SLAMF1 regulation of chemotaxis and autophagy determines CLL patient response
Cinzia Bologna, … , Cox Terhorst, Silvia Deaglio
Cinzia Bologna, … , Cox Terhorst, Silvia Deaglio
Published November 30, 2015
Citation Information: J Clin Invest. 2016;126(1):181-194. https://doi.org/10.1172/JCI83013.
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Research Article Oncology

SLAMF1 regulation of chemotaxis and autophagy determines CLL patient response

Abstract

Chronic lymphocytic leukemia (CLL) is a variable disease; therefore, markers to identify aggressive forms are essential for patient management. Here, we have shown that expression of the costimulatory molecule and microbial sensor SLAMF1 (also known as CD150) is lost in a subset of patients with an aggressive CLL that associates with a shorter time to first treatment and reduced overall survival. SLAMF1 silencing in CLL-like Mec-1 cells, which constitutively express SLAMF1, modulated pathways related to cell migration, cytoskeletal organization, and intracellular vesicle formation and recirculation. SLAMF1 deficiency associated with increased expression of CXCR4, CD38, and CD44, thereby positively affecting chemotactic responses to CXCL12. SLAMF1 ligation with an agonistic monoclonal antibody increased ROS accumulation and induced phosphorylation of p38, JNK1/2, and BCL2, thereby promoting the autophagic flux. Beclin1 dissociated from BCL2 in response to SLAMF1 ligation, resulting in formation of the autophagy macrocomplex, which contains SLAMF1, beclin1, and the enzyme VPS34. Accordingly, SLAMF1-silenced cells or SLAMF1lo primary CLL cells were resistant to autophagy-activating therapeutic agents, such as fludarabine and the BCL2 homology domain 3 mimetic ABT-737. Together, these results indicate that loss of SLAMF1 expression in CLL modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, and suggest that these effects underlie unfavorable clinical outcome experienced by SLAMF1lo patients.

Authors

Cinzia Bologna, Roberta Buonincontri, Sara Serra, Tiziana Vaisitti, Valentina Audrito, Davide Brusa, Andrea Pagnani, Marta Coscia, Giovanni D’Arena, Elisabetta Mereu, Roberto Piva, Richard R. Furman, Davide Rossi, Gianluca Gaidano, Cox Terhorst, Silvia Deaglio

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Figure 6

SLAMF1-deficient cells are resistant to drugs that activate autophagy.

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SLAMF1-deficient cells are resistant to drugs that activate autophagy. (...
(A) ROS production upon 5 hour culture of Mec-1/Ctrlsh (white bars) and Mec-1/SLAMF1sh (gray bars) in the presence of fludarabine (100 μM) or ABT-737 (10 μM). ROS were measured using the CellROX assay and are shown as mean fluorescence intensity (MFI) values. Statistical significance was calculated using Friedman’s test followed by Dunn’s multiple comparison using data from 5 independent experiments performed in triplicate. *P < 0.05; **P < 0.01. (B and C) Induction of autophagy after 5-hour cultures with fludarabine or ABT-737 was shown in Mec-1/Ctrlsh and Mec-1/SLAMF1sh by staining for LC3B (green) and LAMP-2 (red), followed by confocal microscopy analysis (original magnification, ×63; zoom factor of 2) (B) or by Western blot for LC3B and p62 (C). Cells were pretreated with chloroquine for 1 hour to block the autophagic flux. ERK1/2 was used as internal loading control. ΔLC3B-II was calculated as indicated in Figure 3D. The fold-change was calculated over the untreated condition. The p62 fold-change was calculated after normalizing p62 over ERK1/2, with the untreated condition set to 1. Data from 6 experiments. (D) Bar graph showing apoptosis levels in Mec-1/Ctrlsh and Mec-1/SLAMF1sh cultured in the presence of fludarabine (100 μM) or ABT-737 (10 μM) for 48 hours. The percentage of apoptosis was calculated as the sum of cells stained by Annexin V and by propidium iodide (PI). (E) Primary CLL cells (n = 5) with a bimodal distribution of SLAMF1 were separated by cell sorting in a SLAMF1hi and a SLAMF1lo subset. The 2 sorted subpopulations were cultured for 24 hours in the presence of fludarabine 5 μM or ABT-737 (10 nM), and cell death was determined by Annexin V/PI staining. Statistical analyses were performed using Mann-Whitney test.