Chk1 Activation Research Articles

RECQL4 regulates DNA damage response and redox homeostasis in esophageal cancer.

Objective:RECQL4 (a member of the RECQ helicase family) upregulation has been reported to be associated with tumor progression in several malignancies. However, whether RECQL4 sustains esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we determined the functional role for RECQL4 in ESCC progression.Methods:RECQL4 expression in clinical samples of ESCC was examined by immunohistochemistry. Cell proliferation, cellular senescence, the epithelial-mesenchymal transition (EMT), DNA damage, and reactive oxygen species in ESCC cell lines with RECQL4 depletion or overexpression were analyzed. The levels of proteins involved in the DNA damage response (DDR), cell cycle progression, survival, and the EMT were determined by Western blot analyses.Results:RECQL4 was highly expressed in tumor tissues when compared to adjacent non-tumor tissues in ESCC (P < 0.001) and positively correlated with poor differentiation (P = 0.011), enhanced invasion (P = 0.033), and metastasis (P = 0.048). RECQL4 was positively associated with proliferation and migration in ESCC cells. Depletion of RECQL4 also inhibited growth of tumor xenografts in vivo. RECQL4 depletion induced G0/G1 phase arrest and cellular senescence. Importantly, the levels of DNA damage and reactive oxygen species were increased when RECQL4 was depleted. DDR, as measured by the activation of ATM, ATR, CHK1, and CHK2, was impaired. RECQL4 was also shown to promote the activation of AKT, ERK, and NF-kB in ESCC cells.Conclusions:The results indicated that RECQL4 was highly expressed in ESCC and played critical roles in the regulation of DDR, redox homeostasis, and cell survival.

EZH2 activates CHK1 signaling to promote ovarian cancer chemoresistance by maintaining the properties of cancer stem cells.

Background: Ovarian cancer is a fatal malignant gynecological tumor. Ovarian cancer stem cells (OCSCs) contribute to resistance to chemotherapy. The polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a key role in maintaining CSCs. Here, we aimed to investigate the specific mechanism by which EZH2 regulates CSCs to result in chemoresistance and poor prognosis of ovarian cancer.Methods: We used a nude mouse model to obtain a cell line enriched for OCSCs, named SK-3rd cells. The CRISPR and Cas9 endonuclease system was used to establish an EZH2-knockout SK-3rd ovarian cancer cell line. High-throughput PCR array and bioinformatics methods were used to screen the EZH2 target involved in CSC stemness. A luciferase reporter assay and chromatin immunoprecipitation assay were performed to identify activation of CHK1 by EZH2. We evaluated associations between EZH2/CHK1 expression and the chemoresistance and prognosis of ovarian cancer patients.Results: EZH2 plays a critical role in maintaining ovarian CSC stemness and chemo-resistance. CHK1 is an EZH2 target involved in CSC stemness. Knockdown of EZH2 in ovarian CSCs decreased CHK1 expression, while CHK1 overexpression was sufficient to reverse the inhibitory effect on spheroid formation and chemoresistance caused by repression of EZH2. In addition, EZH2 was also shown to play a unique role in activating rather than repressing CHK1 signaling through binding to the CHK1 promoter in epithelial ovarian cancer cells. Finally, in clinical samples, ovarian cancer patients with high levels of EZH2 and CHK1 not only were more resistant to platinum but also had a poorer prognosis.Conclusions: Our data revealed a previously unidentified functional and mechanistic link between EZH2 levels, CHK1 signaling activation, and ovarian CSCs and provided strong evidence that EZH2 promotes ovarian cancer chemoresistance and recurrence.

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress

F-box proteins β-TrCP1 and β-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of β-TrCP1 and β-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that β-TrCP1 and β-TrCP2 communicate during genotoxic stress. We found that expression levels of β-TrCP1 are significantly increased while levels of β-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of β-TrCP1 and β-TrCP2 via the phosphorylation of β-TrCP1 at Ser158. Phosphorylated β-TrCP1 potently promotes the proteasomal degradation of β-TrCP2 and MDM2, resulting in the activation of p53. Additionally, β-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by β-TrCP2. Thus, β-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of β-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed β-TrCP1 as a key player in maintaining the genome integrity through the attenuation of β-TrCP2 levels in response to genotoxic stress.

CBMS-02 Phosphoglycerate mutase 1 (PGAM1) controls DNA damage repair via regulation of WIP1 activity

Phosphoglycerate Mutase 1 (PGAM1) is overexpressed in different forms of cancer and has been suggested to have additional functions beyond its role in metabolism. We here report that PGAM1 is overexpressed in GBMs and indirectly regulates activation of ATM, Chk1 and Chk2 but not ATR, thereby increasing the efficiency of DNA damage repair and resistance to radiation (IR) and temozolomide (TMZ) treatment. Genetic suppression of PGAM1 in multiple GBM cell lines resulted in decrease proliferation, apoptosis and colony formation after radiation and temozolomide treatment compared to parental cells. Moreover, parental cells demonstrated DNA damage (gH2AX foci) whereas isogenic PGAM1 knockdown cells exhibited no DNA damage repair activation and a significant increase in sub-G0 apoptotic cells that expressed annexin-V, cleaved caspase-3 and cleaved PARP-1. Mechanistically, suppression of PGAM1 expression inhibited phosphorylation of ATM at s1981 and the subsequent downstream phosphorylation of Chk2 and cdc25C. Moreover, PGAM1 co-immunoprecipitated with WIP1, a phosphatase reported to bind and dephosphorylate ATM, Chk1, and Chk2. Cytoplasmic binding of WIP1 with PGAM1 prevented nuclear localization of WIP1, leaving ATM and its downstream substrates phosphorylated, which is required for DNA damage repair activity. Consistent with these observations, mice intracranially implanted with PGAM1 knockdown GBM cells and treated with TMZ and IR had longer survival than similarly treated mice implanted with matched control cells. These results therefore define PGAM1 as an activator of DNA damage repair pathway and link tumor metabolism to drug response in GBM.

Autophagic feedback-mediated degradation of IKKα requires CHK1- and p300/CBP-dependent acetylation of p53.

In our previous report, we demonstrated that one of the catalytic subunits of the IκB kinase (IKK) complex, IKKα (encoded by CHUK), performs an NF-κB-independent cytoprotective role in human hepatoma cells under the treatment of the anti-tumor therapeutic reagent arsenite. IKKα triggers its own degradation, as a feedback loop, by activating p53-dependent autophagy, and therefore contributes substantially to hepatoma cell apoptosis induced by arsenite. Interestingly, IKKα is unable to interact with p53 directly but plays a critical role in mediating p53 phosphorylation (at Ser15) by promoting CHK1 activation and CHK1-p53 complex formation. In the current study, we found that p53 acetylation (at Lys373 and/or Lys382) was also critical for the induction of autophagy and the autophagic degradation of IKKα during the arsenite response. Furthermore, IKKα was involved in p53 acetylation through interaction with the acetyltransferases for p53, p300 (also known as EP300) and CBP (also known as CREBBP) (collectively p300/CBP), inducing CHK1-dependent p300/CBP activation and promoting p300-p53 or CBP-p53 complex formation. Therefore, taken together with the previous report, we conclude that both IKKα- and CHK1-dependent p53 phosphorylation and acetylation contribute to mediating selective autophagy feedback degradation of IKKα during the arsenite-induced proapoptotic responses.

Abemaciclib-Based Combinational Therapies to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

We have previously discovered that HDAC6 regulates the DNA damage response (DDR) via modulating the homeostasis of a DNA mismatch repair protein, MSH2, through HDAC6’s ubiquitin E3 ligase activity. Here, we have reported HDAC6’s second potential E3 ligase substrate, a critical cell cycle checkpoint protein, Chk1. We have found that HDAC6 and Chk1 directly interact, and that HDAC6 ubiquitinates Chk1 in vivo and in vitro. Specifically, HDAC6 interacts with Chk1 via the DAC1 domain, which contains its ubiquitin E3 ligase activity. During the cell cycle, Chk1 protein levels fluctuate, peaking at the G2 phase, subsequently resolving via the ubiquitin-proteasome pathway, and thereby allowing cells to progress to the M phase. However, in HDAC6 knockdown non-small cell lung cancer (NSCLC) cells, Chk1 is constitutively active and fails to resolve post-ionizing radiation (IR), and this enhanced Chk1 activity leads to preferential G2 arrest in HDAC6 knockdown cells accompanied by a reduction in colony formation capacity and viability. Depletion or pharmacological inhibition of Chk1 in HDAC6 knockdown cells reverses this radiosensitive phenotype, suggesting that the radiosensitivity of HDAC6 knockdown cells is dependent on increased Chk1 kinase activity. Overall, our results highlight a novel mechanism of Chk1 regulation at the post-translational level, and a possible strategy for sensitizing NSCLC to radiation via inhibiting HDAC6’s E3 ligase activity.

Fumonisin B1 Epigenetically Regulates PTEN Expression and Modulates DNA Damage Checkpoint Regulation in HepG2 Liver Cells.

Fumonisin B1 (FB1), a Fusarium-produced mycotoxin, is found in various foods and feeds. It is a well-known liver carcinogen in experimental animals; however, its role in genotoxicity is controversial. The current study investigated FB1-triggered changes in the epigenetic regulation of PTEN and determined its effect on DNA damage checkpoint regulation in human liver hepatoma G2 (HepG2) cells. Following treatment with FB1 (IC50: 200 µM; 24 h), the expression of miR-30c, KDM5B, PTEN, H3K4me3, PI3K, AKT, p-ser473-AKT, CHK1, and p-ser280-CHK1 was measured using qPCR and/or Western blot. H3K4me3 enrichment at the PTEN promoter region was assayed via a ChIP assay and DNA damage was determined using an ELISA. FB1 induced oxidative DNA damage. Total KDM5B expression was reduced, which subsequently increased the total H3K4me3 and the enrichment of H3K4me3 at PTEN promoters. Increased H3K4me3 induced an increase in PTEN transcript levels. However, miR-30c inhibited PTEN translation. Thus, PI3K/AKT signaling was activated, inhibiting CHK1 activity via phosphorylation of its serine 280 residue preventing the repair of damaged DNA. In conclusion, FB1 epigenetically modulates the PTEN/PI3K/AKT signaling cascade, preventing DNA damage checkpoint regulation, and induces significant DNA damage.

Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts.

Larval tracheae of Drosophila harbour progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. Here we investigate developmental regulation of Chk1 activation. We report that Wnt signaling is high in tracheoblasts and this is necessary for high levels of activated (phosphorylated) Chk1. We find that canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signaling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and are downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, we show that downregulation of Wnt signaling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signaling, driving tracheoblast proliferation.

Effects of Sclareol Against Small Cell Lung Carcinoma and the Related Mechanism: In Vitro and In Vivo Studies.

This study aimed to investigate the anticancer effects and potential mechanisms of sclareol in a human small cell lung carcinoma (SCLC) cell line. Cell viability was determined by the MTT assay. Cell cycle, apoptosis and caspase activity were evaluated by flow cytometry. Cell cycle and DNA damage related protein expression was determined by western blotting. In vivo evaluation of sclareol was carried out in xenografted tumor mice models. Sclareol significantly reduced cell viability, induced G1 phase arrest and subsequently triggered apoptosis in H1688 cells. In addition, this sclareol-induced growth arrest was associated with DNA damage as indicated by phosphorylation of H2AX, activation of ATR and Chk1. Moreover, in vivo evaluation of sclareol showed that it could inhibit tumor weight and volume in a H1688 xenograft model. Sclareol might be a novel and effective therapeutic agent for the treatment of SCLC patients.

Autophagy Contributes to the Maintenance of Genomic Integrity by Reducing Oxidative Stress.

Autophagy has been well documented to play an important role in maintaining genomic stability. However, in addition to directly engulfing and digesting the damaged organelles and chromatin fragments, autophagy can affect many cellular processes including DNA damage response, regulation of redox homeostasis, and cell division; it remains to be determined to what extent each of those processes contributes to the maintenance of genomic stability. We here examined the role of autophagy-dependent redox regulation in the maintenance of genomic stability in two cancer cell lines (HT1080 and U2OS) and mesenchymal stem cells (MSCs) using micronuclei MN, also referred to as cytoplasmic chromatin fragments, as a marker. Our results showed that the spontaneous and genotoxic stress-induced frequencies of MN in cancer cells were significantly reduced by autophagy activators rapamycin and Torin1, and the reduction in MN was accompanied by a reduction in reactive oxygen species (ROS). Increased micronucleation in senescent MSCs, in which autophagic flux is blocked, was also attenuated by rapamycin, together with a reduction in ROS. Inhibition of autophagy by chloroquine (CQ) or ATG5 depletion, on the other hand, resulted in an increased frequency of MN, though a ROS elevation in response to autophagy inhibition was only observed in MSCs. Importantly, the induction of MN by autophagy inhibition in MSCs could be abrogated by antioxidant N-acetylcysteine (NAC). In contrast to the reported impairment of CHK1 activation in Atg7-deficient mouse embryonic fibroblasts, we found that the level of phosphorylated CHK1 was increased by CQ or ATG5 depletion but decreased by rapamycin or Torin1, suggesting that the increased genomic instability by defective autophagy is not caused by insufficient activation of CHK1-homologous recombination cascade. Together, our findings suggest that redox homeostasis regulated by autophagy contributes substantially to the maintenance of genomic stability in certain contexts.

Implications of CLSPN Variants in Cellular Function and Susceptibility to Cancer.

Claspin is a multifunctional protein that participates in physiological processes essential for cell homeostasis that are often defective in cancer, namely due to genetic changes. It is conceivable that Claspin gene (CLSPN) alterations may contribute to cancer development. Therefore, CLSPN germline alterations were characterized in sporadic and familial breast cancer and glioma samples, as well as in six cancer cell lines. Their association to cancer susceptibility and functional impact were investigated. Eight variants were identified (c.-68C>T, c.17G>A, c.1574A>G, c.2230T>C, c.2028+16G>A, c.3595-3597del, and c.3839C>T). CLSPN c.1574A>G (p.Asn525Ser) was significantly associated with breast cancer and was shown to cause partial exon skipping and decreased Claspin expression and Chk1 activation in a minigene splicing assay and in signalling experiments, respectively. CLSPN c.2028+16G>A was significantly associated with familial breast cancer and glioma, whereas c.2230T>C (p.Ser744Pro), was exclusively detected in breast cancer and glioma patients, but not in healthy controls. The remaining variants lacked a significant association with cancer. Nevertheless, the c.-68C>T promoter variant increased transcriptional activity in a luciferase assay. In conclusion, some of the CLSPN variants identified in the present study appear to modulate Claspin’s function by altering CLSPN transcription and RNA processing, as well as Chk1 activation.

Abstract 1375: Investigating synergy between CHK1 and PARP inhibitors in BRCA2 mutant and restored cells

Abstract Introduction: PARP inhibitors (PARPi) effectively target BRCA mutated cancers by exploiting defects in homologous recombination repair (HRR) by synthetic lethality. PARPi cause replication stress, activating ATR, which signals to HRR and to S and G2/M cell cycle checkpoints via CHK1 and WEE1. Our aim was to determine if a CHK1 inhibitor (CHK1i) would be synthetically lethal with PARPi and to identify the underlying mechanism. Methods: Effects of the PARPi rucaparib and the CHK1i PF-477736 were investigated in V-C8 (BRCA2 mutant Chinese hamster fibroblasts) and V-C8.B2 (BRCA2 restored) cell lines. PARP activity was determined by immunoblot densitometry of the PAR product. CHK1, ATR and WEE1 activity was measured by CHK1S296, CHK1S345 and CDK1y15 phosphorylation, respectively, by Western blot. HRR activity was determined by γH2AX and RAD51 foci formation by immunofluorescence microscopy. Cytotoxicity was determined by colony formation assay and cell cycle analysis by flow cytometry using propidium iodide. Results: V-C8 cells had higher baseline PARP activity than V-C8.B2 cells (1.6 and 0.7 nmol/106 cells, respectively), and in both cell lines PARP activity was suppressed 99% by 1 µM rucaparib. ATR, CHK1 and WEE1 were activated by rucaparib (10 µM) in both V-C8 cells (2.1, 1.5 and 1.8-fold) and V-C8.B2 cells (1.7, 1.4 and 1.5-fold) respectively. Conversely, activation of CHK1 and WEE1 was completely inhibited by 50 nM PF-477736 in both cell lines, while upstream activation of ATR by PF-477736 was almost 2-fold in both cell lines. Rucaparib was 550x more cytotoxic to HRR-defective V-C8 cells compared to HRR-competent V-C8.B2 cells (LC50 &amp;lt;0.1 µM vs 8.8 µM; p&amp;lt;0.001 respectively). Combination treatment with PF-477736 (50 nM) sensitised V-C8.B2 cells to rucaparib cytotoxicity 4.9-fold but did not sensitise V-C8 cells. In V-C8.B2 cells, 10 µM rucaparib caused a 6-fold increase in γH2AX and a 4.8-fold increase in RAD51 foci in γH2AX positive cells, demonstrating both increased replication stress and HRR activity in response to DNA damage. Combination treatment with PF-477736 (50 nM) increased γH2AX foci 2-fold compared to rucaparib single agent and inhibited the rucaparib induced RAD51 foci formation entirely, showing complete inhibition of HRR in V-C8.B2 γH2AX positive cells. Conclusion: The CHK1i PF-477736 sensitised HRR competent, but not HRR defective V-C8 cells to rucaparib suggesting that the primary mechanism was through PF-477736 abrogation of HRR mediated by its direct inhibition of CDK1. Cell cycle analysis is ongoing to determine the impact of rucaparib plus PF-477736 modulation of CHK1 and WEE1 activities on S and G2/M checkpoint control. Citation Format: Hannah L. Smith, Lisa Prendergast, Nicola J. Curtin. Investigating synergy between CHK1 and PARP inhibitors in BRCA2 mutant and restored cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1375.

NRF2 preserves genomic integrity by facilitating ATR activation and G2 cell cycle arrest.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a well-characterized transcription factor that protects cells against oxidative and electrophilic stresses. Emerging evidence has suggested that NRF2 protects cells against DNA damage by mechanisms other than antioxidation, yet the mechanism remains poorly understood. Here, we demonstrate that knockout of NRF2 in cells results in hypersensitivity to ionizing radiation (IR) in the presence or absence of reactive oxygen species (ROS). Under ROS scavenging conditions, induction of DNA double-strand breaks (DSBs) increases the NRF2 protein level and recruits NRF2 to DNA damage sites where it interacts with ATR, resulting in activation of the ATR–CHK1–CDC2 signaling pathway. In turn, this leads to G2 cell cycle arrest and the promotion of homologous recombination repair of DSBs, thereby preserving genome stability. The inhibition of NRF2 by brusatol increased the radiosensitivity of tumor cells in xenografts by perturbing ATR and CHK1 activation. Collectively, our results reveal a novel function of NRF2 as an ATR activator in the regulation of the cellular response to DSBs. This shift in perspective should help furnish a more complete understanding of the function of NRF2 and the DNA damage response.

Fractionated radiation promotes proliferation and radioresistance in bystander A549 cells but not in bystander HT29 cells

AimsRecent studies suggest that direct exposure of cells to fractionated radiotherapy might induce radioresistance. However, the effects of fractionated radiotherapy on the non-irradiated bystander cells remain unclear. We hypothesized that fractionated radiotherapy could enhance radioresistance and proliferation of bystander cells. Main methodsHuman tumor cell lines, including A549 and HT29 were irradiated (2 Gy per day). The irradiated cells (either A549 or HT29) were co-cultured with non-irradiated cells of the same line using transwell co-culture system. Tumor cell proliferation, radioresistance and apoptosis were measured using MTT assay, clonogenic survival assay and Annexin-V in bystander cells, respectively. In addition, activation of Chk1 (Ser 317), Chk2 (Thr 68) and Akt (Ser473) were measured via western blot. Key findingsIrradiated HT29 cells induced conventional bystander effects detected as modulation of clonogenic survival parameters (decreased area under curve, D10 and ED50 and increased α) and proliferation in recipient neighbors. While, irradiated A549 cells significantly enhanced the radioresistance and proliferation of bystander cells. These changes were accompanied with enhanced activation of Chk1, Chk2 and Akt in non-irradiated bystander A549 cells. Moreover, both bystander effects (damaging and protective) were mediated through secreted factors. SignificanceThese findings suggest that fractionated radiotherapy could promote proliferation and radioresistance of bystander cells probably through survival and proliferation pathways.

Resistance to the CHK1 inhibitor prexasertib involves functionally distinct CHK1 activities in BRCA wild-type ovarian cancer

High grade serous ovarian cancer (HGSOC) is a fatal gynecologic malignancy in the U.S. with limited treatment options. New therapeutic strategies include targeting of the cell cycle checkpoints, e.g., ATR and CHK1. We recently reported a promising clinical activity of the CHK1 inhibitor (CHK1i) prexasertib monotherapy in BRCA wild-type (BRCAwt) HGSOC patients. In this study, biopsies of treated patients and cell line models were used to investigate possible mechanisms of resistance to CHK1i. We report that BRCAwt HGSOC develops resistance to prexasertib monotherapy via a prolonged G2 delay induced by lower CDK1/CyclinB1 activity, thus preventing cells from mitotic catastrophe and cell death. On the other hand, we noted CHK1’s regulation on RAD51-mediated homologous recombination (HR) repair was not altered in CHK1i-resistant cells. Therefore, CHK1i sensitizes CHK1i-resistant cells to DNA damaging agents such as gemcitabine or hydroxyurea by inhibition of HR. In summary, our results demonstrate new mechanistic insights of functionally distinct CHK1 activities and highlight a potential combination treatment approach to overcome CHK1i resistance in BRCAwt HGSOC.

PTEN and DNA-PK determine sensitivity and recovery in response to WEE1 inhibition in human breast cancer.

Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): 'PTEN low' BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while 'PTEN high' BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.

Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells.

Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication. Indeed, Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs, and effects associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here, we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated, and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.

Protoflavone-Chalcone Hybrids Exhibit Enhanced Antitumor Action through Modulating Redox Balance, Depolarizing the Mitochondrial Membrane, and Inhibiting ATR-Dependent Signaling.

Hybrid compounds combine fragments with complementary targets to achieve a common pharmacological goal. This approach represents an increasingly popular strategy for drug discovery. In this work, we aimed to design antitumor hybrid compounds based on an inhibitor of ataxia-telangiectasia and Rad3-related protein (ATR)-dependent signaling, protoapigenone, and a pro-oxidant ferrocene or chalcone fragment. Four new triazole-coupled hybrids were prepared. The compounds were cytotoxic against human breast cancer cell lines in vitro, showing IC50 values in the sub-micromolar range. The nature of interactions between relevant fragments of the hybrids was evaluated by the Chou–Talalay method. Experimental combination treatment with the fragments showed additive effects or slight/moderate synergism, while strong synergism was observed when the fragments were virtually combined into their hybrids, suggesting a relevant pharmacological benefit of the coupling. All hybrids were strong inhibitors of the ATR-mediated activation of Chk1, and they interfered with the redox balance of the cells leading to mitochondrial membrane depolarization. Additionally, they induced late apoptosis and primary necrosis in MDA-MB-231 and MCF-7 breast cancer cells, respectively. Our results demonstrate that coupling the ATR-dependent signaling inhibitor protoflavone with a pro-oxidant chalcone dramatically increases the antitumor activity compared with either fragment alone. Such compounds may offer an attractive novel strategy for the treatment of various cancers.

Mitochondrial superoxide contributes to oxidative stress exacerbated by DNA damage response in RAD51-depleted ovarian cancer cells

Ovarian cancer is the most lethal gynecological malignancy. Abnormal homologous recombination repair, high level of reactive oxygen species (ROS) and upregulation of antioxidant genes are characteristic features of ovarian cancer. However, the molecular mechanisms governing the redox homeostasis in ovarian cancer cells remain to be fully elucidated. We here demonstrated a critical role of RAD51, a protein essential for homologous recombination, in the maintenance of redox homeostasis. We found that RAD51 is overexpressed in high grade serous ovarian cancer and is associated with poor prognosis. Depletion or inhibition of RAD51 results in G2/M arrest, increased production of reactive oxygen species and accumulation of oxidative DNA damage. Importantly, antioxidant N-acetylcysteine (NAC) significantly attenuated the induction of DNA damage and the perturbation of proliferation caused by RAD51 depletion. We further demonstrated that RAD51 inhibition or depletion led to elevated production of mitochondrial superoxide and increased accumulation of mitochondria. Moreover, CHK1 activation is required for the G2/M arrest and the generation of mitochondrial stress in response to RAD51 depletion. Together, our results indicate that nuclear DNA damage caused by RAD51 depletion may trigger mitochondria-originated redox dysregulation. Our findings suggest that a vicious cycle of nuclear DNA damage, mitochondrial accumulation and oxidative stress may contribute to the tumor-suppressive effects of RAD51 depletion or inhibition.