Chk1 Expression Research Articles

Panobinostat Synergizes with Chemotherapeutic Agents and Improves Efficacy of Standard-of-Care Chemotherapy Combinations in Ewing Sarcoma Cells

Background: The survival rate of patients with Ewing sarcoma (EWS) has seen very little improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. HDAC2, ALK, JAK1, and CDK4 were identified as potential targets using RNA sequencing performed on EWS patient tumors with the bioinformatic analysis of gene expression. Methods/Results: The pan-HDAC inhibitor Panobinostat was cytotoxic to all the Ewing sarcoma cell lines tested. Mechanistically, Panobinostat decreases the expression of proteins involved in the cell cycle, including Cyclin D1 and phospho-Rb, and DNA damage repair, including CHK1. Further, Panobinostat induces a G1 cell cycle arrest. The combination of Panobinostat with Doxorubicin or Etoposide, both of which are used as standard of care in upfront treatment, leads to a synergistic effect in EWS cells. The combination of Panobinostat and Doxorubicin induces an accumulation of DNA damage, a decrease in the expression of DNA damage repair proteins CHK1 and CHK2, and an increase in caspase 3 cleavage. The addition of Panobinostat to standard-of-care chemotherapy combinations significantly reduces cell viability compared to that of chemotherapy alone. Conclusions: Overall, our data indicate that HDAC2 is overexpressed in many EWS tumor samples and HDAC inhibition is effective in targeting EWS cells, alone and in combination with standard-of-care chemotherapy agents. This work suggests that the addition of an HDAC inhibitor to upfront treatment may improve response.

TLK1 Inhibition Enhances the Anticancer Effect of Deep UV Irradiation Through CHK1 Activation.

A deep ultraviolet (DUV) light-emitting diode (LED) is a device that can irradiate electromagnetic waves from 250 nm to 350 nm. Tousled-like kinase 1 (TLK1) encodes a nuclear serine/threonine kinase, which is thought to influence the effects of DUV irradiation in cancer. The aim of this study was to clarify the interaction of TLK1 with DUV irradiation-induced DNA damage in cancer cells. Pancreatic cancer cell lines were treated with or without DUV. TLK1 expression and phosphorylation in the two groups were examined. Then, these cancer cell lines were treated with thioridazine (THD), DUV or both. Thereafter, cytomorphology and apoptosis were assessed. Several proteins related to DNA damage, were analyzed in cancer cells treated with DUV and THD. Tumors in a subcutaneous xenograft model were treated with THD, DUV, or both for six weeks. DUV irradiation induced the phosphorylation of TLK1 in pancreatic cancer cell lines. Cytomorphology was significantly changed in pancreatic cancer cells treated with DUV and THD. TLK1 inhibition enhanced DUV irradiation-induced apoptosis in cancer cells. Interestingly, CHK1 and pCHK1 expression was suppressed after TLK1 inhibition. In addition, inhibition of MRE11 led to a decrease in the expression of CHK1 and pCHK1, accompanied by a notable increase in apoptosis. In the subcutaneous xenograft models, the tumor volume in the DUV and THD groups was lower than that in the other groups. TLK1 phosphorylation is an important event in DUV irradiation. DUV irradiation combined with TLK1 inhibition has therapeutic potential in pancreatic cancer cells.

MDB-64. NOVEL TRANSCRIPTS ASSOCIATE WITH SURVIVAL IN CHILDHOOD MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma is the commonest malignant brain tumour in childhood, with progression-free survival (PFS) <65% in high-risk disease. Novel biomarkers, independent of current clinicopathological risk factors, could enhance understanding of biology and support improved treatment approaches. METHODS Using high-depth RNA sequencing of 217 patients, we identified novel multi-exonic medulloblastoma transcripts using a de novo analysis. We evaluated these alongside known/curated gene transcripts, including correlative survival analysis for 170 patients (aged 3-16 years and treated with curative intent). Multivariable survival analyses of the overall cohort (n=145) and a MBGroup3/MBGroup4 (Group3/Group4)-specific subcohort (n=95) was undertaken to identify prognostic transcripts, adjusting for current risk criteria and correcting for multiple testing. RESULTS From 3,150 novel transcripts identified, expression of 217 varied significantly and were further assessed. One was prognostic for PFS independent of subgroup (located on chromosome 20) while three were independently prognostic within Group3/Group4 (located on chromosome 1, 4 and 21 respectively). Transcript presence was validated by qPCR. From >60,000 known transcripts identified, 9693 were assessed. Four were independently prognostic for PFS in the whole-cohort (MRPS12, SLC25A34, MLYCD and ROGD1). In Group3/Group4, the top upregulated canonical pathways associated with PFS were: polo-like kinase, cyclin/cell cycle regulation, BRCA1/ DNA damage response, and glutamate receptor signalling. MELK and NQO2 were among the top 10 transcripts most highly associated with Group3/Group4 PFS. The prognostic significance of six transcripts (MRPS12, MLYCD, MELK, CDK4, NQO2, ROGD1) was validated in an independent Group3/Group4 dataset (n=470) while ROGD1 was prognostic independent of subgroup. Immunohistochemical validation showed low CDK4 expression, low MLYCD expression and high CHK1 expression were independent prognosticators in Group3/Group4. CONCLUSION De novo transcripts represent a significant component of the medulloblastoma transcriptome, providing prognostic information, and enhancing biological understanding of Group3/Group4 disease. CHK1 and MELK represent promising validated candidates for further evaluation as prognostic biomarkers in Group3/Group4 medulloblastoma.

Apelin-13 improves pulmonary epithelial barrier function in a mouse model of LPS-induced acute lung injury by inhibiting Chk1-mediated DNA damage

Apelin-13, a type of active peptide, can alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the specific mechanism is unclear. Cell cycle checkpoint kinase 1 (Chk1) plays an important role in DNA damage. Here, we investigated the regulatory effect of Apelin on Chk1 in ALI. Chk1-knockout and −overexpression mice were used to explore the role of Chk1 in LPS-induced ALI mice treated with or without Apelin-13. In addition, A549 cells were also treated with LPS to establish a cell model. Chk1 knockdown inhibited the destruction of alveolar structure, the damage of lung epithelial barrier function, and DNA damage in the ALI mouse model. Conversely, Chk1 overexpression had the opposite effect. Furthermore, Apelin-13 reduced Chk1 expression and DNA damage to improve the impaired lung epithelial barrier function in the ALI model. However, the high expression of Chk1 attenuated the protective effect of Apelin-13 on ALI. Notably, Apelin-13 promoted Chk1 degradation through autophagy to regulate DNA damage in LPS-treated A549 cells. In summary, Apelin-13 regulates the expression of Chk1 by promoting autophagy, thereby inhibiting epithelial DNA damage and repairing epithelial barrier function.

Abstract LB259: Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer

Abstract Epigenetic regulation plays an important role in prostate cancer progression and metastasis. KDM5D is a male-specific histone-modifying enzyme encoded on the Y chromosome that demethylates di- and tri-methylated forms of lysine 4 in histone H3, resulting in transcriptional repression of certain genes. In this study, we investigated the role of KDM5D in resistance to inhibitors targeting the G2/M cell cycle checkpoint kinase CHK1. Loss of KDM5D expression was associated with reduced cell proliferation and resistance to the CHK1 inhibitor SRA737 in castration-resistant (CRPC) and neuroendocrine (NEPC) prostate cancer cell lines. In KDM5D expressing cells, depletion of KDM5D differentially impacted the proliferation of prostate cancer cell lines. The androgen-sensitive LNCaP cell line was particularly sensitive to the knockdown of KMD5D. On the other hand, in CRPC cell line 22Rv1, the knockdown of KDM5D caused resistance to both inhibitors, indicating a dual role of KDM5D in mediating ATR and CHK1 inhibitor sensitivity and cell proliferation in prostate cancer cells. Further analysis demonstrated a self-regulatory cycle between CHK1 and KDM5D in 22Rv1 cells, where depletion of KMD5D reduced CHK1 mRNA, while decreased CHK1 caused upregulation of KDM5D and overexpression of KDM5D resulted in increased CHK1 expression. The relevance of this regulatory mechanism to ATR and CHK1 inhibitor sensitivity is currently under investigation, along with several potential mediators of the differential responses to these inhibitors. Additional ATR and CHK1 inhibitors will also be examined in this context. This study may uncover novel biomarkers for ATR and CHK1 inhibitors that are currently in clinical trials for various cancers. KDM5D may be used to stratify prostate cancer patients receiving these highly potent anticancer agents. Citation Format: Wenxiao Zheng, Yuzhou Chen, Qiming J. Wang. Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB259.

Photoactivation Inducing Multifunctional Coupling of Fluorophore for Efficient Tumor Therapy In Situ.

Photoactivatable molecules, with high-precision spatialtemporal control, have largely promoted bioimaging and phototherapy applications of fluorescent dyes. Here, the first photoactivatable sensor (BI) is described that can be triggered by broad excitation light (405-660 nm), which further undergoes intersystem crossing and H-atom transfer processes to forming superoxide anion radicals (O2 -• ) and carbon radicals. Particularly, the photoinduced gain of carbon-centered radicals (BI•) allows for radical-radical coupling to afford the combined crosslink product (BI─BI), which would be oxidized in the presence of O2 -• to produce an extended conjugate system with near infrared emission (820 nm). Besides, the photochemically generated product (Cy─BI) possesses ultra-high photothermal conversion efficiency up to 90.9%, which optimized phototherapy potential. What's more, Western Blot assay reveals that both BI and the photoproduct Cy─BI can efficiently inhibit the expression of CHK1, and the irradiation of BI and Cy─BI further induces apoptosis and ultimately enhances the phototherapeutic effects. Thus, the combination of cell cycle block inducing apoptosis, photodynamic therapy and photothermal therapy treatments significantly suppress solid tumor in vivo antitumor efficacy explorations. This is a novel finding in developing photoactivatable molecules, as well as the broad applicability of photoimaging and phototherapy in tumor-related areas.

Relationship between the Expression of CHK2 and p53 in Tumor Tissue and the Course of Papillary Thyroid Cancer in Patients with CHEK2 Germline Mutations.

The aim of this study was to determine whether the expression of CHK2 and p53 in tumor tissue in carriers of germline CHEK2 mutations can serve as a prognostic marker for PTC, and whether CHEK2 and TP53 copy numbers correlates with the course of PTC disease. This study included 156 PTC patients previously tested for the presence of CHEK2. Clinicopathological features, treatment response, disease outcome, and germline mutation status of the CHEK2 gene were assessed with respect to CHK2 and p53 expression, and CHEK2 and TP53 gene copy statuses. In patients with and without a germline mutation in CHEK2 and with higher CHK2 expression, the chances of an excellent treatment response and no evidence of disease were lower than in patients without or with lower CHK2 expression. TP53 deletion was associated with angioinvasion. In patients with a truncating mutation, the chance of a CHEK2 deletion was higher than in patients with WT CHEK2 alone or those with WT CHEK2 and with the missense I157T mutation. Higher CHK2 expression was associated with poorer treatment responses and disease outcomes. Higher CHK2 expression and positive p53 together with a TP53 deletion could be a prognostic marker of unfavorable disease outcomes in patients with germline truncating mutations in CHEK2.

Natural pentacyclic triterpenoid from Pristimerin sensitizes p53-deficient tumor to PARP inhibitor by ubiquitination of Chk1

Inhibition of checkpoint kinase 1 (Chk1) has shown to overcome resistance to poly (ADP-ribose) polymerase (PARP) inhibitors and expand the clinical utility of PARP inhibitors in a broad range of human cancers. Pristimerin, a naturally occurring pentacyclic triterpenoid, has been the focus of intensive studies for its anticancer potential. However, it is not yet known whether low dose of pristimerin can be combined with PARP inhibitors by targeting Chk1 signaling pathway. In this study, we investigated the efficacy, safety and molecular mechanisms of the synergistic effect produced by the combination olaparib and pristimerin in TP53-deficient and BRCA-proficient cell models. As a result, an increased expression of Chk1 was correlated with TP53 mutation, and pristimerin preferentially sensitized p53-defective cells to olaparib. The combination of olaparib and pristimerin resulted in a more pronounced abrogation of DNA synthesis and induction of DNA double-strand breaks (DSBs). Moreover, pristimerin disrupted the constitutional levels of Chk1 and DSB repair activities. Mechanistically, pristimerin promoted K48-linked polyubiquitination and proteasomal degradation of Chk1 while not affecting its kinase domain and activity. Importantly, combinatorial therapy led to a higher rate of tumor growth inhibition without apparent hematological toxicities. In addition, pristimerin suppressed olaparib-induced upregulation of Chk1 and enhanced olaparib-induced DSB marker γΗ2ΑΧ in vivo. Taken together, inhibition of Chk1 by pristimerin has been observed to induce DNA repair deficiency, which may expand the application of olaparib in BRCA-proficient cancers harboring TP53 mutations. Thus, pristimerin can be combined for PARP inhibitor-based therapy.

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.

New insights into ATR inhibition in muscle invasive bladder cancer: The role of apolipoprotein B mRNA editing catalytic subunit 3B.

Apolipoprotein B mRNA editing catalytic polypeptide (APOBEC), an endogenous mutator, induces DNA damage and activates the ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) pathway. Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer (MIBC), it has a poor survival rate. Therefore, this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B (APOBEC3B) expressing MIBC. Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC. The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis. Western blot analysis was performed to confirm differences in phosphorylated Chk1 (pChk1) expression according to the APOBEC3B expression. Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin. There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC. Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels. Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression. Compared to cisplatin single treatment, combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression. Conclusion: Our study shows that APOBEC3B's higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition. This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

LncRNA SPRY4-IT1 regulates 16HBE cell malignant transformation induced by particulate matter through DUSP6-ERK1/2-Chk1 signaling pathway

Particulate matter (PM), one of the most serious air contaminants, could easily pass through the airway and deposit at the deep alveoli. Thus, it might trigger respiratory diseases like inflammation, asthma and lung cancer on human. Long non-coding RNAs (lncRNAs) are considered as important regulator in promotion and progression of diverse cancers. However, the molecular mechanism of lncRNAs mediating PM-induced lung carcinogenesis remains unclear. In this study, we established a 16HBE malignant transformed cell induced by PM (Cells were treated with 20 μg/ml PM, which named PM-T cells) and explored the roles and mechanisms of lncRNAs in the malignant transformation induced by PM. Compared with 16HBE cells, various biological functions were changed in PM-T cells, such as cell proliferation, migration, cell cycle and apoptosis. LncRNA SPRY4-IT1 was significant down-regulated expression and associated with these biological effects. Our results showed that lncRNA SPRY4-IT1 overexpression reversed these functional changes mentioned above. The further studies indicated that lncRNA SPRY4-IT1 involved in PM-induced cell transformation by modulating Chk1 expression via negative regulation of DUSP6-ERK1/2. In conclusion, our studies suggested that lncRNA SPRY4-IT1 played the role as a tumor suppressor gene and might mediate 16HBE cells malignant transformation induced by PM through regulating DUSP6-ERK1/2-Chk1 signaling pathway.

RAD54B inhibits vascular endothelial senescence via suppression of CHK1/p53/p21 pathway.

RAD54B belongs to the SNF2/SWI2 superfamily, participating in homologous recombination repair. DNA damage is the central driver of aging, but there is no direct evidence of an association between RAD54B and vascular aging. The present study sought to investigate the role and mechanisms of RAD54B in endothelial senescence. In senescent animal models, including spontaneously hypertensive rats, normal aging mice, and D-gal-induced senescent mice, and senescent cell models induced by H2O2, D-gal, and culture, RAD54B was remarkably downregulated. Knockdown of RAD54B increased the expression of p53 and p21, increased the ratio of SA-β-gal-positive cells, and decreased the proportion of EdU-positive cells. Conversely, overexpression of RAD54B reversed the senescent phenotypes stimulated by H2O2 and delayed replicative endothelial senescence. Mechanistically, silencing RAD54B compensatorily increased the expression of RAD51/XRCC4, which remained unchanged in H2O2-induced senescence. RAD54B lacking the SNF2 domain could still reverse the increasing expression of p53/p21 induced by H2O2. RAD54B reduced γH2A.X expression and inhibited the expression and phosphorylation of CHK1. In conclusion, RAD54B exerts a direct protective effect against DNA damage through enhancing homologous recombination repair in endothelial senescence, resulting in inhibition of the downstream CHK1/p53/p21 pathway, suggesting that RAD54B may be a potential therapeutic target for vascular aging-associated diseases.

AZD7762 induces CRBN dependent BAG3 degradation through ubiquitin-proteasome pathway.

Protein degraders are currently under rapid development as a promising modality for drug discovery. They are compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting intracellular protein degradation through proteasomal pathway. More protein degraders identification will greatly promote the development of this field. BAG3 is widely recognized as an excellent therapeutic target in cancer treatments. Exploring protein degraders that target BAG3 degradation has profound implications. Herein, molecular docking was applied to assess binding energy between 81 clinical phase I kinase inhibitors and BAG3. BAG3 protein and mRNA level were detected by western blot and quantitative real-time PCR. CCK8 assay and colony formation assay were applied to detect the cell viability and proliferation rate. Cell death was accessed using flow cytometry combined with PI and Annexin V double staining. AZD7762, a Chk1 kinase inhibitor, was identified to induce BAG3 degradation in a ubiquitin-proteasome pathway. AZD7762-induced BAG3 degradation was not dependent on Chk1 expression or activity. CRBN, an E3 ligase, was identified to bind to BAG3 and mediated BAG3 ubiquitination in the presence of AZD7762. By targeting Chk1 and BAG3, two ideal therapeutic targets in cancer treatment, AZD7762 would be a powerful chemotherapy agent in the future.

Betulinic acid, a major therapeutic triterpene of Celastrus orbiculatus Thunb., acts as a chemosensitizer of gemcitabine by promoting Chk1 degradation.

Celastrus orbiculatus Thunb., also called as oriental bittersweet vine or climbing spindle berry, a traditional Chinese herbal medicine has been used to treat a spectrum of painful and inflammatory diseases for centuries. Explored for their unique medicinal properties, C.orbiculatus offers additional therapeutic effects on cancerous diseases. The effect of single-agent gemcitabine on survival has not long been encouraging, combination therapies provide patients multiple chances of benefit for improved clinical response. This study aims at expounding the chemopotentiating effects and underlying mechanisms of betulinic acid, a primary therapeutic triterpene of C. orbiculatus in combination with gemcitabine chemotherapy. The preparation of betulinic acid was optimized using ultrasonic-assisted extraction method. Gemcitabine-resistant cell model was established by induction of the cytidine deaminase. MTT, colony formation, EdU incorporation and Annexin V/PI staining assays were used to evaluate cytotoxicity, cell proliferation and apoptosis in BxPC-3 pancreatic cancer cell line and H1299 non-small cell lung carcinoma cell line. Comet assay, metaphase chromosome spread and γH2AX immunostaining were applied for DNA damage assessment. Western blot and co-immunoprecipitation was used to detect the phosphorylation and ubiquitination of Chk1. Mode of action of gemcitabine in combination with betulinic acid was further captured in BxPC-3-derived mouse xenograft model. We noticed that the extraction method had an impact on the thermal stability of C. orbiculatus. Ultrasound-assisted extraction at room temperature in shorter processing time could maximize the overall yields and biological activities of C. orbiculatus. The major constituent was identified as betulinic acid, and the pentacyclic triterpene represented the prominent anticancer activity of C. orbiculatus. Forced expression of cytidine deaminase conferred acquired resistance to gemcitabine, while betulinic acid displayed equivalent cytotoxicity toward gemcitabine-resistant and sensitive cells. A combination therapy of gemcitabine with betulinic acid produced synergistic pharmacologic interaction on cell viability, apoptosis and DNA double-strand breaks. Moreover, betulinic acid abrogated gemcitabine-triggered Chk1 activation by destabilizing Chk1 loading via proteasomal degradation. The combination of gemcitabine and betulinic acid significantly retarded BxPC-3 tumor growth in vivo compared to single-agent gemcitabine treatment alone, accompanied with reduced Chk1 expression. These data provide evidence that betulinic acid is a potential candidate for chemosensitization as a naturally occurring Chk1 inhibitor and warrants further preclinical evaluation.

MiR-34 by targeting p53 induces apoptosis and DNA damage in paclitaxel-resistant human oral squamous carcinoma cells.

MicroRNA-34 (miR-34) is one the most important tumor suppressor miRNAs involving in the various aspects of oral cancer. The present study aimed to evaluate the effects of miR-34 restoration in OECM-1 oral cancer resistant to paclitaxel (OECM-1/PTX) and its underlying mechanisms through p53-mediated DNA damage and apoptosis. OECM-1 and OECM-1/PTX were transfected with miR-34 mimic and inhibitor. Cellular proliferation and apoptosis were evaluated through MTT assay and flow cytometry, respectively. The mRNA and protein expression levels of p53, p-glycoprotein (P-gp), ATM, ATR, CHK1, and CHK2 were assessed through qRT-PCR and western blotting. Rhodamin123 uptake assay was used to measure the P-gp activities. P53 expression was also suppressed by sing a siRNA transfection of cells. The expression levels of miR-34 were downregulated in OECM-1/PTX. Restoration of miR-34 led to increase in cytotoxic effects of paclitaxel in cells. In addition, the expression levels and activities of P-gp were reduced following miR-34 transfection. miR-34 transfection upregulated the p53, ATM, ATR, CHK1, and CHK2 expression levels in OECM-1/PTX cells. Furthermore, cells transfected with miR-34 showed higher levels of apoptosis. miR-34 restoration reverses paclitaxel resistance in OECM-1 oral cancer. The chemosensitive effects of miR-34 is mediated through increasing DNA damage and apoptosis in a p53 depended manner.

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.

OR06-1 Cyclophosphamide Depletes Primordial Follicles via the p63 Apoptotic Pathway

Abstract Cancer therapies often cause serious side effects, affecting the quality of life for cancer survivors. The ovary can be affected by cancer therapies, causing premature ovarian insufficiency, leading to endocrine dysfunction and infertility. Thus, maintaining ovarian function against cancer treatment is an unmet need for female cancer patients. Cyclophosphamide (CPA), a common chemotherapeutic agent, is metabolized in vivo and forms DNA crosslinks, inducing apoptosis in rapidly proliferating tumor cells. However, the underlying mechanism of the CPA-induced oocyte death in ovarian reserve remains unclear. This study aims to investigate the mechanism of oocyte death in primordial follicles by generating oocyte-specific Abl1 and p63 knockout and Pik3ca* knockin mouse models using Gdf9-iCre+. Postnatal day 7 female mice were i.p. injected w/wo CPA. Notably, we found CPA drastically reduced the number of primordial follicles without increasing the number of growing follicles. The quantification of surviving follicles validated that 90% of the primordial follicles from oocyte-specific Abl1 knockout mice were lost following CPA treatment in vivo and in vitro. Concurrently, high expression of CHK2 was detected in the oocytes of the ovary cultured with CPA metabolite in vitro. This implied CHK2 rather than ABL is a possible tyrosine kinase for CPA-induced oocyte death. Most of all, p63 was a key player for CPA-induced oocyte death, supported by the result that p63 knockout oocytes were rescued after CPA treatment. To clarify whether the ovaries lose follicles via activation or apoptosis pathway, PIK3CA* mice were examined with CPA. As expected, primordial follicles and primordial follicles with activated oocytes in the ovary of PIK3CA* mice survived against CPA due to constitutive PI3K activity in them. This indicates activated follicles resist gonadotoxic reagents, although p63 is expressed inside their oocytes. Accordingly, the apoptosis markers, BAX and cleaved PARP were highly induced with CPA injection in a time-dependent manner in the ovaries from wild-type female mice but not from the p63 knockout. The double-strand break also occurred in the nucleus of oocytes post CPA administration as γH2AX was detected. Interestingly, OPA1, a protein required for mitochondrial fusion, was highly induced inside the oocyte cytoplasm of p63 knockout, while oocytes in wild-type mice time-dependently lost the OPA1 expression by CPA treatment. This indicates that oocytes without p63 in the nucleus survive and induce mitochondrial fusion to escape apoptosis by mitochondrial damages. In conclusion, CPA induces depletion of oocytes of primordial follicles through the CHK2-TAp63 apoptotic pathway. Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.

Knockdown of Chk1 inhibits proliferation and promotes apoptosis in mouse granulosa cells and its regulation mechanism by miR-15a and miR-16.

Checkpoint kinase 1 (Chk1) is a protein kinase which preserves the genome integrity, and works as an evolutionally conserved DNA damage response and cell cycle checkpoint. However, the functional roles and regulatory mechanism of Chk1 in mouse granulosa cells (GCs) have not been fully elucidated. In this study, by RNA interfering, Chk1 gene was knocked down in GCs. Knockdown of Chk1 inhibited proliferation and increased apoptosis of GCs (p < 0.05), respectively; in addition, cell cycle of GCs was arrested at S and G2/M phases. Further qRT-PCR results showed that cell cycle factors (Cyclin B1 and Cyclin D 1) and a marker gene of proliferation (PCNA) were downregulated (p < 0.001), while apoptotic factors (p53b, p21, caspase-3, and Bax) were upregulated (p < 0.01), which suggested that knockdown of Chk1 may inhibit proliferation, regulate cell cycle, and promote apoptosis at the transcriptional level in GCs. In vitro studies showed a negative correlation between Chk1 mRNA and miR-16 expression during follicular development. To elucidate the relationship between Chk1 and miR-15a/16, luciferase reporter plasmids were constructed and luciferase assays revealed that both miR-15a and miR-16 could bind to the 3' UTR of Chk1 mRNA, and significantly downregulate the protein level of Chk1 (p < 0.01), while miR-16, not miR-15a, could significantly decrease the mRNA level of Chk1 (p < 0.05). This result indicated that miR-16 directly induced Chk1 mRNA destabilization, while miR-15a regulated Chk1 expression through translational repression. Taken together, this study uncovered the roles of Chk1 in mouse granulosa cells and its regulation by miR-15a and miR-16 through different mechanisms.

Benzothiazole and Chromone Derivatives as Potential ATR Kinase Inhibitors and Anticancer Agents.

Despite extensive studies and the great variety of existing anticancer agents, cancer treatment remains an aggravating and challenging problem. Therefore, the development of novel anticancer drugs with a better therapeutic profile and fewer side effects to combat this persistent disease is still necessary. In this study, we report a novel series of benzothiazole and chromone derivatives that were synthesized and evaluated for their anticancer activity as an inhibitor of ATR kinase, a master regulator of the DDR pathway. The cell viability of a set of 25 compounds was performed using MTT assay in HCT116 and HeLa cell lines, involving 72 h incubation of the compounds at a final concentration of 10 µM. Cells incubated with compounds 2c, 7h and 7l were found to show viability ≤50%, and were taken forward for dose–response studies. Among the tested compounds, three of them (2c, 7h and 7l) showed higher potency, with compound 7l exhibiting the best IC50 values in both the cell lines. Compounds 2c and 7l were found to be equally cytotoxic towards both the cell lines, namely, HCT116 and HeLa, while compound 7h showed better cytotoxicity towards HeLa cell line. For these three compounds, an immunoblot assay was carried out in order to analyze the inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells. Compound 7h showed inhibition of pChk1 at Ser 317 in HeLa cells at a concentration of 3.995 µM. Further analysis for Chk1 and pChk1 expression was carried out in Hela cells by treatment against all the three compounds at a range of concentrations of 2, 5 and 10 µM, wherein compound 7h showed Chk1 inhibition at 2 and 5 µM, while pChk1 expression was observed for compound 7l at a concentration of 5 µM. To support the results, the binding interactions of the compounds with the ATR kinase domain was studied through molecular docking, wherein compounds 2c, 7h and 7l showed binding interactions similar to those of Torin2, a known mTOR/ATR inhibitor. Further studies on this set of molecules is in progress for their specificity towards the ATR pathway.

Role of Chk1 gene in molecular classification and prognosis of gastric cancer using immunohistochemistryand LORD-Q assay.

Purpose: The aim of the current study was to assess the prognostic value of theChk1 gene in theDNA damage responsepathway in gastric cancer (GC). Methods: Expression levels of the Chk1 were measured in 220 GC tumor tissues and adjacent healthy/noncancerous tissues using real-time PCR and immunohistochemical staining. Genomic instability in GC patients was measured using the long-run real-time PCR technique for DNA-damage quantificationassay and comet assay. Results: Significantly downregulated expression of Chk1 was observed at themRNA level (p<0.0001) and protein level (p<0.0001). Significantly increased frequency of lesions/10kb and comets was observed in tumor tissues compared with control tissues. Conclusion: The data suggest that downregulated expression of Chk1and positiveHeliobacter pyloriinfection status may haveprognostic significance in GC.