Top Differentially Expressed Genes Research Articles

Integrative bioinformatics analysis of ACS enzymes as candidate prognostic and diagnostic biomarkers in colon adenocarcinoma.

Acyl-CoA synthetase (ACS) enzymes play an important role in the activation of fatty acids. While many studies have found correlations between the expression levels of ACS enzymes with the progression, growth, and survival of cancer cells, their role and expression patterns in colon adenocarcinoma are still greatly unknown and demand further investigation. The expression data of colon adenocarcinoma samples were downloaded from the Cancer Genome Atlas (TCGA) database. Normalization and differential expression analysis were performed to identify differentially expressed genes (DEGs). Gene set enrichment analysis was applied to identify top enriched genes from ACS enzymes in cancer samples. Gene ontology and protein-protein interaction analyses were performed for the prediction of molecular functions and interactions. Survival analysis and receiver operating characteristic test (ROC) were performed to find potential prognostic and diagnostic biomarkers. ACSL6 and ACSM5 genes demonstrated more significant differential expression and LogFC value compared to other ACS enzymes and also achieved the highest enrichment scores. Gene ontology analysis predicted the involvement of top DEGs in fatty acids metabolism, while protein-protein interaction network analysis presented strong interactions between ACSLs, ACSSs, ACSMs, and ACSBG enzymes with each other. Survival analysis suggested ACSM3 and ACSM5 as potential prognostic biomarkers, while the ROC test predicted stronger diagnostic potential for ACSM5, ACSS2, and ACSF2 genes. Our findings revealed the expression patterns, prognostic, and diagnostic biomarker potential of ACS enzymes in colon adenocarcinoma. ACSM3, ACSM5, ACSS2, and ACSF2 genes are suggested as possible prognostic and diagnostic biomarkers.

Spatial Transcriptional Mapping Reveals Site-Specific Pathways Underlying Human Atherosclerotic Plaque Rupture

BackgroundAtherosclerotic plaque ruptures, triggered by blood flow–associated biomechanical forces, cause most myocardial infarctions and strokes. ObjectivesThis study aims to investigate the exact location and underlying mechanisms of atherosclerotic plaque ruptures, identifying therapeutic targets against cardiovascular events. MethodsHistology, electron microscopy, bulk and spatial RNA sequencing on human carotid plaques were studied in proximal, most stenotic, and distal regions along the longitudinal blood flow direction. Genome-wide association studies were used to examine heritability enrichment and causal relationships of atherosclerosis and stroke. Associations between top differentially expressed genes (DEGs) and preoperative and postoperative cardiovascular events were examined in a validation cohort. ResultsIn human carotid atherosclerotic plaques, ruptures predominantly occurred in the proximal and most stenotic regions but not in the distal region. Histologic and electron microscopic examination showed that proximal and most stenotic regions exhibited features of plaque vulnerability and thrombosis. RNA sequencing identified DEGs distinguishing the proximal and most stenotic regions from the distal region which were deemed as most relevant to atherosclerosis-associated diseases as shown by heritability enrichment analyses. The identified pathways associated with the proximal rupture-prone regions were validated by spatial transcriptomics, firstly in human atherosclerosis. Of the 3 top DEGs, matrix metallopeptidase 9 emerged particularly because Mendelian randomization suggested that its high circulating levels were causally associated with atherosclerosis risk. ConclusionsOur findings show plaque site–specific transcriptional signatures associated with proximal rupture-prone regions of carotid atherosclerotic plaques. This led to the geographical mapping of novel therapeutic targets, such as matrix metallopeptidase 9, against plaque rupture.

Transcriptome differential display of a drought‐tolerant early flowering spineless mutant of safflower (Carthamus tinctorius) and identification of candidate genes

AbstractOne of the best candidates to face the increasing environmental stresses is safflower (Carthamus tinctorius L.) with natural tolerance to multiple abiotic stresses including drought. In the present study, transcriptome profile of a spineless drought‐tolerant mutant of safflower with early flowering was compared to its spiny wild type. In differential RNA‐Seq analyses more than 400 differentially expressed genes (DEGs) were identified in spineless drought‐tolerant mutant. The expression of 8 top DEGs, mostly related to the contrasting traits, was confirmed by real‐time quantitative reverse transcription PCR. Transcription factors (TFs) followed by protein kinases had the highest frequency among the identified DEGs. TF families involved in the regulation of cell cycle, development, and environmental responses were prevalent in DEGs of spineless drought‐tolerant mutant, including NAC, bHLH, MYB, HD‐ZIP, and ERF. Three Kyoto Encyclopedia of Genes and Genomes pathways with direct or indirect role in cell/tissue development and metabolic processes were enriched for DEGs. Our results suggest that phenotypic differences between the two contrasting genotypes can be governed via regulation of hormone signal transduction/or cell cycle and stress‐related pathways. Several candidate genes for spine formation, early flowering, and drought tolerance were introduced, important instances are ZHD8 and TBL21 (candidates for spininess), three novel transcripts, respectively, encoding a CCT type protein, a MADS‐box TF and a CCA1 (candidates for early flowering), and CAT2, ZAT10, and CAMTA (candidates for drought response). These findings facilitate the isolation of key genes controlling drought‐tolerance, spininess, and early flowering in safflower and can be applied for developing value‐added varieties through genome‐based breeding programs.

Abstract LB299: Identification of key regulators for nuclear protein export inhibitor and chemotherapy combination in pancreatic cancer using spatial genomics approach

Abstract Background: All patients with pancreatic ductal adenocarcinoma (PDAC) experience disease progression after gemcitabine-nab-paclitaxel (GemPac) treatment with a median time to progression of just under 6 months. There is an urgent need for more effective combination therapies for this highly recalcitrant disease. Previously we showed that the selective inhibitor of nuclear export (SINE) selinexor (Sel) against XPO1 in combination with GemPac enhanced the survival of LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mice. The Sel-GemPac combination also showed evaluable response in PDAC patients in a Phase I study. Here we identified key regulatory molecules involved in the synergy of the Sel-GemPac combination, utilizing KPC mouse tumors and patient biopsy samples. We also evaluated the efficacy of this combination in a Phase II study. Methods: The activity of Sel-GemPac was evaluated in KPC mouse derived 2D cell line and 3D spheroid models. RNA-seq was performed to evaluate transcriptional differences associated with synergy. The identified differentially expressed genes (DEG) were validated in vitro through siRNA mediated silencing, qPCR, cytotoxicity, and related assays. The molecular mechanism of synergy was also evaluated in KPC mice tumors and in biopsies from patients enrolled in the Phase I portion of the trial using spatial transcriptomic and proteomic analysis. The efficacy of Sel-Gem was evaluated in a Phase II study in patients with metastatic PDAC (NCT02178436). Results: In a KPC tumor derived cell line, Sel inhibited cell growth and suppressed spheroid formation. Sel synergized with GemPac (CI<1) leading to superior inhibition of cell proliferation and suppressed long term growth (colonies). GSEA of RNA-seq showed inhibition of protein transport mechanism after treatment. In spatial transcriptomic analysis, marked suppression of stroma supporting markers were observed in tumor and stromal compartments of KPC tumors. In the treated cancer and stromal cells, 197 and 42 genes were differentially expressed respectively out of a total 11,650 genes, affecting 16 pathways. The top upregulated genes included Mt2, Nrn1, Gpihbp1 and Pals2 and the top downregulated genes include Chil3, Pglyrp1, Ear10 and Prap1. In the Phase II portion of the trial, 4 patients were given Sel (60 mg oral) with Gem (1000 mg/m2 i.v.) on days 1, 8, and 15 of the 28-day cycle. One patient from Phase II showed stable reduction of tumor lesion from 38.3 × 33.2 cm (before) to 33.2 × 26.9 cm (after). The best response based on RECIST 1.1 was minus 25%. This patient had an overall survival of 16 months. In patient biopsies, spatial transcriptomic analysis of malignant ductal tissues, detected 346 and 487 DEG between a responder and a non-responder at baseline, affecting more than 29 pathways. Most important the top DEG included S100P, B2M, CXCL5 and HP. Conclusions: Collectively, our studies show that XPO1 may be a valid therapeutic target in PDAC and Sel-GemPac is a combination warranting further investigation. Citation Format: Md. Hafiz Uddin, Sahar F. Bannoura, Husain Y. Khan, Amro Aboukameel, Yiwei Li, Ibrahim Azar, Eliza W. Bael, Miguel Tobon, Donald Weaver, Steve Kim, Tanya Odisho, Amr Mohammed, Gregory Dyson, Rafic Beydoun, Ramzi M. Mohammad, Herbert Chen, Bassel El-Rayes, Philip A. Philip, Mohammad N. Al-Hallak, Asfar S. Azmi. Identification of key regulators for nuclear protein export inhibitor and chemotherapy combination in pancreatic cancer using spatial genomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB299.

Abstract 5797: NTX301, a novel DNA hypomethylating agent, targets lipid metabolism in ovarian cancer

Abstract Background: DNA methylation causes the silencing of tumor suppressor and differentiation-associated genes being linked to chemoresistance and stemness. Previous studies demonstrated that hypomethylating agents (HMA) re-sensitize ovarian cancer (OC) cells to chemotherapy, however, translation to the clinic has been slow. NTX 301 (PinotBio) is a novel, highly potent, and orally bioavailable HMA, in early clinical development. Methods: We assessed the anti-tumor effects of NTX301 in OC models and studied the underlying molecular mechanisms by using cell proliferation, stemness, and ferroptosis assays, RNA sequencing, and validation. Results: OC cells (SKOV3, IC50=5.089nM; OVCAR5 IC50=3.664nM) were highly sensitive to NTX301 (p<0.05) compared to immortalized fallopian tube epithelial cells (FT-190) (IC50=103.3nM). Treatment with NTX301 induced significant downregulation of the DNA methyltransferases (DNMTs) 1-3 expression in SKOV3 and OVCAR5 cells compared with decitabine (p<0.05). Treatment with low dose NTX301 (100nM) induced significant transcriptomic reprogramming with 15,000 differentially expressed genes (DEGs) compared to DMSO (p<0.05). Among the NTX301 down-regulated DEGs, Gene Ontology (GO) enrichment analysis identified pathways related to the regulation of alcohol, cholesterol, and fatty acid biosynthetic process and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase activity, known features of cancer stem cells (CSCs). Indeed, treatment with low dose NTX301 (100nM) reduced the ALDH(+) cell population and expression of stemness-associated transcription factors (Oct4 and Sox2). Additionally, stearoyl-Coenzyme A desaturase 1 (SCD), a key enzyme that regulates unsaturated fatty acid homeostasis in the lipogenesis process was found among the top DEGs downregulated in response to NTX3 (fold change=9.36, FDR<0.05). As blockade of SCD had been shown to induce ferroptosis, oxidized lipids levels were measured by C11-BODIPY staining. NTX301 treatment increased the levels of oxidized lipids compared to DMSO and this was blunted by deferoxamine, indicating that NTX301 promoted cell death via ferroptosis. NTX301-induced ferroptosis was successfully rescued by the addition of oleic acid (200μM) into the culture medium, supporting that it was mediated through SCD depletion. In vivo, monotherapy with NTX301 (0.5mg/kg), significantly inhibited subcutaneous OC xenograft tumor growth (p<0.05). Decreased SCD levels and increased oxidized lipids were recorded in NTX301-treated xenografts. Conclusions: NTX301 is a potent HMA active in OC models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death. Citation Format: Yinu Wang, Xiaolei Situ, Horacio Cardenas, Daniela Matei, Ellie Siu, Sayedabdulrazzaq A. Alhunayan. NTX301, a novel DNA hypomethylating agent, targets lipid metabolism in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5797.

The temporal transcriptomic signature of cartilage formation.

Chondrogenesis is a multistep process, in which cartilage progenitor cells generate a tissue with distinct structural and functional properties. Although several approaches to cartilage regeneration rely on the differentiation of implanted progenitor cells, the temporal transcriptomic landscape of in vitro chondrogenesis in different models has not been reported. Using RNA sequencing, we examined differences in gene expression patterns during cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. Principal component and trajectory analyses revealed a progressively different and distinct transcriptome during chondrogenesis. Differentially expressed genes (DEGs), based on pairwise comparisons of samples from consecutive days were classified into clusters and analysed. We confirmed the involvement of the top DEGs in chondrogenic differentiation using pathway analysis and identified several chondrogenesis-associated transcription factors and collagen subtypes that were not previously linked to cartilage formation. Transient gene silencing of ATOH8 or EBF1 on day 0 attenuated chondrogenesis by deregulating the expression of key osteochondrogenic marker genes in micromass cultures. These results provide detailed insight into the molecular mechanism of chondrogenesis in primary micromass cultures and present a comprehensive dataset of the temporal transcriptomic landscape of chondrogenesis, which may serve as a platform for new molecular approaches in cartilage tissue engineering.

Distinct genome-wide DNA methylation and gene expression signatures in classical monocytes from African American patients with systemic sclerosis

BackgroundSystemic sclerosis (SSc) is a multisystem autoimmune disorder that has an unclear etiology and disproportionately affects women and African Americans. Despite this, African Americans are dramatically underrepresented in SSc research. Additionally, monocytes show heightened activation in SSc and in African Americans relative to European Americans. In this study, we sought to investigate DNA methylation and gene expression patterns in classical monocytes in a health disparity population.MethodsClassical monocytes (CD14+ + CD16−) were FACS-isolated from 34 self-reported African American women. Samples from 12 SSc patients and 12 healthy controls were hybridized on MethylationEPIC BeadChip array, while RNA-seq was performed on 16 SSc patients and 18 healthy controls. Analyses were computed to identify differentially methylated CpGs (DMCs), differentially expressed genes (DEGs), and CpGs associated with changes in gene expression (eQTM analysis).ResultsWe observed modest DNA methylation and gene expression differences between cases and controls. The genes harboring the top DMCs, the top DEGs, as well as the top eQTM loci were enriched for metabolic processes. Genes involved in immune processes and pathways showed a weak upregulation in the transcriptomic analysis. While many genes were newly identified, several other have been previously reported as differentially methylated or expressed in different blood cells from patients with SSc, supporting for their potential dysregulation in SSc.ConclusionsWhile contrasting with results found in other blood cell types in largely European-descent groups, the results of this study support that variation in DNA methylation and gene expression exists among different cell types and individuals of different genetic, clinical, social, and environmental backgrounds. This finding supports the importance of including diverse, well-characterized patients to understand the different roles of DNA methylation and gene expression variability in the dysregulation of classical monocytes in diverse populations, which might help explaining the health disparities.

Haptoglobin Gene Expression and Anthracycline-Related Cardiomyopathy in Childhood Cancer Survivors: A COG-ALTE03N1 Report

BackgroundAnthracycline-related cardiomyopathy is a leading cause of premature death in childhood cancer survivors. The high interindividual variability in risk suggests the need to understand the underlying pathogenesis. ObjectivesThe authors interrogated differentially expressed genes (DEGs) to identify genetic variants serving regulatory functions or genetic variants not easily identified when using genomewide array platforms. Using leads from DEGs, candidate copy number variants (CNVs) and single-nucleotide variants (SNVs) were genotyped. MethodsMessenger RNA sequencing was performed on total RNA from peripheral blood of 40 survivors with cardiomyopathy (cases) and 64 matched survivors without cardiomyopathy (control subjects). Conditional logistic regression analysis adjusting for sex, age at cancer diagnosis, anthracycline dose, and chest radiation was used to assess the associations between gene expression and cardiomyopathy and between CNVs and SNVs and cardiomyopathy. ResultsHaptoglobin (HP) was identified as the top DEG. Participants with higher HP gene expression had 6-fold greater odds of developing cardiomyopathy (OR: 6.4; 95% CI: 1.4-28.6). The HP2-specific allele among the HP genotypes (HP1-1, HP1-2, and HP2-2) had higher transcript levels, as did the G allele among SNVs previously reported to be associated with HP gene expression (rs35283911 and rs2000999). The HP1-2 and HP2-2 genotypes combined with the G/G genotype for rs35283911 and/or rs2000999 placed the survivors at 4-fold greater risk (OR: 3.9; 95% CI: 1.0-14.5) for developing cardiomyopathy. ConclusionsThese findings provide evidence of a novel association between HP2 allele and cardiomyopathy. HP binds to free hemoglobin to form an HP-hemoglobin complex, thereby preventing oxidative damage from free heme iron, thus providing biological plausibility to the mechanistic basis of the present observation.

Mechanistic effects of microwave radiation on pupal emergence in the leafminer fly, Liriomyza trifolii.

Liriomyza trifolii is a significant pest of vegetable and ornamental crops across the globe. Microwave radiation has been used for controlling pests in stored products; however, there are few reports on the use of microwaves for eradicating agricultural pests such as L. trifolii, and its effects on pests at the molecular level is unclear. In this study, we show that microwave radiation inhibited the emergence of L. trifolii pupae. Transcriptomic studies of L. trifolii indicated significant enrichment of differentially expressed genes (DEGs) in 'post-translational modification, protein turnover, chaperones', 'sensory perception of pain/transcription repressor complex/zinc ion binding' and 'insulin signaling pathway' when analyzed with the Clusters of Orthologous Groups, Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes databases, respectively. The top DEGs were related to reproduction, immunity and development and were significantly expressed after microwave radiation. Interestingly, there was no significant difference in the expression of genes encoding heat shock proteins or antioxidant enzymes in L. trifolii treated with microwave radiation as compared to the untreated control. The expression of DEGs encoding cuticular protein and protein takeout were silenced by RNA interference, and the results showed that knockdown of these two DEGs reduced the survival of L. trifolii exposed to microwave radiation. The results of this study help elucidate the molecular response of L. trifolii exposed to microwave radiation and provide novel ideas for control.

Integrative single‐nucleus RNAseq analysis identifies cell‐type‐specific expression perturbations in progressive supranuclear palsy

AbstractBackgroundProgressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by cell‐type‐specific tau lesions in neurons and glia. Using bulk brain RNAseq, we previously identified PSP‐associated genes and co‐expression networks that are enriched with cell‐type marker genes. We extended our work to identify cell‐type‐specific expression perturbations in PSP using a systems approach and in vivo validations in a model system.MethodSingle‐nucleus RNAseq (snRNAseq) was performed using 33 frozen brain temporal cortex (TCX) samples from PSP and control brains. After quality control (QC), integration, and clustering of nuclei, each cluster was annotated for its cell type. We identified differentially expressed genes (DEG) in PSP and further investigated them using an independent bulk RNAseq dataset comprising 408 PSP and control TCX samples for replication and assessing co‐expression patterns. Top DEGs were identified and then validated using external bulk RNAseq datasets from a tau transgenic mouse model. Further, the ability of the validated DEGs to rescue tau‐mediated cellular toxicity was evaluated in a tau transgenic Drosophila model.ResultAfter QC, 30 clusters comprised of 27,284 nuclei were obtained, encompassing two neuronal and six glial cell types. DEGs of each cluster revealed cell‐type‐specific PSP associations. Notably, many of the up‐regulated DEGs in the astroglial clusters were members of the bulk RNAseq co‐expression modules that were also up‐regulated in PSP and enriched with astrocytic markers. There were also consistent findings for the up‐regulated DEGs in the endothelial and microglial clusters, and for down‐regulated DEGs in the oligodendroglial clusters. Subsequently, we prioritized 240 DEGs with congruent cell‐type‐specific perturbations in snRNAseq and bulk RNAseq and validated 29 of those using transgenic mouse model data. Lastly, a significant number of the up‐regulated glial genes, when knocked down in a tau transgenic Drosophila model, led to reduced tau‐mediated cellular toxicity, suggesting novel potential therapeutic avenues.ConclusionPSP brains demonstrated robust cell‐type‐specific gene expression perturbations in the glial cells populations that were validated across different datasets. Importantly, inhibiting the up‐regulated expression perturbations rescued tau‐mediated toxicity in a Drosophila model. Our findings highlight the cell‐type‐specific transcriptome changes in PSP and provide potential therapeutic options for primary tauopathies.

Role of pericytes in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes of endothelial cells. It is characterized by pericyte deficiency. However, the role of pericytes in CCMs is not yet clarified. We found pericytes in Cdh5Cre ERT2 ;Ccm1 fl/fl (Ccm1 ECKO ) mice had a high expression of PDGFRβ. The inhibition of pericyte function by CP-673451 aggravated the CCM lesion development. RNA-sequencing analysis revealed the molecular traits of pericytes, such as highly expressed ECM-related genes, especially Fn1. Furthermore, KLF4 coupled with phosphorylated SMAD3 (pSMAD3) promoted the transcription of fibronectin in the pericytes of CCM lesions. RGDS peptide, an inhibitor of fibronectin, decreased the lesion area in the cerebella and retinas of Ccm1 ECKO mice. Also, human CCM lesions had abundant fibronectin deposition, and pSMAD3- and KLF4-positive pericytes. These findings indicate that pericytes are essential for CCM lesion development, and fibronectin intervention may provide a novel target for therapeutic intervention in such patients.

Hepatopancreatic transcriptome response of Penaeus vannamei to dietary ulvan

The study aimed to determine the effect of feeding dietary ulvan on some genes and pathways of Penaeus vannamei juveniles. Feeding a diet containing ulvan at 1.0 g·kg-1 resulted in differentially expressed genes (DEGs) between the treatments and the control group. Ulvan resulted in 53 DEGs with 26 up and 27-down-regulated DEGs. The DEGs were immune-related, while several affected the energy and substrate metabolic pathways. Specific genes upregulated were Glycerol-3-phosphate acyltransferase (GPAT) of the glycerolipid synthesis, centrosomal protein 120 involved in cell cycling and other activities, RNA helicase, an enzyme involved in opening up DNA molecules, ATP-binding cassette, TATA-binding protein, taurine transporter, transcriptional enhancer factor (TEF), C-type lectin among others. Down-regulated genes included gamma-crystallin, which acts as molecular chaperones; catenin alpha, which is involved in adhesion complex; dystrophin, which is involved in scaffolding for several signaling molecules; and maintenance muscle integrity, among others. Top DEGs that affected significantly important pathways include TEAD (transcriptional enhancer factor domain), GSK3B (glycogen synthase Kinase 3 beta), SLC6A1 (i.e., solute carrier Family 6 Member 1), and ADCY2, which affected signal transduction, environmental adaptation as well as the endocrine, immune, nervous, and digestive systems. In conclusion, dietary ulvan resulted in the up-regulation of immune-related DEGs, which could probably be used to adapt to unfavorable conditions and also affected some energy and substrate metabolic pathways that could potentially be used to direct the overall metabolism.

Synergistic Anti-Tumor Effect on Novel Combination of Selinexor with Azacitidine in AML By Targeting XPO1/eIF4E/C-MYC Signaling

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with high mortality and poor outcome. Highly effective therapy options are always needed for AML. Selinexor (KPT-330) is an oral bioactive selective CRM1 (XPO1) inhibitor, and the clinical trial showed the combination of KPT-330 over standard induction therapy benefits much for AML patients. Azacitidine (AZA), a DNA hypomethylation agent, can induce cell apoptosis and exhibit potential therapeutic efficacy against AML. However, it has not yet determined the anti-tumor effect of KPT-330 with AZA in AML. Here we examined the anti-leukemia effect and potential mechanism of KPT-330 combined with AZA in AML cells to provide evidence for further clinical trials. Methods: A total of 64 AML patients’ BM samples and 64 normal control from healthy volunteers were obtained from Zhongda Hospital Southeast University, in which the 2 patients’ samples were further treated with indicated drugs in vitro. Cell Counting Kit-8 (CCK-8) was used for cell viability assay, Annexin-V/PI staining following flow cytometry analysis for apoptosis, and qPCR and Western Blot for gene expression in AML cell lines and patient samples. CalcuSyn analysis was performed for a synergistic effect. RNA-seq was performed in U937 cells treated with 0.7uM AZA for 48 hrs, and the significantly differentially expressed genes (DEGs) were identified and gene set enrichment analysis (GSEA) was performed. Results: Cell proliferation arrest and apoptosis were significantly observed in U937 and MV4-11 cells treated with the combination of KPT-330 with AZA compared to single drug control (data not shown). Then, we further explored the synergistic effect of the combination in patients’ samples. Results showed that cell growth arrest and apoptosis significantly increased in primary samples from two de novo AML patients compared to the single drug controls, and CalcuSyn analysis showed a synergistic effect of the combination on cell growth arrest (Fig. 1A &1B). To understand the mechanisms underlying the synergy, RNA-seq analysis was performed in U937 cells treated by 0.7uM AZA and 4655 DEGs were identified. XPO1, eIF4E, and MYC are in the top DEGs upon AZA treatment, and GSEA analysis indicated MYC_TARGETS and apoptosis pathway are enriched in DEGs (Fig. 1C &1D ). Moreover, the mRNA level of XPO1 and eIF4E was increased in our AML patient cohort compared with healthy controls (Fig. 1E), and the high expression of XPO1 or elF4E are associated with poor outcome (Fig. 1F). XPO1 is the key component of nuclear-exporting machinery; elF4E, the cargo of XPO1 is translation initiation factor and plays a critical role in translational machinery, which regulates the C-MYC translation. It is also reported that AZA regulates the transcription of C-MYC by affecting methylation. We observed a combination of KPT-330 with AZA significantly down-regulated the mRNA and protein level of oncogene eIF4E and C-MYC in two of the patients’ samples compared to single drug controls (Fig. 1G &1H ). Also, C-MYC knock-down was not only significantly increased the apoptosis in U937 cells compared to scramble shRNA (shCTL) control, but also significantly enhanced the combination-mediated apoptosis compared to that of single drug control in U937 cells (Fig. 1I). these data indicated that XPO1/eIF4E/C-MYC signaling plays critical roles in the combination-mediated anti-leukemia, and the mechanistic model is summarized in Fig.1J. Conclusions: Our data for the first time showed the synergistic effect of a novel combination of KPT-330 with AZA on cell growth arrest and apoptosis in AML patients and also identified the underlying mechanism by targeting XPO1/eIF4E/C-MYC signaling. Our results also highlighted the feasibility of the clinical trial for the combination in the therapy of AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LBODP028 Identifying Biomarkers For Predicting Response To Mineralocorticoid Receptor Antagonist Treatment In Heart Failure

Abstract Despite significant improvements to patient care, heart failure (HF) remains a major public health burden. Mineralocorticoid receptor antagonists (MRAs) reduce hospitalisation and improve survival in patients with HF with reduced ejection fraction (HFrEF) but the widespread use of MRAs is limited by adverse effects including hyperkalaemia. The use of biomarkers to guide therapy may enable earlier treatment or minimise side effects from unnecessary therapies. We have previously described mineralocorticoid receptor responsive genes in monocytes/macrophages and hypothesised that response to MRAs can be detected in this cell population. Therefore, we aimed to identify biomarkers for predicting response to MRAs to facilitate personalised treatment of HF. Microarray-based transcriptome profiling was performed on monocytes obtained from six patients with HFrEF, before and after three months of MRA treatment. Three patients demonstrated improvement in left ventricular ejection fraction (LVEF) after 12 months of MRA treatment (Group 1), whereas the other three patients exhibited minimal functional response to MRAs (Group 2). To visualise sample similarity, multidimensional scaling (MDS) was employed which revealed distinct separation of males from females. To understand the changes in monocyte gene expression following MRA treatment, differential expression analysis was performed. Paired analysis of Group 1 samples (before vs after MRA treatment) identified 319 unique differentially expressed genes (DEGs) with an estimated fold-change ≥ 2 and q-value < 0. 05; 1 upregulated, 318 downregulated, and paired analysis of Group 2 identified 1 unique DEG. RT-qPCR was used validate top DEGs implicated in cardiovascular pathophysiology which confirmed downregulation of 12 of the top DEGs in Group 1. Additionally, cluster of differentiation (CD) encoding genes CD14, CD163, and CD68 were upregulated in Group 1 but downregulated in Group 2. Additional differential expression analyses were also performed by stratifying patients according to additional metrics of functional response including change in B-type natriuretic peptide (BNP). However, these analyses did not identify statistically significant DEGs. Our results indicate MRA treatment can significantly alter the human monocyte transcriptomic profile. Further research is needed to establish the utility of these DEGs as biomarkers for early prediction of MRA treatment-response in patients with HF, and to define sex-specific differences. Presentation: No date and time listed

A Novel Immunomodulatory Mechanism by Which Vitamin D Influences Folate Receptor 3 Expression to Reduce COVID-19 Severity.

Identify potential mechanisms involving gene expression changes through which vitamin D supplementation could be beneficial in preventing adverse COVID-19 outcomes. We performed a literature review to identify differentially expressed genes (DEGs) in the blood between severe and mild COVID-19 patients. We compared these with the top DEGs induced by 6 months of 10,000 IU/day vitamin D supplementation in healthy adults who were vitamin D deficient/insufficient. We used bioinformatic tools to look for a vitamin D response element (VDRE) in DEGs. FOLR3, RGS1, GPR84, and LRRN3 were the most significantly altered genes by 6 months of 10,000 IU/day vitamin D supplementation whose expression levels were also involved in COVID-19 severity. FOLR3 and GPR84 were found to be consistently up-regulated and RGS1 and LRRN3 consistently down-regulated in severe COVID-19 infection. FOLR3 and LRRN3 were down-regulated and RGS1 and GPR84 were up-regulated by 10,000 IU/day vitamin D supplementation. FOLR3 and RGS1 are expressed in neutrophils and lymphocytes, respectively. Vitamin D supplementation may decrease the neutrophil-lymphocyte ratio as has been reported in patients admitted with severe symptoms. There is evidence that vitamin D directly influences the expression of the RGS1 gene through vitamin D receptor binding. A potential negative VDRE (nVDRE) in an intron of the FOLR3 gene was found, which was homologous with two known nVDREs. Combined with other transcription factor elements near the newly identified nVDRE, these observations may explain the mechanism by which vitamin D regulates these genes, thus influencing COVID-19 outcomes.

Radiation-response in primary fibroblasts of long-term survivors of childhood cancer with and without second primary neoplasms: the KiKme study

BackgroundThe etiology and most risk factors for a sporadic first primary neoplasm in childhood or subsequent second primary neoplasms are still unknown. One established causal factor for therapy-associated second primary neoplasms is the exposure to ionizing radiation during radiation therapy as a mainstay of cancer treatment. Second primary neoplasms occur in 8% of all cancer survivors within 30 years after the first diagnosis in Germany, but the underlying factors for intrinsic susceptibilities have not yet been clarified. Thus, the purpose of this nested case–control study was the investigation and comparison of gene expression and affected pathways in primary fibroblasts of childhood cancer survivors with a first primary neoplasm only or with at least one subsequent second primary neoplasm, and controls without neoplasms after exposure to a low and a high dose of ionizing radiation.MethodsPrimary fibroblasts were obtained from skin biopsies from 52 adult donors with a first primary neoplasm in childhood (N1), 52 with at least one additional primary neoplasm (N2+), as well as 52 without cancer (N0) from the KiKme study. Cultured fibroblasts were exposed to a high [2 Gray (Gy)] and a low dose (0.05 Gy) of X-rays. Messenger ribonucleic acid was extracted 4 h after exposure and Illumina-sequenced. Differentially expressed genes (DEGs) were computed using limma for R, selected at a false discovery rate level of 0.05, and further analyzed for pathway enrichment (right-tailed Fisher’s Exact Test) and (in-) activation (z ≥|2|) using Ingenuity Pathway Analysis.ResultsAfter 0.05 Gy, least DEGs were found in N0 (n = 236), compared to N1 (n = 653) and N2+ (n = 694). The top DEGs with regard to the adjusted p-value were upregulated in fibroblasts across all donor groups (SESN1, MDM2, CDKN1A, TIGAR, BTG2, BLOC1S2, PPM1D, PHLDB3, FBXO22, AEN, TRIAP1, and POLH). Here, we observed activation of p53 Signaling in N0 and to a lesser extent in N1, but not in N2+. Only in N0, DNA (excision-) repair (involved genes: CDKN1A, PPM1D, and DDB2) was predicted to be a downstream function, while molecular networks in N2+ were associated with cancer, as well as injury and abnormalities (among others, downregulation of MSH6, CCNE2, and CHUK). After 2 Gy, the number of DEGs was similar in fibroblasts of all donor groups and genes with the highest absolute log2 fold-change were upregulated throughout (CDKN1A, TIGAR, HSPA4L, MDM2, BLOC1SD2, PPM1D, SESN1, BTG2, FBXO22, PCNA, and TRIAP1). Here, the p53 Signaling-Pathway was activated in fibroblasts of all donor groups. The Mitotic Roles of Polo Like Kinase-Pathway was inactivated in N1 and N2+. Molecular Mechanisms of Cancer were affected in fibroblasts of all donor groups. P53 was predicted to be an upstream regulator in fibroblasts of all donor groups and E2F1 in N1 and N2+. Results of the downstream analysis were senescence in N0 and N2+, transformation of cells in N0, and no significant effects in N1. Seven genes were differentially expressed in reaction to 2 Gy dependent on the donor group (LINC00601, COBLL1, SESN2, BIN3, TNFRSF10A, EEF1AKNMT, and BTG2).ConclusionOur results show dose-dependent differences in the radiation response between N1/N2+ and N0. While mechanisms against genotoxic stress were activated to the same extent after a high dose in all groups, the radiation response was impaired after a low dose in N1/N2+, suggesting an increased risk for adverse effects including carcinogenesis, particularly in N2+.

Transcriptome-Guided Identification of Pectin Methyl-Esterase-Related Enzymes and Novel Molecular Processes Effectuating the Hard-to-Cook Defect in Common Bean (Phaseolus vulgaris L.).

The hard-to-cook defect in common beans is dictated by the ability to achieve cell separation during cooking. Hydrolysis of pectin methyl-esters by the pectin methyl-esterase (PME) enzyme influences cell separation. However, the contributions of the PME enzyme and the cell wall to the hard-to-cook defect have not been studied using molecular tools. We compared relevant molecular processes in fast- and slow-cooking bean varieties to understand the mechanisms underpinning the hard-to-cook defect. A PME spectrophotometric assay showed minor differences in enzyme activity between varieties. Meanwhile, a PME HMMER search in the P. vulgaris genome unveiled 113 genes encoding PMEs and PME inhibitors (PMEIs). Through RNA sequencing, we compared the gene expression of the PME-related genes in both varieties during seed development. A PME (Phvul010g080300) and PMEI gene (Phvul005g007600) showed the highest expression in the fast- and slow-cooking beans, respectively. We further identified 2132 differentially expressed genes (DEGs). Genes encoding cell-wall-related enzymes, mainly glycosylphosphatidylinositol mannosyltransferase, xyloglucan O-acetyltransferase, pectinesterase, and callose synthase, ranked among the top DEGs, indicating novel relations to the hard-to-cook defect. Gene ontology mapping revealed hydrolase activity and protein phosphorylation as functional categories with the most abundant upregulated DEGs in the slow-cooking bean. Additionally, the cell periphery contained 8% of the DEGs upregulated in the slow-cooking bean. This study provides new insights into the role of pectin methyl-esterase-related genes and novel cell wall processes in the occurrence of the hard-to-cook defect.

Single Cell/Nucleus Transcriptomics Comparison in Zebrafish and Humans Reveals Common and Distinct Molecular Responses to Alzheimer's Disease.

Neurogenesis is significantly reduced in Alzheimer’s disease (AD) and is a potential therapeutic target. Contrary to humans, a zebrafish can regenerate its diseased brain, and thus is ideal for studying neurogenesis. To compare the AD-related molecular pathways between humans and zebrafish, we compared single cell or nuclear transcriptomic data from a zebrafish amyloid toxicity model and its controls (N = 12) with the datasets of two human adult brains (N = 10 and N = 48 (Microglia)), and one fetal brain (N = 10). Approximately 95.4% of the human and zebrafish cells co-clustered. Within each cell type, we identified differentially expressed genes (DEGs), enriched KEGG pathways, and gene ontology terms. We studied synergistic and non-synergistic DEGs to point at either common or uniquely altered mechanisms across species. Using the top DEGs, a high concordance in gene expression changes between species was observed in neuronal clusters. On the other hand, the molecular pathways affected by AD in zebrafish astroglia differed from humans in favor of the neurogenic pathways. The integration of zebrafish and human transcriptomes shows that the zebrafish can be used as a tool to study the cellular response to amyloid proteinopathies. Uniquely altered pathways in zebrafish could highlight the specific mechanisms underlying neurogenesis, which are absent in humans, and could serve as potential candidates for therapeutic developments.

Differential Retinal Ganglion Cell Vulnerability, A Critical Clue for the Identification of Neuroprotective Genes in Glaucoma.

Retinal ganglion cells (RGCs) are the neurons in the retina which directly project to the brain and transmit visual information along the optic nerve. Glaucoma, one of the leading causes of blindness, is characterized by elevated intraocular pressure (IOP) and degeneration of the optic nerve, which is followed by RGC death. Currently, there are no clinical therapeutic drugs or molecular interventions that prevent RGC death outside of IOP reduction. In order to overcome these major barriers, an increased number of studies have utilized the following combined analytical methods: well-established rodent models of glaucoma including optic nerve injury models and transcriptomic gene expression profiling, resulting in the successful identification of molecules and signaling pathways relevant to RGC protection. In this review, we present a comprehensive overview of pathological features in a variety of animal models of glaucoma and top differentially expressed genes (DEGs) depending on disease progression, RGC subtypes, retinal regions or animal species. By comparing top DEGs among those different transcriptome profiles, we discuss whether commonly listed DEGs could be defined as potential novel therapeutic targets in glaucoma, which will facilitate development of future therapeutic neuroprotective strategies for treatments of human patients in glaucoma.

Determinants of epigenetic resistance to HDAC inhibitors in dystrophic fibro-adipogenic progenitors.

Pharmacological treatment of Duchenne muscular dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials; however, pre‐clinical studies indicated that the beneficial effects of HDACi are restricted to early stages of disease. We show that FAPs from late‐stage mdx mice exhibit aberrant HDAC activity and genome‐wide alterations of histone acetylation that are not fully reversed by HDACi. In particular, combinatorial H3K27 and/or H3K9/14 hypo‐acetylation at promoters of genes required for cell cycle activation and progression, as well as glycolysis, are associated with their downregulation in late‐stage mdx FAPs. These alterations could not be reversed by HDACi, due to a general resistance to HDACi‐induced H3K9/14 hyperacetylation. Conversely, H3K9/14 hyper‐acetylation at promoters of Senescence Associated Secretory Phenotype (SASP) genes is associated with their upregulation in late‐stage mdx FAPs; however, HDACi could reduce promoter acetylation and blunt SASP gene activation. These data reveal that during DMD progression FAPs develop disease‐associated features reminiscent of cellular senescence, through epigenetically distinct and pharmacologically dissociable events. They also indicate that HDACi might retain anti‐fibrotic effects at late stages of DMD.