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 5

Use of selective inhibitors to highlight sequential steps in the SLAMF1-induced autophagy.

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Use of selective inhibitors to highlight sequential steps in the SLAMF1-...
(A) Effects of the pretreatment with SP600125 (JNK1/2 inhibitor, 15 μM), NAC (ROS scavenger, 5 mM), or DPI (NOX2 inhibitor, 0.05 μM) in the phosphorylation of BCL2, JNK1/2, and the appearance of lipidated LC3B induced upon SLAMF1 ligation in Mec-1 cells. Representative gel from 8 different experiments. The percentage of inhibition was calculated as: 100 (intensity of phosphorylated protein/control protein), after considering 100% of the value obtained in the SLAMF1-alone condition. (B and C) Confocal microscopy of primary CLL cells showing the effects of SP600125 (5 μM), NAC (2.5 mM) and DPI (0.05 μM) on the phosphorylation of BCL2 (B) and on LC3B (C) induced upon SLAMF1 ligation. Original magnification, ×63; zoom factor of 2. The box plot shows mean pixel intensity of BCL2 signal or cumulative numbers of LC3B puncta (calculated as average number of puncta/cell) in purified CLL cells from 3 different patients treated as described. (D) Bar graph showing the percentage of inhibition in ROS accumulation following SLAMF1 cross-linking in the presence of NAC and DPI, considering the level of ROS in the SLAMF1-alone condition to be 100%. ROS accumulation was measured with the CellROX probe. (E) Immunoprecipitation experiments performed in Mec-1 cells after cross-linking SLAMF1 with the A12 antibody (30 minutes, 37°C). Where indicated, SP600125 (SP) was added. The right image is representative of an immunoprecipitation experiment with an anti-SLAMF1 or an isotype-matched irrelevant antibody and subsequent immunoblot for beclin1 and VPS34. Whole cell lysate (WCL) is shown in the first lane. Representative images from 3 independent experiments. Statistical significance was calculated using Friedman’s test followed by Dunn’s multiple comparison. ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05.