Targeting Chk1 Research Articles

Chronic stress induces Alzheimer's disease-like pathologies through DNA damage-Chk1-CIP2A signaling.

Stress is an important initiating factor in promoting Alzheimer's disease (AD) pathogenesis. However, the mechanism by which stress induces AD-like cognitive impairment remains to be clarified. Here, we demonstrate that DNA damage is increased in stress hormone Corticotropin-releasing factor (CRF)-treated cells and in brains of mice exposed to chronic restraint stress. Accumulation of DNA damage drives activation of cell cycle checkpoint protein kinase 1 (Chk1), upregulation of cancerous inhibitor of PP2A (CIP2A), tau hyperphosphorylation, and Aβ overproduction, eventually resulting in synaptic impairment and cognitive deficits. Pharmacological intervention targeting Chk1 by specific inhibitor and DNA damage by vitamin C, suppress DNA damage-Chk1-CIP2A signaling pathway in chronic stress animal model, which in turn attenuate AD-like pathologies, synaptic impairments and cognitive deficits. Our study uncovers a novel molecular mechanism of stress-induced AD-like pathologies and provides effective preventive and therapeutic strategies targeting this signaling pathway.

Inhibition of Chk1 stimulates cytotoxic action of platinum-based drugs and TRAIL combination in human prostate cancer cells.

Checkpoint kinase 1 (Chk1) plays an important role in regulation of the cell cycle, DNA damage response and cell death, and represents an attractive target in anticancer therapy. Small-molecule inhibitors of Chk1 have been intensively investigated either as single agents or in combination with various chemotherapeutic drugs and they can enhance the chemosensitivity of numerous tumor types. Here we newly demonstrate that pharmacological inhibition of Chk1 using potent and selective inhibitor SCH900776, currently profiled in phase II clinical trials, significantly enhances cytotoxic effects of the combination of platinum-based drugs (cisplatin or LA-12) and TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in human prostate cancer cells. The specific role of Chk1 in the drug combination-induced cytotoxicity was confirmed by siRNA-mediated silencing of this kinase. Using RNAi-based methods we also showed the importance of Bak-dependent mitochondrial apoptotic pathway in the combined anticancer action of SCH900776, cisplatin and TRAIL. The triple drug combination-induced cytotoxicity was partially enhanced by siRNA-mediated Mcl-1 silencing. Our findings suggest that targeting Chk1 may be used as an efficient strategy for sensitization of prostate cancer cells to killing action of platinum-based chemotherapeutic drugs and TRAIL.

OTUB1 Targets CHK1 for Deubiquitination and Stabilization to Facilitate Lung Cancer Progression and Radioresistance

PurposeRadioresistance of lung cancer poses a significant challenge when it comes to the treatment of advanced, recurrent, and metastatic cases. OTUB1, known as Ovarian tumor domain ubiquitin aldehyde binding 1, is a key member of the deubiquitinase OTU superfamily. This protein is involved in various cellular functions, including cell proliferation, iron death, lipid metabolism and cytokine secretion, as well as immune response processes. However, its specific role and molecular mechanism in lung cancer radioresistance remain to be clarified. Methods and MaterialsThe expression levels of OTUB1 in paired lung cancer tissues were determined by immunohistochemistry (IHC). In vitro and in vivo experiments were conducted to investigate the impact of OTUB1 on the growth and proliferation of lung cancer. Coimmunoprecipitation and Western blotting techniques were performed to examine the interaction between OTUB1 and CHK1. The DNA damage response was measured by comet tailing and immunofluorescence staining. KEGG pathways and GO terms were analyzed based on RNA-seq. ResultsOur findings reveal a high frequency of OTUB1 overexpression, which is associated with an unfavorable prognosis in patients diagnosed with lung cancer. Through comprehensive investigations, we demonstrate that OTUB1 depletion impairs the process of DNA damage repair and overcomes radioresistance. In terms of the underlying mechanism, our study uncovers that OTUB1 deubiquitinates and stabilizes CHK1, which enhances CHK1 stability, thereby regulating DNA damage and repair. Additionally, we identify CHK1 as the primary downstream effector responsible for mediating the functional effects exerted by OTUB1 specifically in lung cancer. Importantly, OTUB1 has the potential to be a valuable marker for improving the efficacy of radiotherapy for lung adenocarcinoma. ConclusionsThese findings unveil a novel role for OTUB1 in enhancing radioresistance by deubiquitination and stabilizing the expression of CHK1 in lung cancer and indicate that targeting OTUB1 holds great potential as an effective therapeutic approach for enhancing the efficacy of radiotherapy in lung cancer.

Inhibition of RRM2 radiosensitizes glioblastoma and uncovers synthetic lethality in combination with targeting CHK1

Glioblastoma (GBM) is an aggressive malignant primary brain tumor. Radioresistance largely contributes to poor clinical outcomes in GBM patients. We targeted ribonucleotide reductase subunit 2 (RRM2) with triapine to radiosensitize GBM. We found RRM2 is associated with increasing tumor grade, is overexpressed in GBM over lower grade gliomas and normal tissue, and is associated with worse survival. We found silencing or inhibition of RRM2 by siRNA or triapine sensitized GBM cells to ionizing radiation (IR) and delayed resolution of IR-induced γ-H2AX nuclear foci. In vivo, triapine and IR reduced tumor growth and increased mouse survival. Intriguingly, triapine led to RRM2 upregulation and CHK1 activation, suggesting a CHK1-dependent RRM2 upregulation following RRM2 inhibition. Consistently, silencing or inhibition of CHK1 with rabusertib abolished the triapine-induced RRM2 upregulation. Accordingly, combining rabusertib and triapine resulted in synthetic lethality in GBM cells. Collectively, our results suggest RRM2 is a promising therapeutic target for GBM, and targeting RRM2 with triapine sensitizes GBM cells to radiation and independently induces synthetic lethality of GBM cells with CHK1 inhibition. Our findings suggest combining triapine with radiation or rabusertib may improve therapeutic outcomes in GBM.

Alternative Lengthening of Telomeres in Pediatric High-Grade Glioma and Therapeutic Implications

Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG), are highly aggressive tumors with dismal prognoses despite multimodal therapy including surgery, radiation therapy, and chemotherapy. To achieve cellular immortality cancer cells must overcome replicative senescence and apoptosis by activating telomere maintenance mechanisms (TMMs) through the reactivation of telomerase activity or using alternative lengthening of telomere (ALT) pathways. Although the ALT phenotype is more prevalent in pHGGs compared to adult HGGs, the molecular pathway and the prognostic significance of ALT activation are not well understood in pHGGs. Here, we report the heterogeneity of TMM in pHGGs and their association with genetic alterations. Additionally, we show that sensitivity to the protein kinase ataxia telangiectasia- and RAD3-related protein (ATR) inhibitor and the ATR downstream target CHK1 is not specific to pHGG ALT-positive cells. Together, these findings underscore the need for novel therapeutic strategies to target ALT in pHGG tumors.

P130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis

Ataxia-telangiectasia mutated and Rad3-related (ATR) kinase is a crucial regulator of the cell cycle checkpoint and activated in response to DNA replication stress by two independent pathways via RPA32-ETAA1 and TopBP1. However, the precise activation mechanism of ATR by the RPA32-ETAA1 pathway remains unclear. Here, we show that p130RB2, a member of the retinoblastoma protein family, participates in the pathway under hydroxyurea-induced DNA replication stress. p130RB2 binds to ETAA1, but not TopBP1, and depletion of p130RB2 inhibits the RPA32-ETAA1 interaction under replication stress. Moreover, p130RB2 depletion reduces ATR activation accompanied by phosphorylation of its targets RPA32, Chk1, and ATR itself. It also causes improper re-progression of S phase with retaining single-stranded DNA after cancelation of the stress, which leads to an increase in the anaphase bridge phenotype and a decrease in cell survival. Importantly, restoration of p130RB2 rescued the disrupted phenotypes of p130RB2 knockdown cells. These results suggest positive involvement of p130RB2 in the RPA32-ETAA1-ATR axis and proper re-progression of the cell cycle to maintain genome integrity.

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors' combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

Splice-switch oligonucleotide-based combinatorial platform prioritizes synthetic lethal targets CHK1 and BRD4 against MYC-driven hepatocellular carcinoma.

Deregulation of MYC is among the most frequent oncogenic drivers in hepatocellular carcinoma (HCC). Unfortunately, the clinical success of MYC-targeted therapies is limited. Synthetic lethality offers an alternative therapeutic strategy by leveraging on vulnerabilities in tumors with MYC deregulation. While several synthetic lethal targets of MYC have been identified in HCC, the need to prioritize targets with the greatest therapeutic potential has been unmet. Here, we demonstrate that by pairing splice-switch oligonucleotide (SSO) technologies with our phenotypic-analytical hybrid multidrug interrogation platform, quadratic phenotypic optimization platform (QPOP), we can disrupt the functional expression of these targets in specific combinatorial tests to rapidly determine target-target interactions and rank synthetic lethality targets. Our SSO-QPOP analyses revealed that simultaneous attenuation of CHK1 and BRD4 function is an effective combination specific in MYC-deregulated HCC, successfully suppressing HCC progression in vitro. Pharmacological inhibitors of CHK1 and BRD4 further demonstrated its translational value by exhibiting synergistic interactions in patient-derived xenograft organoid models of HCC harboring high levels of MYC deregulation. Collectively, our work demonstrates the capacity of SSO-QPOP as a target prioritization tool in the drug development pipeline, as well as the therapeutic potential of CHK1 and BRD4 in MYC-driven HCC.

Abstract 1509: Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition

Abstract Background: ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. We have previously demo started that ATR inhibition is associated with anti-tumor activity in pre-clinical models of soft-tissue sarcomas (STS) (Laroche et al., 2020). We decided to investigate the mechanisms of resistance to ATR inhibition in sarcomas. Methods: We used a CRISPRa was to identify genes that mediate resistance to AZD6738 in STS cells. The anti-tumor activity of AZD6738 (ATR inhibitor) and AZD0156 (ATM inhibitor) was investigated in a panel for 10 STS cell lines and two patient-derived xenografts (PDX) established at Institut Bergonié (Bordeaux, France). Results: We used a CRISPR approach in a STS cell line resistant to AZD6738 and we made a negative-selection screen, to identify genes whose silencing conferred sensitivity to low doses of ATR inhibition. Among these genes, we identified THRAP3 which encodes a protein phosphorylated in response to DNA damage and primarily by ATR. THRAP3 is involved in the maturation and further export of transcripts encoding the ATM and its depletion results in deficient processing of transcripts encoding the ATM kinase. We validate the result of our CRISPR analysis by demonstrating that selective THRAP3 inhibition reduce significantly ATRi IC50. Given the role of role of THRAP3 on the regulation of ATM activity, we decided to investigate the potential synergy between ATR and ATM inhibition in vivo and in vitro. AZD6738 and AZD0156 displayed a synergistic or additive effect in above all the STS cell lines tested. We noted that ATRi treatment reduced phosphorylation of the ATR kinase target CHK1 but increased ATM phosphorylation, suggesting that ATM pathway takes over for DNA Damage Response and cells survival. In contrast, both ATR and ATM pathway were inhibited when AZD6738 and AZD0156 were used in combination, as shown by reduction of CHK1 and ATM phosphorylation. We validated our in vitro results in an in vivo model of liposarcoma xenograft in which AZD6738 and AZD0156 had synergistic anti-tumor activity. Conclusion: Our study demonstrates synthetically lethal interactions between ATM and ATR in vitro and in vivo. This combination deserves further investigaitons in sarcomas. Citation Format: Mariella Spalato, Audrey Laroche-Clary, Vanessa Chaire, Antoine Italiano. Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition [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 1509.

Reality CHEK: Understanding the biology and clinical potential of CHK1.

The DNA damage response enables cells to cope with various stresses that threaten genomic integrity. A critical component of this response is the serine/threonine kinase CHK1 which is encoded by the CHEK1 gene. Originally identified as a regulator of the G2/M checkpoint, CHK1 has since been shown to play important roles in DNA replication, mitotic progression, DNA repair, and overall cell cycle regulation. However, the potential of CHK1 as a cancer therapy has not been realized clinically. Herein we expound our current understanding of the principal roles of CHK1 and highlight different avenues for CHK1 targeting in cancer therapy.

Cotargeting CHK1 and PI3K Synergistically Suppresses Tumor Growth of Oral Cavity Squamous Cell Carcinoma in Patient-Derived Xenografts.

Oral cavity squamous cell carcinomas (OSCCs) are aggressive tumors, and their recurrence leads to poor prognosis and reduced survival rates. This study aimed to identify therapeutic targets and to evaluate the efficacy of targeted inhibitors in OSCC patient-derived xenograft (PDX) models. Herein, we reported that OSCC PDXs recapitulated the genomic signatures of their paired primary tumors and the expression of CHEK1, PIK3CA, and PIK3CD was significantly upregulated in OSCC. The antitumor efficacy of CHK1 inhibitors (PF477736, AZD7762, LY2606368) and PI3K inhibitors (BYL719, GDC0941, GSK1059615) was investigated in OSCC cell lines and PDX models. Targeting either CHK1 or PI3K effectively inhibited cell proliferation and colony formation by inducing cell cycle arrest and apoptosis in in vitro cell-based assays. Cisplatin-based chemotherapy combined with CHK1 inhibitor treatment synergistically inhibited cell proliferation by suppressing CHK1 phosphorylation and inducing PARP cleavage. Furthermore, compared with monotherapy, cotreatment with CHK1 and PI3K inhibitors exerted synergistic anticancer effects by suppressing CHK1, AKT, and 4E-BP1 phosphorylation. In summary, our study identified CHK1 and PI3K as promising targets, especially in a dual treatment strategy combining a CHK1 inhibitor with cisplatin or a PI3K inhibitor as a novel therapeutic approach for OSCC patients with aberrant cell cycle regulation and PI3K signaling activation.

Abstract B15: The oral Chk1 inhibitor, SRA737, synergizes with immune checkpoint blockade in small-cell lung cancer (SCLC)

Abstract Background: Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer. Despite the recent success of immunotherapy in other indications, only a minority of SCLC patients respond to immune checkpoint blockade (ICB) targeting programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) either as a monotherapy or combination. Therefore, there is a strong need to develop strategies to enhance the efficacy of immunotherapy in SCLC. Our group previously discovered that SCLC exhibits high expression of checkpoint kinase 1 (Chk1) and showed that preclinical in vivo models of SCLC respond to Chk1 inhibition. Based on data from others and our group, we hypothesized that targeting Chk1 can enhance antitumor immunity and synergize with ICB. Results: SRA737 treatment decreased cell viability with a range of potencies in a panel of SCLC cell lines in vitro. This was accompanied by an induction of double-strand breaks in sensitive cell lines as demonstrated by increased γ-H2AX. Intriguingly, SRA737 also led to an increase in micronuclei formation and STING activation in cells in vitro. Cell surface and total PD-L1 protein were increased following SRA737 treatment in vitro, further supporting a potential benefit of combining the drug with immune checkpoint blockade therapy in vivo. As hypothesized, SRA737 showed strong synergy with anti-PD-L1 antibody in an immunocompetent xenograft SCLC model. Triple-knockout SCLC cells generated from a GEMM mouse model with conditional deletion of Trp53, Rb1 and p130 were implanted into the flank of B6129F1 mice. The mice were treated for three weeks with either IgG (control), SRA737 (100mg/kg, either 3/7 or 5/7 days), anti-PD-L1 (300ug, 1/7 days) or the combination. While anti-PD1 antibody treatment was largely ineffective, SRA737 significantly delayed tumor growth (at Day 21: T/C=0.30 for 3/7 days and T/C=0.28 for 5/7 days). Combination treatment with SRA737 and anti-PD-L1 demonstrated remarkable antitumor efficacy, resulting in stable disease following SRA737 schedule of 3/7 days (T/C=0.12) and tumor regressions following SRA737 schedule of 5/7 days (T/C=0.1). These effects were sustained after treatment cessation and the long-term survival benefit is being assessed. Discussion: The intrinsic antitumor activity of the Chk1 inhibitor, SRA737, was significantly enhanced by addition of an anti-PD-L1 antibody, leading to tumor regressions in an immunocompetent SCLC model. Preliminary evidence suggests SRA737 induces micronuclei formation, STING activation and PD-L1 expression in tumor cells. Further studies to elucidate the mechanism of Chk1 inhibition-induced antitumor immunity in SCLC are ongoing. SRA737 is currently being tested in clinical trials both as a monotherapy and in combination with other agents. Given that the anti-PD-L1 antibody opdivo is now approved for SCLC, our data suggest intriguing possibilities for therapeutic synergy between the oral Chk1 inhibitor, SRA737, and ICB therapy that warrant further clinical investigation. Citation Format: Triparna Sen, Snezana Milutinovic, Robert J. Cardnell, Lixia Diao, Youhong Fan, Ryan J. Hansen, Bryan Strouse, Michael P. Hedrick, Christian Hassig, Jing Wang, Lauren A. Byers. The oral Chk1 inhibitor, SRA737, synergizes with immune checkpoint blockade in small-cell lung cancer (SCLC) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B15.

Survival-Based Gene Expression Analysis of Chinese HCC Specimens Revealed Prognostic Biological Determinants Regulating Cancer Growth and Tumor Immunity

To identify important druggable targets and immunological determinants associated with the overall survival (OS) of hepatocellular carcinoma (HCC) patients, we interrogated 279 Chinese HCC samples, mostly in early stage, with the NanoString platform assessing the expression of 1,173 genes involved in cell-growth signaling pathways, cellular machineries and tumor immunity. A prognostic model was constructed using 17 genes with bootstrap-corrected C-index of 0.81 for the OS. Among the genes associated with worse OS, cell-growth related druggable targets CHK1 and EZH2 were selected for in vitro and in vivo tumor growth studies. HCC cell line clonogenic assays and mice xenograft models demonstrated that the combinatorial application of CHK1 inhibitor and EZH2 inhibitor resulted in significant suppression of HCC tumor growth. On the tumor immunity front, we discovered that genes enriched in regulating CD8+ T cells and activating NK cells contributed to prolonged OS. Our findings provide potential rationale for investigating combinatorial targeted therapies and immunotherapies in specific subsets of early-stage HCC with distinct gene expression characteristics. Funding: This work was supported by National Natural Science Fund of China (Grant numbers: No.81672330; No. 81871916; No. 81472218), Talent Development Program of Zhongshan Hospital (2015ZSYXGG), and by Genentech Inc., a member of the Roche Group. Declaration of Interest: Chun Sun, Bonnie Liu, Kwame Okrah, Xu Cao, Jian-Jun Guo, Shi-Jing Fu, Su-Chun Li, Hang-jun Dai, Mark R. Lackner, Astrid Kiermaier, Shih-Min A. Huang and Gang Cheng are employees of F. Hoffmann-La Roche/Genentech Inc. All other authors declare no conflict of interest. Ethical Approval: This study was approved, and the collection of written informed consent forms was waived, by the institutional review board of SZH under the condition that all samples were anonymized in this study.

Abstract 3837: JQ1 induces cell death and disrupts BET protein binding to the MYC locus in cholangiocarcinoma cells

Abstract Cholangiocarcinoma (CCA) is an aggressive bile duct neoplasm which is typically not diagnosed until late stage. The current standard of care, resection followed by gemcitabine with or without cisplatin, is relatively ineffective. Additionally, most patients are ineligible for surgical resection, and postoperative chemotherapy rarely prolongs overall survival. No previously identified mutations (KRAS, SMAD4 and p53) have been shown to drive disease progression. We and others have shown that the proto-oncogene c-Myc is expressed at relatively high levels in CCA cells compared to uninvolved normal tissue, suggesting this oncogene as a prospective therapeutic target in CCA. To evaluate whether c-Myc inhibition affected the proliferation of CCA cells, we evaluated the efficacy of the bromodomain and extraterminal domain (BET) inhibitor JQ1 on c-Myc expression and cell proliferation of CCA cells. JQ1 functions as an acetyl lysine (K-Ac) mimetic which binds to the K-Ac binding pocket of BET protein family members (BRD2, BRD3, BRD4 and BRDT). This binding competitively inhibits the association of BET proteins with K-Ac residues of nuclear proteins and inhibits c-Myc expression. Using a patient-derived xenograft (PDX) model of CCA, we showed previously that JQ1 downregulated c-Myc expression and inhibited tumor growth. Therefore, we hypothesized that JQ1 induces CCA cell death through disruption of BET protein binding to the promoter region of the MYC locus, inducing its downregulation. Our data show that JQ1 decreased c-Myc RNA and protein expression as well as the expression c-Myc transcriptional targets CHK1 and BRCA2. JQ1 also decreased CCA cell viability, clonogenic potential and induced apoptosis of KKU-055 CCA cells in vitro. The likely mechanism of JQ1-mediated c-Myc reduction has been postulated to involve competitive inhibition of the K-Ac binding function of the BET protein BRD4. Our current chromatin immunoprecipitation (ChIP) assays indicate that BRD2 binds to the MYC transcriptional start sites (TSS) P1 and P2, while BRD4 primarily binds MYC TSS P1 in KKU-055 cells. JQ1 precludes these BET proteins from binding their respective sites. When exposed to the IC50 of JQ1, ChIP pulldown with an anti-BRD2 antibody displays a near 100% reduction in binding of BRD2 to the MYC TSS while an anti-BRD4 antibody displays a 70% reduction in binding. In addition, stable BRD2 knockdown models of KKU-055 display reduced c-Myc protein expression by 60 to 80%, while BRD4 knockdown models showed no such reduction. The data demonstrate that in addition to BRD4, BRD2 contributes to regulation of expression of c-Myc in CCA cells. Research funded by NIH/NCI R21 CA205501 Citation Format: Samuel C. Fehling, Aubrey L. Miller, Karina J. Yoon. JQ1 induces cell death and disrupts BET protein binding to the MYC locus in cholangiocarcinoma cells [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 3837.

The BRUCE-ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

Trisenox disrupts MDM2-DAXX-HAUSP complex and activates p53, cell cycle regulation and apoptosis in acute leukemia cells.

Trisenox (TX) has been used in the treatment of both de novo and relapsed acute promyelocytic leukemia (APL) patients. Using in vitro APL cell lines model in this research, we report on a new target of TX action through disruption of MDM2-DAXX-HAUSP complex, degradation of MDM2, and activation of p53 expression. TX–induced stress signal was transmitted by protein kinase (ATM & ATR) and phosphorylation of its downstream targets CHK1, CHK2, ATM, and ATR, respectively at the Ser 345, Thr68, Ser1981 and Ser 428 residues involved in complex disruption and p53 up-regulation. TX-activated p53 led to cell cycle arrest and apoptosis in APL cells. Our results showed that TX inhibited cell proliferation, disrupted complex molecules expression and association in APL cells. Our functional studies indicated that TX-induced down-regulation of complex molecules expression was mostly neutralized in both p53 knockdown NB4 cells and nutilin-3 treated KG1a cells. Hence our findings provide a functional evidence of TX action on cell cycle regulation and apoptosis in APL cells. This novel target of TX activity may be useful for designing new APL drugs.

A PIM-CHK1 signaling pathway regulates PLK1 phosphorylation and function during mitosis.

Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have recently provided evidence of DDR-independent roles of CHK1, in particular following phosphorylation of its S280 residue. Here, we demonstrate that CHK1 S280 phosphorylation is cell cycle-dependent and peaks during mitosis. We found that this phosphorylation was catalyzed by the kinase PIM2, whose protein expression was also increased during mitosis. Importantly, we identified polo-like kinase 1 (PLK1) as a direct target of CHK1 during mitosis. Genetic or pharmacological inhibition of CHK1 reduced the activating phosphorylation of PLK1 on T210, and recombinant CHK1 was able to phosphorylate T210 of PLK1 in vitro Accordingly, S280-phosphorylated CHK1 and PLK1 exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with S280-phosphorylated CHK1 from mitotic cell extracts. Moreover, CHK1-mediated phosphorylation of PLK1 was dependent on S280 phosphorylation by PIM2. Inhibition of PIM proteins reduced cell proliferation and mitotic entry, which was rescued by expressing a T210D phosphomimetic mutant of PLK1. Altogether, these data identify a new PIM-CHK1-PLK1 phosphorylation cascade that regulates different mitotic steps independently of the CHK1 DDR function.This article has an associated First Person interview with the first author of the paper.

Evaluation of CHK1 activation in vulvar squamous cell carcinoma and its potential as a therapeutic target in vitro.

CHK1 is an important regulator of the cell cycle and DNA damage response, and its altered expression has been identified in various tumors. Chk1 inhibitors are currently being evaluated as monotherapy and as potentiators of chemotherapy in clinical settings. However, to our knowledge, no previous study has investigated either the activation status or the therapeutic potential of CHK1 targeting in vulvar cancer. Therefore, we examined the expression status of activated CHK1 forms pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in 294 vulvar squamous cell carcinomas (VSCC) using immunohistochemistry and analyzed their relationships with various clinicopathological variables and clinical outcome. To aid translation of preclinical studies, we also assessed cell sensitivity to the Chk1 inhibition in two vulvar cancer cell lines. Compared to the levels of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in normal vulvar squamous epithelium, high nuclear pCHK1Ser345 expression was found in 57% of vulvar carcinomas, whereas low nuclear pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 expressions were observed in 58%, 64%, and 40% of the cases, respectively. Low levels of pCHK1Ser317 and pCHK1Ser280 in the nucleus correlated significantly with advanced tumor behaviors and aggressive features. None of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 forms were identified as prognostic factors. In vitro inhibition of CHK1 by small molecular inhibitors or siRNA reduced viability by inducing DNA damage and apoptosis of vulvar cancer cell lines. In summary, we conclude that cellular functions regulated by CHK1 are phosphorylation/localization‐dependent and deregulation of CHK1 function occurs in VSCC and might contribute to tumorigenesis. Targeting CHK1 might represent as a useful antitumor strategy for the subgroup of VSCC harboring p53 mutations.

Abstract 5824: The BET inhibitor JQ1 sensitizes cholangiocarcinoma cells to PARP inhibitors

Abstract Cholangiocarcinoma (CCA) is an aggressive bile duct neoplasm, and the second most common primary hepatic malignancy. CCA is usually diagnosed at a late stage where the current standard of care, resection followed by gemcitabine with or without cisplatin, is not effective. Up to 90% of CCA patients are ineligible for resection and, of those eligible, postoperative chemotherapy does not significantly prolong overall survival. The limited number of CCA models hinders drug development and discovery of new therapeutic targets. Mutations have been identified in KRAS (17% of CCA tumors), p53 (44%) and SMAD4 (17%), but are not recognized as essential to CCA progression. c-Myc protein is highly expressed in 94% of CCA cases while low to undetectable in normal adult liver, suggesting c-Myc overexpression may contribute to CCA tumor progression but this hypothesis has not been tested. Toward developing novel effective therapies for CCA, we evaluated the effect of the bromodomain and extraterminal domain (BET) inhibitor JQ1 on c-Myc expression and on the proliferation of CCA cells. JQ1 inhibits c-Myc transcription by binding to the acetylated lysine (K-Ac) binding pockets of BET protein family members (BRD2, BRD3, BRD4 and BRDT), preventing their interaction with K-Ac residues on nuclear proteins, disrupting the formation of functional transcriptional complexes and inhibiting c-Myc transcription. We have shown that JQ1 inhibited tumor growth in a patient-derived xenograft (PDX) model of CCA and downregulated expression of c-Myc and its transcriptional targets BRCA2 and Chk1, both involved in DNA damage response. Our current data show that JQ1 decreased c-Myc protein expression, CCA cell viability, and clonogenic potential in KKU-055 CCA cell line. Of note, the literature suggests that tumors deficient in BRCA2 or Chk1 are relatively sensitive to PARP inhibitors (PARPi). Therefore, we hypothesize that JQ1-induced downregulation of c-Myc and its transcriptional targets BRCA2 and Chk1 would potentiate the efficacy of PARPi. We exposed CCA cell line KKU-055 with a range of JQ1 and PARPi (olaparib and veliparib) concentrations as single agent or combination therapy. Each combination of JQ1 with either PARPi was more effective than any of the three drugs as single agents. Combination indices (CI), calculated using CompuSyn 1.0, ranged from 0.05 to 0.09 which indicated strong synergy for JQ1 with olaparib or veliparib. Further, synergy with JQ1 was not dependent on a single PARPi. JQ1 with olaparib (1:1) also decreased the clonogenic potential of KKU-055 cells, compared to each drug as a single agent. These data suggest that inhibition of BET protein function sensitizes CCA cells to PARPi, coincident with downregulation of c-Myc and its transcriptional targets BRCA2 and Chk1. Research funded by NIH/NCI R21 CA205501 Citation Format: Samuel C. Fehling, Aubrey L. Miller, James E. Bradner, Karina J. Yoon. The BET inhibitor JQ1 sensitizes cholangiocarcinoma cells to PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5824.

The ATM and Rad3-Related (ATR) Protein Kinase Pathway Is Activated by Herpes Simplex Virus 1 and Required for Efficient Viral Replication.

The ATM and Rad3-related (ATR) protein kinase and its downstream effector Chk1 are key sensors and organizers of the DNA damage response (DDR) to a variety of insults. Previous studies of herpes simplex virus 1 (HSV-1) showed no evidence for activation of the ATR pathway. Here we demonstrate that both Chk1 and ATR were phosphorylated by 3 h postinfection (h.p.i.). Activation of ATR and Chk1 was observed using 4 different HSV-1 strains in multiple cell types, while a specific ATR inhibitor blocked activation. Mechanistic studies point to early viral gene expression as a key trigger for ATR activation. Both pATR and pChk1 localized to the nucleus within viral replication centers, or associated with their periphery, by 3 h.p.i. Significant levels of pATR and pChk1 were also detected in the cytoplasm, where they colocalized with ICP4 and ICP0. Proximity ligation assays confirmed that pATR and pChk1 were closely and specifically associated with ICP4 and ICP0 in both the nucleus and cytoplasm by 3 h.p.i., but not with ICP8 or ICP27, presumably in a multiprotein complex. Chemically distinct ATR and Chk1 inhibitors blocked HSV-1 replication and infectious virion production, while inhibitors of ATM, Chk2, and DNA-dependent protein kinase (DNA-PK) did not. Together our data show that HSV-1 activates the ATR pathway at early stages of infection and that ATR and Chk1 kinase activities play important roles in HSV-1 replication fitness. These findings indicate that the ATR pathway may provide insight for therapeutic approaches.IMPORTANCE Viruses have evolved complex associations with cellular DNA damage response (DDR) pathways, which sense troublesome DNA structures formed during infection. The first evidence for activation of the ATR pathway by HSV-1 is presented. ATR is activated, and its downstream target Chk1 is robustly phosphorylated, during early stages of infection. Both activated proteins are found in the nucleus associated with viral replication compartments and in the cytoplasm associated with viral proteins. We also demonstrate that both ATR and Chk1 kinase activities are important for viral replication. The findings suggest that HSV-1 activates ATR and Chk1 during early stages of infection and utilizes the enzymes to promote its own replication. The observation may be exploitable for antiviral approaches.