Wee1 Research Articles

Arabidopsis DNA glycosylase MBD4L improves recovery of aged seeds.

DNA glycosylases initiate the base excision repair (BER) pathway by catalyzing the removal of damaged or mismatched bases from DNA. The Arabidopsis DNA glycosylase methyl-CpG-binding domain protein 4 like (MBD4L) is a nuclear enzyme triggering BER in response to the genotoxic agents 5-fluorouracil and 5-bromouracil. To date, the involvement of MBD4L in plant physiological processes has not been analyzed. To address this, we studied the enzyme functions in seeds. We found that imbibition induced the MBD4L gene expression by generating two alternative transcripts, MBD4L.3 and MBD4L.4. Gene activation was stronger in aged than in non-aged seeds. Seeds from mbd4l-1 mutants displayed germination failures when maintained under control or ageing conditions, while 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 seeds reversed these phenotypes. Seed nuclear DNA repair, assessed by comet assays, was exacerbated in an MBD4L-dependent manner at 24 h post-imbibition. Under this condition, the BER genes ARP, APE1L, and LIG1 showed higher expression in 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 than in mbd4l-1 seeds, suggesting that these components could coordinate with MBD4L to repair damaged DNA bases in seeds. Interestingly, the ATM, ATR, BRCA1, RAD51, and WEE1 genes associated with the DNA damage response (DDR) pathway were activated in mbd4l-1, but not in 35S:MBD4L.3/mbd4l-1 or 35S:MBD4L.4/mbd4l-1 seeds. These results indicate that MBD4L is a key enzyme of a BER cascade that operates during seed imbibition, whose deficiency would cause genomic damage detected by DDR, generating a delay or reduction in germination.

Comprehensive profiling of clock genes expression in hepatocellular carcinoma (HCC).

4108 Background: The circadian clock mechanism controls the physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Recent evidence unveiled core clock proteins as novel therapeutic targets in cancer. Our group showed that clock regulators BMAL1 and CLOCK can promote proliferation of liver cancer cells by modulating the cell cycle checkpoint kinase Wee1. Here we further evaluated the molecular landscape of clock pathway alterations in HCC leveraging multi-platform profiling of patient tumor samples. Methods: 780 HCC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Clock gene Score (CS) was determined using expression of core clock genes Z scores (positives of CLOCK, ARNTL, RORA/B/C and negatives of repressors CRY1/2, PER1/2/3, REVERBA/B) stratified by quartiles. xCell was used to quantify cell infiltration in the tumor microenvironment (TME). Significance was determined as P-values and adjusted for multiple testing ( q) of < .05. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy including the T cell inflamed score (TIS) and IFG score. Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for comparison. Results: CS was higher in metastatic sites than primary tumors (median transcripts per million [TPM]: 0.81 vs 0.37, P < .05). No significant differences in patient age and sex were observed between CS Q1 (lowest) and Q4 (highest) cohorts, although a trend towards a higher frequency of males was observed in Q4 (76% vs 68%, Q4 vs Q1, P = .07). CS was positively associated with telomerase subunit TERT mutations (64% vs 52%, Q4 vs Q1, P = .04) and negatively correlated with FGF3 copy number amplification (2% vs 6%, P = .04) and WEE1 gene expression (median TPM: 15 vs 28, q < .05). No dMMR/MSI-H tumors were observed in our series and there were no significant associations with tumor mutational burden and PD-L1 protein expression. Expression of immune related genes was lower in tumors with high CS, including IDO1, CD80, PD-L1, LAG3, CD86, TIM3, PD-1 and PD-L2 (fold change: 0.57-0.67 q < .05). NK cell infiltration in the TME and the TIS score were also significantly lower in CS-high HCC ( q < .05). Individually, lower CLOCK and CRY1 tumor mRNA expression were associated with longer OS (Q1 vs Q4: CLOCK HR 0.71, 95%CI [0.51-0.98], P = .04 and CRY1 HR 0.70 [0.51-0.95], P = .02, respectively). Conclusions: This is the most extensive profiling study to investigate the expression of clock genes in HCC. Our data show that clock genes expression impacts patient survival and is associated with alterations in immune-related gene expression and TIS score which suggest a role in the modulation of anti-tumor immunity. These results support the clock pathway role as a oncogenic driver and its potential as a therapeutic target in HCC.

Iron accumulation and partitioning in hydroponically grown wild and cultivated chickpea (Cicer arietinum L).

Chickpea (Cicer arietinum L.) is a staple food in many developing countries where iron (Fe) deficiency often occurs in their population. The crop is a good source of protein, vitamins, and micronutrients. Fe biofortification in chickpea can be part of long-term strategy to enhance Fe intake in human diet to help to alleviate Fe deficiency. To develop cultivars with high Fe concentration in seeds, understanding the mechanisms of absorption and translocation of Fe into the seeds is critical. An experiment was conducted using a hydroponic system to evaluate Fe accumulation in seeds and other organs at different growth stages of selected genotypes of cultivated and wild relatives of chickpea. Plants were grown in media with Fe zero and Fe added conditions. Six chickpea genotypes were grown and harvested at six different growth stages: V3, V10, R2, R5, R6, and RH for analysis of Fe concentration in roots, stems, leaves, and seeds. The relative expression of genes related to Fe-metabolism including FRO2, IRT1, NRAMP3, V1T1, YSL1, FER3, GCN2, and WEE1 was analyzed. The results showed that the highest and lowest accumulation of Fe throughout the plant growth stages were found in the roots and stems, respectively. Results of gene expression analysis confirmed that the FRO2 and IRT1 were involved in Fe uptake in chickpeas and expressed more in roots under Fe added condition. All transporter genes: NRAMP3, V1T1, YSL1 along with storage gene FER3 showed higher expression in leaves. In contrast, candidate gene WEE1 for Fe metabolism expressed more in roots under Fe affluent condition; however, GCN2 showed over-expression in roots under Fe zero condition. Current finding will contribute to better understanding of Fe translocation and metabolism in chickpea. This knowledge can further be used to develop chickpea varieties with high Fe in seeds.

Abstract 1132: A systems biology approach combining ProLiFiler and Cancer Data Miner for an enhanced preclinical characterization of the WEE-1 inhibitor Adavosertib

Abstract Adavosertib (AZD1775, MK-1775), a clinical stage inhibitor of the tyrosine kinase WEE1, was investigated in a cell proliferation and survival assay with 140 human cancer cell lines (CLs) representing all major tumor types (ProLiFiler platform of Reaction Biology) followed by mechanism of action (MoA) and biomarker analyses using 4HF Biotec’s Cancer Data Miner in silico platform. Adavosertib exhibited a broad anti-cancer activity across all hematological and solid tumor types with IC50 values ranging from 0.06 to 10 µM (median: 0.38 µM), matching the consistently high expression of the WEE1 gene. Among 900 reference compounds, the activity profile of adavosertib correlated best with the profiles of compounds targeting the replication stress response including other WEE1 inhibitors but also inhibitors of checkpoint kinase 1 and 2 (CHK1/2) or ataxia telangiectasia-mutated (ATM). Significant correlations were also seen with compounds blocking mitosis, DNA replication and DNA repair. Interestingly, we observed a subset of cell lines that were resistant to both DNA synthesis and PARP inhibitors but were sensitive to WEE1 inhibition. Moreover, by using multiple datasets of WEE1 inhibitors connected to the molecular annotations of CLs for a data driven biomarker screening, we identified MYC mutations as a predictive marker of sensitivity and PIK3CA or ERBB2 gene amplifications as predictors of resistance. Transcriptome analysis identified up to 900 genes for which higher expression in CLs was associated with CL sensitivity to the compound. Preliminary pathway analysis indicated that these genes are well represented among nuclear factor and Myc-regulated genes. In conclusion, our studies demonstrate broad anticancer activity of adavosertib and confirm its proposed MoA. The biomarkers we identified will facilitate the selection of pre-clinical in vivo tumor models and, if confirmed, even patient selection for clinical trials. The combined use of the ProLiFiler and Cancer Data Miner Platforms has the potential to accelerate and de-risk the development of anti-cancer agents. Citation Format: Vincent Vuaroqueaux, Daniel Feger, Anne-Lise Peille, Oliver Siedentopf, Sadhana Panzade, Sarah Ulrich, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. A systems biology approach combining ProLiFiler and Cancer Data Miner for an enhanced preclinical characterization of the WEE-1 inhibitor Adavosertib [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 1132.

IDENTIFICATION OF WEE GENES IN DICTYOSTELIUM DISCOIDEUM

Cell cycle is a basic cellular mechanism that allows for cellular growth and development, regeneration or repair, and wear of tissues or even regeneration of the whole organism in certain cases. A failure during the cell cycle process leads to various dire consequences. Various proteins and kinases play a vital role in the regulation of the cell cycle to meet cellular demands and needs. Amongst the kinases, Wee1 is a key regulator of cell cycle progression which inhibits mitotic entry by phosphorylating the tyrosine residue on the M-phase-inducing kinase, Cdk1, at the G2/M transition. In Dictyostelium discoideum, wee1 genes that regulate the cell cycle progression at the G2/M transition have not been identified and studied yet. Our findings support the existence of the Wee1 homolog in D. discoideum, which could play a role in cell division at G2/M.

Arthrospira (Spirulina) platensis feeding reduces the early stage of chemically induced rat colon carcinogenesis.

Colorectal cancer is the third most diagnosed cancer worldwide and linked to dietary/lifestyle factors. Arthrospira (Spirulina) platensis (AP) contains bioactive compounds with beneficial effects in vivo/in vitro. We evaluated the effects of AP feeding against 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Male Sprague Dawley rats were given subcutaneous injections of DMH (4 × 40 mg/kg body weight) (G1-G3) or vehicle (G4-G5) twice a week (weeks 3-4). During weeks 1-4, animals were fed a diet containing 1 % (G2) or 2 % (G3-G4) AP powder (w/w). After this period, all groups received a balanced diet until week 12. Some animals were euthanised after the last DMH injection (week 4) for histological, immunohistochemical (Ki-67, γ-H2AX and caspase-3) and molecular analyses (real time-PCR for 91 genes), while other animals were euthanised at week 12 for preneoplastic aberrant crypt foci (ACF) analysis. Both AP treatments (G2-G3) significantly decreased the DMH-induced increase in γ-H2AX (DNA damage) and caspase 3 (DNA damage-induced cell death) in colonic crypts at week 4. In addition, Cyp2e1 (Drug metabolism), Notch1, Notch2 and Jag1 genes (Notch pathway) and Atm, Wee1, Chek2, Mgmt, Ogg1 and Xrcc6 genes (DNA repair) were also down-regulated by 2 % AP feeding (G3) at week 4. A significant reduction in ACF development was observed in both AP-treated groups (G2-G3) at week 12. In conclusion, findings indicate that AP feeding reduced acute colonic damage after DMH, resulting in fewer preneoplastic lesions. Our study provided mechanistic insights on dietary AP-preventive effects against early colon carcinogenesis.

CLL-393: Knockdown of WEE1 Gene Induces Cytotoxic Effects in Hairy Cell Leukemia

Background: Hairy cell leukemia (HCL) is a rare B-cell lymphoid neoplasm with morphological variations that can be difficult to distinguish. A BRAFV600E mutation is found in 80% of patients. Despite the fact that purine analogs (PNA) with or without anti-CD20 antibodies remain the first-line treatment, resistance is still a major issue that has a severe impact on patient outcomes. The successful therapeutic application of short interfering RNA (siRNA) in tumor suppression is being paved by gene silencing. In the cell cycle, the WEE1 gene is the master inhibitor of cyclin-dependent kinase 1. Cumulative data suggests that the WEE1 gene plays a definite function in cell biological processes and that silencing the WEE1 gene could have a variety of implications in cancer cells. Objectives: The purpose of this study was to examine how ex vivo knockdown of the WEE1 gene affects the viability of hairy cell leukemia (HCL) cells in order to see if it has therapeutic potential. Methods: The HCL cell line [Mo T] was used in an experimental in vi’ro investigation. To silence the WEE1 gene ex vivo, cells were transfected using siRNA. Furthermore, the effect of gene knockdown on cell viability was assessed using the MTT test, as well as cell cycle and apoptosis assays performed by flow cytometry. Furthermore, the WEE1 gene expression level in silenced cells was measured and correlated with cellular protein expression. Results: Silencing the WEE1 gene resulted in a significant decrease in cell viability, increased cell apoptosis, and arrested the cell cycle transition from the G1 to the S phase of the cell cycle. In addition, transfected cells showed a significant reduction in WEE1 gene expression, demonstrating the efficiency of gene silencing. Conclusion: In primary hairy cells, silencing the WEE1 gene resulted in significant cytotoxic effects, suggesting that knockdown of the WEE1 gene could be a promising therapeutic target in HCL. Hairy cell leukemia (HCL) is a rare B-cell lymphoid neoplasm with morphological variations that can be difficult to distinguish. A BRAFV600E mutation is found in 80% of patients. Despite the fact that purine analogs (PNA) with or without anti-CD20 antibodies remain the first-line treatment, resistance is still a major issue that has a severe impact on patient outcomes. The successful therapeutic application of short interfering RNA (siRNA) in tumor suppression is being paved by gene silencing. In the cell cycle, the WEE1 gene is the master inhibitor of cyclin-dependent kinase 1. Cumulative data suggests that the WEE1 gene plays a definite function in cell biological processes and that silencing the WEE1 gene could have a variety of implications in cancer cells. The purpose of this study was to examine how ex vivo knockdown of the WEE1 gene affects the viability of hairy cell leukemia (HCL) cells in order to see if it has therapeutic potential. The HCL cell line [Mo T] was used in an experimental in vi’ro investigation. To silence the WEE1 gene ex vivo, cells were transfected using siRNA. Furthermore, the effect of gene knockdown on cell viability was assessed using the MTT test, as well as cell cycle and apoptosis assays performed by flow cytometry. Furthermore, the WEE1 gene expression level in silenced cells was measured and correlated with cellular protein expression. Silencing the WEE1 gene resulted in a significant decrease in cell viability, increased cell apoptosis, and arrested the cell cycle transition from the G1 to the S phase of the cell cycle. In addition, transfected cells showed a significant reduction in WEE1 gene expression, demonstrating the efficiency of gene silencing. In primary hairy cells, silencing the WEE1 gene resulted in significant cytotoxic effects, suggesting that knockdown of the WEE1 gene could be a promising therapeutic target in HCL.

Treatment of Melanoma by Nano-conjugate-Delivered Wee1 siRNA.

Small interfering RNA (siRNA)-based drugs have shown tremendous potential to date in cancer gene therapy. Despite the considerable efforts in siRNA design and manufacturing, unsatisfactory delivery systems persist as a limitation for the application of siRNA-based drugs. In this work, the cholesterol, cell-penetrating peptide conjugate cRGD (R8-cRGD), and polyethylene glycol (PEG) were introduced into low-molecular-weight polyethyleneimine (LMW PEI) to form cRGD-R9-cholesterol-PEI-PEG (RRCPP) nanoparticles with specific targeting and highly penetrating abilities. The enhanced siRNA uptake efficiency of the RRCPP delivery system benefited from R8-cRGD modification. Wee1 is an oncogenic nuclear kinase that can regulate the cell cycle as a crucial G2/M checkpoint. Overexpression of Wee1 in melanoma may lead to a poor prognosis. In the present study, RRCPP nanoparticles were designed for Wee1 siRNA delivery to form an RRCPP/siWee1 complex, which significantly silenced the expression of the WEE1 gene (>60% inhibition) and induced B16 tumor cell apoptosis by abrogating the G2M checkpoint and DNA damage in vitro. Furthermore, the RRCPP/siWee1 complex suppressed B16 tumor growth in a subcutaneous xenograft model (nearly 85% inhibition rate) and lung metastasis (nearly 66% inhibition rate) with ideal in vivo safety. Briefly, our results support the validity of RRCPP as a potential Wee1 siRNA carrier for melanoma gene therapy.

Combined Inhibition of Chk1 and Wee1 As a New Therapeutic Strategy for High-Risk Multiple Myeloma Patients

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite introduction of novels agents that have significantly improved clinical outcomes, most patients relapse and develop drug resistance. Therefore, novel therapeutic strategies to overcome chemotherapy resistance are needed in clinical settings.MM is characterized by genomic instability and high level of replicative stress that arise during the disease's pathogenesis. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways that include the kinases ATR, Chk1 and Wee1. Chk1 is activated by ATR after DNA damage or replicative stress and promote cell cycle arrest through the regulation of S and G2 checkpoints. Wee1 kinase plays a central role in the proper timing of cell division cycle controlling entry into mitosis and DNA replication during S phase. Interestingly, experimental studies have identified that Chk1 inhibitors are strongly synergistic with Wee1 inhibitors in various cancer models. Furthermore, Chk1 and Wee1 inhibitors are currently under clinical investigation.Here, we investigated the therapeutic interest of Chk1 and Wee1 in MM.First we investigated the prognostic value of Chk1 and/or Wee1 gene expression in MM patients. Both Chk1 and Wee1 were significantly overexpressed in human myeloma cell lines (HMCLs, n= 40) compared to bone marrow plasma cells (BMPCs, n=5) (P<001 and P<001 respectively). Chk1 and Wee1 expression were significantly higher in the poor prognosis “proliferation” MM subgroup (P<001 and P<001 respectively).High Chk1 expression and high Wee1 expression could predict for shorter overall survival (OS) in 4 independent cohorts of newly-diagnosed patients (P<0.001 and P=0.007 in the HM cohort (N=206), P<0.001 and P=0.008 in UAMS-TT2 cohort (N=345), P<0.001 and P<0.001 in Hovon cohort (N=282) and P=0.002 and P<0.001 in UAMS-TT3 cohort (N=186)). Same results were obtained for event free survival (EFS). Moreover, Chk1 and Wee1 high expression are associated with a poor prognosis in a cohort of 242 patients treated by Bortezomib monotherapy (Mulligan Cohort, P<0.001 and P=0.001 respectively).Interestingly, combined high expression of Chk1 and Wee1 are associated to significantly shorter survival compared to high expression of Chk1 or Wee1 alone in the 4 independent cohorts investigated. Same results were obtained for event free survival (EFS).According, to these results, we investigated the therapeutic interest of Chk1 and Wee1 inhibitors (AZD7762 and MK-1775) alone and in combination in MM.Chk1 inhibitor (AZD7762) induced a dose dependent cell growth inhibition in 13 HMCLs with a median IC50 of 179nM (range 69-366nM). A correlation between HMCLs sensitivity and P53 mutation was observed (r =0.7, P<0.01).Wee1 inhibitor (MK-1775) induced also a dose dependent inhibition of cell growth in all investigated HMCLs (n=10) with a median of IC50 of 450nM (range 100-800nM).Combination treatment of Wee1 inhibitor IC20 with various doses of Chk1 inhibitor was performed in 4 HMCLs. We identified a high synergy (combination index range 0.4-0.7) associated with a significant and remarkable reduction of Chk1 inhibitor (AZD7762) IC50 (198nM to 20nM for XG6; 166nM to 25nM for AMO1; 386nM to 18nM for XG7 and 163nM to 21nM for OPM2) in all the tested HMCLs. Chk1 and Wee1 inhibitors combination demonstrated a significant increase in apoptosis induction, proliferation inhibition and DNA damage induction compared to Chk1i or Wee1i alone in 3 HMCLs.We investigated the effects of Chk1i, Wee1i and Chk1i/Wee1i combination on primary MM cells of patients (n = 5) co-cultured with their bone marrow microenvironment. After 4 days of treatment, cells were enumerated and the fraction of viable myeloma cells (CD138+) and non-myeloma cells (CD138-) were determined by flow cytometry. Chk1i/Wee1i combination significantly and specifically induced a higher toxicity on myeloma cells of patients compared to Chk1i or Wee1i alone. This toxicity is specific of MM cells since normal bone marrow cells were significantly less affected by the combination treatment (P < 0.01).Taken together, these data suggest that association of Chk1i and Wee1i may represent a promising therapeutic approach in high-risk MM patients characterized by high Chk1 and Wee1 expression. DisclosuresBruyer:Diag2tec: Employment. Martin:Diag2tec: Employment. Cartron:Celgene: Consultancy, Employment; Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:EngMab: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding.

Abstract 23: Precision medicine approaches to Fanconi anemia oral cancer personalized prevention and treatment

Abstract There is an increased risk of oral cavity and other head and neck cancers as Fanconi patients live longer post transplantation. DNA repair defects in Fanconi patients contribute to “field effect” in this population, which causes frequent recurrences and often fatal, second primary oral malignancies. These developments show a need for translation of new basic science concepts for this patient population. We are examining the effects of both Metformin and Pioglitazone, and Polo kinase and Wee Kinase inhibitors in Fanconi anemia oral cancer cell lines, and leukoplakia control cells. We employed 3 FA oral cancer cell lines for the current study. We performed clonogenic and cell proliferation (MTT) experiments in replicates and repeated those experiments to establish initial dose responses of the inhibitors at biologically relevant concentrations. We used ANOVA analysis with a P&amp;lt;0.05 as our level of significance. Concurrently, we are examining pioglitazone-metformin signaling pathways in biopsies from precancerous oral lesion (non-FA) patients treated with pioglitazone in our multiple chemoprevention clinical trials via confocal fluorescence microscopy. We found both the Wee Kinase inhibitor and POLO kinase inhibitor resulted in dose dependent decreases in proliferation and clonogenic potential in several experiments in all cell lines. We observed decreased proliferation at 72 hours at clinically achievable serum levels of 75%. However, we also observed cytotoxic effects as well, as judged by MTT reading at 72 hours being lower than the original Day 0 MTT readings. Pioglitazone treatment also resulted in dose dependent decreases in cell proliferation and clonogenic potential at clinically achievable serum levels. Similar effects can be achieved with metformin, however, at higher doses. Additionally, we have identified surrogate endpoints as suitable pharmacodynamic readouts by IHC analysis of the cell lines. There are precision medicine treatments for FA-associated oral carcinoma and these are feasible, based on earlier promising results we present here. We are attempting to discover new treatment methodologies for these patients, as conventional agents and radiation therapy are often unacceptable, due to the DNA fragility of the patient population. In combination, these efforts support our future goals of chemoprevention in this underserved prevention population. Citation Format: Daniel K. Swenson, Beverly R. Wuertz, Mustafa M. Ali, Gretchen M. Unger, Frank G. Ondrey. Precision medicine approaches to Fanconi anemia oral cancer personalized prevention and treatment [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 23.

Knockdown of long non-coding RNA VIM-AS1 inhibits glioma cell proliferation and migration, and increases the cell apoptosis via modulation of WEE1 targeted by miR-105-5p.

Glioma including glioblastoma is the main type of primary brain tumors worldwide. LncRNAs have participated in glioma formation. This study aims to investigate the underlying mechanism for VIM-AS1/miR-105-5p/WEE1 signaling in glioma. The clinical tumors and adjacent tissues were collected from 24 patients with glioma in the Shang Luo Central Hospital. Then, the clinical samples were subjected to hematoxylin-eosin staining (H&E). VIM-AS1, miR-105-5p, and WEE1 levels were measured using real-time PCR. The protein levels of WEE1, Cyclin A1, PCNA, N-cadherin, Vimentin, and Bcl-2, E-cadherin, and Bax were analyzed using Western blot. The overall survival of glioma patients was evaluated using the Kaplan-Meier analysis. The interaction between VIM-AS1 and miR-105-5p was determined using RIP assay and Dual-Luciferase reporter assay, and the binding between miR-105-5p and WEE1 was also detected by Dual-Luciferase reporter assay. Cell proliferation, colony formation, cell cycle, apoptosis, and migration were confirmed using CCK-8, colony formation assay, flow cytometry, and transwell assay, respectively. VIM-AS1 was elevated in cancer tissues, and high level of VIM-AS1 was positively correlated with poor overall survival. Then, VIM-AS1 could bind to and downregulate miR-105-5p. Furthermore, the knockdown of VIM-AS1 significantly suppressed tumor growth in vivo. The knockdown of VIM-AS1/overexpression of miR-105-5p inhibited glioma cell growth, colony formation, and migration, and enhanced the cell apoptosis by inhibiting expression of Cyclin A1, PCNA, Vimentin, N-cadherin, and Bcl-2, and by increasing the expression of Bax and E-cadherin. Interestingly, the overexpression of VIM-AS1 reversed the tumor-suppressing role of miR-105-5p in glioma cells. Besides, the expression of WEE1 was synergistically regulated by VIM-AS1 and miR-105-5p. Consequently, VIM-AS1 promoted glioma progression via upregulating WEE1 or downregulating miR-105-5p. VIM-AS1/miR-105-5p/WEE1 signaling may be a promising target for glioma treatment.

Cell cycle period control through modulation of clock inputs.

In this work, we study period control of the mammalian cell cycle via coupling with the cellular clock. For this, we make use of the oscillators' synchronization dynamics and investigate methods of slowing down the cell cycle with the use of clock inputs. Clock control of the cell cycle is well established via identified molecular mechanisms, such as the CLOCK:BMAL1-mediated induction of the wee1 gene, resulting in the WEE1 kinase that represses the active form of mitosis promoting factor (MPF), the essential cell cycle component. To investigate the coupling dynamics of these systems, we use previously developed models of the clock and cell cycle oscillators and center our studies on unidirectional clock [Formula: see text] cell cycle coupling. Moreover, we propose an hypothesis of a Growth Factor (GF)-responsive clock, involving a pathway of the non-essential cell cycle complex cyclin D/CDK4. We observe a variety of rational ratios of clock to cell cycle period, such as: 1:1, 3:2, 4:3, and 5:4. Finally, our protocols of period control are successful in effectively slowing down the cell cycle by the use of clock modulating inputs, some of which correspond to existing drugs.

Pinostrobin suppresses the Ca2+-signal-dependent growth arrest in yeast by inhibiting the Swe1-mediated G2 cell-cycle regulation.

Pinostrobin, a flavonoid compound known for its diverse pharmacological actions, including anti-leukemic and anti-inflammatory activities, has been repeatedly isolated by various screenings, but its action mechanism is still obscure. Previously, pinostrobin was rediscovered in our laboratory using a yeast-based assay procedure devised specifically for the inhibitory effect on the activated Ca2+ signaling that leads the cells to severe growth retardation in the G2 phase. Here, we attempted to identify target of pinostrobin employing the genetic techniques available in the yeast. Using various genetically engineered yeast strains in which the Ca2+-signaling cascade can be activated by the controlled expression of the various signaling molecules of the cascade, its target was narrowed down to Swe1, the cell-cycle regulatory protein kinase. The Swe1 kinase is situated at the downstream of the Ca2+-signaling cascade and downregulates the Cdc28/Clb complex by phosphorylating the Cdc28 moiety of the complex in the G2 phase. We further demonstrated that pinostrobin inhibits the protein kinase activity of Swe1 in vivo as estimated by the decreased level of Cdc28 phosphorylation at Tyr-19. Since the yeast SWE1 gene is an ortholog for the human WEE1 gene, our finding implied a potentiality of pinostrobin as the G2 checkpoint abrogator in cancer chemotherapy.

Five Genes Associated With Survival in Patients With Lower-grade Gliomas Were Identified by Information-theoretical Analysis.

Understanding of the molecular events associated with progression and survival differences in patients with lower-grade gliomas (LGGs) is still unclear. The comparison of findings across studies using different datasets and methods is essential for a new molecular-based classification system. The aim of the study was to identify biomarkers for prognostic classification of patients with LGGs, and furthermore to lay a foundation for future development of targeted therapies for LGGs. Using information-theoretic and statistical approaches, we analyzed mRNA expression data for 18,413 genes from LGG samples in order to identify candidate biomarkers for survival. The candidate genes were then evaluated for their potential as prognostic biomarkers using multivariable Cox regression analyses that adjusted for the effects of age and grade. WEE1, EMP3, E2F7, CD58 and NSUN7 genes were identified as candidate biomarkers of LGGs and their high expression was associated with significantly shorter survival. The hazard ratios for mortality were 5.02 (95% CI=3.40-7.40) for WEE1, 5.45 (95% CI=3.63-8.18) for EMP3, 4.49 (95% CI=3.03-6.66) for E2F7, 4.77 (95% CI=3.22-7.06) for CD58 and 4.38 (95% CI=2.97-6.47) for NSUN7. In addition, the expression pattern of these genes, associated with shorter survival in LGGs, was also observed in glioblastoma multiforme. Identification of genes associated with poor outcomes will provide insights into novel biological mechanisms that may lead to improvement in progression and survival for patients with LGGs.

Targeting replication stress response using polypurine reverse hoogsteen hairpins directed against WEE1 and CHK1 genes in human cancer cells

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.

Fruit Architecture in Polyamine-Rich Tomato Germplasm Is Determined via a Medley of Cell Cycle, Cell Expansion, and Fruit Shape Genes.

Shape and size are important features of fruits. Studies using tomatoes expressing yeast Spermidine Synthase under either a constitutive or a fruit-ripening promoter showed obovoid fruit phenotype compared to spherical fruit in controls, suggesting that polyamines (PAs) have a role in fruit shape. The obovoid fruit pericarp exhibited decreased cell layers and pericarp thickness compared to wild-type fruit. Transgenic floral buds and ovaries accumulated higher levels of free PAs, with the bound form of PAs being predominant. Transcripts of the fruit shape genes, SUN1 and OVATE, and those of CDKB2, CYCB2, KRP1 and WEE1 genes increased significantly in the transgenic ovaries 2 and 5 days after pollination (DAP). The levels of cell expansion genes CCS52A/B increased at 10 and 20 DAP in the transgenic fruits and exhibited negative correlation with free or bound forms of PAs. In addition, the cell layers and pericarp thickness of the transgenic fruits were inversely associated with free or bound PAs in 10 and 20 DAP transgenic ovaries. Collectively, these results provide evidence for a linkage between PA homeostasis and expression patterns of fruit shape, cell division, and cell expansion genes during early fruit development, and suggest role(s) of PAs in tomato fruit architecture.

Cloning, transformation and expression of cell cycle-associated protein kinase OsWee1 in indica rice (Oryza sativa L.).

The development process of seed in plants is a cycle of cells which occur gradually and regularly. One of the genes involved in controling this stage is the Wee1 gene. Wee1 encode protein kinase which plays an important role in phosphorylation, inactivation of cyclin-dependent kinase 1 (CDK1)-cyclin (CYC) and inhibiting cell division at mitotic phase. The Overexpression of Wee1 leads to delaying entry into mitotic phase, resulting in enlargement of cell size due to suppression of cell division. Accordingly, the cloning and overexpressing of Wee1 in rice plant is important aim of this research in achieving better quantity and quality of future rice. The main objective of this present study is to cloning and generate transgenic rice plants overexpressing of Wee1 gene. Wee1 was isolated from cDNA of indica rice (Oryza sativa), called OsWee1. The full length of OsWee1 was 1239 bp in size and successfully inserted into plant expression vector pRI101ON. Seven-day-old rice seedlings were prepared for transformation of OsWee1 gene using Agrobacterium-mediated transformation method. Four positive transgenic lines were identified through the presence of kanamycin resistance gene (nptII) using genomic PCR analysis. Southern blot analysis result provides evidence that four independent rice transformants contained one to three rearranged transgene copies. Further screening in transgenic rice generation is needed in order to obtain stable expression of OsWee1.

Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts.

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was over-expressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

Molecular biology progress of small cell lung cancer

Small cell lung cancer (SCLC) is typified by early recurrence and metastasis associated with many genetic changes. The drug ROVA-T composed of the DLL3 antibody Rovalpituzumab and the cytoto-xic drug Tesirine achieves the tumor-killing effect by releasing the Tesirine when bound to DLL3. Nfib promotes the SCLC metastasis by altering the structure of the chromosome. The PRAP, EZH2 and Wee1 inhibitors inhibit the DNA damage repair to improve the antitumor activity of chemotherapeutic drugs. Combination of Ipilimumab and Nivolumab can activate the human immune system to exert antitumor effect. Key words: Carcinoma, small cell; Immunotherapy; Targeted therapy

Regulation of G2/M Transition by Inhibition of WEE1 and PKMYT1 Kinases.

In the cell cycle, there are two checkpoint arrests that allow cells to repair damaged DNA in order to maintain genomic integrity. Many cancer cells have defective G1 checkpoint mechanisms, thus depending on the G2 checkpoint far more than normal cells. G2 checkpoint abrogation is therefore a promising concept to preferably damage cancerous cells over normal cells. The main factor influencing the decision to enter mitosis is a complex composed of Cdk1 and cyclin B. Cdk1/CycB is regulated by various feedback mechanisms, in particular inhibitory phosphorylations at Thr14 and Tyr15 of Cdk1. In fact, Cdk1/CycB activity is restricted by the balance between WEE family kinases and Cdc25 phosphatases. The WEE kinase family consists of three proteins: WEE1, PKMYT1, and the less important WEE1B. WEE1 exclusively mediates phosphorylation at Tyr15, whereas PKMYT1 is dual-specific for Tyr15 as well as Thr14. Inhibition by a small molecule inhibitor is therefore proposed to be a promising option since WEE kinases bind Cdk1, altering equilibria and thus affecting G2/M transition.