Public Gene Expression Datasets Research Articles

Abstract 823: Gene expression analysis reveals a pitfall in the molecular research of prostate tumors relevant to Gleason score

Abstract Gleason score (GS) is a powerful prognostic factor in prostate cancer (PCa). A GS-7 tumor typically has the primary architectural pattern and secondary prevalent one being graded as 3 and 4 or 4 and 3, respectively. Due to the well-known multifocal occurrence of different patterns, a biological sample from a GS-7 tumor used in a molecular experiment will be uncertain regarding the actually represented pattern if no special attention is given to specimen preparation. In this study, by an integrative analysis of public gene expression datasets for PCa, we demonstrate that such an uncertainty is not uncommon. First, based on a unique dataset (Sowalsky, et.al, Clin. Cancer Res., 2017) of paired Gleason pattern 3 (GP-3) and Gleason pattern 4 (GP- 4) specimens of 13 GS-7 tumors, we identify 303 differentially expressed genes with respect to those two architectural patterns. Then, using the TCGA data, we show that the profile of these genes is a strong signature for distinguishing the GS-6 (3+3) tumors from the GS-8 (4+4) ones (AUC = 0.96, p-value = 9*10E-17), implying that the differences of transcription program between the GS-8 (4+4) and GS-6 (3+3) samples resemble the differences between the GP-3 and GP-4 specimens. We further demonstrate that the co-expression pattern of the signature genes in the subset of the GS-7 samples resembles that present in the aggregate of GS-6 and GS-8 samples. Finally, we verify the findings using the GSE21032 data. Our results suggest that the GS-7 specimens used to generate two frequently-cited genome-wide expression datasets largely are individual GP-3 or GP-4 specimens rather than the “intermediate” specimens of GP-3 and GP-4. This provides molecular evidence for the aforementioned uncertainty of GS-7 tumor samples, indicating a potential pitfall in the existing molecular and translational research of prostate tumors relevant to GS. Citation Format: Wensheng Zhang, Yan Dong, Kun Zhang. Gene expression analysis reveals a pitfall in the molecular research of prostate tumors relevant to Gleason score [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 823.

Genomic and transcriptomic analyses in Drosophila suggest that the ecdysteroid kinase-like (EcKL) gene family encodes the ‘detoxification-by-phosphorylation’ enzymes of insects

Phosphorylation is a phase II detoxification reaction that, among animals, occurs near exclusively in insects, but the enzymes responsible have never been cloned or otherwise identified. We propose the hypothesis that members of the arthropod-specific ecdysteroid kinase-like (EcKL) gene family encode detoxicative kinases. To test this hypothesis, we annotated the EcKL gene family in 12 species of Drosophila and explored their evolution within the genus. Many ancestral EcKL clades are evolutionarily unstable and have experienced repeated gene gain and loss events, while others are conserved as single-copy orthologs. Leveraging multiple published gene expression datasets from D. melanogaster, and using the cytochrome P450s—a classical detoxification family—as a test case, we demonstrate relationships between xenobiotic induction, detoxification tissue-enriched expression and evolutionary instability in the EcKLs and the P450s. We devised a systematic method for identifying candidate detoxification genes in large gene families that is concordant with experimentally determined functions of P450 genes in D. melanogaster. Applying this method to the EcKLs suggested a significant proportion of these genes play roles in detoxification, and that the EcKLs may constitute a detoxification gene family in insects. Additionally, we estimate that between 11 and 16 uncharacterised D. melanogaster P450s are strong detoxification candidates. Lastly, we also found previously unreported genomic and transcriptomic variation in a number of EcKLs and P450s associated with toxic stress phenotypes using a targeted phenome-wide association study (PheWAS) approach in D. melanogaster, presenting multiple future avenues of research for detoxification genetics in this species.

Identification of transcription factors MYC and C/EBP\u03b2 mediated regulatory networks in heart failure based on gene expression omnibus datasets

BackgroundHeart failure is one of leading cause of death worldwide. However, the transcriptional profiling of heart failure is unclear. Moreover, the signaling pathways and transcription factors involving the heart failure development also are largely unknown. Using published Gene Expression Omnibus (GEO) datasets, in the present study, we aim to comprehensively analyze the differentially expressed genes in failing heart tissues, and identified the critical signaling pathways and transcription factors involving heart failure development.MethodsThe transcriptional profiling of heart failure was identified from previously published gene expression datasets deposited in GSE5406, GSE16499 and GSE68316. The enriched signaling pathways and transcription factors were analyzed using Database for Annotation, Visualization and Integrated Discovery (DAVID) website and gene set enrichment analysis (GSEA) assay. The transcriptional networks were created by Cytoscape.ResultsCompared with the normal heart tissues, 90 genes were particularly differentially expressed in failing heart tissues, and those genes were associated with multiple metabolism signaling pathways and insulin signaling pathway. Metabolism and insulin signaling pathway were both inactivated in failing heart tissues. Transcription factors MYC and C/EBPβ were both negatively associated with the expression profiling of failing heart tissues in GSEA assay. Moreover, compared with normal heart tissues, MYC and C/EBPβ were down regulated in failing heart tissues. Furthermore, MYC and C/EBPβ mediated downstream target genes were also decreased in failing heart tissues. MYC and C/EBPβ were positively correlated with each other. At last, we constructed MYC and C/EBPβ mediated regulatory networks in failing heart tissues, and identified the MYC and C/EBPβ target genes which had been reported involving the heart failure developmental progress.ConclusionsOur results suggested that metabolism pathways and insulin signaling pathway, transcription factors MYC and C/EBPβ played critical roles in heart failure developmental progress.

Airways Expression of SARS-CoV-2 Receptor, ACE2, and TMPRSS2 Is Lower in Children Than Adults and Increases with Smoking and COPD

It has been reported that angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are the main cell entry proteins for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and play a critical role in causing coronavirus disease 2019 (COVID-19). To investigate the expression level of these SARS-CoV-2 host cell entry genes in the lung airway, we used public gene expression datasets. We have found a differential expression of ACE2 and TMPRSS2 in nasal and bronchial airways relative to age and diseases status. Children were found to have significantly lower expression of COVID-19 receptors in the upper and lower airways (nasal and bronchial). Moreover, the lung airway expression of both ACE2 and TMPRSS2 was found to be significantly upregulated in smokers compared with non-smokers, and in patients with chronic obstructive pulmonary disease (COPD) compared with healthy subjects. No difference was observed in the blood expression levels of ACE2 and TMPRSS2 between children and adults, or in COPD or diabetic patients. However, a significant increase in blood expression levels of these genes was observed in patients with essential hypertension, whereas only ACE2 was upregulated in the blood of asthmatics. These results suggest that the observed difference in COVID-19 severity between children and adults could, in part, be attributed to the difference in ACE2 and TMPRSS2 airways tissue expression levels.

AGXT2L1 is downregulated in carcinomas of the digestive system

Alanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1) is a modulator of phospholipid metabolism, and its role in tumor biology is obscure. Previously, significant downregulation of AGXT2L1 has been observed in hepatocellular carcinoma. The aim of the present study was to investigate AGXT2L1 expression and its association with the clinical characteristics of common carcinomas of the digestive system. In the present study, the expression levels of AGXT2L1 were detected by immunohistochemical staining in colorectal cancer (CRC), gastric cancer and pancreatic cancer tissues. The associations between AGXT2L1 expression and clinicopathological features were analyzed using public gene expression datasets. Small interfering RNA was transfected into SW480 and HCT116 cells to explore the role of AGXT2L1 in CRC cells. AGXT2L1 expression was significantly decreased in cancerous tissues compared with in normal tissues, and low AGXT2L1 expression was associated with an unfavorable prognosis in patients. Furthermore, it was revealed that AGXT2L1 may regulate phosphatidylinositol and phosphatidylserine metabolism in cancerous tissues, and that decreased AGXT2L1 expression could induce autophagy in CRC cells. Overall, the present study provides a basis for further understanding of the role of AGXT2L1 and its association with autophagy in cancer.

Computational Approach to Identifying Universal Macrophage Biomarkers.

Macrophages engulf and digest microbes, cellular debris, and various disease-associated cells throughout the body. Understanding the dynamics of macrophage gene expression is crucial for studying human diseases. As both bulk RNAseq and single cell RNAseq datasets become more numerous and complex, identifying a universal and reliable marker of macrophage cell becomes paramount. Traditional approaches have relied upon tissue specific expression patterns. To identify universal biomarkers of macrophage, we used a previously published computational approach called BECC (Boolean Equivalent Correlated Clusters) that was originally used to identify conserved cell cycle genes. We performed BECC analysis using the known macrophage marker CD14 as a seed gene. The main idea behind BECC is that it uses massive database of public gene expression dataset to establish robust co-expression patterns identified using a combination of correlation, linear regression and Boolean equivalences. Our analysis identified and validated FCER1G and TYROBP as novel universal biomarkers for macrophages in human and mouse tissues.

Maternal blood EBF1-based microRNA transcripts as biomarkers for detecting risk of spontaneous preterm birth: a nested case-control study

Objective Both genetic variants and maternal blood mRNA levels of EBF1 gene have been linked to sPTB. Animal and human studies suggest that specific EBF1-based miRNAs are involved in various physiological and pathophysiological processes. However, to date, we did not find any reports of EBF1-based miRNAs or miRNA transcripts in relation to sPTB. We therefore aimed to examine whether maternal blood early B cell factor 1 (EBF1) gene-based microRNA (miRNA) transcripts can be used for detecting risk of spontaneous preterm birth (sPTB). Methods We conducted a nested case-control study within a Canadian cohort consisting of 1878 singleton pregnancies enrolled from May 2008 to December 2010 in Calgary, Alberta, Canada. We used a public gene expression dataset (GSE59491) derived from maternal blood in trimesters 2–3 that included women with sPTB (n = 51) and term births (n = 106) matched for maternal age, race/ethnicity, pre-pregnancy body mass index, smoking during pregnancy, and parity within the Canadian cohort. Two bioinformatics tools, miRWalk and STarMirDB, with different algorithms were applied to retrieve miRNA transcripts that putatively target the EBF1 gene (i.e. EBF1-based). Limma moderated t-tests were used to examine differentially expressed (DE) miRNA transcripts (sPTB vs term) within trimesters. Logistic regression models with miRNA transcript tertiles were applied to assess threshold associations between candidate miRNA transcripts’ levels and sPTB. Receiver operating characteristic (ROC) analyses were used to identify the maximum Youden Index and its corresponding optimal sensitivity/specificity cut-point of EBF1-based miRNA transcripts for classifying sPTB, and to compare the classification performance of a linear combination (score) of miRNA transcripts with that of individual miRNA transcripts. A five-fold cross-validation was applied to examine the possible overfitting problem of the final ROC model. Results Four maternal blood EBF1-based miRNA transcripts (MIR4266, MIR1251, MIR601, MIR3612) in the 3rd trimester were significantly associated with sPTB. The odds ratios (95%CIs) for highest versus lowest tertile of the four miRNA transcripts were 3.01–5.25(1.21–13.14, p ≤ .018). The combined 4-miRNA transcripts’ score significantly improved the classification of sPTB compared to individual miRNA transcripts (AUC increased from 0.65–0.69 to 0.82, p ≤ .0034) and showed a sensitivity for sPTB of 0.81 and a specificity of 0.72. The final ROC model of the EBF1-based 4 miRNA transcripts’ score in cases and controls had no significant overfitting issue. Conclusions Maternal blood EBF1-based miRNA transcripts may, along with other biomarkers, be useful in screening for sPTB risk in 3rd trimester. Our results also provide clues for further study of potential molecular mechanisms underlying the relationship between EBF1 gene and sPTB, e.g. connecting genetic variants, mRNA expression, and miRNA regulation.

In silico analysis of the immunological landscape of pituitary adenomas.

Immunotherapy has gained traction in the treatment of solid tumors but the immunological landscape of pituitary adenomas is not well defined. We sought to investigate the immunological composition in pituitary adenomas using RNA deconvolution (CIBERSORTx) on an existing gene expression dataset for pituitary adenomas. We applied an established computational approach (CIBERSORTx) on 134 pituitary adenomas from a previously published gene expression dataset to infer the proportions of 22 subsets of immune cells. We investigated associations between each immune cell type and tumor subtype. We found that the majority of infiltrating immune cells within pituitary adenomas were comprised of M2 macrophages followed by resting CD4+ memory T cells and mast cells. Silent pituitary tumors have higher M2 macrophage fractions when compared to other subtypes. In contrast, Cushing pituitary tumors, both overt and subclinical cases, had higher CD8+ T cells fractions than GH tumors, prolactinomas, hyperthyroid tumors, and silent tumors. RNA deconvolution of the immune infiltrates of pituitary adenomas using CIBERSORTx suggests that most pituitary adenomas comprise of M2 macrophages, but each adenoma subtype has a unique immune landscape. This may have implications in targeting each adenoma subtype with different immunotherapies.

Development of a Bioinformatics Framework for Identification and Validation of Genomic Biomarkers and Key Immunopathology Processes and Controllers in Infectious and Non-infectious Severe Inflammatory Response Syndrome.

Sepsis is defined as dysregulated host response caused by systemic infection, leading to organ failure. It is a life-threatening condition, often requiring admission to an intensive care unit (ICU). The causative agents and processes involved are multifactorial but are characterized by an overarching inflammatory response, sharing elements in common with severe inflammatory response syndrome (SIRS) of non-infectious origin. Sepsis presents with a range of pathophysiological and genetic features which make clinical differentiation from SIRS very challenging. This may reflect a poor understanding of the key gene inter-activities and/or pathway associations underlying these disease processes. Improved understanding is critical for early differential recognition of sepsis and SIRS and to improve patient management and clinical outcomes. Judicious selection of gene biomarkers suitable for development of diagnostic tests/testing could make differentiation of sepsis and SIRS feasible. Here we describe a methodologic framework for the identification and validation of biomarkers in SIRS, sepsis and septic shock patients, using a 2-tier gene screening, artificial neural network (ANN) data mining technique, using previously published gene expression datasets. Eight key hub markers have been identified which may delineate distinct, core disease processes and which show potential for informing underlying immunological and pathological processes and thus patient stratification and treatment. These do not show sufficient fold change differences between the different disease states to be useful as primary diagnostic biomarkers, but are instrumental in identifying candidate pathways and other associated biomarkers for further exploration.

Highly expressed ribosomal protein L34 predicts poor prognosis in acute myeloid leukemia and could be a potential therapy target

B ackground: Acute myeloid leukemia (AML) is a devastating malignancy with great heterogeneity, novel prognostic biomarkers and therapy targets are needed to improve the precise management of AML patients. Increasing evidence has shown the role of RPL34, a ribosomal protein, in tumorigenesis and progression. However, the detailed expression status and clinical significance of RPL34 in AML are largely unknown. M ethods: The expression level of RPL34 was detected in bone marrow samples from both AML patients and AML cell lines. Then using recombinant shRNA-lentiviral vector, we analyzed the impact of RPL34 knockdown on cell proliferation, apoptosis and cell cycle distribution. Lastly, by analyzing public gene expression datasets (GSE12417 and GSE2191), we determined the prognostic role of RPL34 in AML. Results: The mRNA level of RPL34 was significantly elevated in AML bone marrow samples and cell lines. Patients with high level of RPL34 had inferior survival outcomes than their counterparts, and upregulation of RPL34 may mediate chemoresistance in AML. Through knockdown of RPL34 in HL-60 cell line, we found cell proliferation was inhibited, cell apoptosis was triggered, and cell cycle was arrested in S phase. Conclusions: The present study demonstrated that downregulation of RPL34 could inhibit cell proliferation, promote cell apoptosis, and induce cell cycle arrest in AML cell line HL-60. Also, its expression and clinical significance in AML patients was confirmed. All these findings suggest that RPL34 may be a potential novel therapy target in AML. Key words: Acute myeloid leukemia; ribosomal protein L34; biomarker; therapy target; chemoresistance

HYAL1 Is Downregulated in Idiopathic Pulmonary Fibrosis and Inhibits HFL-1 Fibroblast Proliferation When Upregulated.

Background Idiopathic pulmonary fibrosis (IPF), the most common interstitial lung disease, arises from transforming growth factor beta 1- (TGFβ1-) induced aberrant fibroproliferation in response to epithelial injury. The TGFβ1-) induced aberrant fibroproliferation in response to epithelial injury. The TGFMethods We first performed microarray data mining of previously published gene expression datasets to identify key gene signatures in IPF lung tissues. HYAL1 expression levels in IPF and normal lung tissues were then characterized using immunohistochemistry followed by real-time quantitative reverse transcription-PCR (qRT-PCR) and western blot analysis on isolated fibroblasts from fresh lung tissues of IPF and healthy donors. A human fetal lung fibroblast HFL-1 cell line, which was used in place of primary lung fibroblasts, was used to assess the proliferative or apoptotic effects associated with lentiviral-induced HYAL1 overexpression using CCK-8 cell proliferation assay and Annexin V-APC staining. The identification of potentially associated molecular pathways was performed using microarray analysis followed by qRT-PCR and western blot analysis. Results Lung tissue microarray data mining and immunohistochemistry revealed significantly downregulation of HYAL1 in IPF lung tissue. However, HYAL1 in IPF lung tissue. However, HYAL1 in IPF lung tissue. However, HYAL1 in IPF lung tissue. However, β1-) induced aberrant fibroproliferation in response to epithelial injury. The TGFβ1-) induced aberrant fibroproliferation in response to epithelial injury. The TGFConclusions We showed that HYAL1 overexpression could prevent HFL-1 fibroproliferation. Furthermore, our findings suggest that transcriptional regulators and BMP receptor signaling may be involved in HYAL1 modulation in IPF therapy.HYAL1 in IPF lung tissue. However,

Abstract P3-05-09: mRNA expression level and prognostic significance of different immune-related biomarkers in early breast cancer

Abstract Background: The combination of an anti-Programmed Cell Death-Ligand-1 (anti-PD-L1) agent, atezolizumab, plus chemotherapy (CT) as first line treatment in triple negative (TN) advanced breast cancer (ABC), resulted into a significant improvement in Progression Free Survival (PFS) as compared to Nab-paclitaxel alone, and a significant improvement in Overall Survival (OS) in the PD-L-1 positive subgroup (> 1% by IHC). With these results, the IMpassion 130 study met its primary endpoint, although its impact on the prognosis of TN ABC is less than expected, suggesting that innovative approaches are needed to maximize the role of immunotherapy in this disease subset. In this perspective, the potential role of addressing innate immunity as opposed to adaptive immunity has not yet been evaluated. It has been shown that Tumor-Associated Macrophages (TAMs) may express the immune checkpoint inhibitor PD-L1, thus contributing to immunosuppression. In addition, in response to microenvironmental signals they acquire a tumor-promoting M2-like phenotype, that supports angiogenesis, tissue remodeling and metastasis formation. They also express the Signal-regulatory protein (SIRPα) which provides a “don’t eat” me signal that impairs phagocytosis of CD47 positive cancer cells. CD47 is expressed in all human solid tumor cells; its role is to protect cancer cells from being recognized by innate immune surveillance. With these premises, the aim of this study is to evaluate the expression and prognostic value of different biomarkers related to innate and adaptive immunity, by using publicy available gene expression datasets, to assess if double immune co-targeting (i.e. innate and adaptive) might represent a viable strategy to optimise immunotherapy in ABC. Methods: We used previously published gene expression datasets by two online tools: GOBO and GEPIA. GOBO is an ultrafast tool including 1881 early BC patients generated on Affymetrix U133A microarrays, with a median follow up of 120 months. Expression of CD47, SIRP, CD163 and CD204 (M2-like phenotype) mRNA were evaluated as continuous variables. Kaplan Meier survival curves were used to assess the prognostic ability of the identified biomarkers. GEPIA is a web server for analysing the RNA sequencing expression data of tumours and normal samples from the TCGA and the GTEx projects, by a standard processing pipeline. Results: CD47 and CD204 mRNAs were significantly overexpressed in BC samples with respect to normal tissues (p < 0.05). Furthermore, CD47, SIRPalpha and CD163 were differentially modulated in the different BC subtypes (p<0.00001), showing an increased expression in the basal like group. Finally, the expression of mRNA CD47, SIRPa and CD163 were associated with a significanly worse OS only in the luminal A subtype (p<0.008, p<0.05 and p<0.01, respectively). Conclusions: These results indicate that innate immunity is involved in BC prognosis, and might be considered a target for therapy, in particular in those subtypes such as the Luminal A group, characterised by a favourable outcome; conversely, no effect was observed in subtypes characterised by a worse prognosis such as the basal-like tumors. Further investigation on the role of innate immunity in BC is warranted. Citation Format: Veronica Martini, Francesco Favero, Davide Corà, Feba Varughese, Antonio Sica, Alessandra Gennari. mRNA expression level and prognostic significance of different immune-related biomarkers in early breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-09.

SGDB: A Sports Gene Database for Visualization of Sports Effects on Human Skeletal Muscle Gene Expression

The relationship between physical exercise and human health has been widely recognized by sports-related professionals and the public. However, the molecular mechanism behind sports remains unclear. A microarray sequencing technology has been widely used to detect gene changes after physical exercise and researchers have accumulated a great deal of data. These data provide a large amount of information for researchers to understand the relationship between genetics and exercise and study changes in genes after different forms of exercise, and their relations with human, gender and age. We integrate eight published gene expression datasets from human skeletal muscle and construct an online web server called SGDB standing for Sports Gene DataBase. SGDB can be used to search for genes that are expressed after exercise and no exercise. One can determine the effects of physical exercise on gene expression while allowing for visualization of changes in female and male. In addition, different forms of exercise and relationships with age are also available. Currently, SGDB contains 54613 annotated gene probes based on the Affymetrix Human Genome U133 Plus 2 Affay (GPL570). It has become one of the most important resources for researchers to study sports and genes. The online access address for SGDB is http://www.sportsgene.top.

EBF1 Gene mRNA Levels in Maternal Blood and Spontaneous Preterm Birth.

Genetic variants of six genes (EBF1, EEFSEC, AGTR2, WNT4, ADCY5, and RAP2C) have been linked recently to gestational duration and/or spontaneous preterm birth (sPTB). Our goal was to examine sPTB in relation to maternal blood mRNA levels of these genes. We used a public gene expression dataset (GSE59491) derived from maternal blood in trimesters 2 and 3 that included women with sPTB (n = 51) and term births (n = 106) matched for maternal age, race/ethnicity, pre-pregnancy body mass index, smoking during pregnancy, and parity. T tests were used to examine mRNA mean differences (sPTB vs term) within and across trimesters, and logistic regression models with mRNA quartiles were applied to assess associations between candidate gene mRNA levels and sPTB. Based on these analyses, one significant candidate gene was used in a Gene Set Enrichment Analysis (GSEA) to identify related gene sets. These gene sets were then compared with the ones previously linked to sPTB in the same samples. Our results indicated that among women in the lowest quartile of EBF1 mRNA in the 2nd or 3rd trimester, the odds ratio for sPTB was 2.86 (95%CI 1.08, 7.58) (p = 0.0349, false discovery rate (FDR) = 0.18) and 4.43 (95%CI 1.57, 12.50) (p = 0.0049, FDR = 0.06), respectively. No other candidate gene mRNAs were significantly associated with sPTB. In GSEA, 24 downregulated gene sets were correlated with 2nd trimester low EBF1 mRNA and part of previous sPTB-associated gene sets. In conclusion, mRNA levels of EBF1 in maternal blood may be useful in detecting increased risk of sPTB as early as 2nd trimester. The potential underlying mechanism might involve maternal-fetal immune and cell cycle/apoptosis pathways.

Neutrophils suppress tumor‐infiltrating T cells in colon cancer via matrix metalloproteinase‐mediated activation of TGFβ

High T‐cell infiltration in colorectal cancer (CRC) correlates with a favorable disease outcome and immunotherapy response. This, however, is only observed in a small subset of CRC patients. A better understanding of the factors influencing tumor T‐cell responses in CRC could inspire novel therapeutic approaches to achieve broader immunotherapy responsiveness. Here, we investigated T cell‐suppressive properties of different myeloid cell types in an inducible colon tumor mouse model. The most potent inhibitors of T‐cell activity were tumor‐infiltrating neutrophils. Gene expression analysis and combined in vitro and in vivo tests indicated that T‐cell suppression is mediated by neutrophil‐secreted metalloproteinase activation of latent TGFβ. CRC patient neutrophils similarly suppressed T cells via TGFβ in vitro, and public gene expression datasets suggested that T‐cell activity is lowest in CRCs with combined neutrophil infiltration and TGFβ activation. Thus, the interaction of neutrophils with a TGFβ‐rich tumor microenvironment may represent a conserved immunosuppressive mechanism in CRC.

Functional Impact of MacroH2A Histone Variants in Acute Myeloid Leukemia

Histone variants are emerging as key regulatory molecules in cancer. Recent data provided first evidence of how transcriptional deregulation and changes in the deposition of histone variants may affect malignant transformation. H2A variants are highly conserved and consist of 8 members. Among all histone variants, macroH2A variants hold a unique C-terminal macro domain, which is approximately twice the size of its H2A-like histone domain. In mammals, there are 3 macroH2A variants encoded by H2AFY (macroH2A1.1, macroH2A1.2) and H2AFY2 (macroH2A2). MacroH2A variants occupy large repressive domains throughout the genome, exerting a repressive role on transcription. So far, the function of macroH2A proteins in hematopoiesis and leukemic transformation is incompletely understood. Here, we report on a functional role of macroH2A histone variants that may influence cell competition in leukemia and normal hematopoietic stem and progenitor cell (HSPC) function. In published gene expression datasets, macroH2A variants H2AFY and H2AFY2 are highly expressed in hematopoietic stem cells (HSCs) compared to mononuclear cells (both p<0.0001****). Moreover, expression of H2AFY and H2AFY2 appeared even higher in acute myeloid leukemia (AML) (p=0.0750 and 0.0037**, respectively) when compared to the expression level in HSCs. This high expression is independent of different genetic subtypes and AML risk groups. To assess for the functional dependency of leukemic cells on the expression of macroH2A variants, we performed an in vitro CRISPR-Cas9 dropout screen focusing on macroH2A variants in MOLM-13 cells. Genetic inactivation of H2AFY but not H2AFY2 resulted in outcompetition of infected cells against non-infected competitors. This finding could be recapitulated in 3 additional AML cell lines (OCI-AML3, THP-1, HL-60), indicating a broader dependency on H2AFY irrespective of the oncogenic background or underlying driver mutation. To validate the functional impact of H2AFY on leukemia development in vivo, we generated a novel conditional knockout mouse model for H2afy and induced leukemia through retroviral infection of sorted Lineage-Sca1+cKit+ (LSK) cells with the oncogene MLL-AF9. Results of these functional studies will be presented. To understand the functional impact of macroH2A variants on normal HSPC function in vivo, we performed competitive repopulation studies. We used conventional knockout mouse models for the 2 H2afy isoforms: macroH2A1.1 and macroH2A1.2 In addition, for inactivation of both isoforms, we used the conditional macroH2A knockout mouse model. H2afy2 was either inactivated by RNAi or conditionally knockout through Mx1-Cre recombinase activation. In brief, whole bone marrow cells (WBMCs) were transplanted into primary recipient mice in a 1:1 competitive manner and compared to the respective wildtype littermate controls. Peripheral blood (PB) chimerism was monitored on a monthly basis 4 weeks post-transplantation for a total of 16 weeks. We observed no significant difference in the recipients of macroH2A1.1 (n=5/5) or macroH2A1.2 (n=10/9), suggesting both macroH2A isoforms are dispensable for intrinsic functions of HSCs. Remarkably, we found macroH2A2-deficient cells (n=6) competed significantly worse than macroH2A2+/+ (n=5) controls in PB (p=0.0043**) and in WBM (p=0.0043**), indicating a requirement for macroH2A2 in maintenance of HSPCs. These findings could be recapitulated by RNA-mediated inactivation of H2afy2 in sorted HSCs. We observed significant reduction of macroH2A2-depleted hematopoiesis after 16 weeks in WBM (p=0.0286*) and PB (p=0.0079**). Importantly, this functional impairment was less pronounced when investigating multipotent progenitor and committed progenitor cell function in spleen colony-forming unit (CFU) assay in vivo or CFU assay in vitro. Taken together, our data indicate distinct requirements for macrohistone variants in leukemic and normal hematopoietic cells. Dependency of leukemic cells but not normal HSPCs on macroH2A may indicate a potential therapeutic index. Disclosures No relevant conflicts of interest to declare.

Targeting the Plasma Cell Specific Purine Metabolic Enzyme, AMPD1, Induces Multiple Myeloma Cell Death Accompanying NAD Depletion

Introduction. Novel agents have improved the prognosis of multiple myeloma (MM) patients. However, MM is still an incurable disease. In order to achieve a cure, it is necessary to develop novel therapeutic agents that are highly specific to MM cells and target different pathways from the present anti-MM agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and MM cells, and to examine whether it can be a novel target for MM therapy. Materials and methods. Public available gene expression websites, GenomicScape and Genevestigator were utilized to study genes specifically expressed in human plasma cells and MM cells. To examine gene expression in MM cell lines, gene expression data set from the Cancer Cell Line Encyclopedia (CCLE) was analyzed. AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression in MM cell lines and patient derived MM cells were examined by western blot and immunohistochemistry. Genes co-expressed with AMPD1 in MM cells were identified using public available gene expression datasets. Molecular pathway analysis was conducted using Molecular Signatures Database. Cell viability of MM cell lines, peripheral blood mononuclear cells (PBMCs) and patient derived bone marrow mononuclear cells treated by AMPD1 inhibitor (compound #3) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed by flow-cytometry after staining with 7AAD. Intracellular NAD and NADH concentrations in MM cell lines were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Z-VAD-FMK (Caspase inhibitor) and Nec-1 (RIP1 kinase inhibitor) were used in combination with compound #3 to study the mechanism of AMPD1 inhibition induced MM cell death. Results. We identified several genes specifically expressed in human plasma cells and MM cells using public available gene expression websites. Among the identified genes, we focused on AMPD1, a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), since this gene has not been previously studied in MM. We found that AMPD1 gene expression was limited to MM cell lines and patient derived MM cells through CCLE analysis and RT-PCR. AMPD1 protein expression was detected only in MM cell lines, bone marrow MM cells and extramedullary plasmacytomas. Genes associated with AMPD1 expression were related to hypoxic pathways. MM cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to those cultured under normoxia, indicating that AMPD1 plays a significant role in MM cell surviving under hypoxic condition such as the bone marrow microenvironment. Compound #3 induced cell death in MM cell lines and patient derived MM cells, while toxicity against PBMCs and non-MM cells were minimal. Compound #3 was more effective against MM cell lines cultured under hypoxia compared to those under normoxia, reflecting the higher AMPD1 expression under hypoxia. Since, AMPD1 is associated with purine metabolism, we analyzed the intracellular concentration of NAD and NADH, which are major cellular metabolites, in MM cell lines post compound #3 treatment. Marked decrease of NAD concentration and NAD / NADH ratio was observed in compound #3 treated MM cell lines compared to control, demonstrating that AMPD1 inhibition depletes intracellular NAD leading to MM cell death. Compound #3 treated MM cell lines showed increase in Annexin V- PI+ and Annexin V+ PI+ fractions. Additionally, Z-VAD-FMK and Nec-1 treatment did not reverse cell death induced by compound #3, indicating a distinct mechanism of cytotoxicity by compound #3 from other anti-MM agents inducing apoptosis and necroptosis. Conclusions. This is the first report so far that AMPD1 is specifically expressed in MM cells. AMPD1 inhibitor showed specific cytotoxicity against MM cells, while toxicities against non-MM cells were minimal. Additionally, AMPD1 inhibitor was more effective under hypoxic condition, suggesting that AMPD1 inhibition works more efficiently in the bone marrow microenvironment. Considering the specificity against MM cells and its distinct mechanism of action from the present anti-MM agents, AMPD1 inhibition is a potent novel therapeutic strategy for MM. Disclosures Matsuoka: Bristol-Myers Squibb Corp.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Identification of ZNF217 As an Essential Oncogenic Gene in B-Cell Acute Lymphoblastic Leukemia By CRISPR/Cas9-Based Library Screening

Background and Significance Although the prognosis of pediatric B-cell acute lymphoblastic leukemia (B-ALL) has been significantly improved in recent years, adult patients continue to have dismal survival. This is partially due to the fact that adult patients tend to have more unfavorable cytogenetic characteristics such as MLL-AF4 fusion and BCR-ABL1 fusion. N6-methyladenosine (m6A) is the most prevalent epigenetic modification on eukaryotic messenger RNA (mRNA), which plays important roles in many fundamental bioprocesses. The aberrant regulation of m6A modification is crucial for the initiation and progression of various cancers including acute myeloid leukemia (AML). However, the studies of m6A modification in B-ALL have been limited. Therefore, we began with the screening of essential m6A regulators in B-ALL with unfavorable cytogenetic characteristics. Zinc Finger Protein 217 (ZNF217) is not only a Kruppel-like family of transcription factor but also a versatile m6A regulator. Although ZNF217 has been identified as a candidate oncogene and therapeutic target in many solid tumors, the potential function of ZNF217 in leukemia, especially in B-ALL, remains unknown. Experimental Approach and Results To determine which m6A machinery associated genes play essential roles in the survival/viability of B-ALL cells, we performed CRISPR/Cas9-based library screening using single-cloned B-ALL cells expressing Cas9. The sgRNA library contained sgRNAs targeting all the reported m6A machinery associated genes (including METTL3, METTL14, FTO; 25 sgRNAs per gene), 11 common essential genes, and scramble controls. The screening results suggested a set of m6A machinery associated genes, especially ZNF217, might be essential for the survival of B-ALL cells. To further rank these genes by their essentiality in B-ALL, we performed Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK). In our MAGeCK negative-selection ranking, ZNF217 was ranked as the top 1 candidate, indicating ZNF217 might be the most essential m6A regulator in B-ALL. In agreement with these results, ZNF217 is also highly expressed in B-ALL. We analyzed the published gene expression datasets and found ZNF217 is highly expressed in B-ALL patients with different cytogenetic changes, including MLL-AF4 fusion and BCR-ABL1 fusion, compared to normal CD19+CD10+ B-cell progenitors. To further validate the function of ZNF217, we perform growth competition experiments using B-ALL cells with either MLL-AF4 fusion or BCR-ABL1 fusion. We found that knockout of ZNF217 significantly decreased the competitive fitness of both B-ALL subtypes. In addition, we also found that knockout of ZNF217 in AML cells did not show significant effect on cell survival, suggesting the function of ZNF217 may be specific in B-lineage. Conclusions In summary, by use of CRISPR-Cas9 screening, we identified ZNF217 as an essential gene in B-ALL cells. We found ZNF217 is highly expressed in various subtypes of B-ALL patients, and our data suggest that ZNF217 is required for the survival of B-ALL cells, but not that of AML cells. Overall, our data indicate that ZNF217 plays an essential oncogenic role in B-ALL. Figure Legend A. Experimental scheme of CRISPR/Cas9-based library screening B. Growth competition assays using B-ALL cells (left) and AML cells (right) Figure Disclosures Chen: Genovel Biotech Corp: Other: scientific founder and Chairman.

Transcriptional Regulation of Autophagy Genes via Stage-Specific Activation of CEBPB and PPARG during Adipogenesis: A Systematic Study Using Public Gene Expression and Transcription Factor Binding Datasets

Autophagy is the cell self-eating mechanism to maintain cell homeostasis by removing damaged intracellular proteins or organelles. It has also been implicated in the development and differentiation of various cell types including the adipocyte. Several links between adipogenic transcription factors and key autophagy genes has been suggested. In this study, we tried to model the gene expression and their transcriptional regulation during the adipocyte differentiation using high-throughput sequencing datasets of the 3T3-L1 cell model. We applied the gene expression and co-expression analysis to all and the subset of autophagy genes to study the binding, and occupancy patterns of adipogenic factors, co-factors and histone modifications on key autophagy genes. We also analyzed the gene expression of key autophagy genes under different transcription factor knockdown adipocyte cells. We found that a significant percent of the variance in the autophagy gene expression is explained by the differentiation stage of the cell. Adipogenic master regulators, such as CEBPB and PPARG target key autophagy genes directly. In addition, the same factor may also control autophagy gene expression indirectly through autophagy transcription factors such as FOXO1, TFEB or XBP1. Finally, the binding of adipogenic factors is associated with certain patterns of co-factors binding that might modulate the functions. Some of the findings were further confirmed under the knockdown of the adipogenic factors in the differentiating adipocytes. In conclusion, autophagy genes are regulated as part of the transcriptional programs through adipogenic factors either directly or indirectly through autophagy transcription factors during adipogenesis.

Gene Expression Signature in Diagnosis of Endometriosis

Study Objective To develop a prototype of a complex gene expression biomarker for the diagnosis of endometriosis based on differences between molecular signatures of endometrium from women with and without endometriosis. Design Prospective observational cohort study. II-1. Evidence obtained from a well-designed, controlled trial without randomization. Setting Department of Reproductive Medicine and Surgery at the A.I. Evdokimov Moscow State Medical and Dental University. Patients or Participants 30 women with endometriosis and 15 women without endometriosis (control group). Interventions Laparoscopic excision of endometriotic foci, hysteroscopy with endometrial sampling. RNA was isolated from all samples and stored in RNA Later. RNA sequencing was performed using Illumina HiSeq 3000 equipment for single-end sequencing. Unique bioinformatics algorithms were developed and validated using experimental and public gene expression datasets. Measurements and Main Results We performed gene expression analysis of dataset containing 83 samples (30 endometrial and 53 endometriotic) and 15 samples (endometrial) of patients with and without endometriosis respectively. We extracted a complex molecular signature of 38 genes and found that the expression of 26 genes in it was significantly increased while the expression of 12 genes was significantly repressed in the endometrium of patients with endometriosis. This endometrial genetic signature successfully differentiated 53 samples of endometriotic lesions from 15 endometrial samples of healthy women (area under the ROC curve (AUC) =1. The comparison of our dataset of 83 samples of endometrial and endometriotic tissue with preexisting dataset containing 134 samples of other tissues (cervix, ovary, stomach, lung) revealed high sensitivity (94%) and specificity (97%) in the ability of studied molecular signature to equally identify endometrium and endometriotic tissue of patients with endometriosis. Conclusion We obtained a complex molecular biomarker that could be used as a basis for early diagnosis of endometriosis via utilization of endometrial biopsy. Our findings indicate that easily accessible eutopic endometrium can be potentially used as a non-surgical marker for the presence of endometriosis.