scholarly journals Exploration and Validation of A Novel Inflammatory Response-Associated Gene Signature to Predict Osteosarcoma Prognosis and Immune Infiltration

2021 ◽  
Author(s):  
Yucheng Fu ◽  
Guoyu He ◽  
Zhuochao Liu ◽  
Jun Wang ◽  
Zhusheng Zhang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Cell Lines ◽  
Cox Regression ◽  
Functional Enrichment ◽  
Molecular Signature ◽  
Risk Scores ◽  
Pcr Analysis ◽  
Osteosarcoma Cell ◽  
Immune Infiltration ◽  
Qrt Pcr

Abstract Background: Inflammatory response took part in the progression of tumor and was regarded as the hallmark of cancer. However, the prognostic relationship between osteosarcoma and inflammatory response-associated genes (IRGs) was unclear. This research aimed to explore the correlations between osteosarcoma prognosis and IRG signature.Methods: The inflammatory response-associated differentially expressed messenger RNAs (DEmRNAs) were screened out through Gene Expression Omnibus (GEO) and Molecular Signature Database (MSigDB) databases. Univariate and multivariate cox regression analyses were utilized to construct the IRG signature. The prognostic value of signature was investigated through Kaplan–Meier (KM) survival curve and nomogram. DEmRNAs among high and low inflammatory response-associated risks were identified and functional enrichment analyses were conducted. ESTIMATE, CIBERSORT and single-sample gene set enrichment analyses (ssGSEA) were implemented to reveal the alterations in immune infiltration. All above results were validated in Target database. Quantitative real-time PCR (qRT-PCR) analysis was applied to detect the expression of the IRGs in human osteoblast and osteosarcoma cell linesResults: The IRG signature that consisted of two genes (MYC, CLEC5A) was established. In training and validation datasets, patients with lower risk scores lived longer and the IRG signature was confirmed as the independent prognostic factor in osteosarcoma. The nomogram was constructed and the calibration curves indicated the reliability of this model. Functional analysis of risk score-associated DEmRNAs showed that immune-related pathways and functions were significantly enriched. ssGSEA revealed that 14 immune cells and 11 immune functions were significantly dysregulated. The qRT-PCR results indicated IRGs were significantly differently expressed in osteosarcoma and osteoblast cell lines.Conclusions: The novel osteosarcoma inflammatory response-associated prognostic signature was established and validated in this study. This model could serve as the biomarker and therapeutic target for osteosarcoma in the future.

scholarly journals Construction of circRNA-miRNA-mRNA network and identification of novel potential biomarkers for non-small cell lung cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jia Yang ◽  
Ran Hao ◽  
Yunlong Zhang ◽  
Haibin Deng ◽  
Wenjing Teng ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lines ◽  
Target Genes ◽  
Cox Regression ◽  
Small Cell ◽  
Functional Enrichment ◽  
Small Cell Lung ◽  
Immune Infiltration ◽  
Qrt Pcr

Abstract Background The underlying circular RNAs (circRNAs)-related competitive endogenous RNA (ceRNA) mechanisms of pathogenesis and prognosis in non-small cell lung cancer (NSCLC) remain unclear. Methods Differentially expressed circRNAs (DECs) in two Gene Expression Omnibus datasets (GSE101684 and GSE112214) were identified by utilizing R package (Limma). Circinteractome and StarBase databases were used to predict circRNA associated-miRNAs and mRNAs, respectively. Then, protein–protein interaction (PPI) network of hub genes and ceRNA network were constructed by STRING and Cytoscape. Also, analyses of functional enrichment, genomic mutation and diagnostic ROC were performed. TIMER database was used to analyze the association between immune infiltration and target genes. Kaplan–Meier analysis, cox regression and the nomogram prediction model were used to evaluate the prognostic value of target genes. Finally, the expression of potential circRNAs and target genes was validated in cell lines and tissues by quantitative real-time PCR (qRT-PCR) and Human Protein Atlas (HPA) database. Results In this study, 15 DECs were identified between NSCLC tissues and adjacent-normal tissues in two GEO datasets. Following the qRT-PCR corroboration, 7 DECs (hsa_circ_0002017, hsa_circ_0069244, hsa_circ_026337, hsa_circ_0002346, hsa_circ_0007386, hsa_circ_0008234, hsa_circ_0006857) were dramatically downregulated in A549 and SK-MES-1 compared with HFL-1 cells. Then, 12 circRNA-sponged miRNAs were screened by Circinteractome and StarBase, especially, hsa-miR-767-3p and hsa-miR-767-5p were significantly up-regulated and relevant to the prognosis. Utilizing the miRDB and Cytoscape, 12 miRNA-target genes were found. Functional enrichment, genomic mutation and diagnostic analyses were also performed. Among them, FNBP1, AKT3, HERC1, COL4A1, TOLLIP, ARRB1, FZD4 and PIK3R1 were related to the immune infiltration via TIMER database. The expression of ARRB1, FNBP1, FZD4, and HERC1 was correlated with poor overall survival (OS) in NSCLC patients by cox regression and nomogram. Furthermore, the hub-mRNAs were validated in cell lines and tissues. Conclusion We constructed the circRNA-miRNA-mRNA network that might provide novel insights into the pathogenesis of NSCLC and reveal promising immune infiltration and prognostic biomarkers.

scholarly journals Development of A Vitamin-related Gene Signature to Predict the Immune Characteristics and Prognosis of Glioma

2020 ◽  
Author(s):  
Qing Zhang ◽  
Qingyu Liang ◽  
Gefei Guan ◽  
Wen Cheng ◽  
Lianhe Yang ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Cox Regression ◽  
The Body ◽  
Functional Enrichment ◽  
Molecular Signature ◽  
Risk Scores ◽  
Cox Regression Analysis ◽  
Related Risk ◽  
Tcga Dataset ◽  
Genome Atlas

Abstract Background: Vitamins not only play a pivotal role in maintaining homeostasis of the body, but also have complex impacts on the occurrence and progression of tumors. However, the effects of vitamins on glioma and the underlying mechanism have not been fully elucidated. Methods: Vitamin -related genes were extracted from the Molecular Signature Database v7.1 (MSigDB). The overlapping overall survival (OS)-related genes in The Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), and GSE16011 cohorts screened out by univariate COX regression analysis were utilized to construct a risk model based on the TCGA cohort via random survival forest analysis and multivariate COX regression analysis. The powerful prognostic predictive potential of the vitamin-related risk signature was verified by Kaplan–Meier survival analysis and receiver operating characteristic (ROC) analysis in the three datasets. The ssGSEA method of the GSVA package was used for functional enrichment and immune cell component analyses. ESTIMATE score analysis was used for auxiliary analysis of glioma immune characteristics. A nomogram was constructed and assessed based on the TCGA dataset.Results: The vitamin-related six-gene (POSTN, IRX5, EEF2, RAB27A, MDM2, and ENO1) risk signature constructed based on the TCGA dataset accurately predicted the outcomes of glioma patients and credibly distinguished between different levels and molecular subtypes of glioma in the TCGA, CGGA, and GSE16011 cohorts. Gliomas with high risk scores exhibited high immune scores, low tumor purity, and immunosuppressive features. The nomogram constructed by combining the vitamin-related risk signature and clinicopathological factors precisely predicted the 1-, 3-, and 5-year OS of glioma patients.Conclusions: Our study revealed that the vitamin-related six-gene risk signature, as an independent prognostic factor, could accurately distinguish the grade, molecular subtype, and immune characteristics of glioma.

scholarly journals Development of A Vitamin-Related Gene Signature to Predict the Immune Characteristics and Prognosis of Glioma

2020 ◽  
Author(s):  
Qing Zhang ◽  
Qingyu Liang ◽  
Gefei Guan ◽  
Wen Cheng ◽  
Lianhe Yang ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Cox Regression ◽  
The Body ◽  
Functional Enrichment ◽  
Molecular Signature ◽  
Risk Scores ◽  
Cox Regression Analysis ◽  
Related Risk ◽  
Tcga Dataset ◽  
Genome Atlas

Abstract Background: Vitamins not only play a pivotal role in maintaining homeostasis of the body, but also have complex impacts on the occurrence and progression of tumors. However, the effects of vitamins on glioma and the underlying mechanism have not been fully elucidated. Methods: Vitamin -related genes were extracted from the Molecular Signature Database v7.1 (MSigDB). The overlapping overall survival (OS)-related genes in The Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), and GSE16011 cohorts screened out by univariate COX regression analysis were utilized to construct a risk model based on the TCGA cohort via random survival forest analysis and multivariate COX regression analysis. The powerful prognostic predictive potential of the vitamin-related risk signature was verified by Kaplan–Meier survival analysis and receiver operating characteristic (ROC) analysis in the three datasets. The ssGSEA method of the GSVA package was used for functional enrichment and immune cell component analyses. ESTIMATE score analysis was used for auxiliary analysis of glioma immune characteristics. A nomogram was constructed and assessed based on the TCGA dataset.Results: The vitamin-related six-gene (POSTN, IRX5, EEF2, RAB27A, MDM2, and ENO1) risk signature constructed based on the TCGA dataset accurately predicted the outcomes of glioma patients and credibly distinguished between different levels and molecular subtypes of glioma in the TCGA, CGGA, and GSE16011 cohorts. Gliomas with high risk scores exhibited high immune scores, low tumor purity, and immunosuppressive features. The nomogram constructed by combining the vitamin-related risk signature and clinicopathological factors precisely predicted the 1-, 3-, and 5-year OS of glioma patients.Conclusions: Our study revealed that the vitamin-related six-gene risk signature, as an independent prognostic factor, could accurately distinguish the grade, molecular subtype, and immune characteristics of glioma.

A Six CT83-Related Genes Based Prognostic Signature for Lung Adenocarcinoma

2021 ◽  
Vol 24 ◽  
Author(s):  
Yongmei Wang ◽  
Guimin Zhang ◽  
Ruixian Wang
Keyword(s):  
Regression Analysis ◽  
Lung Adenocarcinoma ◽  
Differential Expression ◽  
Risk Score ◽  
Prognostic Model ◽  
Cox Regression ◽  
Differential Expression Analysis ◽  
Functional Enrichment ◽  
Risk Scores ◽  
Cox Regression Analysis

Background: This study aims to explore the prognostic values of CT83 and CT83-related genes in lung adenocarcinoma (LUAD). Methods: We downloaded the mRNA profiles of 513 LUAD patients (RNA sequencing data) and 246 NSCLC patients (Affymetrix Human Genome U133 Plus 2.0 Array) from TCGA and GEO databases. According to the median expression of CT83, the TCGA samples were divided into high and low expression groups, and differential expression analysis between them was performed. Functional enrichment analysis of differential expression genes (DEGs) was conducted. Univariate Cox regression analysis and LASSO Cox regression analysis were performed to screen the optimal prognostic DEGs. Then we established the prognostic model. A Nomogram model was constructed to predict the overall survival (OS) probability of LUAD patients. Results: CT83 expression was significantly correlated to the prognosis of LUAD patients. A total of 59 DEGs were identified, and a predictive model was constructed based on six optimal CT83-related DEGs, including CPS1, RHOV, TNNT1, FAM83A, IGF2BP1, and GRIN2A, could effectively predict the prognosis of LUAD patients. The nomogram could reliably predict the OS of LUAD patients. Moreover, the six important immune checkpoints (CTLA4, PD1, IDO1, TDO2, LAG3, and TIGIT) were closely correlated with the Risk Score, which was also differentially expressed between the LUAD samples with high and low-Risk Scores, suggesting that the poor prognosis of LUAD patients with high-Risk Score might be due to the immunosuppressive microenvironments. Conclusion: A prognostic model based on six optimal CT83 related genes could effectively predict the prognosis of LUAD patients.

Identifying the Molecular Cause of Extreme Endoplasmic Reticulum Dilation in Pediatric Osteosarcoma and Its Relationship to the Disease

2021 ◽  
Author(s):  
◽  
Rachael Wood ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Genomic Instability ◽  
Cell Lines ◽  
Molecular Signature ◽  
Osteosarcoma Cell ◽  
Copii Vesicle ◽  
Molecular Therapeutic ◽  
Molecular Therapeutic Targets ◽  
Pediatric Osteosarcoma ◽  
Crispr Activation

Pediatric osteosarcoma tumors are characterized by an unusual abundance of grossly dilated endoplasmic reticulum and an immense genomic instability that has complicated identifying new effective molecular therapeutic targets. Here we report a novel molecular signature that encompasses the majority of 108 patient tumor samples, PDXs and osteosarcoma cell lines. These tumors exhibit reduced expression of four critical COPII vesicle proteins that has resulted in the accumulation of procollagen-I protein within ‘hallmark’ dilated ER. Using CRISPR activation technology, increased expression of only SAR1A and SEC24D to physiologically normal levels was sufficient to restore both collagen-I secretion and resolve dilated ER morphology to normal.

Toll-like receptor 3 is a potential prognosis marker and associated with immune infiltration in stomach adenocarcinoma

2021 ◽  
pp. 1-17
Author(s):  
Zhihao Huang ◽  
Aoxiao He ◽  
Jiakun Wang ◽  
Hongcheng Lu ◽  
Xiaoyun Xu ◽  
...  
Keyword(s):  
Cox Regression ◽  
Clinical Stage ◽  
Mrna Level ◽  
Positive Association ◽  
Cox Regression Analysis ◽  
Immune Infiltration ◽  
Qrt Pcr ◽  
Tlr3 Expression ◽  
Immune Biomarkers ◽  
Prognosis Marker

BACKGROUND: Toll-like receptors participate in various biological mechanisms, mainly including the immune response and inflammatory response. Nevertheless, the role of TLRs in STAD remains unclear. OBJECTIVE: We aimed to explore the expression, prognosis performance of TLRs in STAD and their relationship with immune infiltration. METHODS: Student’s t-test was used to evaluate the expression of TLRs between STAD tissues and normal tissues. Kaplan-Meier method was applied to explored the prognosis value of TLRs in STAD. And qRT-PCR validated their expression and prognosis value. Spearman’s correlation analysis and Wilcoxon rank-sum test were used to assess the association between TLRs and immune infiltration in STAD. RESULTS: The mRNA level of TLR3 was downregulated in STAD. We summarized genetic mutations and CNV alteration of TLRs in STAD cohort. Prognosis analysis revealed that STAD patients with high TLR3 expression showed better prognosis in OS, FP and PPS. The result of qRT-PCR suggested that TLR3 expression was decreased in STAD tissues and STAD patients with high TLR3 mRNA level had a better OS. Univariate and multivariate cox regression analysis suggested TLR3 expression and clinical stage as independent factors affecting STAD patients’ prognosis. A positive association existed between TLR3 expression and the abundance of immune cells and the expression of various immune biomarkers. Furthermore, key targets related to TLR3 were identified in STAD, mainly including MIR-129 (GCAAAAA), PLK1, and V$IRF1_01. CONCLUSIONS: Our result demonstrated TLR3 as a prognosis marker and associated with immune infiltration in STAD.

scholarly journals Prognostic Modeling of Patients with Metastatic Melanoma Based on Tumor Immune Microenvironment Characteristics

2021 ◽  
Author(s):  
Jing Liu ◽  
Ting Ye ◽  
Xue fang Zhang ◽  
Yong jian Dong ◽  
Wen feng Zhang ◽  
...  
Keyword(s):  
High Risk ◽  
Metastatic Melanoma ◽  
Prognostic Model ◽  
Cox Regression ◽  
Risk Group ◽  
Low Risk ◽  
Risk Scores ◽  
Immune Microenvironment ◽  
Immune Infiltration ◽  
Tumor Immune Microenvironment

Abstract Most of the malignant melanomas are already in the middle and advanced stages when they are diagnosed, which is often accompanied by the metastasis and spread of other organs.Besides, the prognosis of patients is bleak. The characteristics of the local immune microenvironment in metastatic melanoma have important implications for both tumor progression and tumor treatment. In this study, data on patients with metastatic melanoma from the TCGA and GEO datasets were selected for immune, stromal, and estimate scores, and overlapping differentially expressed genes (DEGs) were screened. A nine-IRGs prognostic model (ALOX5AP, ARHGAP15, CCL8, FCER1G, GBP4, HCK, MMP9, RARRES2 and TRIM22) was established by univariate COX regression, LASSO and multivariate COX regression. Receiver operating characteristic (ROC) curves were used to test the predictive accuracy of the model. Immune infiltration was analyzed by using CIBERSORT, Xcell and ssGSEA in high-risk and low-risk groups. The immune infiltration of the high-risk group was significantly lower than that of the low-risk group. Immune checkpoint analysis revealed that the expression of PDCD1, CTLA4, TIGIT, CD274, HAVR2 and LAG3 were significantly different in groups with different levels of risk scores. WGCNA analysis found that the yellow-green module contained seven genes (ALOX5AP, FCER1G, GBP4, HCK, MMP9, RARRES2 and TRIM22) from the nine-IRG prognostic model, of which the yellow-green module had the highest correlation with risk scores. The results of GO and KEGG suggested that the genes in the yellow-green module were mainly enriched in immune-related biological processes. Finally, we analyzed the prognostic ability and expression characteristics of ALOX5AP, ARHGAP15, CCL8, FCER1G, GBP4, HCK, MMP9, RARRES2 and TRIM22 in metastatic melanoma. Overall, a prognostic model for metastatic melanoma based on the characteristics of the tumor immune microenvironment was established, which was helpful for further studies.It could function well in helping people to understand the characteristics of the immune microenvironment in metastatic melanoma and to find possible therapeutic targets.

scholarly journals An 8-lncRNA Signature Predicts Survival of Triple-Negative Breast Cancer Patients Without Germline BRCA1/2 Mutation

2020 ◽  
Author(s):  
Minling Liu ◽  
Wei Dai ◽  
Mengyuan Zhu ◽  
Xueying Li ◽  
Shan Huang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Overall Survival ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Cox Regression ◽  
Validation Cohort ◽  
Functional Enrichment ◽  
Biological Behavior ◽  
Risk Scores ◽  
Training Cohort

Abstract Background: Triple-negative breast cancer (TNBC) is a particular breast cancer subtype with poor prognosis due to its aggressive biological behavior and strong heterogeneity. TNBC with germline BRCA1/2 mutation (gBRCAm) have higher sensitivity to DNA damaging agents including platinum-based chemotherapy and PARP inhibitors. But the treatment of TNBC without gBRCAm remains challenging. This study aimed to develop a long non-coding RNA (lncRNA) signature of TNBC patients without gBRCAm to improve risk stratification and optimize individualized treatment.Methods: 98 TNBC patients without gBRCAm were acquired from The Cancer Genome Atlas (TCGA) database. The univariable Cox regression analysis and LASSO Cox regression model were applied to establish an lncRNA signature in the training cohort (N = 59). Then Kaplan–Meier survival curve and time-dependent ROC curve were used to validate the prognostic ability of the signature. The signature related mRNAs were identified using the Pearson correlation. Functional enrichment analysis of related mRNA was performed using the Metascape. The qPCR assay was performed to confirm the expressions and clinicopathological correlationsof two potential lncRNAs HAGLROS and TONSL-AS1 in 30 paired clinical triple-negative breast cancer samples without gBRCAm.Results:We developed an 8-lncRNA signature in the training cohort including HAGLROS, AL139002.1, AL391244.2, AP000696.1, AL391056.1, AL513304.1, TONSL-AS1 and AL031008.1. In both the training and validation cohort, patients with higher risk scores showed significantly worse overall survival compared to those with lower risk scores(P=0.00018 and P =0.0068 respectively). 1, 5, 8-year AUC in the training cohort were 1.000, 1.000 and 0.908 respectively, in the validation cohort were 0.785, 0.790 and 0.892 respectively indicating that our signature has a good prognostic capacity. Signature related mRNA mainly enriched in terms include RNA metabolic process, DNA repair pathways, and so on. Two potential lncRNAs HAGLROS and TONSL-AS1 were found frequently overexpressed in TNBC without gBRCAm, and significantly associated with tumor grade and invasion.Conclusions: We constructed a novel 8-lncRNA signaturewhich significantly associated with the overall survival of TNBC patients without gBRCAm. Among those 8lncRNAs, HAGLROS and TONSL-AS1 may be potential therapeutic targetswhich function needed further exploration.

scholarly journals Three Immune-Associated Subtypes of Diffuse Glioma Differ in Immune Infiltration, Immune Checkpoint Molecules, and Prognosis

2020 ◽  
Vol 10 ◽  
Author(s):  
Quanwei Zhou ◽  
Xuejun Yan ◽  
Weidong Liu ◽  
Wen Yin ◽  
Hongjuan Xu ◽  
...  
Keyword(s):  
Immune Cell ◽  
Cox Regression ◽  
Expression Profiles ◽  
The Cancer Genome Atlas ◽  
Functional Enrichment ◽  
Immune Checkpoints ◽  
Signal Pathways ◽  
Diffuse Glioma ◽  
Immune Infiltration ◽  
Immune Related Genes

Diffuse glioma is one of the most prevalent malignancies of the brain, with high heterogeneity of tumor-infiltrating immune cells. However, immune-associated subtypes of diffuse glioma have not been determined, nor has the effect of different immune-associated subtypes on disease prognosis and immune infiltration of diffuse glioma patients. We retrieved the expression profiles of immune-related genes from The Cancer Genome Atlas (TCGA) (n = 672) and GSE16011 (n = 268) cohorts and used them to identify subtypes of diffuse glioma via Consensus Cluster Plus analysis. We used the limma, clusterProfiler, ESTIMATE, and survival packages of R for differential analysis, functional enrichment, immune and stromal score evaluation respectively in three subtypes, and performed log-rank tests in immune subtypes of diffuse glioma. The immune-associated features of diffuse glioma in the two cohorts were characterized via bioinformatic analyses of the mRNA expression data of immune-related genes. Three subtypes (C1–3) of diffuse glioma were identified from TCGA data, and were verified using the GSE16011 cohort. We then evaluated their immune characteristics and clinical features. Our mRNA profiling analyses indicated that the different subtypes of diffuse glioma presented differential expression profile of specific genes and signal pathways in the TCGA cohort. Patients with subtype C1, who were mostly diagnosed with grade IV glioma, had poorer outcomes than patients with subtype C2 or C3. Subtype C1 was characterized by a higher degree of immune cell infiltration as estimated by GSVA, and more frequent wildtype IDH1. By contrast, subtype C3 included more grade II and IDH1-mutated glioma, and was associated with more infiltration of CD4+T cells. Most subtype C2 had the features between subtypes C1 and C3. Meanwhile, immune checkpoints and their ligand molecules, including PD1/(PD-L1/PDL2), CTLA4/(CD80/CD86), and B7H3/TLT2, were significantly upregulated in subtype C1 and downregulated in subtype C3. In addition, patients with subtype C1 exhibited more frequent gene mutations. Univariate and multivariate Cox regression analyses revealed that diffuse glioma subtype was an effective, independent, and better prognostic factor. Therefore, we established a novel immune-related classification of diffuse glioma, which provides potential immunotherapy targets for diffuse glioma.

RAD001, An Inhibitor of mTOR, Enhances Monocytic Differentiation Induced by ATRA through Phosphorylation of C/EBPα In AML Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2152-2152
Author(s):  
Hideki Yoshida ◽  
Toshihiko Imamura ◽  
Atsushi Fujiki ◽  
Yoshifumi Hirashima ◽  
Mitsuru Miyachi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Microarray Analysis ◽  
Combination Treatment ◽  
Annexin V ◽  
Resistant Cell ◽  
Pcr Analysis ◽  
Monocytic Differentiation ◽  
Cd11b Expression ◽  
Qrt Pcr ◽  
Induced Apoptosis

Abstract Abstract 2152 Background: CCAAT/ enhancer binding protein alpha (C/EBPα) is a critical transcription factor that controls monocytic and granulocytic differentiation. Several recent studies have reported that C/EBPα expression is down-regulated in acute myeloid leukemia (AML), leading to suppression of monocytic differentiation. All-trans-retinoic acid (ATRA) induces numerous transcriptional factors, including C/EBPα; however, ATRA alone is not sufficient to induce monocytic differentiation in AML. The purpose of this study was to identify agents that increase the efficacy of ATRA. RAD001 (Everolimus; provided by Novartis), a rapamycin analog, is a relatively new drug that inhibits the Akt/ PI3K/ mTOR pathway. To assess the utility of differentiation therapy as a treatment for types of AML other than acute promyelocytic leukemia, we evaluated the effects of RAD001 and ATRA combination treatment in several AML cell lines. Methods: Three AML cell lines (U-937, THP-1, and KOCL48) and two primary AML samples were treated with 2.5–5.0 nM RAD001 and 1 μM ATRA for five days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis and flow cytometric analysis (FCM) of CD11b expression. An Annexin V assay was carried out to measure apoptosis. Microarray analysis using an Agilent expression array was employed to determine changes in gene expression associated with ATRA and RAD001 combination treatment. Quantitative RT-PCR (qRT-PCR) analysis was performed to validate the microarray results. Western blotting was carried out to measure the phosphorylation of C/EBPα at Ser 21. Results: We determined that ATRA and RAD001 treatment induced morphological changes characteristic of monocytic differentiation. Microarray analysis of THP-1 revealed that ATRA and RAD001 induced expression of a set of genes associated with monocytic differentiation, including MPEG1, CD11b, CD115 and CD14. FCM analysis confirmed that ATRA and RAD001 intensified CD11b expression in the three cell lines tested, especially in the two ATRA-resistant cell lines (KOCL48 and U937). qRT-PCR analysis also revealed that ATRA and RAD001 treatment increased expression of C/EBPα and C/EBPε, which is involved in the terminal stages of monocytic differentiation, in all three cell lines and two primary samples compared to treatment with ATRA only. Expression of PU.1 was also increased by combination treatment in all cells tested except the U937 cell line. Western blot analysis revealed that ATRA and RAD001 decreased phosphorylation of C/EBPα at serine 21. ATRA and RAD001 combination treatment also suppressed cell growth in two ATRA-resistant cell lines (growth inhibition rate: 70–80%). The Annexin V assay demonstrated that ATRA and RAD001 combination treatment strongly induced apoptosis in the three cell lines tested. Microarray analysis revealed that FasL,FADD, and caspase 8, which are associated with apoptotic pathways, showed the greatest degree of up-regulation in THP-1 cells treated with ATRA and RAD001. qRT-PCR analysis confirmed up-regulation of these genes in all three cell lines and in both primary AML samples, indicating that ATRA and RAD001 induce apoptosis in AML cells through the extrinsic cell death signaling pathway. Conclusions: RAD001 induced monocytic differentiation through induction of a set of genes associated with monocytic differentiation and phosphorylation of C/EBPα at Ser 21 when combined with ATRA. This combination therapy also induced apoptosis in AML cells through activation of the extrinsic cell death signaling pathway. Disclosures: No relevant conflicts of interest to declare.

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