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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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 3

Cross-linking of SLAMF1 in CLL cells stabilizes the autophagic flux.

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Cross-linking of SLAMF1 in CLL cells stabilizes the autophagic flux. (A)...
(A) qPCR and Western blot showing vimentin and VPS34 mRNA and protein expression by Western blot in primary SLAMF1+ (n = 9) or SLAMF1– (n = 14) CLL cells. (B) Reconstitution of SLAMF1 expression in Mec-1/SLAMF1sh cells by transfection of a shRNA-resistant SLAMF1 mutant (mut2) or an empty vector (mock). Data are presented as the ratio between expression of the target gene in Mec-1/Ctrlsh cells over that of Mec-1/SLAMF1sh cells (gray bars) or the ratio between Mec-1/SLAMF1sh/mut2 cells over Mec-1/SLAMF1sh/mock transfected cells (open bars). The graph shows results from 4 independent transfection experiments. (C) Confocal microscopy analysis of LC3B (green) and LAMP-2 (red) staining in purified CLL cells treated with the agonistic anti-SLAMF1 monoclonal antibody A12, followed by a cross-linker (6 hours, 37°C). Nuclei were counterstained with DAPI (blue). Original magnification, ×63; zoom factor of 2. Representative images from 5 different experiments. Scatter plots represent colocalization analyses between LC3B and LAMP-2 using LAS AF Version Lite 2.4 software. Pearson coefficient (R) and Overlap coefficient (R[r]) are listed. The box plot shows cumulative numbers of LC3B puncta (calculated as average number of puncta/cell) in purified CLL cells from 5 different patients. LAMP-2 mean pixel intensity was analyzed using ImageJ software. (D) Western blot analysis of LC3B-I/II expression, following SLAMF1 cross-linking in the presence or absence of chloroquine (Chloro, 15 μM). ΔLC3B-II levels were calculated as the difference of LC3B-II protein levels between chloroquine-treated and untreated states. Data from 8 independent experiments. (E) Electron microscopy analysis of CLL cells treated with SLAMF1 antibody as in C. Upon SLAMF1 ligation, autophagosomes became visible as double membranes surrounding cytoplasmic material (full arrow). Late autophagosomes were visualized as a single membrane surrounding a more electron-dense interior (open arrow), in proximity to multivesicular bodies (MVB, arrowhead). Scale bars: 0.5 μm. UT, untreated. Mann-Whitney U test (A, C) or Wilcoxon signed rank test (D) were used.