DNA Replication Pathways Research Articles

Comprehensive Insights into the Remarkable Function and Regulatory Mechanism of FluG during Asexual Development in Beauveria bassiana.

Asexual development is the main propagation and transmission mode of Beauveria bassiana and the basis of its pathogenicity. The regulation mechanism of conidiation and the key gene resources for utilization are key links to improving the conidia yield and quality of Beauveria bassiana. Their clarification may promote the industrialization of fungal pesticides. Here, we compared the regulation of morphology, resistance to external stress, virulence, and nutrient utilization capacity between the upstream developmental regulatory gene fluG and the key genes brlA, abaA, and wetA in the central growth and development pathway. The results showed that the ΔbrlA and ΔabaA mutants completely lost the capacity to conidiate and that the ΔwetA mutant had seriously reduced conidiation capacity. Although the deletion of fluG did not reduce the conidiation ability as much as deletions of brlA, abaA, and wetA, it significantly reduced the fungal response to external stress, virulence, and nutrient utilization, while the deletion of the three other genes had little effect. Via transcriptome analysis and screening the yeast nuclear system library, we found that the differentially expressed genes in the ΔfluG mutants were concentrated in the signaling pathways of ABC transporters, propionate metabolism, tryptophan metabolism, DNA replication, mismatch repair, and fatty acid metabolism. FluG directly acted on 40 proteins that were involved in various signaling pathways such as metabolism, oxidative stress, and cell homeostasis. The analysis indicated that the regulatory function of fluG was mainly involved in DNA replication, cell homeostasis, fungal growth and metabolism, and the response to external stress. Our results revealed the biological function of fluG in asexual development and the responses to several environmental stresses as well as its influence on the asexual development regulatory network in B. bassiana.

Overexpression of zinc finger DHHC-type containing 1 is associated with poor prognosis and cancer cell growth and metastasis in uterine corpus endometrial carcinoma.

The zinc finger DHHC-type containing 1 (ZDHHC1) gene is implicated in the pathogenesis and progression of various malignant tumors, but its precise involvement in uterine corpus endometrial carcinoma (UCEC) remains unknown. Thus, this study investigated ZDHHC1 expression in UCEC using publicly available TCGA and Xena databases and elucidated the functions and mechanisms of the ZDHHC1 gene in UCEC progression using bioinformatics and in vitro experiments. The correlation between ZDHHC1 expression and prognosis, clinical features, immune cells, and RNA modifications of UCEC was evaluated using nomograms, correlation, ROC, and survival analyses. The impacts of ZDHHC1 overexpression on UCEC progression and mechanisms were explored with bioinformatics and in vitro experiments. Our study revealed that ZDHHC1 expression was significantly downregulated in UCEC and correlated with poor prognosis, cancer diagnosis, clinical stage, age, weight, body mass index, histological subtypes, residual tumor, tumor grade, and tumor invasion. Notably, Cox regression analysis and constructed nomograms showed that downregulated ZDHHC1 expression was a prognostic factor associated with poor prognosis in patients with UCEC. Conversely, above-normal ZDHHC1 expression inhibited the cell growth, cell cycle transition, migration, and invasion of UCEC cells, which may be related to the cell cycle, DNA replication, PI3K-AKT, and other pathways that promote tumor progression. Altered ZDHHC1 expression in UCEC was significantly associated with RNA modifications and the changes in cancer immune cell populations, such as CD56 bright NK cells, eosinophils, Th2 cells, and cell markers. In conclusion, considerably reduced ZDHHC1 expression in UCEC is associated with cancer cell growth, metastasis, poor prognosis, immune infiltration, and RNA modifications, revealing the promising potential of ZDHHC1 as a prognostic marker for UCEC.

Deciphering the Antibacterial Mechanisms of 5-Fluorouracil in Escherichia coli through Biochemical and Transcriptomic Analyses.

The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU against Escherichia coli was 32-64 µg/mL, including strains carrying blaNDM-5, which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU against E. coli by using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding gene upp increased the MIC of 5-FU against E. coli by 32-fold, indicating the role of the upp gene in 5-FU resistance. Additionally, transcriptomic analysis of E. coli treated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD+/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects on E. coli through multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.

Depletion of histone H3K27 demethylase exerts anti-tumor activity as a monotherapy and in combination therapy with ceralasertib in non-small cell lung carcinoma.

e20035 Background: Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide. Non-small cell lung carcinoma (NSCLC) represents 85% of lung cancers and is the most common type with poor prognosis. The lysine-specific demethylase UTX (gene name KDM6A) demethylates H3K27me2/3 at genes and enhancers and have been shown to support oncogenic transcription factors. We hypothesize that UTX is a highly pleiotropic factor in tumorigenesis and may plays a critical role in controlling DNA replication-associated gene activation as well as in replication stress-related DNA damage repair. Methods: To evaluate the role of UTX in tumorigenesis, shRNA-mediated UTX knockdown was employed in NCI-H1975 cells followed by cell cycle and cell proliferation assays. In the UTX-knocked down NCI-H1975 cells, target genes involved in DNA replication and DNA damage repair were discovered by RNA sequencing and further validated with RT-qPCR. Effects of UTX depletion on DNA replication and radiation-induced DNA repair were analyzed by immunofluorescence. Effects of UTX depletion on cell cycle arrest and the ATR–CHK1 checkpoint signaling pathway were analyzed by flow cytometry and immunoblotting. In vivo response to UTX inhibition monotherapy (UTX knockdown) and combination therapy of UTX knockdown with ceralasertib, a selective ATR kinase inhibitor, was evaluated in dox-inducible UTX knockdown NCI-H1975 xenografts. Results: Induction of UTX knockdown significantly reduced the NCI-H1975 tumor volume compared to the control group, showing anti-tumor activity. Furthermore, loss of UTX induced hyperactivation of ATR–CHK1 checkpoint signaling pathway, suggesting the UTX-depleted tumor cells attempt to alleviate replication stress to escape from apoptosis. To inhibit the UTX-depletion induced ATR–CHK1 pathway, ceralasertib (formerly AZD6738), an oral potent selective inhibitor of ATR, was combined with UTX-knockdown for in vitro and in vivo evaluation. Consistent with in vitro cell-based assay, combination therapy of ceralasertib with UTX depletion showed enhanced tumor growth inhibition and regression in the NCI-H1975 xenograft model. Conclusions: Our findings provide new insights into a pro-oncogenic role of UTX in supporting tumor maintenance via engaging DNA replication and repair pathway. Depletion of UTX alone may serve as a new therapeutic target in non-small cell lung carcinoma; and further combination with ceralasertib enhances the anti-tumor activity of UTX knockdown by inhibiting the ATR–CHK1 checkpoint signaling pathway.

Rosa roxburghii Fruit Extracts Upregulate Telomerase Activity and Ameliorate Cell Replicative Senescence.

Anti-aging functional foods benefit the elderly. Telomeres are chromosomal ends that maintain genome stability extended by telomerase catalytic subunit TERT. Due to the end-replication problem, telomeres shorten after each cell cycle without telomerase in most human cells, and eventually the cell enters the senescence stage. Natural products can attenuate the aging process by increasing telomerase activity, such as TA-65. However, TA-65 is expensive. Other Chinese natural products may achieve comparable effects. Here, we found that Rosa roxburghii fruit extracts effectively increase TERT expression and telomerase activity in cultured human mesenchymal stem cells. Both R. roxburghii fruit extracts obtained by freeze-drying and spray-drying increased the activity of telomerase. R. roxburghii fruit extracts were able to reduce reactive oxygen species levels, enhance superoxide dismutase activity, and reduce DNA damage caused by oxidative stress or radiation. R. roxburghii fruit extracts promoted cell proliferation, improved senescent cell morphology, delayed replicative cellular senescence, attenuated cell cycle suppressors, and alleviated the senescence-associated secretory phenotype. Transcriptome and metabolic profiling revealed that R. roxburghii fruit extracts promote DNA replication and telomere maintenance pathways and decrease triglyceride levels. Overall, we provide a theoretical basis for the application of R. roxburghii fruit as an anti-aging product.

NGEF is a potential prognostic biomarker and could serve as an indicator for immunotherapy and chemotherapy in lung adenocarcinoma.

Neuronal guanine nucleotide exchange factor (NGEF) plays a key role in several cancers; however, its role in lung adenocarcinoma (LUAD) remains unclear. The aim of this study was to evaluate the efficacy of NGEF as a prognostic biomarker and potential therapeutic target for LUAD. NGEF expression data for multiple cancers and LUAD were downloaded from multiple databases. The high- and low-NGEF expression groups were constructed based on median NGEF expression in LUAD samples, and then performed Kaplan-Meier survival analysis. Differentially expressed genes (DEGs) from the two NGEF expression groups were screened and applied to construct a protein-protein interaction network. The primary pathways were obtained using gene set enrichment analysis. The associations between NGEF expression and clinical characteristics, immune infiltration, immune checkpoint inhibitors (ICIs), sensitivity to chemotherapy, and tumor mutation burden (TMB) were investigated using R. Levels of NGEF expression in the lung tissue was validated using single-cell RNA sequencing, quantitative polymerase chain reaction (qPCR), immunohistochemical staining, and western blot analysis. The expression of NGEF mRNA was upregulated in multiple cancers. mRNA and protein expression levels of NGEF were higher in patients with LUAD than in controls, as validated using qPCR and western blot. High NGEF expression was an independent prognostic factor for LUAD and was associated with advanced tumor stage, large tumor size, more lymph node metastasis, and worse overall survival (OS). A total of 182 overlapping DEGs were screened between The Cancer Genome Atlas and GSE31210, among which the top 20 hub genes were identified. NGEF expression was mainly enriched in the pathways of apoptosis, cell cycle, and DNA replication. Moreover, elevated NGEF expression were associated with a high fraction of activated memory CD4+ T cells and M0 macrophages; elevated expression levels of the ICIs: programmed cell death 1 and programmed cell death 1 ligand 1 expression; higher TMB; and better sensitivity to bortezomib, docetaxel, paclitaxel, and parthenolide, but less sensitivity to axitinib and metformin. NGEF expression is upregulated in LUAD and is significantly associated with tumor stages, OS probability, immune infiltration, immunotherapy response, and chemotherapy response. NGEF may be a potential diagnostic and prognostic biomarker and therapeutic target in LUAD.

Transcriptomic characteristics according to tumor size and SUVmax in papillary thyroid cancer patients

The SUVmax is a measure of FDG uptake and is related with tumor aggressiveness in thyroid cancer, however, its association with molecular pathways is unclear. Here, we investigated the relationship between SUVmax and gene expression profiles in 80 papillary thyroid cancer (PTC) patients. We conducted an analysis of DEGs and enriched pathways in relation to SUVmax and tumor size. SUVmax showed a positive correlation with tumor size and correlated with glucose metabolic process. The genes that indicate thyroid differentiation, such as SLC5A5 and TPO, were negatively correlated with SUVmax. Unsupervised analysis revealed that SUVmax positively correlated with DNA replication(r = 0.29, p = 0.009), pyrimidine metabolism(r = 0.50, p < 0.0001) and purine metabolism (r = 0.42, p = 0.0001). Based on subgroups analysis, we identified that PSG5, TFF3, SOX2, SL5A5, SLC5A7, HOXD10, FER1L6, and IFNA1 genes were found to be significantly associated with tumor aggressiveness. Both high SUVmax PTMC and macro-PTC are enriched in pathways of DNA replication and cell cycle, however, gene sets for purine metabolic pathways are enriched only in high SUVmax macro-PTC but not in high SUVmax PTMC. Our findings demonstrate the molecular characteristics of high SUVmax tumor and metabolism involved in tumor growth in differentiated thyroid cancer.

Synthesis and antitumor activities of novel 3-(6-aminopyridin-3-yl)benzamide derivatives: Inducing cell cycle arrest and apoptosis via AURKB transcription inhibition

Here, a series of 3-(6-aminopyridin-3-yl) benzamide derivatives were designed and synthesized. Cell viability assay indicated that most compounds exhibited potent antiproliferative activity against all the tested cancer cells. Among them, compound 7l displayed the best antiproliferative activity particularly in A549 cells, with an IC50 value of 0.04 ± 0.01 μM. RNA-seq analysis was employed to explore the potential pathways related to the antiproliferative activity of compound 7l. The data revealed that 7l exerted antiproliferative activity mainly by regulating cell cycle, DNA replication and p53 signaling pathway. Indeed, compound 7l induced G2/M phase arrest by AURKB transcription inhibition and resulted in cell apoptosis via p53 signaling pathway. Most importantly, compound 7l demonstrated potent antitumor activity in A549 xenograft tumor model. Collectively, 7l might be a promising lead compound for the development of new therapeutic agents for AURKB overexpressed or mutated cancers.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling

BackgroundBone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive.MethodsTo assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients.ResultsIn the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis.ConclusionsBMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Adaptive use of error-prone DNA polymerases provides flexibility in genome replication during tumorigenesis.

Human cells possess many different polymerase enzymes, which collaborate in conducting DNA replication and genome maintenance to ensure faithful duplication of genetic material. Each polymerase performs a specialized role, together providing a balance of accuracy and flexibility to the replication process. Perturbed replication increases the requirement for flexibility to ensure duplication of the entire genome. Flexibility is provided via the use of error-prone polymerases, which maintain the progression of challenged DNA replication at the expense of mutagenesis, an enabling characteristic of cancer. This review describes our recent understanding of mechanisms that alter the usage of polymerases during tumorigenesis and examines the implications of this for cell survival and tumor progression. Although expression levels of polymerases are often misregulated in cancers, this does not necessarily alter polymerase usage since an additional regulatory step may govern the use of these enzymes. We therefore also examine how the regulatory mechanisms of DNA polymerases, such as Rad18-mediated PCNA ubiquitylation, may impact the functionalization of error-prone polymerases to tolerate oncogene-induced replication stress. Crucially, it is becoming increasingly evident that cancer cells utilize error-prone polymerases to sustain ongoing replication in response to oncogenic mutations which inactivate key DNA replication and repair pathways, such as BRCA deficiency. This accelerates mutagenesis and confers chemoresistance, but also presents a dependency that can potentially be exploited by therapeutics.

Mechanistic insights into FEN1-mediated drug sensitivity and risk signature in colon cancer: An integrative bioinformatics study.

The overexpression of Flap endonuclease 1 (FEN1) has been implicated in drug resistance and prognosis across various cancer types. However, the precise role of FEN1 in colon cancer remains to be fully elucidated. In this study, we employed comprehensive datasets from The Cancer Genome Atlas, Gene Expression Omnibus, and Human Protein Atlas to examine FEN1 expression and assess its correlation with clinical pathology and prognosis in colon cancer. We utilized the pRRophetic algorithm to evaluate drug sensitivity and performed differential expression analysis to identify genes associated with FEN1-mediated drug sensitivity. Gene set enrichment analysis was conducted to further investigate these genes. Additionally, single-cell sequencing analysis was employed to explore the relationship between FEN1 expression and functional states. Cox regression analysis was implemented to construct a prognostic model, and a nomogram for prognosis was developed. Our analysis of The Cancer Genome Atlas and Gene Expression Omnibus datasets revealed a significant upregulation of FEN1 in colon cancer. However, while FEN1 expression showed no notable correlation with prognosis, it displayed associations with metastasis. Single-cell sequencing analysis further confirmed a positive correlation between FEN1 expression and colon cancer metastasis. Furthermore, we detected marked discrepancies in drug responsiveness between the High_FEN1 and Low_FEN1 groups, identifying 342 differentially expressed genes. Enrichment analysis showed significant suppression in processes related to DNA replication, spliceosome, and cell cycle pathways in the Low_FEN1 group, while the calcium signaling pathway, cAMP signaling pathway, and other pathways were activated. Of the 197 genes differentially expressed and strongly linked to FEN1 expression, 39 were significantly implicated in colon cancer prognosis. Finally, we constructed a risk signature consisting of 5 genes, which, when combined with drug treatment and pathological staging, significantly improved the prediction of colon cancer prognosis. This study offers novel insights into the interplay among FEN1 expression levels, colon cancer metastatic potential, and sensitivity to therapeutic agents. Furthermore, we successfully developed a multi-gene prognostic risk signature derived from FEN1.

Abstract 140: Distinct pathways regulated by activated β-Catenin and YAP in hepatoblastoma pathogenesis

Abstract Hepatoblastoma (HB) is a rare, yet the most common primary malignant hepatic liver in children. A significantly portion of children with HB have tumors that are not resected at diagnosis, leading to the dismal prognosis in these patients. Concomitant YAP and β-catenin activation could be seen 80% of HB. The signaling pathways regulated by YAP and β-catenin in HB pathogenesis remain unclear. Here, we engineered a inducible HB murine model using hydrodynamic injection to deliver transposon plasmids encoding constitutive YAP and doxycycline (Dox) inducible ΔN90-β-Catenin (Yap/TRE-β-Catenin). When fed with Dox, HB formed rapidly in the mice. Upon Dox withdraw, HB regressed over the long term. We analyzed gene expression patterns in mouse HB lesions upon short term Dox withdrawal, i.e, tumors still existed yet ΔN90-β-Catenin was turned off. This approach assisted to identify genes regulated by the activated β-Catenin in HB tumors. We compared these genes with the previously identified YAP regulated genes and found the distinct gene expression programs regulated by YAP and β-Catenin in HB. Specifically, YAP off down-regulated Hippo pathway and up-regulated metabolism-related pathways, whereas β-Catenin off down-regulated protein export, DNA replication pathway and up-regulated immune-related pathways. Finally, we identified ONECUT1 as a gene down-regulated by activated β-Catenin in HB. ONECUT1 expression was down-regulated in human HBs, and co-expression of ONECUT1 strongly suppressed Yap/β-Catenin HB formation in mice. Mechanically, ONECUT1 could regulate proliferation-related downstream signaling. Altogether our studies demonstrated that suppression of activated CTNNB1 mutations could repress HB progression in vivo, and we identified distinct pathways regulated by activated β-catenin and YAP in HB. Suppression of ONECUT1 expression by β-catenin is a key genetic event leading to HB formation. Citation Format: Weiting Liao, Xin Chen. Distinct pathways regulated by activated β-Catenin and YAP in hepatoblastoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 140.

Abstract 2958: Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype

Abstract Multiple myeloma (MM) is an incurable plasma cell (PC) cancer in which all patients are expected to relapse. Cytotoxic agents such as high-dose melphalan (HDM), which is used as a myeloablative agent prior to autologous stem cell transplant (ASCT) in MM, drive therapy-induced senescence (TIS) in non-cancerous tissues. We hypothesized that HDM similarly activates TIS pathways in surviving MM cells and this response may correlate with patient outcomes post-ASCT. To test this, we developed an in vitro model in which 5TGM1 mouse MM cells are cultured with 10uM melphalan (HDM) or vehicle (Veh) for 6 hours, followed by co-culture in normal media with primary mouse bone marrow stromal cells. Cultures were maintained for 10 days and imaged to quantify cumulative population doublings (CPDs). 5TGM1 were then isolated by FACS to measure telomere-associated DNA damage foci (TAFs), sensitivity to senolytic drugs, and senescence gene expression (RT-qPCR and scRNA-seq). To translate these studies, scRNA-seq was used to analyze CD138+ PCs from MM patient bone marrow post-ASCT. Additionally, longitudinal bone biopsies from MM patients at diagnosis and post-ASCT were stained for CD138+ PCs; PC burden was quantified using artificial intelligence assisted histology (HALO). HDM-5TGM1 exhibited stable growth arrest (decreased CPDs) and sustained DNA damage (increased TAFs) compared to Veh-5TGM1. HDM-5TGM1 were also significantly more sensitive to senolytic ablation (Dasatinib+Quercetin or Fisetin). HDM-5TGM1 showed increased senescence (Cdkn1a, Cdkn1c, Glb1), anti-apoptosis (Bcl2l1), and senescence associated secretory phenotype (Ccl5, Icam1, Mmp13) genes, as well as myeloid markers found to be increased in MM dormancy (Axl, Fcer1g, Mpeg1). Analysis by scRNA-seq revealed that HDM-5TGM1 were G0/G1 arrested, and gene set enrichment analysis (GSEA) confirmed upregulation of senescence pathways with downregulation of DNA replication and cell cycle pathways. Consistent with RT-qPCR results, these cells exhibited myeloid gene expression. Analysis of patient CD138+ PCs post-ASCT by scRNA-seq also revealed a myeloid-like cluster. Of interest, of the 491 genes upregulated in this cluster, 268 were also upregulated in HDM-5TGM1. CD138+ PCs could also be detected in patient bone biopsies post-ASCT, although PC burden was significantly reduced post-ASCT. Surprisingly, the percent reduction in PC burden post-ASCT was greater in relapsed (≤3 years) vs non-relapsed patients. Further, in relapsed patients, PC burden post-ASCT positively correlated with time in remission. Altogether, these findings demonstrate that HDM can induce senescence features and a myeloid-dormancy signature in surviving MM cells. This phenotype is associated with longer durable response, suggesting that activation of TIS may be a strategy to extend PFS. Further, senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent relapse. Citation Format: Angelo Jose Guilatco, Marta Diaz-del-Castillo, Gabriel Alvares Borges, Neal I. Sannuli, Megan L. Ritting, Syed Mohammed Aalam, Nagarajan Kannan, Tamar Tchkonia, James L. Kirkland, Yi Lin, Taxiarchis Kourelis, Matthew T. Drake, Thomas Levin Andersen, Megan Weivoda. Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2958.

Abstract 6014: PLK1-dependent phosphorylation of PRMT5 promotes DNA damage response in prostate cancer

Abstract Prostate cancer stands out as the most frequently diagnosed cancer in men and is projected to be the second leading cause of cancer-related deaths among men in the US. Consequently, there is an urgent need for studies focusing on innovative therapeutic approaches. DNA Damage Response (DDR) is critical for cell survival, as it promotes genomic stability and reduces the risk of inheriting damage. DDR also promotes cancer cells' survival, making it a therapeutic target in cancer. PRMT5, an enzyme in the methyltransferase family, is frequently activated and overexpressed in various cancers, including prostate cancer. Furthermore, research has revealed that PRMT5 has been implicated in DDR in prostate cancer, and the regulation of PRMT5-dependent DDR is influenced by its phosphorylation. Polo-like kinase 1 (PLK1) is a serine/threonine kinase also reportedly involved in DDR in prostate cancer. Our preliminary study found that PRMT5 is positively correlated with PLK1 in prostate cancer based on the TCGA database. Nonetheless, the mechanism through which PRMT5 is regulated needs to be clarified. Here, we found that PLK1 phosphorylates PRMT5 at the S470 site, and this specific phosphorylation is required for maintaining the enzymatic activity of PRMT5. Moreover, Plk1-associated phosphorylation of PRMT5 subsequently promotes DNA damage repair, indicated by the decreasing level of γH2AX after DNA double-strand breaks (DSB). Our RNA-seq analyses of prostate cancer cells with wild-type or mutant S470 sites indicate that Plk1-associated phosphorylation on the S470 site exerts an influence on DNA replication and DDR pathways. These findings shed light on a novel perspective regarding the roles of PLK1-mediated PRMT5 phosphorylation in DDR, offering potential therapeutic strategies in prostate cancer. Citation Format: Jia Peng, Yanquan Zhang, Jianlin Wang, Qianjin Li, Daheng He, Xinyi Wang, Xiongjian Rao, Meng Wu, Pingli Mo, Lei Wang, Sai Wu, Ruixin Wang, Jinghui Liu, Mansoureh Nouri, Izumi Tadahide, Xiaoqi Liu. PLK1-dependent phosphorylation of PRMT5 promotes DNA damage response in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6014.

Comparative Analysis of the Growth, Physiological Responses, and Gene Expression of Chinese Soft-Shelled Turtles Cultured in Different Modes.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture turtle due to its taste and nutritional and medicinal value. More ecological culturing modes, such as rice-turtle co-culture, should be developed to meet the ecological benefit demand. We compared growth, physiological parameters, and transcriptome data to detect the physiological responses and regulatory mechanisms of pond-cultured turtles as compared to co-cultured turtles. The co-cultured turtles grew slower than pond-cultured turtles. The gonadosomatic index of co-cultured male turtles was lower than that of pond-cultured male turtles, and both the mesenteric fat index and limb fat index were lower in co-cultured turtles than in pond-cultured turtles (p < 0.05). The blood GLU of the co-cultured turtles was significantly lower than the GLU of the pond-cultured turtles (p < 0.05), while the values of CRE, UA, BUN, AKP, ACP, GOT, and CAT were higher in the co-cultured turtles than in the pond-cultured turtles (p < 0.05). In total, 246 and 598 differentially expressed genes (DEGs) were identified in the brain and gut from turtles cultured in the two different modes, respectively. More DEGs were related to environmental information processing, metabolism, and human diseases. In the brain, the top enriched pathways of DEGs included the longevity regulating pathway, glycerolipid metabolism, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway, while in the gut, the top enriched pathways of DEGs included the cell cycle, DNA replication, cellular senescence, and p53 signaling pathway. The turtles acclimated to the different culturing conditions by adjusting their growth, physiological, and biochemical characteristics and related gene expression during a short culture period.

Comparative transcriptome combined with physiological analyses revealed key genes and pathways for cadmium tolerance in wild-type and mutant microalgae Dunaliella salina

Cadmium (Cd) has become one of the major contaminants in aquatic ecosystems. The microalgae Dunaliella salina is known as a cadmium (Cd) accumulator and shows a high level of tolerance to Cd, but improved Cd tolerance and relevant mechanisms are still required for investigation. In this study, two mutant strains (M1 and M2) of D. salina with high Cd resistance were obtained by chemical mutagenesis. In the normal medium, there were no differences between the two mutant strains and the wild-type strain in biochemical and physiological indicators. However, the two mutant strains showed high Cd resistance, rapid growth rate, and good Cd accumulation capacity in the medium containing high levels of Cd compared with the wild strain, which may be involved in increases in protein content, accumulation of carotenoids and chlorophylls, and glutathione reductase (GR) activity. We identified 829, 587, and 740 differentially expressed genes (DEGs) from the Cd/Control, Cd + M1/Cd, and Cd + M2/Cd sample groups by transcriptome analysis, respectively. The main DEGs were found in the ribosome, DNA replication, photosynthesis-antenna, and photosynthesis pathways after KEGG enrichment. There were 39 DEGs involved in the photosynthesis and photosynthesis-antenna pathways, of which 28 DEGs were down-regulated by Cd in the wild-type strain but remained relatively unchanged in the mutant strains. Under Cd exposure, the mutant strains showed higher mRNA levels of most DEGs involved in the ribosome and DNA replication pathways than the wild strain. Taken together, the present study will help us understand the mechanism of Cd tolerance in microalgae, and provide candidate genes and key pathways for phytoremediation.

Genome-Wide and Expression Pattern Analysis of the HIT4 Gene Family Uncovers the Involvement of GHHIT4_4 in Response to Verticillium Wilt in Gossypium hirsutum.

Chromatin remodelers are essential for regulating plant growth, development, and responses to environmental stresses. HIT4 (HEAT-INTOLERANT 4) is a novel stress-induced chromatin remodeling factor that has been less studied in abiotic stress and stress resistance, particularly in cotton. In this study, we conducted a comprehensive analysis of the members of the HIT4 gene family in Gossypium hirsutum using bioinformatics methods, including phylogenetic relationships, gene organization, transcription profiles, phylogenetic connections, selection pressure, and stress response. A total of 18 HIT4 genes were identified in four cotton species, with six HIT4 gene members in upland cotton. Based on the evolutionary relationships shown in the phylogenetic tree, the 18 HIT4 protein sequences were classified into four distinct subgroups. Furthermore, we conducted chromosome mapping to determine the genomic locations of these genes and visually represented the structural characteristics of HIT4 in G. hirsutum. In addition, we predicted the regulatory elements in HIT4 in G. hirsutum and conducted an analysis of repetitive sequences and gene collinearity among HIT4 in four cotton species. Moreover, we calculated the Ka/Ks ratio for homologous genes to assess the selection pressure acting on HIT4. Using RNA-seq, we explored the expression patterns of HIT4 genes in G. hirsutum and Gossypium barbadense. Through weighted gene co-expression network analysis (WGCNA), we found that GHHIT4_4 belonged to the MEblue module, which was mainly enriched in pathways such as DNA replication, phagosome, pentose and glucuronate interconversions, steroid biosynthesis, and starch and sucrose metabolism. This module may regulate the mechanism of upland cotton resistance to Verticillium wilt through DNA replication, phagosome, and various metabolic pathways. In addition, we performed heterologous overexpression of GH_D11G0591 (GHHIT4_4) in tobacco, and the results showed a significant reduction in disease index compared to the wild type, with higher expression levels of disease resistance genes in the transgenic tobacco. After conducting a VIGS (virus-induced gene silencing) experiment in cotton, the results indicated that silencing GHHIT4_4 had a significant impact, the resistance to Verticillium wilt weakened, and the internode length of the plants significantly decreased by 30.7% while the number of true leaves increased by 41.5%. qRT-PCR analysis indicated that GHHIT4_4 mainly enhanced cotton resistance to Verticillium wilt by indirectly regulating the PAL, 4CL, and CHI genes. The subcellular localization results revealed that GHHIT4_4 was predominantly distributed in the mitochondria and nucleus. This study offers preliminary evidence for the involvement of the GHHIT4_4 in cotton resistance to Verticillium wilt and lays the foundation for further research on the disease resistance mechanism of this gene in cotton.

MAD2L2 as a novel prognostic biomarker and its correlation with immune infiltrates in glioma

Background: The most common type of central nervous system tumor is glioma, the identification of Glioma biomarkers is essential. MAD2L2, which is abbreviated as MAD2-Like2, collaborates with a number of proteins to carry out numerous essential cellular functions. Uncertain is the role of MAD2L2 in glioma. The role of MAD2L2 expression is examined for the first time in relation to gliomas occurrence and development in this study Methods: A bioinformatics and clinicopathological analysis, immune infiltration analysis, and enrichment analysis were conducted based on TCGA and additional gene expression analysis. Using ssGSEA and TIMER, the immune response to MAD2L2 expression in glioma was analyzed statistically. This study also analyzed the effect of MAD2L2 overexpression on major chemotherapeutic medicines and the methylation status of glioma patients. CGGA, HPA data analysis, and K-M survival were also utilized to validate the results. Results: MAD2L2 was a crucial independent prognostic factor for glioma patients. Correlations were found between MAD2L2 expression and age, IDH status, WHO grades and 1p/19q codeletion. MAD2L2 is intimately connected to the DNA replication, transcription, cell cycle, immune system, and signal transduction pathways, as determined by the GSEA. In glioma, The majority of MAD2L2 DNA methylation sites were hypomethylated, and the degree of methylation was associated with patient outcomes. Chemotherapy would benefit MAD2L2 overexpression patients. according to these results. The expression of MAD2L2 was associated with partial immune cell infiltration and co-expressed with immune-related genes and immune checkpoints. Conclusion: Glioma is characterized by higher MAD2L2 expression, and high MAD2L2 expression is linked with a poor prognosis. MAD2L2 might participate in tumor development by regulating tumor-infiltrating cells inside the TME. MAD2L2 could possibly be an immunotherapeutic target.

Transcriptome analysis of the response of Lactiplantibacillus plantarumIMAU10120 to infection by phage P2

Bacteriophage infection of lactic acid bacteria remains a significant problem in the food fermentation industry. To improve understanding about the underlying mechanism of phage–host infection, we used RNA sequencing technology to investigate the effect of phage infection on the expression of host genes based on the one‐step growth curve. Results identified a total of 3207 genes, of which 178 showed a significant differential expression in phage‐infected host. Moreover, bioinformatic analyses unravelled that these genes regulated many processes in host bacteria, including ribosome, RNA polymerase, DNA replication and metabolic pathways. This study further elucidates the response of host bacteria to phage infection. These results provide valuable data for developing effective phage control measures and improving the success rate of fermentation in the dairy industry.

Abstract A011: DPY30 regulates immunoediting by suppressing uncoordinated DNA replication in pancreatic cancer

Abstract DNA replication is a tightly regulated, multi-step process orchestrated by licensing and replication factors that ensure the faithful duplication of the entire genome exclusively during S phase of the cell cycle. Although the mechanisms preventing uncoordinated DNA replication are well characterized, it remains unclear how cells counteract its potentially harmful effects when it does occur, especially in a context of oncogenic stress. Here, we identify DPY30, an understudied component of all COMPASS-like complexes, as a modulator of DNA replication. We found upregulated DPY30 expression in pancreatic ductal adenocarcinoma (PDAC) as well as a correlation between increasing DPY30 expression level and higher tumor grade and poor clinical outcomes. To investigate the role of DPY30 in DNA replication, we generated and characterized human- and mouse-derived PDAC cells silenced or knockout for DPY30 expression in vivo, within a context of increasing immune pressure. ATAC-Seq and ChIP-Seq were used to inform on the epigenetic landscape of PDAC cells silenced for DPY30 or WDR5 expression. Additionally, we conducted unbiased proteomics and transcriptomics analyses to uncover the underlying molecular mechanisms. The canonical function of COMPASS complexes is to regulate histone 3 lysine 4 (H3K4) methylation. Unexpectedly, DPY30 loss in PDAC cells did not affect H3K4 methylation patterns compared to WDR5 loss, the core member of the COMPASS-like complex. Transcriptomic data unveil a dysregulation of genes belonging to DNA replication and repair pathways in DPY30-silenced PDAC cells. This prompted unbiased proteomics analysis, which showed that DPY30 interacts with members of the DNA helicase, possibly stabilizing the DNA replication fork progression. We found that DPY30 overexpression, by preventing Geminin ubiquitylation, strongly stabilizes its expression, which in turn suppresses Cdt1 activity in late S-phase, buffering the risk of uncoordinated DNA replication in G2/M phase. Consistently, DPY30 loss resulted in DNA re-replication and chromosomal instability, as manifested by DNA fiber and metaphasic spread. In vivo, whole-exome sequencing analysis of DPY30-knockout tumors revealed that DPY30 loss markedly increased mutational burden in immunodeficient mice. In syngeneic models, DPY30 loss strongly inhibited tumor growth, prolonged survival, and increased T cell infiltration of tumors. Moreover, the immune system selectively cleared tumor cells with complex karyotypes, and we observed improved anti-tumor efficacy with DPY30 knockdown and anti-PD-1 treatment compared to either condition alone. In summary, our study has unveiled a previously unrecognized role for DPY30 in stabilizing the replisome machinery by preventing excessive DNA replication. In the context of PDAC, DPY30 sustains genome stability, providing a strong rationale for exploring DPY30 or its effector proteins as potential targets and suggesting that patients whose tumors carry low DPY30 expression might be successfully treated with immune checkpoint inhibitors. Citation Format: Francesca Citron, Luigi Perelli, Chiara Balestrieri, Luca Cecchetto, Yanshuo Chu, I-Lin Ho, Li Zhang, Er-Yen Yen, Luis Castillo Montanez, Rutvi Shah, Sergio Attanasio, Sanjana Srinivasan, Charles Dyke, Ko-Chien Chen, Shan Jiang, Jing Pan, Sisi Gao, Huamin Wang, Wantong Yao, Linghua Wang, Mauro Di Pilato, Giannicola Genovese, Andrea Viale, Giulio Draetta. DPY30 regulates immunoediting by suppressing uncoordinated DNA replication in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A011.