DNA Replication Process Research Articles

Expression of MCMs in Endometrial Cancer and its Biological Correlation Analysis.

Minichromosome maintenance (MCM) has been demonstrated to be involved in tumorigenesis and pathogenesis of many cancer types. However, the role of MCMs in endometrial cancer (EC) has not been elucidated. We first employed GEPIA, cBioPortal, and R software to perform the differential expression analysis, survival analysis, and gene alteration analysis of the MCMs family. Then, GSE17025 and GSE63678 datasets and CTPAC were used to verify the mRNA and protein expression levels of MCM4. In addition, the internal mechanism of the MCM4 was investigated by comparing MCM4 expression-correlated differentially expressed genes (DEGs) from GEPIA and MCM4-interacted genes from STRING. Last, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify MCM4-related biological processes. Compared with normal tissues, only MCM2 and MCM4 expression were significantly upregulated in EC tissues. High expression of MCM4 was related to worse clinicopathological features and poor prognosis in EC cohorts. Additionally, a certain degree of gene alterations in the MCM2-7 gene was observed. By comparing MCM4 expression-correlated DEGs and MCM4- interacted genes, six genes were obtained: SSRP1, ORC1, GINS1, CDK2, DBF4 and GINS3. Functional enrichment analysis suggested that MCM4 may be involved in regulating the biological processes of DNA replication and the p53 signaling pathway. This was the first comprehensive study to disclose the biological effects of MCMs in EC, indicating that MCM4 could be used as a new prognostic biomarker and potential therapeutic target for EC.

Transcriptome Analysis Reveals That C17 Mycosubtilin Antagonizes Verticillium dahliae by Interfering with Multiple Functional Pathways of Fungi.

Verticillium wilt is a kind of soil-borne plant fungal disease caused by Verticillium dahliae (Vd). Vd 991 is a strong pathogen causing cotton Verticillium wilt. Previously, we isolated a compound from the secondary metabolites of Bacillus subtilis J15 (BS J15), which showed a significant control effect on cotton Verticillium wilt and was identified as C17 mycosubtilin. However, the specific fungistatic mechanism by which C17 mycosubtilin antagonizes Vd 991 is not clear. Here, we first showed that C17 mycosubtilin inhibits the growth of Vd 991 and affects germination of spores at the minimum inhibitory concentration (MIC). Morphological observation showed that C17 mycosubtilin treatment caused shrinking, sinking, and even damage to spores; the hyphae became twisted and rough, the surface was sunken, and the contents were unevenly distributed, resulting in thinning and damage to the cell membrane and cell wall and swelling of mitochondria of fungi. Flow cytometry analysis with ANNEXINV-FITC/PI staining showed that C17 mycosubtilin induces necrosis of Vd 991 cells in a time-dependent manner. Differential transcription analysis showed that C17 mycosubtilin at a semi-inhibitory concentration (IC50) treated Vd 991 for 2 and 6 h and inhibited fungal growth mainly by destroying synthesis of the fungal cell membrane and cell wall, inhibiting its DNA replication and transcriptional translation process, blocking its cell cycle, destroying fungal energy and substance metabolism, and disrupting the redox process of fungi. These results directly showed the mechanism by which C17 mycosubtilin antagonizes Vd 991, providing clues for the mechanism of action of lipopeptides and useful information for development of more effective antimicrobials.

Prospects of POLD1 in Human Cancers: A Review.

Cancer is the second leading cause of death globally, exceeded only by cardiovascular disease. Despite the introduction of several survival-prolonging treatment modalities, including targeted therapy and immunotherapy, the overall prognosis for the metastatic disease remains challenging. Therefore, the identification of new molecular biomarkers and therapeutic targets related to cancer diagnosis and prognosis is of paramount importance. DNA polymerase delta 1 (POLD1), a catalytic and proofreading subunit of the DNA polymerase δ complex, performs a crucial role in DNA replication and repair processes. Recently, germline and somatic mutations of the POLD1 gene have been acknowledged in several malignancies. Moreover, diversified POLD1 expression profiles have been reported in association with clinicopathological features in a variety of tumor types. With this review, we aim to summarize the current knowledge on the role of POLD1 in cancers. In addition, we discuss the future prospects and clinical applications of the assessment of POLD1 mutation and expression patterns in tumors.

Electrochemical Determination of Flap Endonuclease 1 Activity Amplified by CRISPR/Cas12a Trans‐Cleavage**

AbstractFlap endonuclease 1 (FEN1) participates in the DNA replication and repair processes, which plays an important role in maintaining genome stability. Determination of FEN1 level is highly required for biochemical studies and clinical diagnosis. Herein, a novel electrochemical approach for the detection of FEN1 is developed based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated (Cas) proteins trans‐cleavage. The dumbbell structured DNA substrate, single‐stranded DNA labelled with electrochemical species and three‐dimensional tetrahedral DNA are designed and involved in the reactions. This method displays high sensitivity and good specificity, which can not only monitor FEN1 in various biological samples directly, but also help screen FEN1 inhibitors with potential use for anti‐cancer drug discovery.

Detection and characterization of constitutive replication origins defined by DNA polymerase epsilon

BackgroundDespite the process of DNA replication being mechanistically highly conserved, the location of origins of replication (ORI) may vary from one tissue to the next, or between rounds of replication in eukaryotes, suggesting flexibility in the choice of locations to initiate replication. Lists of human ORI therefore vary widely in number and location, and there are currently no methods available to compare them. Here, we propose a method of detection of ORI based on somatic mutation patterns generated by the mutator phenotype of damaged DNA polymerase epsilon (POLE).ResultsWe report the genome-wide localization of constitutive ORI in POLE-mutated human tumors using whole genome sequencing data. Mutations accumulated after many rounds of replication of unsynchronized dividing cell populations in tumors allow to identify constitutive origins, which we show are shared with high fidelity between individuals and tumor types. Using a Smith–Waterman-like dynamic programming approach, we compared replication origin positions obtained from multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods. Many DNA features co-localized with the consensus set of ORI, including chromatin loop anchors, G-quadruplexes, S/MARs, and CpGs. Among all features, the H2A.Z histone exhibited the most significant association.ConclusionsOur results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

Analytical and therapeutic profiles of DNA methylation alterations in cancer; an overview of changes in chromatin arrangement and alterations in histone surfaces.

DNA methylation is the most important epigenetic element that activates the inhibition of gene transcription and is included in the pathogenesis of all types of malignancies. Remarkably, the effectors of DNA methylation are DNMTs (DNA methyltransferases) that catalyze denovo or keep methylation of hemimethylated DNA after the DNA replication process. DNA methylation structures incancer are altered, with three procedures by which DNA methylation helps cancer development which are including direct mutagenesis, hypomethylation of the cancer genome, and also focal hypermethylation of the promoters of TSGs (tumor suppressor genes). Conspicuously, DNA methylation, nucleosome remodeling, RNA-mediated targeting, and histone modification balance modulate many biological activities that are essential and indispensable to the genesis of cancer and also can impact many epigenetic changes including DNA methylation and histone modifications as well as adjusting of non-coding miRNAs expression in prevention and treatment of many cancers. Epigenetics points to heritable modifications in gene expression that do not comprise alterations in the DNA sequence. The nucleosome is the basic unit of chromatin, consisting of 147 base pairs (bp) of DNA bound around a histone octamer comprised of one H3/H4 tetramer and twoH2A/H2B dimers. DNA methylation is preferentially distributed over nucleosome regions and is less increased over flanking nucleosome-depleted DNA, implying a connection between nucleosome positioning and DNA methylation. In carcinogenesis, aberrations in the epigenome may also include in the progression of drug resistance. In this report, we report the rudimentary notes behind these epigenetic signaling pathways and emphasize the proofs recommending that their misregulation can conclude in cancer. These findings in conjunction with thepromising preclinical and clinical consequences observed with epigenetic drugs against chromatin regulators, confirm the important role of epigenetics in cancer therapy.

Transcriptome analysis of hepatopancreas of Chinese grass shrimp, Palaemonetes sinensis, infected by Enterocytospora artemiae

The emergence of the microsporidian, Enterocytospora artemiae, has caused serious economic losses to the aquaculture industry of Palaemonetes sinensis. The hepatopancreas is the main digestive and immune organ of P. sinensis, and the main site of E. artemiae infection. We used next-generation sequencing to determine the effects of E. artemiae parasitism on the hepatopancreas of P. sinensis at the transcriptome level. The hepatopancreas of P. sinensis was parasitized by E. artemiae, and 881 differentially expressed genes (DEGs) were obtained, of which 643 were upregulated and 238 were downregulated. These DEGs are mainly involved in DNA replication, transcription, translation, immunity, and metabolism. Among them, the cellular processes of DNA replication, transcription and translation are significantly strengthened, which may be related to the use of host ATP and nucleic acid by E. artemiae to achieve proliferation and damage to host cells to enhance DNA replication and repair. Moreover, to defend against E. artemiae, some immune genes related to antioxidation, such as glutathione metabolism, seleno compound metabolism, and cytochrome p450 2L1, were significantly upregulated, but simultaneously, tumor necrosis factor, NF-κB inhibitor α, and other immune-related genes were significantly down regulated, indicating that the parasitism of E. artemiae led to a significant decline in the immune defense ability of P. sinensis. From the perspective of metabolism, the metabolism-related DEGs of retinol, glycine, serine, and threonine metabolism, were significantly downregulated, resulting in insufficient nutrient absorption and decreased energy supply of the P. sinensis, which in turn affected their growth. The differential genes and pathways identified in this study can provide a reference basis to further elucidate the pathogenic mechanism of P. sinensis infected with E. artemiae and the prevention and control of microsporidia disease.

Implication of E3 ligase RAD18 in UV-induced mutagenesis in human induced pluripotent stem cells and neuronal progenitor cells.

Pluripotent stem cells (PSCs) have the potential to differentiate to any of the other organs. The genome DNA integrity of PSCs is maintained by a high level of transcription for a number of genes involved in DNA repair, cell cycle and apoptosis. However, it remains unclear how high the frequency of genetic mutation is and how these DNA repair factors function in PSCs. In this study, we employed Sup F assay for the measurement of mutation frequency after UV-C irradiation in induced pluripotent stem cells (iPSCs) as PSC models and neural progenitor cells (NPCs) were derived from iPSCs as differentiated cells. iPSCs and NPCs exhibited a lower mutation frequency compared with the original skin fibroblasts. In RNA-seq analysis, iPSCs and NPCs showed a high expression of RAD18, which is involved in trans-lesion synthesis (TLS) for the emergency tolerance system during the replication process of DNA. Although RAD18 is involved in both error free and error prone TLS in somatic cells, it still remains unknown the function of RAD18 in PSCs. In this study we depleted of the RAD18 by siRNA knockdown resulted in decreased frequency of mutation in iPSCs and NPCs. Our results will provide information on the genome maintenance machinery in PSCs.

A facile enzyme-assisted multiple recycling amplification strategy for ultrasensitive fluorescence detection of HIV-1 DNA

We developed an enzyme-assisted multiple signal amplification reaction for target nucleic acid detection. In this strategy, the target nucleic acid as triggers, and in the presence of two specific primers, DNA polymerase and DNA nicking endonuclease act in concert to promote the repetitive cycle DNA replication process, leading to an exponential increase in nucleic acid sequence. Both ends of each sequence of the amplification products and the specific primer could from a unit of Mg2+-DNAzyme which in the presence of cofactor Mg2+ could recognize and cyclically cleave the hairpin probes in the solution and thus realize real-time fluorescence detection of HIV-1 DNA. Under optimal conditions, the detection limit of this method is 10−17 M. The simple in design, isothermal conditions and easy fluorescence measurement of this method exhibits superior sensitivity and specificity, and thus holds great potential to be a promising assay for quantitative detection of DNA or RNA in theoretical and clinic studies.

Characterization of the gene signature correlated with favorable response to chemoradiotherapy in rectal cancer: A hypothesis-generating study.

This study aimed to define the gene signature associated with response to neoadjuvant chemoradiotherapy (nCRT), or chemoradiosensitivity (CRS) signature, in rectal cancer, and investigate the correlation between the CRS signature and characteristics of tumor. Three public microarray datasets of pre-nCRT rectal cancer were used to discover and validate the CRS signature, and the pathway analysis of the CRS signature was performed. Patients in The Cancer Genome Atlas (TCGA) dataset were stratified according to the CRS signature enrichment score, and mutational profile and proportions of infiltrated immune cells were compared. In the discovery dataset (GSE53781), 95 genes were upregulated in complete responders compared to non-complete responders and defined as the CRS signature. Pathways regarding DNA replication and repair processes as well as inflammatory response were enriched in the CRS signature. In the validation datasets (GSE35452 and GSE45404), patients with favorable response to nCRT exhibited higher enrichment score of the CRS. In TCGA-READ cohort, patients with high CRS signature harbored KRAS mutation in lower frequency than those with low CRS signature. In addition, proportions of proinflammatory immune cells were higher, but proportion of immunosuppressive M2 macrophages was lower in patients with high CRS signature than those with low CRS signature. The current integrative bioinformatic analysis suggests the CRS signature and showed that the CRS signature is associated with dissimilar mutational profile and increased immune response. The discovered CRS signature and related characteristics may serve as candidate of stratification factor in upcoming studies for rectal cancer.

Pol32, an accessory subunit of DNA polymerase delta, plays an essential role in genome stability and pathogenesis of Candida albicans

ABSTRACT Candida albicans is a pathobiont that inflicts serious bloodstream fungal infections in individuals with compromised immunity and gut dysbiosis. Genomic diversity in the form of copy number alteration, ploidy variation, and loss of heterozygosity as an adaptive mechanism to adverse environments is frequently observed in C. albicans. Such genomic variations also confer a varied degree of fungal virulence and drug resistance, yet the factors propelling these are not completely understood. DNA polymerase delta (Polδ) is an essential replicative DNA polymerase in the eukaryotic cell and is yet to be characterized in C. albicans. Therefore, this study was designed to gain insights into the role of Polδ, especially its non-essential subunit Pol32, in the genome plasticity and life cycle of C. albicans. PCNA, the DNA clamp, recruits Polδ to the replication fork for processive DNA replication. Unlike in Saccharomyces cerevisiae, the PCNA interaction protein (PIP) motif of CaPol32 is critical for Polδ’s activity during DNA replication. Our comparative genetic analyses and whole-genome sequencing of POL32 proficient and deficient C. albicans cells revealed a critical role of Pol32 in DNA replication, cell cycle progression, and genome stability as SNPs, indels, and repeat variations were largely accumulated in pol32 null strain. The loss of pol32 in C. albicans conferred cell wall deformity; Hsp90 mediated azoles resistance, biofilm development, and a complete attenuation of virulence in an animal model of systemic candidiasis. Thus, although Pol32 is dispensable for cell survival, its function is essential for C. albicans pathogenesis; and we discuss its translational implications in antifungal drugs and whole-cell vaccine development.

Compound probiotics can improve intestinal health by affecting the gut microbiota of broilers.

Probiotics, as a widely used additive, have played a unique advantage in replacing antibiotic products. As a result, the probiotic effects on broiler development, intestinal flora, intestinal barrier, and immunity were assessed by this investigation. Four hundred and eighty 1-day-old Arbor Acres broilers were randomly allotted to 4 groups of 5 replicates with 24 broilers each. The control was fed only a basal corn-soybean meal diet. Probiotics I, probiotics II, and probiotics III were fed basal diet and 1, 5, and 10 g/kg compound probiotics (Lactobacillus casei: Lactobacillus acidophilus: Bifidobacterium = 1:1:2), respectively. We found that broilers in the compound probiotic group exhibited better growth performance and carcass characteristics compared with control, especially among probiotics III group. The intestinal barrier-related genes relative expression of Claudin, Occludin, MUC2, and ZO-1 mRNA in the probiotic group increased at 21 and 42 d compared with control, especially among probiotics III group (P < 0.05). The early gut immune-related genes (TLR2, TLR4, IL-1β, and IL-2) mRNA increased compared with control, while the trend at 42 d was completely opposite to that in the earlier stage (P < 0.05). Among them, probiotics III group showed the most significant changes compared to probiotics II group and probiotics I group. Select probiotics III group and control group for 16S rDNA amplicon sequencing analysis. The 16S rDNA amplicon sequencing results demonstrated that probiotics increased the relative abundance of beneficial microbes such as o_Bacteroidales, f_Rikenellaceae, and g_Alistipes and improved the cecum's gut microbiota of 42-day-old broilers. Additionally, adding the probiotics decreased the relative abundance of harmful microbes such as Proteobacteria. PICRUSt2 functional analysis revealed that most proteins were enriched in DNA replication, transcription, and glycolysis processes. Therefore, this study can provide theoretical reference value for probiotics to improve production performance, improve intestinal barrier, immunity, intestinal flora of broilers, and the application of probiotics.

Doxorubicin induces cardiomyocyte death owing to the accumulation of dysfunctional mitochondria by inhibiting the autophagy fusion process

Doxorubicin (Dox), an anthracycline antibiotic, is an anticancer drug that inhibits DNA replication and cellular metabolic processes in cancer cells with high proliferative potential. However, Dox causes severe side effects, including myocardial damage and heart failure, but the molecular mechanism underlying Dox-induced myocardial injury remains uncertain. In the present study, we evaluated the effects of Dox on the mitochondrial quality control system and regulation of mitochondrial respiration and autophagy in an in vitro rat myoblast H9c2 cell culture model using western blotting, immunohistochemistry, the Seahorse XF24 system, and flow cytometry. Our results showed that Dox did not impair the initiation of autophagic flux or the functions of lysosomes; however, Dox affected the mitochondrial quality control system, leading to a fission-dominant morphology and impaired regulation of mitochondrial respiration, thereby increasing oxidative stress and inhibited progression of autophagy, particularly the fusion of autophagosomes with lysosomes. This inhibition caused a significant decrease in the formation of autolysosomes and was responsible for the accumulation of dysfunctional mitochondria and subsequent increase in oxidative stress, eventually leading to increased myocardial cell death.

Changes in the intra- and extra-mechanical environment of the nucleus in Saos-2 osteoblastic cells during bone differentiation process: Nuclear shrinkage and stiffening in cell differentiation

Osteogenic differentiation has been reportedly regulated by various mechanical stresses, including fluid shear stress and tensile and compressive loading. The promotion of osteoblastic differentiation by these mechanical stresses is accompanied by reorganization of the F-actin cytoskeleton, which is deeply involved in intracellular forces and the mechanical environment. However, there is limited information about the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be directly involved in various cellular functions. Here, we investigated the changes in the intracellular force applied to the nucleus and the effect on nuclear morphology and mechanical properties during osteogenic differentiation in human osteoblast-like cells (Saos-2). We carried out cell morphological analyses with confocal fluorescence microscopy, nuclear indentation test with atomic force microscopy (AFM), and fluorescence recovery after photobleaching (FRAP) for intranuclear DNA. The results revealed that a significant reorganization of the F-actin cytoskeleton from the nuclear surfaces to the cell periphery occurred in the osteogenic differentiation processes, simultaneously with the reduction of compressive forces to the nucleus. Such changes also facilitated nuclear shrinkage and stiffening, and further intranuclear chromatin compaction. The results indicate that the reduction of the intracellular compressive force due to reorganization of the F-actin cytoskeleton affects the intra- and extra-mechanical environment of the nucleus, and this change may affect gene expression and DNA replication in the osteogenic differentiation process.

Incidence of Hereditary Gastric Cancer May Be Much Higher than Reported.

Hereditary gastric cancers (HGCs) are supposed to be rare and difficult to identify. Nonetheless, many cases of young patients with gastric cancer (GC) fulfill the clinical criteria for considering this diagnosis but do not present the defined pathogenic mutations necessary to meet a formal diagnosis of HGC. Moreover, GC in young people is a challenging medical situation due to the usual aggressiveness of such cases and the potential risk for their relatives when related to a germline variant. Aiming to identify additional germline alterations that might contribute to the early onset of GC, a complete exome sequence of blood samples from 95 GC patients under 50 and 94 blood samples from non-cancer patients was performed and compared in this study. The number of identified germline mutations in GC patients was found to be much higher than that from individuals without a cancer diagnosis. Specifically, the number of high functional impact mutations, including those affecting genes involved in medical diseases, cancer hallmark genes, and DNA replication and repair processes, was much higher, strengthening the hypothesis of the potential causal role of such mutations in hereditary cancers. Conversely, classically related HGC mutations were not found and the number of mutations in genes in the CDH1 pathway was not found to be relevant among the young GC patients, reinforcing the hypothesis that existing alternative germline contributions favor the early onset of GC. The LILRB1 gene variants, absent in the world's cancer datasets but present in high frequencies among the studied GC patients, may represent essential cancer variants specific to the Amerindian ancestry's contributions. Identifying non-reported GC variants, potentially originating from under-studied populations, may pave the way for additional discoveries and translations to clinical interventions for GC management. The newly proposed approaches may reduce the discrepancy between clinically suspected and molecularly proven hereditary GC and shed light on similar inconsistencies among other cancer types. Additionally, the results of this study may support the development of new blood tests for evaluating cancer risk that can be used in clinical practice, helping physicians make decisions about strategies for surveillance and risk-reduction interventions.

Mitochondrial genome maintenance - the kinetoplast story.

Mitochondrial DNA replication is an essential process in most eukaryotes. Similar to the diversity in mitochondrial genome size and organization in the different eukaryotic supergroups, there is considerable diversity in the replication process of the mitochondrial DNA. In this review, we summarize the current knowledge of mitochondrial DNA replication and the associated factors in trypanosomes with a focus on Trypanosoma brucei, and provide a new model of minicircle replication for this protozoan parasite. The model assumes the mitochondrial DNA (kinetoplast DNA, kDNA) of T. brucei to be loosely diploid in nature and the replication of the genome to occur at two replication centers at the opposing ends of the kDNA disc (also known as antipodal sites, APS). The new model is consistent with the localization of most replication factors and in contrast to the current model, it does not require the assumption of an unknown sorting and transport complex moving freshly replicated DNA to the antipodal sites. In combination with the previously proposed sexual stages of the parasite in the insect vector, the new model provides a mechanism for maintenance of the mitochondrial genetic diversity.

Tyroviruses are a new group of temperate phages that infect Bacillus species in soil environments worldwide

BackgroundBacteriophages are widely considered to be highly abundant and genetically diverse, with their role in the evolution and virulence of many pathogens becoming increasingly clear. Less attention has been paid on phages preying on Bacillus, despite the potential for some of its members, such as Bacillus anthracis, to cause serious human disease.ResultsWe have isolated five phages infecting the causative agent of anthrax, Bacillus anthracis. Using modern phylogenetic approaches we place these five new Bacillus phages, as well as 21 similar phage genomes retrieved from publicly available databases and metagenomic datasets into the Tyrovirus group, a newly proposed group named so due to the conservation of three distinct tyrosine recombinases. Genomic analysis of these large phages (~ 160–170 kb) reveals their DNA packaging mechanism and genomic features contributing to virion morphogenesis, host cell lysis and phage DNA replication processes. Analysis of the three tyrosine recombinases suggest Tyroviruses undergo a prophage lifecycle that may involve both host integration and plasmid stages. Further we show that Tyroviruses rely on divergent invasion mechanisms, with a subset requiring host S-layer for infection.ConclusionsUltimately, we expand upon our understanding on the classification, phylogeny, and genomic organisation of a new and substantial phage group that prey on critically relevant Bacillus species. In an era characterised by a rapidly evolving landscape of phage genomics the deposition of future Tyroviruses will allow the further unravelling of the global spread and evolutionary history of these Bacillus phages.

Pharmacological targeting of type phosphodiesterase 4 inhibits the development of acute myeloid leukemia by impairing mitochondrial function through the Wnt/β-catenin pathway

Acute myeloid leukemia (AML) is prone to drug-resistant relapse with a low 5-year survival rate. New therapeutic modalities are sorely needed to provide hope for AML relapse patients. Herein, we demonstrated a specific inhibitor of type 4 phosphodiesterase (PDE4), Zl-n-91, could significantly reduce the proliferation of AML cells, block DNA replication process, and increase AML cell death. Zl-n-91 also impeded the growth of subcutaneous xenograft and prolonged the survival of the MLL-AF9-driven AML model. Bioinformatic analysis revealed that elevated mitochondrial gene signatures inversely correlate with the survival of AML patients; and importantly, Zl-n-91 strongly suppressed the function of mitochondria. In addition, this PDE4 inhibitor induced alterations in multiple signaling pathways, including the reduction of β-catenin activity. Stimulation of the Wnt/β-catenin pathway could attenuate the inhibitory effect of Zl-n-91 on AML cell proliferation as well as mitochondrial function. Taken together, we revealed for the first time that targeting PDE4 activity could attenuate mitochondrial function through a Wnt/β-catenin pathway, which in turn would block the growth of AML cells. Specific PDE4 inhibitors can potentially serve as a new treatment modality for AML patients.

Therapeutic Targeting of Spliceosome Mutant Myeloid Neoplasms Via PARP1 Inhibition

Somatic mutations in splicing factors (SF) SF3B1, SRSF2, U2AF1 are observed across a spectrum of clonal myeloid disorders including myelodysplastic syndromes (MDS). Previous studies identified increased R-loops in SF-mutant cells. Abnormal R-loop accumulation is associated with increased genomic instability and preferential sensitivity to ATR kinase inhibitor. Motivated by these observations, we set out to identify novel therapeutic targets in SF-mutant myeloid malignancies. Using MLLAF9 Srsf2WT and Srsf2P95Hcells, we performed a focused drug screen using inhibitors targeting different DNA damage response pathways and drugs that inhibit DNA replication or DNA metabolic processes. Unexpectedly, Srsf2P95H cells displayed increased sensitivity to inhibitors targeting PARP1/2 (Fig. 1A-C). Olaparib (PARPi) treatment induced higher apoptosis in Srsf2P95Hcells than in Srsf2WTcells (Fig. 1D). To ensure this phenomenon extends beyond Srsf2 mutation, we generated MLLAF9 leukemia on U2af1WT and U2af1S34Fbackgrounds. PARPi treatment in U2af1S34F cells resulted in reduced cell viability and increased apoptosis compared to U2af1WT cells (Fig. 1E-G). Next, we genetically inactivated Parp1 in Srsf2WT and Srsf2P95H cells. The absence of Parp1 did not affect the growth of Srsf2WT cells (Fig. 1H). In contrast, Parp1 deletion significantly reduced proliferation of Srsf2P95H cells (Fig. 1I).Finally, to confirm this phenotype in vivo, we transplanted established MLLAF9 Srsf2WTor Srsf2P95H leukemia into syngeneic mice. Eleven days post-transplant, mice received either vehicle or PARPi. Mice transplanted with Srsf2WT cells showed no survival advantage when treated with PARPi (Fig. 1J). In contrast, PARPi treatment significantly prolonged survival of mice transplanted with Srsf2P95H cells compared to vehicle-treated mice (Fig. 1K). To examine whether PARPi sensitivity is conserved in humans, we treated K562 cells expressing either SRSF2P95H or SF3B1K700E mutation. SRSF2P95H and SF3B1K700E cells showed increased sensitivity to olaparib and rucaparib (Fig. 1L-M). This observation was further validated in a panel of primary AML blasts carrying SRSF2 and U2AF1 mutations(Fig. 1N). Taken together, our results identify PARP1 as a novel therapeutic target in SF-mutant myeloid neoplasms. PARP inhibitors preferentially kill cells with defective homologous recombination (HR)-mediated DNA repair. We asked if the observed PARPi sensitivity in SF-mutant cells was due to defects in HR. To measure HR activity, we performed a CRISPR-Cas9/mClover assay. The assay measures HR-dependent insertion of an mClover-containing donor cassette into Cas9-mediated double-stranded breaks in the LMNA gene, which results in the expression of a green fluorescent Lamin A/C protein. There was no significant difference in the relative percentage of mClover positivity in SRSF2P95H or U2AF1S34F cells compared to control (Fig. 1O), suggesting that these cells are HR-proficient. Interestingly, SF3B1K700E cells showed significantly reduced HR efficiency, consistent with a published study. We next investigated cellular PARP1 activity in SRSF2- and U2AF1-mutant cells by monitoring Poly-ADP-ribosylated chain (PAR) levels. SRSF2P95H cells had elevated PAR level compared to SRSF2WT cells (Fig. 1P). Since PARP1 is known to associate with R-loops, we wanted to test whether increased PARP1 activity and PARPi sensitivity are R-loop dependent. We generated SRSF2-mutant cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within the RNA:DNA hybrids. Induction of RNaseH1 reduced PAR level and PARPi sensitivity in SRSF2P95H cells, suggesting that SRSF2-mutant cells elicit a PARP1 response to promote survival in an R-loop-dependent manner (Fig. 1Q). Lastly, since SF-mutant cells are also sensitive to ATRi, we tested if combining PARPi and ATRi can further sensitize SF-mutant cells. Combined PARPi + ATRi treatment induced synergistic killing of Srsf2- and U2af1-mutant cells compared to wildtype cells in cell viability and colony forming assays (Fig. 1R-S). In summary, this study provides a pre-clinical rationale for therapeutic targeting of PARP1 in SF-mutant leukemia. Moreover, PARP and ATR inhibitor combination could emerge as a new therapeutic strategy in this genetically distinct disease subtype. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hepatic transcriptome analysis reveals the metabolic strategies of largemouth bass (Micropterus salmoides) under different dissolved oxygen condition

Dissolved oxygen (DO) affects aquatic animals at a fundamental level so that the differences in its metabolic strategies under prolonged hypoxic conditions need an urgent exploration. In this experiment, largemouth bass (Micropterus salmoides) were chronically exposed (6 weeks) to severe hypoxia (S-HYP, DO: 2.0 ± 0.4 mg/L) and mild hypoxia (M-HYP, DO: 5.1 ± 0.4 mg/L). Compared to the control group (CON, DO:8.4 ± 0.4 mg/L), 1196 and 232 differentially expressed genes (DEGs) were obtained in S-HYP and M-HPY groups via transcriptome analysis, respectively. In S-HYP, lipolysis was promoted while anabolism was blocked. Meanwhile, significantly less fat droplet area was observed in the liver histology of S-HYP. Additionally, the cell cycle also responded to hypoxia, being blocked in the G1 phase with the suspension of DNA replication process. In M-HYP, the processing of protein in the endoplasmic reticulum and the synthesis of various aminoacyl t-RNA were inhibited, and a novel balance of the urea cycle might be established in the biosynthesis of arginine. The key DEGs involved in the above metabolic pathways, such as atgl, cpt1, arg1, etc., were validated by Q-PCR yielding results consistent with transcriptome data. This study indicates that the largemouth bass is prone to increase the proportion of lipid as an energy supply to adapt to the reprogramming of energy metabolism, while reducing the rate of cell proliferation to adapt to chronic severe hypoxia. This is also an undescribed observation in fish liver metabolism that largemouth bass may transform the synthesis and processing strategies of protein when exposed to chronic mild hypoxia.