Wee1 Inhibitor Research Articles

Abstract B018: RNR inhibitors enhance chemotherapeutic activity in pancreatic cancer cells through altering replication stress

Abstract Despite extensive study, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal resulting in a need for new effective therapies and, in particular, combination therapies. Gemcitabine that targets ribonucleotide reductase (RNR) is active in PDAC, albeit with modest impacts on long-term outcomes. Inhibition of RNR leads to depletion of dNTP pools, provoking replication stress (RS). RS activates the ATR/CHK1/WEE1 S phase DNA damage checkpoint cascade. Our study provides insights into the dynamic interplay between inhibitors of RRM1 (gemcitabine, clofarabine, fludarabine) and RRM2 (triapine) and targeting of the ATR/WEE1 DNA damage checkpoint cascade. Understanding the mechanisms underlying the combination therapy activity will enable development of optimal dosing approaches that optimize efficacy while limiting toxicity. RNR inhibitors and DNA damage checkpoint inhibitors were assessed on viability and cell growth as mono and combination therapy in human PDAC cell lines and in mouse transgenic PDAC cell lines (KMC cell lines). We detected RS using DNA combing and pRPA32 foci/H2AX staining and correlated these with response to single and combination therapy. Gemcitabine caused greater S phase to G2 cell cycle delay in sensitive cell lines (CFPAC1, HPAFII) compared to resistant cell lines (Miapaca2, PANC1). Delayed S phase progression induced by gemcitabine appears to be due to impairment of the replication machinery leading to a DNA damage response (γH2AX S139) and fork stalling. During fork stalling, RPA32 protects single-strand DNA (ssDNA). Gemcitabine increases γH2AX, as well as double positive staining for γH2AX and pRPA32 foci, and decreases replication fork speed indicative of RS. These changes correlated with the sensitivity to gemcitabine monotherapy. KMC cell lines exhibited lower sensitivity to the ATR inhibitor, AZD6738, and the WEE1 inhibitor, MK1775, than human PDAC cell lines. MK1775 and AZD6738 IC50s in PDAC cell lines were highly correlated (Pearson r=0.888, p value=0.0006), although the correlation was weaker in KMC PDAC cell lines (Pearson r=0.353, p value=0.350). Combined treatment with gemcitabine and ATR/WEE1 inhibitors was effective in gemcitabine-resistant human and KMC PDAC cell lines both in terms of decreased viability and increased RS. Other inhibitors of RRM1 (clofarabine) and also RRM2 (triapine) also effectively decreased cell viability in combination with ATR/WEE1 inhibitors. The viability was recovered with exogenous dNTPs in cells treated with the RRM1 inhibitors gemcitabine and clofarabine, but not the RRM2 inhibitor triapine, consistent with the RS induced by RRM1 inhibitors being due to decreased endogenous dNTP levels. RRM1 and RRM2 inhibitors cause replication stress and are synergistic with inhibition of the ATR/WEE1 pathway that contributes to ability of cells to survive RS-driven cell death. These findings support the design and implementation of in vivo studies that will subsequently drive clinical trials with potential benefit for PDAC patients. Citation Format: Soon Young Park, Kang Jing Jeong, Alfonso Poire, Isabel English, Jonathan R. Brody, Rosalie C. Sears, Gordon B. Mills. RNR inhibitors enhance chemotherapeutic activity in pancreatic cancer cells through altering replication stress [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B018.

AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy.

Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma. The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro . A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry. Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro . AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 + /CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion. This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.

WEE1 Inhibitors Mediate Antitumor Effects on Endometrial Cancer through Activation of Innate Immune Responses.

Introduction: Recurrence signifies the primary mortality factor in patients suffering from endometrial cancer, with few efficacious treatments currently available for recurrent cases. This research investigates the anti-tumoral capacities of WEE1 inhibitors within the context of endometrial cancer, aiming to establish a novel therapeutic avenue for high recurrence-risk patients. Materials and methods: We evaluated WEE1 expression in endometrial cancer patients utilizing immunohistochemistry on paraffin-embedded tissue sections. The cytotoxic potential of WEE1 inhibitors on endometrial cancer cells was assessed by CCK8 assay. Assays to gauge the influence of WEE1 inhibitors on cell proliferation and migration included clonal proliferation, wound healing, and transwell assays. We determined the impacts on apoptosis and cell cycle stages by flow cytometry. Employing qRT-PCR and western blotting, we investigated the mechanistic pathways underlying the anti-tumoral activity of WEE1 inhibitors. In vivo evaluations were executed to ascertain the inhibitory effect of WEE1 inhibitors on tumor growth in mice. Results: WEE1 exhibited high-level expression in endometrial cancer tissues, particularly pronounced in recurrent compared with non-recurrent patients. WEE1 inhibitors effectively eliminated endometrial cancer cells while inhibiting their proliferation and migration. Flow cytometric analyses revealed a significant promotion of apoptosis and an increase in G2/M phase cell proportion upon WEE1 inhibitor treatment. qRT-PCR and western blotting elucidated that WEE1 inhibitors activated the innate immune signaling pathway in endometrial cancer cells. Furthermore, in vivo assessments demonstrated substantial tumor growth suppression due to WEE1 inhibitors. Conclusions: WEE1 inhibitors initiated an innate immune response in endometrial cancer, exhibiting considerable anti-tumoral effects, which was promising for postoperative treatment of endometrial cancer, especially recurrent endometrial cancer patients.

Targeting WEE1 Kinase in Gynecological Malignancies.

WEE1 kinase is involved in the G2/M cell cycle checkpoint control and DNA damage repair. A functional G2/M checkpoint is crucial for DNA repair in cancer cells with p53 mutations since they lack a functional G1/S checkpoint. Targeted inhibition of WEE1 kinase may cause tumor cell apoptosis, primarily, in the p53-deficient tumor, via bypassing the G2/M checkpoint without properly repairing DNA damage, resulting in genome instability and chromosomal deletion. This review aims to provide a comprehensive overview of the biological role of WEE1 kinase and the potential of WEE1 inhibitor (WEE1i) for treating gynecological malignancies. We conducted a thorough literature search from 2001 to September 2023 in prominent databases such as PubMed, Scopus, and Cochrane, utilizing appropriate keywords of WEE1i and gynecologic oncology. WEE1i has been shown to inhibit tumor activity and enhance the sensitivity of chemotherapy or radiotherapy in preclinical models, particularly in p53-mutated gynecologic cancer models, although not exclusively. Recently, WEE1i alone or combined with genotoxic agents has confirmed its efficacy and safety in Phase I/II gynecological malignancies clinical trials. Furthermore, it has become increasingly clear that other inhibitors of DNA damage pathways show synthetic lethality with WEE1i, and WEE1 modulates therapeutic immune responses, providing a rationale for the combination of WEE1i and immune checkpoint blockade. In this review, we summarize the biological function of WEE1 kinase, development of WEE1i, and outline the preclinical and clinical data available on the investigation of WEE1i for treating gynecologic malignancies.

ACOX2 Serves as a Favorable Indicator Related to Lipid Metabolism and Oxidative Stress for Biochemical Recurrence in Prostate Cancer.

Given the heterogeneity of tumors, there is an urgent need for accurate prognostic parameters in prostate cancer (PCa) patients. Lipid metabolism (LM) reprogramming and oxidative stress (OS) play a vital role in the progression of PCa. In this work, we identified five LM-OS-related genes (including ACOX2, PPRAGC1A, PTGS1, PTGS2, and HAO1) associated with the biochemical recurrence (BCR) of PCa. Subsequently, a prognostic signature was established based on these five genes. Kaplan-Meier survival estimates, receiver operating characteristic curves, and relationship analysis between risk score and clinical characters were applied to measure the robustness of the signature in an external cohort. A nomogram of risk score combined with clinical characteristics was constructed for clinical application. Functional enrichment analysis suggested that the underlying mechanism related to the signature included the calcium signaling, lipid transport, and cell cycle signaling pathways. Furthermore, WEE1 inhibitor was identified as a potential agent related to the cell cycle for high-risk patients. The mRNA expression and the prognostic value of the five genes were determined, and ACOX2 was identified as the key gene related to the prognostic signature. The protein expression of ACOX2 was measured in a prostate tissue microarray through an immunohistochemistry assay, confirming the bioinformatics results. By constructing the ACOX2-overexpressing PCa cell lines PC-3 and 22Rv1, the biological function of PCa cells was investigated. The cell viability, colony formation, migration, and invasion ability of PCa cell lines overexpressing ACOX2 were hindered. Decreased cellular lipid content and elevated cellular ROS content were observed in ACOX2-overexpressing PCa cell lines with reduced G2/M phases. In conclusion, this work presents the first prognostic signature specifically focused on LM-OS for PCa. ACOX2 could serve as a favorable indicator for the BCR in PCa. Further experiments are required to identify the potential underlying mechanism.

Fission yeast Wee1 is required for stable kinetochore-microtubule attachment.

Wee1 is a cell cycle regulator that phosphorylates Cdk1/Cdc2 and inhibits G2/M transition. Loss of Wee1 in fission yeast results in an early onset of mitosis. Interestingly, we found that cells lacking Wee1 require the functional spindle checkpoint for their viability. Genetic analysis indicated that the requirement is not attributable to the early onset of mitosis. Live-cell imaging revealed that some kinetochores are not attached or bioriented in the wee1 mutant. Furthermore, Mad2, a component of the spindle checkpoint known to recognize unattached kinetochores, accumulates in the vicinity of the spindle, representing activation of the spindle checkpoint in the mutant. It appears that the wee1 mutant cannot maintain stable kinetochore-microtubule attachment, and relies on the delay imposed by the spindle checkpoint for establishing biorientation of kinetochores. This study revealed a role of Wee1 in ensuring accurate segregation of chromosomes during mitosis, and thus provided a basis for a new principle of cancer treatment with Wee1 inhibitors.

Major clinical research advances in gynecologic cancer in 2023: a tumultuous year for endometrial cancer.

In the 2023 series, we summarized the major clinical research advances in gynecologic oncology based on communications at the conference of Asian Society of Gynecologic Oncology Review Course. The review consisted of 1) Endometrial cancer: immune checkpoint inhibitor, antibody drug conjugates (ADCs), selective inhibitor of nuclear export, CDK4/6 inhibitors WEE1 inhibitor, poly (ADP-ribose) polymerase (PARP) inhibitors. 2) Cervical cancer: surgery in low-risk early-stage cervical cancer, therapy for locally advanced stage and advanced, metastatic, or recurrent setting; and 3) Ovarian cancer: immunotherapy, triplet therapies using immune checkpoint inhibitors along with antiangiogenic agents and PARP inhibitors, and ADCs. In 2023, the field of endometrial cancer treatment witnessed a landmark year, marked by several practice-changing outcomes with immune checkpoint inhibitors and the reliable efficacy of PARP inhibitors and ADCs.

Discovery of potential WEE1 inhibitors via hybrid virtual screening.

G2/M cell cycle checkpoint protein WEE1 kinase is a promising target for inhibiting tumor growth. Although various WEE1 inhibitors have entered clinical investigations, their therapeutic efficacy and safety profile remain unsatisfactory. In this study, we employed a comprehensive virtual screening workflow, which included Schrödinger-Glide molecular docking at different precision levels, as well as the utilization of tools such as MM/GBSA and Deepdock to predict the binding affinity between targets and ligands, in order to identify potential WEE1 inhibitors. Out of ten molecules screened, 50% of these molecules exhibited strong inhibitory activity against WEE1. Among them, compounds 4 and 5 showed excellent inhibitory activity with IC50 values of 1.069 and 3.77nM respectively, which was comparable to AZD1775. Further investigations revealed that compound 4 displayed significant anti-proliferative effects in A549, PC9, and HuH-7 cells and could also induce apoptosis and G1 phase arrest in PC9 cells. Additionally, molecular dynamics simulations unveiled the binding details of compound 4 with WEE1, notably the crucial hydrogen bond interactions formed with Cys379. In summary, this comprehensive virtual screening workflow, combined with in vitro testing and computational modeling, holds significant importance in the development of promising WEE1 inhibitors.

Synthesis and biological evaluation of (2‐aminosulfonylpyridin‐6‐yl)pyrazolopyrimidinone derivatives as Wee1 inhibitors for cancer treatment

AbstractFor an analog‐based design of novel Wee1 inhibitors, we profiled in vitro ADMET and in vivo PK properties of adavosertib. Based on the properties of adavosertib, we aimed to improve its metabolic stability by designing a novel target compound 1a with an aminosulfonyl group instead of the 2‐hydroxypropan‐2‐yl moiety in adavosertib. Derivatives of target compound 1a were synthesized and evaluated for Wee1 enzyme inhibition and liver microsomal phase I stability. We identified compound 1a as a sub‐nanomolar Wee1 inhibitor and 10 additional compounds with one‐digit nanomolar Wee1 inhibitory activity, among which seven compounds including 1a exhibited improved metabolic stabilities compared with adavosertib. However, MDA‐MB‐231 cell growth inhibitory activities of all synthesized compounds and Wee1 substrate phosphorylation inhibitory activities of selected compounds were inferior to adavosertib overall. Moreover, the representative compound 1a exhibited low permeability, which may be the reason for the low cellular activities of compound 1a.

FBH1 deficiency sensitizes cells to WEE1 inhibition by promoting mitotic catastrophe

WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.

Abstract B028: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a poorly immunogenic, high-grade neuroendocrine carcinoma arising in the lung. Immune checkpoint blockade (ICB) added to chemotherapy is now the standard upfront therapy for SCLC but leads to a modest increase in overall survival and progression-free survival. These modest benefits underscore the critical need to identify pathways and targets that can durably enhance the antitumor responses of ICB in SCLC. SCLC is characterized by a ubiquitous loss of TP53 which disrupts the G1-S cell cycle checkpoint and as a result, most SCLC is dependent on G2-M cell cycle checkpoint regulators including WEE1. We and others have shown that DNA damage response (DDR) components are overexpressed in SCLC and targeting DDR could be an effective therapeutic strategy in SCLC. Methods: In this study, we have performed WEE1 inhibition either alone or in combination with PD-L1 blockade in a panel of SCLC models. Results: We demonstrate that inhibition of WEE1 induces G2/M cell cycle arrest, DNA damage, and cytosolic DNA accumulation in SCLC models. We further show that WEE1 targeting activates the STING-TBK1-IRF3 pathway which increases type I interferons (IFN-α and IFN-β) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T-cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade caused remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T-cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Conclusions: Our study demonstrates the cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC. Given the increasing importance of immunotherapy for the management of SCLC and that WEE1 inhibitors are already in clinical trials, combining a WEE1 inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B028.

Abstract A023: Preclinical development of PKMYT1 and WEE1 inhibitor combinations

Abstract Background: CCNE1-amplification or overexpression are prevalent alterations in multiple tumor types, including high-grade serous ovarian, endometrial, and gastro-esophageal cancers. CCNE1-amplification is associated with high levels of replication stress, genomic instability, and resistance to chemotherapy. Previously, CRISPR-enabled synthetic lethal screening identified that loss of the CDK1 regulator Membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) is synthetic lethal with CCNE1-overexpression, leading to the development of lunresertib - a first-in-class, potent, selective, orally bioavailable PKMYT1 inhibitor currently being investigated in phase I clinical trials (NCT04855656, NCT05147272, NCT05147350). In CCNE1 overexpressing cells, lunresertib treatment leads to reduction of inhibitory CDK1-T14 phosphorylation and activated CDK1 in S-phase, triggering premature mitosis, chromosome pulverization, and ultimately cell death. In CCNE1-amplified tumor models, lunresertib exhibits antitumor activity as monotherapy. We hypothesized that further activation of CDK1 by reducing the inhibitory phosphorylation site at CDK1-pY15 by WEE1 inhibition would increase the antitumor activity of lunresertib. Methods: In vitro cell viability studies were used to determine the level of synergy between lunresertib and the WEE1 inhibitor Debio-0123 in engineered as well as tumor-derived CCNE1-high models. The lunresertib/Debio-0123 combination mechanism of action was investigated using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, the pre-clinical anti-tumor activity and tolerability of combination lunresertib/Debio-0123 was evaluated in vivo using CCNE1-amplified tumor xenografts in mice. Results: Combination of low doses of lunresertib with Debio-0123 selectively induced synergistic cytotoxicity and catastrophic DNA damage in CCNE1-overexpressing cells, without affecting wild type counterparts. Increased DNA damage resulted from robust activation of CDK1 in S-phase, causing rapid induction of premature mitosis. Combination of lunresertib and Debio-0123 abrogated phosphorylation of both Thr14 and Tyr15 inhibitory CDK1 sites at doses that led to only partial dephosphorylation in both monotherapy settings. Mechanistically, synergistic CDK1 dephosphorylation was partially mediated by activation of CDC25B phosphatase. In a mouse model, the combination of RP-6306 and Debio-0123 was well-tolerated and demonstrated tumor regressions in a CCNE1-overexpressing tumor xenograft model at sub-therapeutic monotherapy doses. Conclusions: Lunresertib and the WEE1 inhibitor Debio-0123 elicit strong synergistic activity in preclinical models of CCNE1-overexpressoin due to rapid and complete CDK1 activation. In a xenograft tumor model, intermittent dosing of lunresertib/WEE1i lead to tumor regressions at sub-therapeutic monotherapy doses. Together these findings provide mechanistic rationale for clinical development of PKMYT1 and WEE1 inhibitor combinations. Citation Format: David Gallo, Fenil Shah, Rino Stocco, Prasamit Baruah, Jimmy Fourtounis, Rosie Kryczka, Marc L Hyer, Anne Roulston, C Gary Marshall, Michal Zimmermann, Stephen Morris, Michael Zinda. Preclinical development of PKMYT1 and WEE1 inhibitor combinations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A023.

Abstract B144: Discovery and preclinical evaluation of novel oral WEE1 degraders

Abstract Background: WEE1 is a key component of cell cycle checkpoints as it inhibits cell cycle progression through phosphorylation of CDK1. Inhibition of WEE1 could induce premature mitotic entry of cells with damaged DNA, leading to mitotic catastrophe and cell death. WEE1 small molecule inhibitors (SMIs) have demonstrated clinical benefits particularly in gynecological malignancies. However, limited antitumor activity and multifaceted toxicity profile from SMIs warranted the discovery of novel WEE1-targeting modalities to afford better therapeutic index towards a greater patient population. Here, we report the discovery of a novel oral, heterobifunctional WEE1 degrader NXD01 and its preclinical evaluation. Methods: We discovered NXD01 through our AI-empowered, prediction-guided compound optimization platform neoDegrader. We quantified NXD01-mediated WEE1 protein degradation by western blot and evaluated the anti-proliferative activity of NXD01 by CellTiter-Glo assay in multiple human cancer cell lines. We further assessed downstream effects of WEE1 degradation on phosphorylation of its target CDK1, and markers of cell cycle and apoptosis by western blot and flow cytometry. We studied the in vivo antitumor activity of NXD01 by oral gavage in the SK-UT-1 (human uterine leiomyosarcoma cell) xenograft model, followed by PK/PD analysis of the tumor tissues. Results: In SK-UT-1 cells, NXD01 potently degraded WEE1 protein at a DC50 of 1.4 nM with no obvious hook effect and achieved complete degradation within 4 h of treatment (DC90 = 4.5 nM). Marked reduction in CDK1-Y15 phosphorylation and increased γ-H2AX levels (a marker for DNA damage) and PARP cleavage (apoptosis) were all detected for NXD01 at 100 nM, one-tenth the effective concentration for the WEE1 SMI azenosertib (ZN-c3). Treatment of NXD01 (but not ZN-c3) at 100 nM greatly increased the proportion of cells in the G2/M phase indicating mitotic arrest. These results translated into a more potent anti-proliferative effect by NXD01 compared to ZN-c3 in vitro: the IC50 was 1.3 nM vs 37 nM for MOLT4, a T cell leukemia line, as well as across 10 gynecological cancer cell lines (NXD01 IC50: 10-210 nM; ZN-c3 IC50: 100-975 nM). NXD01 exhibited good exposure after oral administration in mice (dose normalized AUC0-t: 765 mg∙h/mL, t1/2: 3 h, F%: 15.7%). NXD01 showed strong in vivo antitumor activity in the SK-UT-1 xenograft model, achieving 96% tumor growth inhibition (TGI) at 30 mg/kg BID, as compared to 81% TGI for ZN-c3 at 60 mg/kg QD. Analysis of tumor samples suggested that 85% of in-tumor WEE1 protein had been degraded by NXD01 at 12 h post dose. Conclusion: We have discovered a structurally-novel, orally bioavailable, cereblon-mediated, heterobifunctional degrader of WEE1 protein NXD01. NXD01 displayed stronger in vitro and in vivo antitumor activity than currently the most advanced WEE1 small molecule inhibitor azenosertib, making NXD01 a differentiated candidate with strong potential for clinical development. Citation Format: Weiqiang Xing, Meixin Tao, Fan Huang, Liuge Gu, Min Cao, Chunhao Wang, Jianxiong Diao, Chunming Du, Xinyu Bai, Mingchen Chen, Xin Zheng, Ying Lei, Yang Xie, Taylor B. Guo. Discovery and preclinical evaluation of novel oral WEE1 degraders [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B144.

Abstract B060: Dual blockade of BRD4 and the ATR/WEE1 pathway exploits ARID1A loss in clear cell ovarian cancer

Abstract Clear Cell Ovarian Cancer (CCOC) is one of the most challenging subtypes of ovarian cancer to treat as advanced stages are often chemo-resistant compared to the more common high-grade serous ovarian cancer (HGSOC). Limited treatment options are available for this devastating disease. Understanding the resistance mechanism and identifying effective therapeutic options for CCOC are clearly needed. Exploiting genetic alterations common in CCOC, such as ARID1A, is a rational strategy for combination therapies. ARID1A, an epigenetic tumor suppressor, is the most common genetic mutation in CCOC, with about 50% of CCOC demonstrating loss of ARID1A. ARID1A regulates the transcription of specific genes (e.g. c-myc) by altering the chromatin structure. We hypothesize that ARID1A loss increases CCOC cell dependency on chromatin remodeling and DNA repair pathways for survival. Through a drug screen, BET inhibitors and DNA damage response inhibitors were the most active monotherapies in ARID1A mutant CCOC. However, monotherapy efficacy needs to be further improved. We demonstrate in our study that the combination of BRD4 inhibitor (BRD4i) with DNA damage response (DDR) inhibitors (ATR or WEE1 inhibitors; e.g. BRD4i-ATRi) were synergistic at low doses leading to decreased survival, and colony formation in an ARID1A dependent manner. Combination BRD4i-ATRi caused significant tumor regression and increased overall survival in ARID1AMUT not ARID1AWT patient-derived xenograft (PDX) models. Combination BRD4i-ATRi significantly increased gH2AX, and decreased RAD51 foci and BRCA1 expression suggesting loss in the ability to repair DNA double-strand-breaks by homologous recombination (HR), more so than with monotherapy in ARID1AMUT cells. These studies demonstrate BRD4i-ATRi is an effective treatment strategy that capitalizes on synthetic lethality with ARID1A loss in CCOC warranting further study. Citation Format: Yasuto Kinose, Haineng Xu, Sushil Kumar, Hyoung Kim, Sergey Medvedev, Xiaolei Wang, Erin George, Xiaoyin Shan, Sarah Gitto, Kurt D'Andrea, Bradley Wubbenhorst, Margaret Whicker, Dorothy Hallberg, Lauren Schwartz, Katherine L Nathanson, Gordon Mills, Victor E Velculescu, Tian-Li Wang, Eric Brown, Ronny Drapkin, Fiona Simpkins. Dual blockade of BRD4 and the ATR/WEE1 pathway exploits ARID1A loss in clear cell ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B060.

Abstract C171: Immune desert microenvironment is associated with poor outcome in Wilms tumors

Abstract Background: Presence of anaplasia is a potent marker for adverse outcome in patients with Wilms tumors with diffuse anaplasia (DAWT). Patients with WT with focal anaplasia (FAWT) are considered as intermediate risk. Besides TP53 mutations thought to arise in anaplastic components, the crosstalk between genomic alterations and immune landscape remains unknown. Methods: We performed whole-exome and/or RNA sequencing on genomic DNA derived from 12 matched tumor-normal WTs and extended analysis in an independent dataset of 9 cases. TP53 mutations and tumor infiltrating lymphocytes (TILs: CD3, CD4 and CD8) were also assessed. Validation was performed using TARGET-WT (n=96) and GEO-WT (n=12) cohorts. Prognostic role of TILs was investigated in another 55 pre-treated WTs. Results: TP53 (50%) and DROSHA (33.3%) were the sole recurrent mutations identified. TP53 mutations occurred in mutually exclusive manner with chromatin-modifying genes which were altered in both FAWT (100%) and DAWT (33.3%). Microenvironment analysis revealed a reproducible immune desert WTs with poor outcome that enriched in DAWT cases and TP53 mutation, which mirrored the inversion of CD4:CD8 ratio in TP53-mutated comparing to TP53-wild tumors (P=0.003). Immune desert WTs were also characterized by overexpression of DNA repair pathways, PRC2, HDCA, and polycomb targets of EZH2, leading to higher sensitivity to HDCA inhibitor of vorinostat. Additionally, the immunophenotypes were tightly associated with replication stress subtypes; WTs with high replication stress were susceptible to ATR and WEE1 inhibitors. High ratio of CD4/CD8 was considered as independent recurrence-free and overall survival factors for WTs (both P<0.05). Conclusions: Our results reveal that WT with anaplastic features are characterized by either TP53 alterations or chromatin-modifying genes. These data implicate TP53 as a tumor cell-intrinsic driver of an immune desert phenotype, in contrast with adult TP53 tumors. TP53 inactivation might therefore causes epigenetic deregulation across cancer sites, and has implications for immunotherapy. Citation Format: Xiaoping Su, Gabriel Malouf. Immune desert microenvironment is associated with poor outcome in Wilms tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C171.

Abstract B086: Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models

Abstract Background: Triple negative breast cancer subtype is associated with poor prognosis and is mostly treated with chemotherapy, including the DNA crosslinking agent Carboplatin. Carboplatin treatment leads to DNA damage and a cell cycle inhibition allowing DNA damage repair. However, many patients are resistant or develop resistance to Carboplatin. The identification of novel treatments against chemoresistant TNBC is a major unmet clinical need. Method: TNBC patient-derived cell lines (PDCs) from patient-derived xenografts (PDXs) generated from chemotherapy resistant tumors were used for shRNA high throughput screens to identify genetic vulnerabilities that could resensitize these cells to Carboplatin. Validation of this target was performed genetically by shRNA silencing and pharmacologically with commercially available target inhibitors both in vitro and in vivo, using Alamar Blue viability assays and calculating synergy using the Chou Talalay method. DNA damage was assessed using yH2AX expression, and apoptosis using c-PARP and Annexin V, by confocal microscopy, flow cytometry and/or western blotting. Results: We identified ataxia telangiectasia and Rad3-related protein (ATR) as a target in one of the cell lines screened and validated it with an ATR shRNA. We tested the combination of the ATR inhibitor (ATRi), Elimusertib, with Carboplatin and found the combination to be synergistic (Average CI=0.30). DNA damage was markedly increased with combination treatment and a pan-nuclear yH2AX staining together with increased pHH3 expression were observed, suggestive of mitotic catastrophe. Cells treated with Carboplatin accumulated in S phase and a shift toward G2/M when Elimusertib was added. Apoptosis was significantly increased with combination treatment. PKMYT1 and WEE1 are two kinases regulating cell cycle entry by inhibiting CDK1, which controls entry into mitosis. Carboplatin treatment resulted in an increase in PKMYT1 expression. PKMYT1 inhibition with RP6306 re-sensitized the cells to Carboplatin more than WEE1 inhibition with Adavosertib, and this synergy was further increased with low doses of Elimusertib. Gene silencing of WEE1 and PKMYT1 both re-sensitized the cells to Carboplatin, but only WEE1 silencing alone significantly prevent proliferation, suggesting the requirement of PKMYT1 activity for resistance but not for cell proliferation without Carboplatin. In vivo validation using the PDXs from which the cell lines have been derived, demonstrated a significant delayed in tumor growth and prolonged survival when combining Carboplatin with ATRi compared to Carboplatin alone in two models. Conclusion: ATR inhibition resensitizes drug resistant TNBC cells to Carboplatin both in vitro and in vivo. The combination of Carboplatin with ATRi led to DNA damage and mitotic catastrophe. PKMYT1 plays a role in Carboplatin-resistant TNBC, collaborating with ATR in managing the cell cycle response to DNA damage, and PKMYT1 inhibition also re-sensitizes cells to Carboplatin, an effect increased with the triple combination of adding ATR inhibition. Citation Format: Juliet Guay, Catherine Chabot, Cédric Darini, Marguerite Buchanan, Adriana Aguilar-Mahecha, Tim Kong, Connie Yang, Mark Basik, Sidong Huang. Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B086.

Abstract C147: The DNA replication checkpoint inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi), are potentially well tolerated and effective cancer treatments

Abstract Previous studies have demonstrated WEE1i and ATR inhibitors (ATRi and WEE1i) to be promising cancer therapeutics through synthetic lethality with various cancer associated mutations. However, a key limitation to the use of these inhibitors as cancer therapies in prior clinical trials has been the occurrence of adverse hematological effects, including anemia and thrombocytopenia. Herein, we describe two novel inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi) that are potentially both effective in tumor suppression in animal models and well tolerated. ATRN-1051 was developed to be both a potent WEE1i and selective for WEE1 over other kinases (PLK1, PLK2 and PLK3). ATRN-1051 has an IC50 of 2.2 nM for WEE1 and limits the proliferation of various cancer cell lines in culture in the 100 nM to 200 nM range. Notably, ATRN-1051 is particularly effective in suppressing the growth of Cyclin E overexpressing cells lines, pinpointing Cyclin E as a potential biomarker for ATRN-1051. In addition, ATRN-1051 has potentially favorable pharmacokinetic properties that permits 3-8 times lower dosing than other clinical WEE1 inhibitors to achieve similar exposure (AUC, 0-24) levels (1). Consistent with the increase selectivity of ATRN-1051 fostering increased tolerability, dose-range finding studies indicate that doses potentially expected to cause significant tumor suppression are hematological well tolerated in mice, with red blood cell and platelet counts remaining in a non-pathogenic range. Importantly, daily oral dosing of ATRN-1051 suppresses the growth of CCNE1-amplified high-grade serous ovarian xenografted tumors over the course of 28 days, providing further evidence of the role of CCNE1-overexpression as a biomarker for ATRN-1051 treatment. Based on these data, ATRN-1051 has entered and is now progressing through IND-enabling studies. As a distinct upstream DNA replication checkpoint inhibitor, ATRN-119 is a macrocyclic ATRi that is highly specific for inhibition of ATR over other phosphatidylinositol kinase-related kinases (PIKKs), such as ATM, DNA-PK, and MTOR, implying the potential for increased tolerability. Supporting this implication, daily dosing of ATRN-119 suppresses tumor growth in xenograft mouse models of colon, pancreatic, and prostate cancers and causes no appreciable loss of body weight or hematologic toxicity. Daily dosing of ATRN-119 in combination with PARP inhibition causes significant tumor reduction in a BRCA2-deficient PDX model of high-grade serous ovarian cancer, again with no appreciable loss of body weight. These findings have led to a biomarker-driven Phase 1/2a clinical trial of ATRN-119 with daily dosing (Simpkins, PI). Together, these findings underscore the promise of ATRN-1051 and ATRN-119 as DNA replication checkpoint inhibitors for the treatment of a variety of cancers. (1) Data from study in A-427 NSCLC xenograft model as reported in Zentalis Corporate Overview, March 2022 Citation Format: Joseph Vacca, Stephen Rocca, Molly Hansbarger, Sonia Ritzmann, Justin Frye, Fiona Simpkins, Eric J. Brown, Oren Gilad. The DNA replication checkpoint inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi), are potentially well tolerated and effective cancer treatments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C147.

Delineating the interplay between oncogenic pathways and immunity in anaplastic Wilms tumors

Wilms tumors are highly curable in up to 90% of cases with a combination of surgery and radio-chemotherapy, but treatment-resistant types such as diffuse anaplastic Wilms tumors pose significant therapeutic challenges. Our multi-omics profiling unveils a distinct desert-like diffuse anaplastic Wilms tumor subtype marked by immune/stromal cell depletion, TP53 alterations, and cGAS-STING pathway downregulation, accounting for one-third of all diffuse anaplastic cases. This subtype, also characterized by reduced CD8 and CD3 infiltration and active oncogenic pathways involving histone deacetylase and DNA repair, correlates with poor clinical outcomes. These oncogenic pathways are found to be conserved in anaplastic Wilms tumor cell models. We identify histone deacetylase and/or WEE1 inhibitors as potential therapeutic vulnerabilities in these tumors, which might also restore tumor immunogenicity and potentially enhance the effects of immunotherapy. These insights offer a foundation for predicting outcomes and personalizing treatment strategies for aggressive pediatric Wilms tumors, tailored to individual immunological landscapes.

The XPO7/Npat Axis Inactivation Is a Therapeutic Vulnerability for TP53-Mutated AML

In acute myeloid leukemia (AML), TP53 mutations are the most prognostically unfavorable genetic alterations, as they confer resistance to various therapeutic agents and form relapsing clones, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes or pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. We employed two distinct KO screen libraries, through which we identified Xpo7, a putative nuclear/cytoplasmic transporter, as a common factor necessary for Trp53-KO AML survival. Interestingly, Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. Further probing this phenomenon, we performed a CRISPR-Cas9 saturation mutagenesis scan on all Xpo7 exons. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT AMLs. In stark contrast, these same sgRNAs were mostly depleted in Trp53-KO AMLs. To determine the role of Xpo7 depletion on the expansion of Trp53 deficient clones, we prepared AML cells containing a small fraction of Trp53-KO cells both in the Xpo7-WT and KO backgrounds and assessed the proportion over time in vitro or in vivo. Remarkably, the expansion of Trp53-KO clones, noticeable in the Xpo7-WT setting, was significantly reduced under the Xpo7-KO background, prolonging the mice's survival. These data suggest that Xpo7 depletion effectively impedes Trp53-KO clone expansion. Since Xpo7 is presumed to mediate the nuclear import and export of proteins, we hypothesized that Xpo7 contributes to retaining Trp53 protein within the nucleus in Trp53-WT AMLs. As expected, nuclear Trp53 protein and mRNA levels of Trp53-targeting genes decreased significantly upon Xpo7 depletion. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AMLs. Next, to elucidate the underlying mechanisms causing the toxic effects in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose localization was potentially regulated by Xpo7 in the Trp53-KO state. Employing mass spectrometry on an immunoprecipitate using anti-Xpo7 and cytoplasmic/nuclear protein extracts from both Xpo7-WT and KO AML cells, we found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and showed a significant shift towards cytoplasmic localization upon Xpo7 depletion in Trp53-KO AMLs. Strikingly, Npat protein levels displayed a notable upregulation by Trp53 depletion in AML cells. To evaluate the relevance of the XPO7/NPAT axis in human TP53-mutated AML cells, we examined the correlation between XPO7 and NPAT expression levels and AML subtypes and genetic background using publicly available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets. Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018; Iacobucci et al. Nat Genet. 2019). The Xpo7 and Npat protein expression evaluation using primary AML samples corroborated these findings, revealing overexpression in TP53-mutated AML cases. Finally, we assessed the therapeutic effect of Npat depletion in Trp53-deficient AML cells, using the dTAG system to induce Npat protein degradation by knock-in FKBP12 F36V into Npat loci. dTAG v-1 treatment immediately degraded endogenous Npat protein and led to delayed G1/S transition progression. Moreover, Npat degradation induced DNA damage, particularly in the presence of a WEE1 inhibitor or etoposide, and subsequent apoptosis. Transplantation of these AML cells into immunocompromised mice followed by dTAGv1 treatment for one week demonstrated suppressing leukemia progression in vivo. Furthermore, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed the proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. We unveiled the novel regulatory mechanism of protein localization by XPO7, depending on TP53 mutational state. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML.

Targeting DNA Damage and Repair Machinery via Delivering WEE1 Inhibitor and Platinum (IV) Prodrugs to Stimulate STING Pathway for Maximizing Chemo-Immunotherapy in Bladder Cancer.

Both cisplatin-based chemotherapy and immune checkpoint blockers (ICBs)-based immunotherapy are the first-line treatments for patients with advanced bladder cancer. Cancer cells can develop resistance to cisplatin through extensive DNA repair, while a low response rate to ICBs is mostly due to the presence of an immunosuppressive microenvironment and low PD-L1 expression. Herein, a glutathione (GSH)-responsive nanoparticle (NP2) loaded with cisplatin prodrug (Pt (IV)) and WEE1 inhibitor (MK1775) is designed. NP2 can be triggered by GSH in cancer cells, and the released MK1775 can inhibit the activity of WEE1 protein, which ultimately increases DNA damage by cisplatin. Genome-wide RNA sequencing first reveals that NP2 can inhibit DNA repair machinery by interfering with the cell cycle and significantly activate the stimulator of interferon genes pathway. Tumor growth is significantly inhibited by NP2 in vivo. As innate and adaptive immune responses are stimulated, the immunosuppressive microenvironment is modified, and the "immune cold tumor" is transformed into an "immune hot tumor". In addition, NP2 can upregulate PD-L1 expression in tumor cells, thereby increasing the response rate of PD-L1 monoclonal antibody (αPD-L1) and eliciting long-term immune responses in both primary and metastatic tumors.