between treatment response and concomitant alterations in RB1 and STK11 (LKB1), whose functional relevance was validated in an in vitro model. Immunoblots showed that WEE1i treatment resulted in downregulation of the ribonucleotide reductase subunit RRM2 and induction of an S-phase DNA damage response (DDR) in vivo. Metabolomics identified a profound change in the purine and pyrimidine synthesis pathways in WEE1i-sensitive PDXs and these changes appear functionally relevant since PDX ex vivo data demonstrate rescue of the WEE1i effects after addition of nucleosides. Another important association with WEE1i sensitivity was increased cyclin E expression. Together, these data suggest that early entry into S phase and an imbalance of origin firing to available dNTPs represent drivers of sensitivity to the WEE1i. Interestingly by contrast, ATRi-sensitive PDXs (representing 10% of the models) were characterized primarily by those tumors harboring ATM alterations, suggesting that synthetic lethality with this major DDR pathway is the primary mechanism of sensitivity to the ATR inhibitor in ovarian and triple negative breast cancer. This study therefore highlights differences between the drivers of sensitivity to inhibitors of these two important RSR proteins. Citation Format: Violeta Serra, Cristina Cruz, Zhongwu Lai, Marta Castroviejo-Bermejo, Marta Palafox, Urszula M. Polanska, Gemma N. Jones, Anderson Wang, Filippos Michopoulos, Rachel Brough, Brian Dougherty, Elaine Cadogan, Susan Critchlow, Alejandra Bruna, J. Carl Barrett, Cristina Saura, Christopher J. Lord, Carlos Caldas, Joaquin Arribas, Judith Balmaña, Mark J. O’Connor. Biomarkers for inhibitors of the replication stress response proteins WEE1 and ATR in triple negative breast cancer [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 3503.

DNA Damage Response Inhibitor Combinations Exert Synergistic Antitumor Activity in Aggressive B-Cell Lymphomas

The DNA damage response (DDR) kinases ATR, Chk1, and Wee1 play vital roles in the response to replication stress and in maintaining cancer genomic stability. Inhibitors of these kinases are currently under clinical investigation. Mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) are aggressive lymphomas whose clinical outcome is still largely unsatisfactory. These cell lymphoma subtypes are highly dependent on both Chk1 and Wee1 for survival. We investigated the activity of the ATR inhibitor AZD6738 as single agent and in combination with either Chk1 (AZD6738) or Wee1 (AZD1775) inhibitors in several preclinical models of MCL and DLBCL. This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, and validation experiments on in vivo models. Cellular and molecular mechanisms of the observed synergistic effect as well as pharmacodynamic analysis of in vivo samples were studied. AZD6738 exerted a strong synergistic cytotoxic effect in combination with both AZD7762 and AZD1775 in the 2 lymphoma subtypes regardless of their TP53, MYC, and ATM mutational status. These DDR inhibitor combinations, similarly to the Chk1/Wee1 inhibitor combination, caused a marked S-phase delay, with an increase in cyclin-dependent kinases (CDK) activity, increased DNA damage, and decreases in Wee1, MYC, and RRM2 protein levels. The synergistic in vitro activity translated to striking in vivo antitumor activity. DDR-DDR inhibitor combinations could potentially offer promising novel therapeutic strategies for patients with B-cell lymphoma.

Abstract 2956: Inhibition of CHK1 sensitizes Ewing sarcoma cells to CDK1-dependent cell death in S-phase

Abstract Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The treatment of Ewing sarcoma has changed very little in the past two decades and novel treatment approaches are needed. We recently identified that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), the rate limiting enzyme in the synthesis of deoxyribonucleotides. We subsequently found that the inhibition of checkpoint kinase 1 (CHK1) increases the sensitivity of Ewing sarcoma cells to inhibitors of RNR, such as gemcitabine. The ATR-CHK1 pathway, from a mechanistic standpoint, regulates multiple stages of the cell cycle, including S-phase, the G2/M transition, and M phase. In the current work, we used cell synchronization to identify that the inhibition of CHK1 in S-phase cells results in premature entry into mitosis and cell death. Similarly, the inhibition of ATR serine/threonine kinase (ATR), the upstream activator of CHK1, also causes Ewing sarcoma cells to inappropriately enter mitosis. Moreover, we have found in Ewing sarcoma cells that this aberrant entry into mitosis is mediated, in part, by cyclin dependent kinase 1 (CDK1). In addition, activation of CDK1 by inhibiting the WEE1 kinase with AZD1775 also results in entry into mitosis and cell death in Ewing sarcoma cells. Currently, ongoing work is focused on the in vivo testing of gemcitabine in combination with CHK1 and WEE1 inhibitors as a novel therapeutic approach for the treatment of Ewing sarcoma. Citation Format: David Gordon, Kelli Goss, Stacia Koppenhafer. Inhibition of CHK1 sensitizes Ewing sarcoma cells to CDK1-dependent cell death in S-phase [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 2956.

Abstract 3499: Dianhydrogalactitol (VAL-083) in combination with AZD1775 increases survival in diffuse intrinsic pontine glioma (DIPG), in vivo

Abstract OBJECTION: VAL-083 is a structurally unique bi-functional DNA targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces interstrand crosslinks leading to DNA double-strand breaks and S/G2 cell cycle arrest. By inhibiting the G2 checkpoint regulator kinase Wee1, AZD1775 allows a cancer cell with DNA damage to progress past the G2 checkpoint and into premature mitosis leading to cancer cell death. Herein we assess the activity of VAL-083 as single agent as well as in combination with Wee1 Kinase inhibitor AZD1775 in patient derived model systems of DIPG. METHODS: DIPG derived cell lines SF8628 and NEM157 (H3.3K27) as well as SF10693 (H3.1K27M) and pediatric glioblastoma cell lines SF188 (H3.3K27 wildtype) were treated with increasing concentrations of single agent VAL-083 as well as in combination with AZD1775. To determine potential synergistic activity, we applied the Chou-Talalay method, which allows the quantitative determination of drug interactions by calculating a combination index (CI). In vivo activity of VAL-083 (3 mg/kg) as single agent as well as in combination with AZD1775 (60 mg/kg) was assessed in an orthotopic engraftment model of pediatric DIPG (SF8628). RESULTS: The IC50 of VAL-083 as single agent ranged from 1µM to 10µM. VAL-083 exhibited synergistic activity in combination with AZD1775 in cell lines SF8628 and SF188 and additive effect in NEM157 with CI values ranging from 0.04 to 0.95, with CI &amp;lt; 1 indicating synergy. In vivo, treatment with VAL-083 as single agent and in combination with AZD1775 conferred significant survival benefit to mice with engrafted DIPG tumors compared to control as well as single agent treatment with AZD1775. The median survival for mice treated with VAL-083 alone was 54.5 days (p=0.0004, VAL-083 vs. control) and for the VAL-083/AZD1775 combination it was 62 days (p&amp;lt;0.0001, VAL-083/AZD1775 vs. control), compared to 44 days for control and 47 days for mice treated with AZD1775 alone (p=0.0839, AZD1775 vs. control). CONCLUSION: Our present study highlights that the combination of VAL-083 and AZD1775 might be a promising new therapeutic strategy for children with DIPG. Ongoing studies continue to assess the in vivo activity in other DIPG models as well explore the underlying mechanism of action of the combination strategy. Citation Format: Anne Steino, Xiaodong Yang, Cassie Kline, Jeffrey Bacha, Dennis M. Brown, Sabine Mueller. Dianhydrogalactitol (VAL-083) in combination with AZD1775 increases survival in diffuse intrinsic pontine glioma (DIPG), in vivo [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 3499.