Aberrant Gene Expression Patterns Research Articles

Bulk RNA sequencing of human pediatric lung cell populations reveals unique transcriptomic signature associated with postnatal pulmonary development.

Postnatal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well-documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human postnatal lung development are yet to be fully understood. In this study, we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and nonendothelial mesenchymal cells, along with lung leukocytes) from 1-day-old to 8-yr-old organ donors with no known lung disease. For analysis, we considered the donors in four age groups [less than 30 days old neonates, 30 days to < 1 yr old infants, toddlers (1 to < 2 yr), and children 2 yr and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared with the number of DEG found during infant to toddler- or toddler to older children-transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.NEW & NOTEWORTHY This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.

Small molecules modulating RNA splicing: a review of targets and future perspectives

The review focuses on small molecules that modulate RNA splicing by interacting with a variety of targets, and in the context of disease treatment.

Single-cell transcriptional profiling reveals aberrant gene expression patterns and cell states in autoimmune diseases

Multiple sclerosis(MS), primary Sjögren syndrome (pSS), and systemic lupus erythematosus (SLE) share numerous clinical symptoms and serological characteristics. We analyzed 153550 cells of scRNA-seq data of 17 treatment-naive patients (5 MS, 5 pSS, and 7 SLE) and 10 healthy controls, and we examined the enrichment of biological processes, differentially expressed genes (DEGs), immune cell types, and their subpopulations, and cell-cell communication in peripheral blood mononuclear cells (PBMCs). The percentage of B cells, megakaryocytes, monocytes, and proliferating T cells presented significant changes in autoimmune diseases. The enrichment of cell types based on gene expression revealed an elevated monocyte. MIF, MK, and GALECTIN signaling networks were obvious differences in autoimmune diseases. Taken together, our analysis provides a comprehensive map of the cell types and states of ADs patients at the single-cell level to understand better the pathogenesis and treatment of these ADs.

Activating NSD2 Mutations Drive Oncogenic Reprogramming By Disturbing Epigenetic Landscape in Mantle Cell Lymphoma

Background: NSD2/MMSET/WHSC1, a histone lysine methyltransferase (HMT), is an oncoprotein first characterized by its overexpression in multiple myeloma (MM). NSD2 mutations within the catalytic SET domain are frequently identified in lymphoid malignancies including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL). We previously demonstrated that NSD2 p.E1099K mutation caused an imbalance of H3K36me2/H3K27me3 and drove glucocorticoid resistance in pediatric ALL. In MCL, NSD2 mutations, especially p.E1099K and p.T1150A, are found in 10-15% of cases of MCL and are enriched in MCL patients who relapse from targeted therapies such as ibrutinib, an inhibitor of B cell signaling. However, the activity of NSD2 mutations in MCL remains unexplored. We hypothesize that NSD2 mutations contribute to disease progression and therapy resistance due to aberrant chromatin modification. Aim: To demonstrate the role of NSD2 mutations in MCL progression due to aberrant chromatin modification. Methods: We created isogenic MCL cell lines by knock-in NSD2 p.E1099K and NSD2 p.T1150A mutation into Z138 and Jeko-1 cell lines using CRISPR/Cas9 gene editing. We then determined H3K36me2/H3K27me3/H3K36me3 using immunoblot and the biological activities including cell growth (IncuCyte), apoptosis (Annexin V/PI staining), cell cycle (BrdU incorporation), and in vivo experiments using luciferase-tagged cell lines in NOD-SCID mice. We determined alteration of gene transcriptome using RNA-Seq and histone modification using CUT&amp;RUN (H3K36me2, H3K27me3 and H3K4me3) and ChIP-Seq (H3K27ac) in isogenic Z138 cell lines. Finally, we integrated multi-omics analysis of our MCL cell lines and human patient samples and to comprehensively disclose the epigenetic landscape in MCL with NSD2 mutations. Results: We successfully created isogenic Z138 cell lines with NSD2 p.E1099K mutation or p.T1150A mutation, and isogenic Jeko-1 cell line with NSD2 p.E1099K mutation. Insertion of either NSD2 p.E1099K mutation or p.T1150A mutation by CRISPR/Cas9 gene editing led to a striking increase of H3K36me2, a decrease of H3K27me3 but no change of H3K36me3. NSD2 p.E1099K mutation significantly increased cellular growth and enhanced S phase entry while decreased G0/G1 phase and apoptosis. In NOD-SCID mouse xenografts, NSD2 p.E1099K mutation resulted in a higher tumor burden and shorter survival lifespan in mice. RNA-Seq analysis demonstrated striking changes in gene expression with 1794 genes upregulated and 1492 genes downregulated in NSD2 mutant MCL cells. Among them, anti-apoptotic genes BCL2 and BCL2L2 were upregulated while pro-apoptotic genes BAX, BID, and BIK were downregulated in NSD2 mutant cells, which was consistent with the biological phenotypes. Surprisingly, CCND1, CCNE1, CCNE2, TP53, and CDKN2C ( p21) were downregulated while CDKN2D, CDKL3, and CDKL5 were upregulated in NSD2 mutant cells. Altered gene expression in isogenic Z138 cells was compared with patient expression profiles, with gene ontology revealing significant overlap and enrichment of cell adhesion, neural pathways, and evidence of activated signaling. NSD2 mutation drove these oncogenic reprogramming by disturbing epigenetic landscape as increased levels of H3K36me2, H3K4me3 and H3K27ac but decreased levels of H3K27me3, which contributed to aberrant gene expression patterns. Conclusions: The NSD2 mutation led to increased tumor cell growth but decreased apoptosis due to the dysregulation of epigenetic landscape and gene expression, suggesting an oncogenic reprogramming driven by activated NSD2 mutations in MCL.

Dissolution of oncofusion transcription factor condensates for cancer therapy.

Cancer-associated chromosomal rearrangements can result in the expression of numerous pathogenic fusion proteins. The mechanisms by which fusion proteins contribute to oncogenesis are largely unknown, and effective therapies for fusion-associated cancers are lacking. Here we comprehensively scrutinized fusion proteins found in various cancers. We found that many fusion proteins are composed of phase separation-prone domains (PSs) and DNA-binding domains (DBDs), and these fusions have strong correlations with aberrant gene expression patterns. Furthermore, we established a high-throughput screening method, named DropScan, to screen drugs capable of modulating aberrant condensates. One of the drugs identified via DropScan, LY2835219, effectively dissolved condensates in reporter cell lines expressing Ewing sarcoma fusions and partially rescued the abnormal expression of target genes. Our results indicate that aberrant phase separation is likely a common mechanism for these PS-DBD fusion-related cancers and suggest that modulating aberrant phase separation is a potential route to treat these diseases.

Effects of Trichostatin A on the Histone Deacetylases (HDACs), Intrinsic Apoptotic Pathway, p21/Waf1/Cip1, and p53 in Human Neuroblastoma, Glioblastoma, Hepatocellular Carcinoma, and Colon Cancer Cell Lines

Background: The aberrant and altered patterns of gene expression play an important role in the biology of cancer and tumorigenesis. DNA methylation and histone deacetylation are the most studied epigenetic mechanisms. Histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA) and trichostatin A (TSA) are a group of anticancer compounds for the treatment of solid and hematological cancers. Previously, we reported the effect of two HDACIs, valproic acid (VPA) and TSA, on colon cancer and hepatocellular carcinoma (HCC), respectively. The aim of the current in vitro study is to investigate the effects of TSA on the intrinsic apoptotic pathway, p21/Waf1/Cip1 (p21), p53, and histone deacetylases (HDACs) 1, 2 and 3 in human neuroblastoma LAN-1, glioblastoma GBM-29, HCC SMMC7721, and colon cancer COLO 201 cell lines.&#x0D; Materials and methods: In this lab-trial study, all three cell lines were seeded at the density of 3 × 105 cells per well and incubated for 24 hours. Then, the cells were treated with TSA based on IC50 values for 24 hours except in the control groups; the control cells were treated with the equal amounts of the DMSO solvent. Subsequently, cell viability, cell apoptosis and gene expression were determined by three techniques including MTT assay, flow cytometry assay, and qRT-PCR.&#x0D; Results: The result of qRT-PCR indicated that TSA could increase the expression levels of Bid, BimEL, Noxa, p21, and p53 genes and decrease those of Bcl-xL, RIP, Mcl-1, XIAP, HDACs 1, 2 and 3 significantly (P &lt; 0.0001) by which it inhibited cell growth and induced significant cell apoptosis in LAN-1, GBM-29, SMMC7721, and COLO 201 cell lines (p value&lt;0.001).&#x0D; Conclusion: TSA can affect cell apoptotic via the intrinsic apoptotic pathway in LAN-1, GBM-29, SMMC7721, and COLO 201 cell lines.

Viewing AML through a New Lens: Technological Advances in the Study of Epigenetic Regulation

Epigenetic modifications, such as histone modifications and DNA methylation, are essential for ensuring the dynamic control of gene regulation in every cell type. These modifications are associated with gene activation or repression, depending on the genomic context and specific type of modification. In both cases, they are deposited and removed by epigenetic modifier proteins. In acute myeloid leukemia (AML), the function of these proteins is perturbed through genetic mutations (i.e., in the DNA methylation machinery) or translocations (i.e., MLL-rearrangements) arising during leukemogenesis. This can lead to an imbalance in the epigenomic landscape, which drives aberrant gene expression patterns. New technological advances, such as CRISPR editing, are now being used to precisely model genetic mutations and chromosomal translocations. In addition, high-precision epigenomic editing using dCas9 or CRISPR base editing are being used to investigate the function of epigenetic mechanisms in gene regulation. To interrogate these mechanisms at higher resolution, advances in single-cell techniques have begun to highlight the heterogeneity of epigenomic landscapes and how these impact on gene expression within different AML populations in individual cells. Combined, these technologies provide a new lens through which to study the role of epigenetic modifications in normal hematopoiesis and how the underlying mechanisms can be hijacked in the context of malignancies such as AML.

Transcriptome Analysis of Protection by Dendrobium Nobile Alkaloids (DNLA) against Chronic Alcoholic Liver Injury in Mice.

To investigate the protective effects of Dendrobium nobile Lindl. alkaloids (DNLA) against chronic alcoholic liver injury. C57BL/6J mice were fed with the Lieber-DeCarli alcohol diet to induce chronic alcoholic liver injury. DNLA (20 mg/kg/day) was gavaged along with the alcohol diet for 28 days. Liver injury was evaluated by serum enzymes. Triglyceride levels, histopathology, and transcriptome changes were examined by RNA-Seq and qPCR. DNLA decreased serum triglyceride levels in mice receiving alcohol. Hepatocyte degeneration and steatosis were ameliorated by DNLA, as evidenced by H&E and Oil-red O staining. DNLA brought the alcohol-induced aberrant gene expression pattern towards normal. Alcohol induced 787 differentially expressed genes (padj &lt; 0.01). DNLA induced 280 differentially expressed genes to a much less extent. Ingenuity pathway analysis showed that DNLA ameliorated alcohol-induced oxidative stress and xenobiotic metabolism disruption. qPCR verified that DNLA alleviated over-activation of Cyp2a4, Cyp2b10, and Abcc4; attenuated oxidative stress (Hmox1, Gstm3, Nupr1), reduced the expression of Nrf2 genes (Nqo1, Gclc, Vldlr); and rescued some metabolic genes (Insig1, Xbp1, Socs3, Slc10a2). In conclusion, DNLA was effective against alcohol-induced fatty liver disease, and the protection may be attributed to alleviated oxidative stress and restored metabolism homeostasis, probably through modulating nuclear receptor CAR-, PXR-, and Nrf2-mediated gene expression pathways.

Aberrant gene expression pattern in the glycolysis-cholesterol synthesis axis is linked with immune infiltration and prognosis in prostate cancer: A bioinformatics analysis.

Aberrant lipid metabolism is an early event in tumorigenesis and has been found in a variety of tumor types, especially prostate cancer (PCa). Therefore, We hypothesize that PCa can be stratified into metabolic subgroups based on glycolytic and cholesterogenic related genes, and the different subgroups are closely related to the immune microenvironment. Bioinformatics analysis of genomic, transcriptomic, and clinical data from a comprehensive cohort of PCa patients was performed. Datasets included the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) dataset, GSE70768, our previously published PCa cohort. The unsupervised cluster analysis was employed to stratify PCa samples based on the expression of metabolic-related genes. Four molecular subtypes were identified, named Glycolytic, Cholesterogenic, Mixed, and Quiescent. Each metabolic subtype has specific features. Among the 4 subtypes, the cholesterogenic subtype exhibited better median survival, whereas patients with high expression of glycolytic genes showed the shortest survival. The mitochondrial pyruvate carriers (MPC) 1 exhibited expression difference between PCa metabolic subgroups, but not for MPCs 2. Glycolytic subtypes had lower immune cell scores, while Cholesterogenic subgroups had higher immune cell scores. Our results demonstrated that metabolic classifications based on specific glycolytic and cholesterol-producing pathways provide new biological insights into previously established subtypes and may guide develop personalized therapies for unique tumor metabolism characteristics.

Battle with Cancer: Prevalence in Different Districts and What Needs to Be Done?

Cancer is characterized by abnormal gene function and aberrant patterns of gene expression. A growing body of data suggests that acquired epigenetic aberrations work in concert with genetic changes to generate this deregulation. According to a WHO research, India saw 1.16 million new cancer cases in 2018, with one out of every ten Indians developing cancer at some point in their life and one out of every 15 dying from it. According to the study, tobacco-related head and neck cancers are frequent in India, especially oral cancer in males and cervical cancer in women, and both cancer types are linked to lower socioeconomic position. Conclusion: Tobacco use is becoming a leading cause of cancer in the North East, and it is estimated that tobacco use is responsible for 22% of cancer deaths worldwide. A comprehensive cancer control program is the need of the hour to prevent this vibrant region from becoming the cancer capital of the nation.

Epigenetic Factors in Eutopic Endometrium in Women with Endometriosis and Infertility.

Eutopic endometrium in patients with endometriosis is characterized by aberrant expression of essential genes during the implantation window. It predisposes to disturbance of endometrial receptivity. The pathomechanism of implantation failures in women with endometriosis remains unclear. This paper aims to summarize the knowledge on epigenetic mechanisms in eutopic endometrium in the group of patients with both endometriosis and infertility. The impaired DNA methylation patterns of gene promoter regions in eutopic tissue was established. The global profile of histone acetylation and methylation and the analysis of selected histone modifications showed significant differences in the endometrium of women with endometriosis. Aberrant expression of the proposed candidate genes may promote an unfavorable embryonic implantation environment of the endometrium due to an immunological dysfunction, inflammatory reaction, and apoptotic response in women with endometriosis. The role of the newly discovered proteins regulating gene expression, i.e., TET proteins, in endometrial pathology is not yet completely known. The cells of the eutopic endometrium in women with endometriosis contain a stable, impaired methylation pattern and a histone code. Medication targeting critical genes responsible for the aberrant gene expression pattern in eutopic endometrium may help treat infertility in women with endometriosis.

EP139: Spatial transcriptomic approaches for characterizing childhood intellectual disability

The genetic characterization of childhood intellectual disability (ID) as a form of developmental delay has undergone extensive study. Genome sequencing (GS) and exome sequencing (ES) have emerged as the standard test of choice for diagnostic uses as a result of evidence based studies. The American College of Medical Genetics and Genomics recently issued clinical guidelines where GS/ES was recommended in first-tier testing confirmation of suspected intellectual disability. This abstract proposes the application of spatial transcriptomic methods to reveal gene expression patterns and cellular and tissue environments in diagnosed pediatric patients with intellectual disability towards establishing potential medically actionable findings for these patients. A review will be performed using keywords “intellectual disability”, AND “whole transcriptomic” and “whole exome sequencing” and “whole genome sequencing” AND “intellectual disability” and “signaling pathways” AND “intellectual disability” and “cellular and tissue heterogeneity” AND “intellectual disability” and “gene expression” AND “intellectual disability” as inclusion criteria to propose a set of standards and methods for spatial transcriptomic approaches for revealing tissue and cellular heterogeneity and gene expression patterns for characterizing childhood intellectual disability. “Single cell sequencing” is also included in the search methods. “Epilepsy” and “autism spectrum disorder” were excluded search criteria. ID is a medical disorder characterized by reduction in cognitive and intellectual function and impaired social adaptation, affecting 1-3% of the worldwide population. No pharmacological therapies are currently available. Approximately 1000 genes have been discovered in ID indicating significant genetic heterogeneity. A significant level of genetic heterogeneity has been demonstrated in ID. In a whole transcriptomic analysis using RNA-Seq in consanguineous families with ID, it was found that SHTN1 was down-regulated while FGFR2 gene was upregulated. Gene expression alterations were also observed in genes affecting neuronal and actin cytoskeletal function in ID families. The Rho GTPase signaling pathway was associated with ID being implicated in the actin-rich nature of dendritic spines, reflecting impaired cytoskeleton remodeling and impacting cell processes such as neuronal migration and synaptic plasticity. Variants in the Rho GTPase family are significant in that they are key regulators of actin dynamics and organization. Systems biology approaches such as spatial transcriptomics focus on the overall interconnected network of individual genes and spatial distribution of mRNA molecules and allow for the revealing of cellular heterogeneity in tissues and the subcellular distribution of transcripts in conditions such as childhood ID. Individual cell functioning is only sufficiently explained in the context of identifying their exact locations and spatial transcriptomics methods connect gene expression to the spatial organization of cells. Techniques include fluorescent in situ hybridization, in situ sequencing and in silico approaches and could be applied to understanding more profoundly the cellular and tissue behavior of ID and suggesting a spatially-informed model of the condition. This is particularly significant since the actin cytoskeleton and molecular pathways such as tyrosine kinase pathways and GTPase signaling have been observed in ID. I will describe how these techniques could potentially apply to ID, and propose a set of spatial transcriptomics approaches to generate insight into the aberrant signaling pathways, cellular networks, tissue heterogeneity and gene expression patterns for this condition, and what information could potentially result of as a result of these methods, which may constitute a new contribution to the literature on ID. Spatial transcriptomics gives the ability to researchers to comprehend the spatial component of cell-to-cell signaling networks that are implicated in neurobiological disorders such as ID.

The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase

Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.

Neuronal and cardiac toxicity of pharmacological compounds identified through transcriptomic analysis of human pluripotent stem cell-derived embryoid bodies

Concurrent with the ‘3R’ principle, the embryonic stem cell test (EST) using mouse embryonic stem cells, developed in 2000, remains the solely accepted in vitro method for embryotoxicity testing. However, the scope and implementation of EST for embryotoxicity screening, compliant with regulatory requirements, are limited. This is due to its technical complexity, long testing period, labor-intensive methodology, and limited endpoint data, leading to misclassification of embryotoxic potential. In this study, we used human induced pluripotent stem cell (hiPSC)-derived embryoid bodies (EB) as an in vitro model to investigate the embryotoxic effects of a carefully selected set of pharmacological compounds. Morphology, viability, and differentiation potential were investigated after exposing EBs to folic acid, all-trans-retinoic acid, dexamethasone, and valproic acid for 15 days. The results showed that the compounds differentially repressed cell growth, compromised morphology, and triggered apoptosis in the EBs. Further, transcriptomics was employed to compare subtle temporal changes between treated and untreated cultures. Gene ontology and pathway analysis revealed that dysregulation of a large number of genes strongly correlated with impaired neuroectoderm and cardiac mesoderm formation. This aberrant gene expression pattern was associated with several disorders of the brain like mental retardation, multiple sclerosis, stroke and of the heart like dilated cardiomyopathy, ventricular tachycardia, and ventricular arrhythmia. Lastly, these in vitro findings were validated using in ovo chick embryo model. Taken together, pharmacological compound or drug-induced defective EB development from hiPSCs could potentially be used as a suitable in vitro platform for embryotoxicity screening.

Canonical Notch Signaling Is Required for KrasG12D -Induced Myeloproliferative Neoplasm

Notch is a highly conserved signaling pathway that regulates cell-fate specification and tissue homeostasis in various contexts. Depending on the context, Notch signaling could be oncogenic or tumor suppressive. The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function (GOF) NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias.To investigate the role of Notch signaling in T cell acute lymphoblastic leukemia/lymphoma (T-ALL) and myeloproliferative neoplasm (MPN) induced by Ras mutation, we downregulated Notch signaling in oncogenic Kras (KrasG12D) mouse model, which develops T-ALL and MPN simultaneously. We took two independent genetic approaches to downregulate Notch signaling. Mx1-Cre-mediated conditional expression of dominant-negative Mastermind-like1 (DNMAML) potently inhibits canonical Notch-mediated transcriptional activation, while knocking out Protein O-fucosyltransferase 1 (Pofut1) reduces interactions of Notch receptors and their ligands and downregulates Notch signaling. To study the cell-autonomous functions of downregulating Notch signaling in oncogenic Kras -induced leukemogenesis, we transplanted either bone marrow cells or splenocytes into lethally irradiated mice.As expected, inhibition of Notch signaling significantly inhibited T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras, preserved intact Notch signaling, and acquired GOF Notch1 mutation(s). Surprisingly, we found that downregulation of Notch signaling inhibited MPN development in a cell-autonomous manner. Analysis of donor-derived hematopoiesis revealed that loss of Notch signaling significantly reduced myeloid compartment in Kras recipients with significant decrease of proliferation. Further mechanistic studies indicate that inhibition of Notch signaling significantly upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. RNA-Seq analysis of control and mutant myeloid progenitors (MPs) demonstrated that KrasG12D MPs exhibited enhanced oxidative phosphorylation and mitochondria respiration, and this aberrant gene expression pattern was largely restored by DNMAML expression. These metabolic changes were further validated in functional assays. Furthermore, we demonstrated that combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target Notch downstream mechanisms in oncogenic Ras-driven myeloid leukemias. DisclosuresPadron:Incyte: Honoraria, Research Funding.

Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that is causally associated with various lymphomas and carcinomas. Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from MM patients. Here, we analyzed two EBV-positive MM-patient-derived cell lines, IM9 and ARH77, and found defective viral genomes and atypical viral gene expression patterns. We performed transcriptome sequencing to characterize the viral and cellular properties of the two EBV-positive cell lines, compared to the canonical MM cell line 8226. Principal-component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226. Immunological Genome Project analysis designated these cells as stem cell and bone marrow derived. IM9 and ARH77 displayed atypical viral gene expression, including leaky lytic cycle gene expression with an absence of lytic DNA amplification. Genome sequencing revealed that the EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 showed EBV genome heterogeneity, suggesting cells harboring multiple and variant viral genomes. We identified atypical high-level expression of lytic genes BLRF1 and BLRF2. We demonstrated that short hairpin RNA (shRNA) depletion of BLRF2 altered viral and host gene expression, including a reduction in lytic gene activation and DNA amplification. These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B cells derived from MM tumors, and they suggest that EBV may contribute to the etiology of MM. IMPORTANCE EBV is an oncogenic herpesvirus, but its mechanisms of oncogenesis are not fully understood. A role for EBV in MM has not yet been established. We analyzed EBV-positive B-cell lines derived from MM patients and found that the cells harbored defective viral genomes with aberrant viral gene expression patterns and cell gene signatures for bone marrow-derived lymphoid stem cells. These findings suggest that aberrant EBV latent infection may contribute to the etiology of MM.

Exploring the Role of a Novel Tumor Suppressor - OSR2 in T-ALL

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, representing &amp;gt;25% of all cancers in children 0-14 years. Despite major advancements in pediatric ALL treatment, it remains the second most lethal childhood cancer, accounting for ~25% of deaths. The two types of ALL are precursor-B, or B-ALL, and precursor-T, or T-ALL, which have distinct molecular landscapes. Of these types, T-ALL comprises about 15% and 25% of pediatric and adult cases, respectively, and is historically considered more aggressive and treatment-resistant, with an inferior prognosis. In the precision medicine era, it is imperative to identify genetic alterations and aberrant gene expression patterns, to better understand tumor biology and improve treatment outcomes by identifying new therapeutic targets. Our study investigates a novel transcription factor, odd-skipped related transcription factor 2 (OSR2), which we hypothesize is a putative T-ALL tumor suppressor. We are using a zebrafish T-ALL model expressing transgenic human MYC (hMYC) regulated by a lymphoblast-specific promoter, rag2. Prior work in zebrafish and human T-ALL found low OSR2 levels in ~95% of T-ALL. Based on this, we then used RNA-seq to analyze 10 hMYC zebrafish T-ALL, confirming low-to-absent osr2 in all 10 T-ALL relative to wild-type (WT) T cells. We further confirmed decreased osr2 expression by qRT-PCR of additional T-ALL and WT thymocytes. We hypothesized that if OSR2 suppresses T-ALL, impaired zebrafish Osr2 function might increase T-ALL incidence and shorten latency. To test this, we bred osr2-mutant fish to rag2:hMYC transgenic animals to create three genotypes: heterozygous osr2-mutant (osr2het) fish, heterozygous hMYC (hMYChet) fish, and compound-heterozygote (osr2het;hMYChet) fish. We screened these genotypes for T-ALL incidence by serial fluorescence microscopy, with T-ALL subsequently confirmed by fluorescence-based flow cytometry. By 7 months of age, we found 9/18 (50%) of double-heterozygous fish developed T-ALL, compared to 0/7 hMYChet fish (p = 0.026); osr2het fish also did not develop T-ALL. Together, our findings suggest osr2 allelic loss accelerates MYC-driven T-ALL, supporting our hypothesis that osr2 is a T-ALL tumor suppressor. Disclosures No relevant conflicts of interest to declare.

Glomerular Endothelial Cells as Instigators of Glomerular Sclerotic Diseases.

Glomerular endothelial cell (GEnC) dysfunction is important in the pathogenesis of glomerular sclerotic diseases, including Focal Segmental Glomerulosclerosis (FSGS) and overt diabetic nephropathy (DN). GEnCs form the first cellular barrier in direct contact with cells and factors circulating in the blood. Disturbances in these circulating factors can induce GEnC dysfunction. GEnC dysfunction occurs in early stages of FSGS and DN, and is characterized by a compromised endothelial glycocalyx, an inflammatory phenotype, mitochondrial damage and oxidative stress, aberrant cell signaling, and endothelial-to-mesenchymal transition (EndMT). GEnCs are in an interdependent relationship with podocytes and mesangial cells, which involves bidirectional cross-talk via intercellular signaling. Given that GEnC behavior directly influences podocyte function, it is conceivable that GEnC dysfunction may culminate in podocyte damage, proteinuria, subsequent mesangial activation, and ultimately glomerulosclerosis. Indeed, GEnC dysfunction is sufficient to cause podocyte injury, proteinuria and activation of mesangial cells. Aberrant gene expression patterns largely contribute to GEnC dysfunction and epigenetic changes seem to be involved in causing aberrant transcription. This review summarizes literature that uncovers the importance of cross-talk between GEnCs and podocytes, and GEnCs and mesangial cells in the context of the development of FSGS and DN, and the potential use of GEnCs as efficacious cellular target to pharmacologically halt development and progression of DN and FSGS.

Identification of PDE7B as a Potential Core Gene Involved in the Metastasis of Clear Cell Renal Cell Carcinoma.

BackgroundMetastasis is the main cause of treatment failure in various cancer, including ccRCC. However, the key genes involved in ccRCC metastasis remain largely unknown.PurposeThe identification of the aberrant gene expression patterns associated with metastatic traits is of great clinical significance. The aim of this study was to investigate the clinical significance and function of PDE7B in ccRCC.Materials and MethodsExpression profiling data for patient-matched primary and metastatic ccRCC tumors were obtained from GEO Dataset. Limma package was used to identify differentially expressed genes (DEGs) between the metastatic and the primary groups. Gene Ontology, Kyoto Encyclopedia of Genes Genomes (KEGG), and PPI network analysis were used to study the interacting activities and the interconnection of the DEGs. CCK-8 assays and Transwell assays were performed to detect the proliferation and migration of renal cancer cells.ResultsWe obtained 163 DEGs, including 132 that were upregulated and 31 that were downregulated in metastatic ccRCC tissues. Both Gene Ontology function and KEGG pathway analysis showed that DEGs were involved in extracellular matrix (ECM) organization and cell adhesion. After utilizing PPI network to explore the interconnection among the DEGs, 22 genes were selected as the hub genes. Subsequently, survival analysis revealed that seven hub genes (SFN, NKX2-1, HP, MAPT, EPHA4, KCNAB1, and PDE7B) were significantly associated with overall survival disease-specific survival, and progression-free interval in ccRCC. Moreover, the low expression of PDE7B was found in clinical ccRCC samples and correlated with TNM stage and histologic grade. We further showed that knockdown of PDE7B increased cell growth and migration of renal cancer cells.ConclusionOur results implicated that PDE7B may play a key role in the development of metastatic RCC.