Chk1 Activation Research Articles

CHK2 activation contributes to the development of oxaliplatin resistance in colorectal cancer

BackgroundColorectal cancer (CRC), the most common cancer type, causes high morbidity and mortality. Patients who develop drug resistance to oxaliplatin-based regimens have short overall survival. Thus, identifying molecules involved in the development of oxaliplatin resistance is critical for designing therapeutic strategies.MethodsA proteomic screen was performed to reveal altered protein kinase phosphorylation in oxaliplatin-resistant (OR) CRC tumour spheroids. The function of CHK2 was characterised using several biochemical techniques and evident using in vitro cell and in vivo tumour models.ResultsWe revealed that the level of phospho-CHK2(Thr68) was elevated in OR CRC cells and in ~30% of tumour samples from patients with OR CRC. We demonstrated that oxaliplatin activated several phosphatidylinositol 3-kinase-related kinases (PIKKs) and CHK2 downstream effectors and enhanced CHK2/PARP1 interaction to facilitate DNA repair. A phosphorylation mimicking CHK2 mutant, CHK2T68D, but not a kinase-dead CHK2 mutant, CHK2D347A, promoted DNA repair, the CHK2/PARP1 interaction, and cell growth in the presence of oxaliplatin. Finally, we showed that a CHK2 inhibitor, BML-277, reduced protein poly(ADP-ribosyl)ation (PARylation), FANCD2 monoubiquitination, homologous recombination and OR CRC cell growth in vitro and in vivo.ConclusionOur findings suggest that CHK2 activity is critical for modulating oxaliplatin response and that CHK2 is a potential therapeutic target for OR CRC.

Human papillomaviruses sensitize cells to DNA damage induced apoptosis by targeting the innate immune sensor cGAS.

The cyclic GMP-AMP synthase (cGAS) is a critical regulator of the innate immune response acting as a sensor of double-strand DNAs from pathogens or damaged host DNA. Upon activation, cGAS signals through the STING/TBK1/IRF3 pathway to induce interferon expression. Double stranded DNA viruses target the cGAS pathway to facilitate infection. In HPV positive cells that stably maintain viral episomes, the levels of cGAS were found to be significantly increased over those seen in normal human keratinocytes. Furthermore the downstream effectors of the cGAS pathway, STING and IRF3, were fully active in response to signaling from the secondary messenger cGAMP or poly (dA:dT). In HPV positive cells cGAS was detected in both cytoplasmic puncta as well as in DNA damage induced micronuclei. E6 was responsible for increased levels of cGAS that was dependent on inhibition of p53. CRISPR-Cas9 mediated knockout of cGAS prevented activation of STING and IRF3 but had a minimal effect on viral replication. A primary function of cGAS in HPV positive cells was in response to treatment with etoposide or cisplatin which lead to increased levels of H2AX phosphorylation and activation of caspase 3/7 cleavage while having only a minimal effect on activation of homologous recombination repair factors ATM, ATR or CHK2. In HPV positive cells cGAS was found to regulate the levels of the phosphorylated non-homologous end-joining kinase, DNA-PK, which may contribute to H2AX phosphorylation along with other factors. Importantly cGAS was also responsible for increased levels of DNA breaks along with enhanced apoptosis in HPV positive cells but not in HFKs. This study identifies an important and novel role for cGAS in mediating the response of HPV positive cells to chemotherapeutic drugs.

Downregulation of ASF1B inhibits tumor progression and enhances efficacy of cisplatin in pancreatic cancer.

Pancreatic cancer is an aggressive and lethal cancer with the highest mortality rate. Hence, the development of new targeting and innovative treatment strategies is needed. Recent studies reported that the histone chaperone anti-silencing function 1B (ASF1B) can be used as a diagnosis and prognosis cancer biomarker. However, functional studies of ASF1B in pancreatic cancer have not been performed. This study compared expression levels of ASF1B in pancreatic cancer specimens with those of normal tissues using publicly available online databases. We found that ASF1B was commonly overexpressed in pancreatic cancer specimens, which is associated with poor prognosis. ASF1B downregulation in pancreatic cancer cells reduced their colony formation, proliferation, migration, and invasion abilities, and inhibited MMP9 activity. Furthermore, ASF1B expression downregulation increased cell cycle S-phase arrest and DNA damage though activation of the checkpoint kinases Chk1 and Chk2 pathways. Additionally, increased caspase (caspases-3 and -9) activation and PARP cleavage led to enhanced caspase-dependent apoptosis and improved cisplatin sensitivity. Collectively, our results indicate that ASF1B may serve as a potential biomarker of pancreatic cancer and a novel therapeutic target.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Cell cycle-dependent radiosensitivity in mouse zygotes

Mammalian zygotes are hypersensitive to radiation exposure compared with later-stage embryos and somatic cells, which may be due to an unusual DNA damage response (DDR). DNA damage checkpoints are an essential part of the DDR, allowing for faithful replication of cells. Although the DDR and radiosensitivity of somatic cells are dependent on the cell cycle phase, it remains largely unclear how the irradiation of zygotes at different phases affects cell cycle progression and preimplantation development. Here, mouse zygotes were irradiated with 10 Gy γ-rays at all four cell cycle phases. DNA damage checkpoints were activated by γ-irradiation at the G2 phase, but not at the G1, S, and M phases. The absence of DNA damage checkpoints at the G1 and M phases seems to be due to the low abundance of phosphorylated CHK2, which plays a key role in checkpoint activation in response to ionizing radiation. The cause of the inoperative S phase checkpoint may lie downstream of CHK2 activation. The inactive DNA damage checkpoints at the G1 and S phases contributed to micronucleus formation in the subsequent 2-cell stage, whereas irradiation at the M phase led to the highest incidence of chromatin bridges. The low developmental rates of embryos irradiated at the G1, S, and M phases suggest that embryos with these two types of chromatin abnormalities are prone to developmental failure. Taken together, these results suggest that the radiosensitivity of zygotes can be ascribed to a defective DDR at the G1, S, and M phases.

Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.

Both phosphorylation and phosphatase activity of PTEN are required to prevent replication fork progression during stress by inducing heterochromatin

PTEN is a tumor suppressor protein frequently altered in various cancers. PTEN-null cells have a characteristic of rapid proliferation with an unstable genome. Replication stress is one of the causes of the accumulation of genomic instability if not sensed by the cellular signaling. Though PTEN-null cells have shown to be impaired in replication progression and stalled fork recovery, the association between the catalytic function of PTEN regulated by posttranslational modulation and cellular response to replication stress has not been studied explicitly. To understand molecular mechanism, we find that PTEN-null cells display unrestrained replication fork progression with accumulation of damaged DNA after treatment with aphidicolin which can be rescued by ectopic expression of full-length PTEN, as evident from DNA fiber assay. Moreover, the C-terminal phosphorylation (Ser 380, Thr 382/383) of PTEN is essential for its chromatin association and sensing replication stress that, in response, induce cell cycle arrest. Further, we observed that PTEN induces HP1α expression and H3K9me3 foci formation in a C-terminal phosphorylation-dependent manner. However, phosphatase dead PTEN cannot sense replication stress though it can be associated with chromatin. Together, our results suggest that DNA replication perturbation by aphidicolin enables chromatin association of PTEN through C-terminal phosphorylation, induces heterochromatin formation by stabilizing and up-regulating H3K9me3 foci and augments CHK1 activation. Thereby, PTEN prevents DNA replication fork elongation and simultaneously causes G1-S phase cell cycle arrest to limit cell proliferation in stress conditions. Thus PTEN act as stress sensing protein during replication arrest to maintain genomic stability.

WASp modulates RPA function on single-stranded DNA in response to replication stress and DNA damage

Perturbation in the replication-stress response (RSR) and DNA-damage response (DDR) causes genomic instability. Genomic instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency disorder, yet the mechanism remains largely uncharacterized. Replication protein A (RPA), a single-strand DNA (ssDNA) binding protein, has key roles in the RSR and DDR. Here we show that human WAS-protein (WASp) modulates RPA functions at perturbed replication forks (RFs). Following genotoxic insult, WASp accumulates at RFs, associates with RPA, and promotes RPA:ssDNA complexation. WASp deficiency in human lymphocytes destabilizes RPA:ssDNA-complexes, impairs accumulation of RPA, ATR, ETAA1, and TOPBP1 at genotoxin-perturbed RFs, decreases CHK1 activation, and provokes global RF dysfunction. las17 (yeast WAS-homolog)-deficient S. cerevisiae also show decreased ScRPA accumulation at perturbed RFs, impaired DNA recombination, and increased frequency of DNA double-strand break (DSB)-induced single-strand annealing (SSA). Consequently, WASp (or Las17)-deficient cells show increased frequency of DSBs upon genotoxic insult. Our study reveals an evolutionarily conserved, essential role of WASp in the DNA stress-resolution pathway, such that WASp deficiency provokes RPA dysfunction-coupled genomic instability.

Acetylation dependent translocation of EWSR1 regulates CHK2 alternative splicing in response to DNA damage.

Ewing sarcoma breakpoint region 1 (EWSR1) is a member of FET (FUS/EWSR1/TAF15) RNA-binding family of proteins. The Ewing sarcoma oncoprotein EWS-FLI1 has been extensively studied, while much less is known about EWSR1 itself, especially the potential role of EWSR1 in response to DNA damage. Here, we found that UV irradiation induces acetylation of EWSR1, which is required for its nucleoli translocation. We identified K423, K432, K438, K640, and K643 as the major acetylation sites, p300/CBP and HDAC3/HDAC10 as the major acetyltransferases and deacetylases, respectively. Mechanically, UV-induced EWSR1 acetylation repressed its interaction with spliceosomal component U1C, which caused abnormal splicing of CHK2, suppressing the activity of CHK2 in response to UV irradiation. Taken together, our findings uncover acetylation as a novel regulatory modification of EWSR1, and is essential for its function in DNA damage response.

Abstract 1285: Chemotherapy induces PD-L1 overexpression in CRC through NF-κBp65

Abstract Background: The programmed cell death ligand (PD-L1) is an immune-inhibitory molecule that suppresses the activation of T cells, leading to the survival and growth of tumors. Overexpression of PD-L1 on cancer cells is associated with poor clinical outcomes in many types of cancers. PD-L1 upregulation contributes to chemoresistance in several types of cancer, but little is known about the mechanisms for alterations in PD-L1 expression associated with chemotherapy, especially in patients with CRC. Therefore, a better understanding of the mechanisms underlying changes in PD-L1 levels in response to standard of care chemotherapeutics in CRC is important. In this study, we examined the effects of thechemotherapeutics that are approved for the treatment of patients with CRC (oxaliplatin, 5FU, and irinotecan whose metabolite is SN38) in inducing PD-L1 expression and determining the molecular pathwaysinvolved in PD-L1 regulation. Methods: PD-L1 expression in CRC cell lines was determined in vitro using western blotting and FACS analysis. Changes in PD-L1 transcript levels were measured using real time PCR. RPPA analyses were performed to identify proteins/pathways affected by chemotherapeutics. Pharmacological inhibitors and siRNAs were used to target specific proteins in determining their role in PD-L1 expression. Results: We observed that oxaliplatin, 5FU, and SN38, resulted in induction of PD-L1 to various degrees. Interestingly this induction was observed in KRAS or BRAF mutated CRC cells but not in CRC cells with wild-type KRAS and BRAF. Increased levels of PD-L1 were due to increased transcription of PD-L1 resulting from exposure to chemotherapeutics. Chemotherapeutics lead to the activation of the DNA damage signaling mediator Chk1 and the downstream transcription activator NF-κBp65. Inhibition of Chk1 or NF-κBp65 resulted in abrogated PD-L1 induction in response to chemotherapeutic treatment. Conclusion: PD-L1 can be induced following treatment with chemotherapy in CRC cells harboring mutations in BRAF or KRAS. Our findings provide a rationale for studies combining inhibitors of NF-κBp65 with standard of care chemotherapeutics in CRC to block PD-L1 expression and enhance the efficacy of chemotherapy damaged-tumor cell clearance by innate immunity that, in turn, should improve patient outcomes. Citation Format: Fan Fan, Susmita Ghosh, Lee Ellis, Rajat Bhattacharya. Chemotherapy induces PD-L1 overexpression in CRC through NF-κBp65 [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 1285.

Abstract 2017: Therapeutic induction of replication stress in the context of salvage therapy in osteosarcoma

Abstract Osteosarcoma (OS) is an aggressive pediatric cancer with ~35% of patients developing metastasis over time. The survival rate for metastatic and relapsed OS patients is <30% and there is currently no standardized salvage therapy. Lack of efficacy is attributed to extensive genetic complexity present in OS that is partly due to moderate levels of replication stress (RS). While high levels of RS can induce cell death, moderate RS levels may cause genomic instability that contributes to progression of OS. Therefore, induction of RS to high levels, especially in genetically complex cancers like OS, could be a promising therapeutic strategy. Bromodomain and extra-terminal domain (BET) proteins (BRD2,3,4) are a family of epigenetic readers that not only regulate gene expression networks, but also regulate DNA replication and RS. BRD4 directly regulates major factors involved in DNA replication and checkpoint signaling. Thus, disruption of BRD4 function should exacerbate RS to levels that cause cell death. The objective of this study is to test the hypothesis that BET inhibition potentiates the efficacy of current salvage therapy through RS induction in aggressive OS. The effects of BET inhibitors (BETi), AZD5153 and OTX-015, as single agents and in combination with drugs used in salvage therapy such as topotecan were evaluated for effects on OS cell growth, PARP cleavage, and the DNA damage repair network. BET knockdown experiments were performed to evaluate target selectivity and dependency. In vivo efficacy and safety studies focused on patient-derived xenografts (PDXs) of relapsed OS. TT2-77 xenoline, Saos2, G292, and U2OS cell lines were selected for in vitro experiments. Combination index and Bliss independence analyses demonstrated additive to synergistic cell growth inhibition upon treatment with clinically relevant concentrations of BETi+topotecan. Significant increase in PARP cleavage was observed in the combination compared to single agent, indicating enhancement of apoptosis. Moreover, Western analyses demonstrated that BETi induces its effect, at least partly, via decreased CHK1 activation and increased DNA damage. Selective siRNA treatments illustrated that transient knockdown of individual BET proteins was not sufficient for potentiation of topotecan-induced cell death in OS cells, indicating that simultaneous knockdown of BETs may be required. Dose-finding studies of AZD5153 in relapsed OS PDXs that harbor replication stress signatures (TT2-77 and PDX96) indicated that daily doses of 1.25 or 2.5 mg/kg AZD5153 were well tolerated and effective in partially suppressing tumor growth compared to vehicle (p<0.05, Two-way ANOVA; Holm-Sidak). In vivo combination treatments of BETi+topotecan are in progress. These data collectively suggest that BET inhibition alongside salvage therapy holds promise as a novel treatment strategy for inducing RS-mediated cell death in aggressive OS. Citation Format: Niknam Riyahi, Pankita H. Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Melissa A. Trowbridge, Kathy Coy, Henry Mang, Reagan K. Wohlford, Anthony L. Sinn, Emily S. Sims, Matt J. Repass, Nuri Damayanti, Farinaz Barghi, Harlan E. Shannon, Michael J. Ferguson, Jamie L. Renbarger, Karen E. Pollok. Therapeutic induction of replication stress in the context of salvage therapy in osteosarcoma [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 2017.

Icaritin inhibits PLK1 to activate DNA damage response in NK/T cell lymphoma and increases sensitivity to GELOX regime

Natural killer/T cell lymphoma (NKTCL) is a highly aggressive subtype of non-Hodgkin lymphoma. Gemcitabine, oxaliplatin, and L-asparaginase (GELOX) is one of the first-line chemotherapy regimens of NKTCL. Yet, the prognosis of NKTCL is poor. Icaritin is an herb-derived monomer from icariin with antitumor effects. We found that icaritin induced proliferation inhibition and apoptosis of NKTCL both in vitro and in vivo. Moreover, icaritin inhibited the dissemination of NKTCL in vivo. RNA sequencing revealed the Polo-like kinase 1 (PLK1) gene and DNA damage response (DDR) as the targets of icaritin. Mechanistically, icaritin inhibited PLK1 to promote checkpoint kinase 2 (Chk2) homodimerization and its T387 phosphorylation, which further activated p53, leading to the activation of the DDR pathway. Moreover, inhibiting PLK1 increased Forkhead box O3a nuclear localization, the latter of which activated ataxia telangiectasia mutated (ATM), an early sensor of DNA damage. Then ATM phosphorylated Chk2 T68 and initiated Chk2 activation. Remarkably, the combined treatment of icaritin and GELOX achieved better antitumor efficacy than single treatment in vivo. In summary, our results proved the efficacy of icaritin treating NKTCL, provided insights into its antitumor molecular mechanism, and revealed the application value of icaritin in facilitating clinical NKTCL treatment.

Molecular insights into the microtubules depolymerizing activity of the IL-8 receptor B antagonist SB225002.

We have previously reported the novel off-target microtubules destabilizing activity of SB225002, a compound that was originally designed as a selective and potent IL-8 receptor B antagonist. In the present study we investigated the reversibility of SB225002 antimitotic effect and provided additional mechanistic insights underlying cell death induction in SW480 human colorectal adenocarcinoma cells. Mitotically arrested cells by SB225002 treatment were isolated by shake-off, and their identity was verified by both flow cytometry and immunoblotting. The reversibility of SB225002 antimitotic effects was investigated by flow cytometry and immunoblotting. Prometaphase arrested cells were imaged via indirect immunofluorescence and confocal microscopy. Activation of CHK1 in mitotically arrested cells was assessed by immunoblotting, and the relationship between CHK1 and mitotic arrest was examined via siRNA-mediated knockdown of CHK1. JNK signaling was evaluated via immunoblotting as well as pharmacological inhibition, followed by flow cytometry. The role of reactive oxygen species (ROS) in cytotoxicity was evaluated by ROS scavenging and flow cytometry. Following SB225002 washout, the mitotic checkpoint was abrogated, and cell cycle perturbations were gradually restored with induction of cell death. Mechanistically, CHK1 checkpoint was activated by SB225002 and occurred downstream of the mitotic checkpoint. In addition, SB225002 activated JNK signaling which contributed to cell death and restrained polyploidy. Furthermore, SB225002 increased intracellular ROS which played a role in mediating SB225002 cytotoxicity. Findings of the present study warrants further development of SB225002 as a lead compound that uniquely targets microtubules dynamics and IL-8 signaling.

Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

Chk1 Inhibition Hinders the Restoration of H3.1K56 and H3.3K56 Acetylation and Reprograms Gene Transcription After DNA Damage Repair.

H3K56 acetylation (H3K56Ac) was reported to play a critical role in chromatin assembly; thus, H3K56ac participates in the regulation of DNA replication, cell cycle progression, DNA repair, and transcriptional activation. To investigate the influence of DNA damage regulators on the acetylation of histone H3 and gene transcription, U2OS cells expressing SNAP-labeled H3.1 or SNAP-labeled H3.3 were treated with ATM, ATR, or a Chk1 inhibitor after ultraviolet (UV) radiation. The levels of H3.1K56ac, H3.3K56ac, and other H3 site-specific acetylation were checked at different time points until 24 h after UV radiation. The difference in gene transcription levels was also examined by mRNA sequencing. The results identified Chk1 as an important regulator of histone H3K56 acetylation in the restoration of both H3.1K56ac and H3.3K56ac. Moreover, compromising Chk1 activity via chemical inhibitors suppresses gene transcription after UV radiation. The study suggests a previously unknown role of Chk1 in regulating H3K56 and some other site-specific H3 acetylation and in reprograming gene transcription during DNA damage repair.

CHK1 phosphorylates PRIMPOL to promote replication stress tolerance.

Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.

Ricolinostat enhances adavosertib‑induced mitotic catastrophe in TP53‑mutated head and neck squamous cell carcinoma cells.

TP53 mutation is one of the most frequent gene mutations in head and neck squamous cell carcinoma (HNSCC) and could be a potential therapeutic target. Recently, the WEE1 G2 checkpoint kinase (WEE1) inhibitor adavosertib (Adv) has attracted attention because of its selective cytotoxicity against TP53-mutated cells and has shown promising activity in early phase clinical trials. In the present study, it was demonstrated that combined treatment with Adv and a selective histone deacetylase 6 (HDAC6) inhibitor, ricolinostat (RCS), synergistically enhanced cell death induction in four out of five HNSCC cell lines with TP53 mutation (CAL27, SAS, HSC-3, and OSC-19), one HNSCC cell line with impaired TP53 function by HPV-infection (UPCI-SCC154), and TP53-knockout human lung cancer cell line (A549 TP53-KO), but not in TP53 wild-type A549 cells. Time-lapse imaging showed that RCS enhanced the Adv-induced mitotic catastrophe. Consistent with this, RCS treatment suppressed checkpoint kinase 1 (Chk1) (Ser345) phosphorylation and co-administration of RCS with Adv suppressed cyclin-dependent kinase 1 (Tyr15) phosphorylation along with increased expression of γ-H2A.X, a marker of DNA double-strand breaks in CAL27 cells. These data showed that RCS enhanced Adv-induced premature mitotic entry and cell death induction in the mitotic phase. However, although HDAC6 knockdown enhanced Adv-induced cell death with γ-H2A.X elevation, HDAC6 knockdown did not repress Chk1 phosphorylation in CAL27 cells. Our data demonstrated that the co-administration of RCS with Adv in HNSCC cells resulted in the suppression of Chk1 activity, leading to synergistically enhanced apoptosis via mitotic catastrophe in a p53-dependent manner. This enhanced cell death appeared to be partially mediated by the inhibition of HDAC6 activity by RCS.

Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome

In human cells, ATR/Chk1 signaling couples S phase exit with the expression of mitotic inducers and prevents premature mitosis upon replication stress (RS). Nonetheless, under-replicated DNA can persist at mitosis, prompting chromosomal instability. To decipher how the DNA replication checkpoint (DRC) allows cells to enter mitosis over time upon RS, we developed a FRET-based Chk1 activity sensor. During unperturbed growth, a basal Chk1 activity level is sustained throughout S phase and relies on replication origin firing. Incremental RS triggers stepwise Chk1 over-activation that delays S-phase, suggesting a rheostat-like role for DRC coupled with the replication machinery. Upon RS, Chk1 is inactivated as DNA replication terminates but surprisingly is reactivated in a subset of G2 cells, which relies on Cdk1/2 and Plk1 and prevents mitotic entry. Cells can override active Chk1 signaling and reach mitosis onset, revealing checkpoint adaptation. Cell division following Chk1 reactivation in G2 results in a p53/p21-dependent G1 arrest, eliminating the daughter cells from proliferation.

Chk1 Inhibition Ameliorates Alzheimer’s Disease Pathogenesis and Cognitive Dysfunction Through CIP2A/PP2A Signaling

Alzheimer’s disease (AD) is the most common neurodegenerative disease with limited therapeutic strategies. Cell cycle checkpoint protein kinase 1 (Chk1) is a Ser/Thr protein kinase which is activated in response to DNA damage, the latter which is an early event in AD. However, whether DNA damage-induced Chk1 activation participates in the development of AD and Chk1 inhibition ameliorates AD-like pathogenesis remain unclarified. Here, we demonstrate that Chk1 activity and the levels of protein phosphatase 2A (PP2A) inhibitory protein CIP2A are elevated in AD human brains, APP/PS1 transgenic mice, and primary neurons with Aβ treatment. Chk1 overexpression induces CIP2A upregulation, PP2A inhibition, tau and APP hyperphosphorylation, synaptic impairments, and cognitive memory deficit in mice. Moreover, Chk1 inhibitor (GDC0575) effectively increases PP2A activity, decreases tau phosphorylation, and inhibits Aβ overproduction in AD cell models. GDC0575 also reverses AD-like cognitive deficits and prevents neuron loss and synaptic impairments in APP/PS1 mice. In conclusion, our study uncovers a mechanism by which DNA damage-induced Chk1 activation promotes CIP2A-mediated tau and APP hyperphosphorylation and cognitive dysfunction in Alzheimer’s disease and highlights the therapeutic potential of Chk1 inhibitors in AD.

P53-NEIL1 co-abnormalities induce genomic instability and promote synthetic lethality with Chk1 inhibition in multiple myeloma having concomitant 17p13(del) and 1q21(gain).

Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.