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Title: Macrophages confer survival signals via CCR1-dependent translational MCL-1 induction in chronic lymphocytic leukemia.      
keywords:
Transcriptome or Gene expression
ID:
PRJNA374356
description:
Protective interactions with bystander cells in micro-environmental niches such as lymph nodes (LNs) contribute to survival and therapy resistance of chronic lymphocytic leukemia (CLL) cells. This is caused by a shift in expression of BCL-2 family members. Pro-survival proteins BCL-XL, BFL-1, and MCL-1 are upregulated by LN-residing T cells through CD40L interaction, presumably via NF-κB signaling. Macrophages also reside in the LN, and are assumed to provide important supportive functions for CLL cells. However, if and how macrophages are able to induce survival is incompletely known. We first established that macrophages induced survival due to an exclusive upregulation of MCL-1. Next, we investigated the mechanism underlying MCL-1 induction by macrophages in comparison with CD40L. Genome-wide expression profiling of in vitro macrophage- and CD40L-stimulated CLL cells indicated activation of the PI3K-AKT-mTOR pathway, which was confirmed in ex vivo CLL LN material. Inhibition of PI3K-AKT-mTOR signaling abrogated MCL-1 upregulation and survival by macrophages as well asCD40 stimulation. MCL-1 can be regulated at multiple levels, and we established that AKT leads to increased MCL-1 translation, but does not affect MCL-1 transcription or protein stabilization. Furthermore, among macrophage-secreted factors that could activate AKT, we found that induction of MCL-1 and survival critically depended on C-C Motif Chemokine Receptor-1 (CCR1). In conclusion, this study indicates that two distinct micro-environmental factors, CD40L and macrophages, signal via CCR1 to induce AKT activation resulting in translational stabilization of MCL-1, and hence can contribute to CLL cell survival. Overall design: Patient material was obtained from three CLL patients, after written informed consent, during routine follow-up or diagnostic procedures at the Academic Medical Center, Amsterdam, the Netherlands. Peripheral blood mononuclear cells (PBMCs) of these CLL patients were isolated and stored in liquid nitrogen. Monocyte derived macrophages were obtained by differentiating monocytes isolated from healthy donor buffy coats after obtaining written informed consent. Monocytes were then differentiated to either M1 macrophages using 10ng/mL IFN-γ or M2 macrophages using 10ng/mL IL-4. NIH-3T3 mouse embryofibroblasts (3T3 cells) were supplied and characterized (for identity control, cytogenetics, and immunophenotype) by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). To mimic T cell induced CD40 signaling, these 3T3 cells were stably transfected with human CD40L (3T40 cells). CLL samples (n=3) were thawed and diluted and, in two different experiments, stimulated for 16h with either 3T40 cells or macrophages (M1/M2) or left untreated (CTRL). CLL cells were sorted to >99% purity using CD5/CD19 staining and FACS. Total cellular RNA was isolated and gene expression levels were measured using Affymetrix Human Genome U133 Plus 2.0 arrays. This study includes two re-analyzed sample data (CLL_CD40L_3T40_rep2; CLL_CD40L_3T40_rep3) available in the GEO database as GSE50572 (GSM1223755 and GSM1223743, respectively). The complete dataset representing: (1) 13 new sample data and (2) the re-analyzed data of GSM1223755 and GSM1223743, is linked below as a supplementary file. The results_combined.xlsx contains the results of the differential expression analysis. The metadata.txt contains additional annotations for the re-analyzed samples (e.g. patient ID no. etc.).
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA374356
authentication:
none
authorization:
none
ID:
pmid:28192408
name:
Homo sapiens
ncbiID:
ncbitax:9606
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject