CD38/CD31 Interactions Activate Genetic Pathways Leading to Proliferation and Migration in Chronic Lymphocytic Leukemia Cells Silvia Deaglio,1,2 Semra Aydin,1,2 Maurizia Mello Grand,3 Tiziana Vaisitti,1,2 Luciana Bergui,4 Giovanni D’Arena,5 Giovanna Chiorino,3 and Fabio Malavasi1,2 1Department of Genetics, Biology and Biochemistry, 2Research Center for Experimental Medicine (CeRMS), 4Department of Medicine and Experimental Oncology, University of Torino Medical School, Turin, Italy; 3Laboratory of Cancer Genomics, Fondo Edo Tempia, Biella, Italy; and 5IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy Human CD38 is a pleiotropic glycoprotein belonging to a family of enzymes/receptors involved in the catabolism of extracel- lular nucleotides. CD38-receptor activities are regulated through binding to the nonsubstrate ligand CD31. CD38 expression above a critical threshold is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients. Activation of CD38 by means of agonistic monoclonal antibodies or the CD31 ligand induces proliferation and immunoblast differentiation of CLL cells. Here we define the genetic signature that follows long-term in vitro interactions between CD38+ CLL lymphocytes and CD31+ cells. The emerging profile confirms that the CD31/CD38 axis activates genetic programs relevant for proliferative responses. It also in- dicates a contribution of this pathway to the processes mediating migration and homing. These results further support the notion that the CD31/CD38 axis is part of a network of accessory signals that modify the microenvironment, favoring localization of leukemic cells to growth-permissive sites. © 2010 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2009.00146 INTRODUCTION tic marker for chronic lymphocytic leuke- We showed that activation of CD38 Human CD38 is a pleiotropic glyco- mia (CLL) patients (5). The working hy- induces proliferation and immunoblast protein belonging to a complex family of pothesis of our group was that CD38 is differentiation of CLL cells (10). These enzymes of the cell surface involved in not merely a marker in CLL, but is also a signals are regulated by physical local- the catabolism of extracellular nu- cell surface receptor and adhesion mole- ization of the CD38 receptor within lipid cleotides (1). During evolution, CD38 ac- cule directly involved in the delivery of microdomains and act in synergy with quired the ability to mediate cell–cell in- growth signals (6). Several pieces of evi- the B-cell receptor/CD19 complex (11). teractions, acting as a receptor and dence support this view. First, CD38 ex- The initial observations were obtained by binding the nonsubstrate ligand CD31. pression is higher within the bone mar- use of agonistic monoclonal antibodies CD31/CD38 interactions drive activation row and the lymph nodes, where the and then extended to a model closely re- and proliferation of distinct lymphocyte proliferative core of the disease resides sembling the physiological environment populations (2). (7). Moreover, within each CLL clone, in which CLL cells grow and expand. In- Along with the absence of mutations cells expressing CD38 are enriched in ex- deed, CD38+ CLL cells can bind to in the immunoglobulin variable region pression of Ki-67, suggesting that CD38+ murine fibroblasts transfected with the heavy chain gene (IgVH) (3) and expres- cells are a cycling subset (8). The CD38+ CD31 ligand, with resulting increased sion of the cytoplasmic kinase ZAP-70 subset is also characterized by a specific growth and survival (12). In vivo, several (4), CD38 expression above a critical genetic profile, showing upregulation of CD31+ cells can be found in lymphoid threshold is a reliable negative prognos- proliferation/survival pathways (9). organs, often in close contact with CD38+ CLL cells (13). This work was undertaken to obtain a Address correspondence and reprint requests to Silvia Deaglio or Fabio Malavasi, De- genome-wide signature of the transcrip- partment of Genetics, Biology and Biochemistry, University of Turin School of Medicine, tional events that follow activation of the via Santena 19, 10126 Torino, Italy. Phone: (+ 39-011) 696-1734; Fax: (+ 39-011) 696-6155; CD31/CD38 axis. It also provides some E-mail: [email protected]; [email protected]. answers to the issues raised by a recent Submitted October 10, 2009; Accepted for publication November 19, 2009; Epub (www. report, in which the relevance of CD31/ molmed. org) ahead of print November 20, 2009. CD38 interactions in the context of CLL MOL MED 16(3-4)87-91, MARCH-APRIL 2010 | DEAGLIO ET AL. | 87 CD31/CD38 AXIS IN CLL CELLS Table 1. Clinical and biological features of the CLL patients. was called into question (14). The emerg- ing genetic profile indicates that Patient Age, IgVH, CD38, ZAP-70, a b CD31/CD38 interactions are indeed fol- no. years Sex Stage YD TX % % % FISH lowed by activation of genetic programs 1 60 F 0/A 10 T 100 54 2 11q– relevant not only for proliferative re- 2 65 F 2/A 7 T 100 35 12 11q–,13q– sponses, as could be anticipated on the 3 72 F 0/A 7 NT — 70 11 +12,17p– basis of in vitro experiments, but also for 4 47 M 2/A 21 T 91.2 75 0 ND migration and homing. This latter point 557M4/C7T97799ND deserves further attention and suggests a 6 83 M 2/A 5 T 99.7 66 23 +12 potential role for CD38 in directing CLL 7 75 F 1/B 6 NT 99 42 12 17p– cells to specific microenvironments. 8 61 M 4/A 8 NT 99.3 78 21 ND 9 48 M 2/B 8 T 99.1 34 35 13q– 10 67 M 2/A 13 T 99 58 14 +12 MATERIALS AND METHODS aStage defined according to Rai and Binet diagnostic criteria. YD, years since diagnosis; Patients and Cells TX, therapy; NT, untreated; —, missing data; ND, none detected. We obtained 10 samples from CLL bShown as percentage similarity to the closest germline gene. patients presenting with typical mor- phology and immunophenotype after oligonucleotide glass arrays representing to the pathway matrix output given by we obtained written informed consent 41,000 human unique genes and tran- PLAGE. in accordance with institutional guide- scripts (Human Whole Genome Oligo lines and the Declaration of Helsinki. Microarray Array, Agilent Technologies, Web Deposition of Data Analyses included CD38 and ZAP-70 Cernusco, Italy) (15). A dye-swap repli- Data in this study have been deposited stainings (cutoff positivity values of cate was performed for each sample. in the Gene Expression Omnibus (GEO) ≥20% and ≥10%, respectively [15]), Data analysis. Data were preprocessed site (http://www.ncbi.nlm.nih.gov/geo), IgVH mutational status (unmutated if using the Rosetta Resolver SE software accession number GSE14063. ≥98% homology to the germline gene) (Rosetta Biosoftware, Seattle, WA, USA) and fluorescence in situ hybridization (15). Error-weighted Student two-sample All supplementary materials are available (FISH) for chromosomes 11, 12, 13 and paired t test with Benjamini and online at www.molmed.org. 17 (Table 1). Hochberg P value adjustment was ap- B cells were purified from peripheral plied to obtain differentially expressed RESULTS AND DISCUSSION blood mononuclear cells by negative sequences in the L-CD31+ versus L-mock, CD38 receptor activities are regulated selection (15). L-CD31+ versus basal and L-mock versus through binding to CD31, a nonsubstrate basal class comparisons, after a sequence ligand surrogated by ligation with ago- Culture Conditions prefiltering step (average expression nistic monoclonal antibodies [reviewed Mouse L-cells transfected with human ratio P value with respect to the Univer- in (1)]. In vitro experiments showed that CD31 (L-CD31+) or with the empty plas- sal Reference less than 0.01 in at least these interactions induce marked prolif- mid (L-mock) were obtained as de- 50% of the patients of each class compar- eration and immunoblast transformation scribed (16). Freshly purified CLL cells ison). Venn diagrams were then created of a fraction of the leukemic clone. We (1.5 × 106) were plated on mitomycin to visualize intersections between class asked whether these results would en- C–inactivated (0.5 mg/mL, 20 min at comparison results and to select the se- able us to find a confirmation by use of a 37°C) L-CD31+ or L-mock fibroblasts (2 × quences of interest. different technological approach, also 105/well in a 24-well plate) for 5 d. Pathway analysis. Significant differ- needed because the relevance of the ential expression at the level of a collec- CD31/CD38 axis in CLL has recently Gene Profiling tion of predefined pathways from the been called into question (14). We ad- Data collection. RNA was obtained Kyoto Encyclopedia of Genes and dressed this issue by obtaining a ge- from freshly purified CLL cells (basal) or Genomes (KEGG) database was identi- nome-wide signature of the events tak- after culture on L-CD31+ (L-CD31+ pro- fied using Pathway-Level Analysis of ing place after CD31/CD38 interactions. file) or L-mock fibroblasts (L-mock pro- Gene Expression (PLAGE) (http://dulci. To this aim, CLL cells from 10 CD38+ pa- file). Amplified and labeled specimens org/pathways/) (17). tients were used immediately (basal pro- from each sample were combined with Hierarchical clustering. Results were file) or cultured for 5 days in the pres- the Human Universal Reference Total represented using Cluster 3.0 and Tree- ence of L-CD31+ transfectants (L-CD31+ RNA (Clontech, Saint-Germain-en-Laye, View (http://rana.lbl.gov/ EisenSoftware. profile) or of mock-transfected cells France), fragmented and hybridized to htm) and applying hierarchical clustering (L-mock profile) used as controls. The 88 | DEAGLIO ET AL. | MOL MED 16(3-4)87-91, MARCH-APRIL 2010 RESEARCH ARTICLE choice of the 5-day time point was based L-CD31+ vs basal on previous results indicating that prolif- (P=0.001) eration and survival are highest after this incubation time (12).