CD38/CD31 Interactions Activate Genetic Pathways Leading to Proliferation and Migration in Chronic Lymphocytic 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 belonging to a family of /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 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 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 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 (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

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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 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 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 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

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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). Moreover, this ap- L-mock vs basal (P=0.01) proach rests on physiological considera- 2,136 tions in that CD31/CD38 static interac- 2,774 1,779 tions are likely to take place within lymphoid organs, in which leukemic cells lie in close and prolonged contact 974 + 1,645 with CD31 residential elements (13). 175 Sample and reference RNAs were co- hybridized to arrays representing 41,000 human unique genes and transcripts. 2,948 The strategy selected to identify the sig- L-CD31+ vs L-mock (P=0.001) nature in response to CD31/CD38 inter- actions relied on identification of genes differentially expressed in the L-CD31+ 1,645 sequences define CD31/CD38 signature: versus L-mock comparison and subse- - 1,238 are annotated quent exclusion of genes modulated in the L-mock versus basal comparison. Re- - 256 are part of KEGG pathways sults are presented in the Venn diagram of Figure 1. Two P value thresholds were - 233 are part of 85 differentially expressed pathways adopted, one to exclude genes that - 122 are part of activation/migration pathways changed ex pression simply as a result of coculture (L-mock versus basal, P value 0.01), and the other to select sequences Figure 1. CD38 engagement by the CD31 ligand triggers genetic pathways leading to with a more significant level of modula- cell proliferation and movement. Venn diagram showing the strategy selected to identify tion (L-CD31+ versus L-mock and L-CD31+ the signature in response to CD31/CD38 interactions. Results were obtained by applying versus basal, P value 0.001) among the error-weighted Student two-sample paired t test with Benjamini and Hochberg P value + + genes differentially expressed as a direct adjustment to L-CD31 versus L-mock, L-CD31 versus basal and L-mock versus basal class consequence of coculture on L-CD31+ comparisons. Circle radii are proportional to the number of transcripts differentially ex- transfectants. pressed in each condition. The gray area defines the CD31/CD38 signature. A cutoff of 0.01 for the adjusted P value was applied to exclude genes that changed expression sim- The result was the identification of ply as a result of coculture (L-mock versus basal), whereas a more stringent threshold (ad- 1645 sequences modulated after CD31/ justed P < 0.001) was applied to the other two comparisons. The resulting 1645 sequences CD38 interactions. The list of genes con- are involved in 85 differentially regulated pathways identified by PLAGE. Of these path- tained in the pathways of the KEGG with ways, 30% are implicated in lymphocyte adhesion/movement and/or signaling. a significant enrichment score (adjusted P value < 0.01) is available as Supplemen- tary Table 1. PLAGE of the sequences to the panel of 30 apoptosis-regulating 120 hours incubation) and/or to charac- identified 85 differentially regulated genes that Tonino and colleagues found teristics intrinsic to the patients selected. pathways (Figure 2 and Supplementary to be unmodulated (14). In our series, 7 of In the context of the control of cell Table 2). Of these, 14 pathways (16%) are 30 genes were differentially regulated movement, relevant examples are up- involved in lymphocyte signaling (black upon CD31/CD38 interactions, including modulation of genes ruling leukocyte arrows in Figure 2), and 12 pathways P19, Bax, Bcl-RAMBO, Bim, PUMA, transendothelial migration, actin cy- (14%) are implicated in / Harakiri and Mcl-1 (Figure 3). All of these toskeleton and focal adhesion on the one movement phenomena (red arrows in genes, with the exception of Mcl-1, are hand, and downregulation of genes influ- Figure 2). Relevant examples of upmodu- upregulated upon CD31/CD38 interac- encing interactions with the extracellular lated genes regulating activation/ tions, suggesting that deregulation of matrix and coding for cell adhesion mole- proliferation pertain to the B-cell receptor, apoptosis is a component of this signa- cules on the other. This genetic signature the mitogen-activated protein kinase– ture. The apparent contrast of these re- implies that long-term CD31/CD38 inter- and the Toll-like receptor–signaling sults with those of Tonino et al. may be actions modulate the competence of CLL pathways (Figure 2 and Supplementary attributable to experimental differences cells to grow and progress in response to Table 2). Specific attention was dedicated (3T3 cells versus L-fibroblasts, 72 versus microenvironmental signals and condi-

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basal L-mock L-CD31+ Pathway name Cell adhesion molecules CAMs 12/132 ECM-receptor interaction 3/87 Antigen processing and presentation 3/82 Type I diabetes mellitus 4/44 Bile acid biosynthesis 3/39 Aminoacyl-tRNA biosynthesis 3/32 Histidine metabolism 3/41 Epithelial cell signaling in Helicobacter pylori infection 5/68 GlycosylphosphatidylinositolGPI-anchor biosynthesis 4/23 Alkaloid biosynthesis II 3/19 1- and 2-Methylnaphthalene degradation 3/24 Benzoate degradation via CoA ligation 4/28 Limonene and pinene degradation 3/28 Glycerophospholipid metabolism 5/66 Adherens junction 14/77 TGF-beta signaling pathway 8/84 Ubiquitin mediated proteolysis 5/45 Tyrosine metabolism 3/59 Inositol phosphate metabolism 5/51 Phosphatidylinositol signaling system 7/78 Oxidative phosphorylation 5/129 Ribosome 7/120 Arachidonic acid metabolism 3/56 Glutathione metabolism 5/42 Metabolism of xenobiotics by cytochrome P450 4/71 Cholera - Infection 4/41 Chondroitin sulfate biosynthesis 3/21 -cytokine receptor interaction 11/256 Glycan structures - biosynthesis 2 7/67 Fructose and mannose metabolism 6/47 Galactose metabolism 3/32 Fatty acid metabolism 5/51 Glycan structures - biosynthesis 1 12/112 Huntington’s disease 4/31 Cell cycle 7/114 Valine, leucine and isoleucine degradation 7/50 Amyotrophic lateral sclerosis ALS 6/19 Glycerolipid metabolism 4/60 DNA polymerase 5/25 Purine metabolism 12/152 Pyrimidine metabolism 10/93 Jak-STAT signaling pathway 11/154 Chronic myeloid leukemia 13/76 Adipocytokine signaling pathway 7/71 receptor signaling pathway 12/63 Fc epsilon RI signaling pathway 11/75 Type II diabetes mellitus 6/44 Focal adhesion 19/194 Axon guidance 11/128 pathway 10/176 Pathogenic Escherichia coli infection - EHEC 7/49 Pathogenic Escherichia coli infection - EPEC 7/49 Long-term potentiation 9/69 MAPK signaling pathway 26/255 Apoptosis 12/84 Regulation of actin cytoskeleton 26/207 Insulin signaling pathway 22/136 Wnt signaling pathway 17/149 VEGF signaling pathway 12/70 Melanogenesis 6/102 Notch signaling pathway 7/48 Glioma 7/63 mediated cytotoxicity 12/131 receptor signaling pathway 10/93 Leukocyte transendothelial migration 16/117 Lysine degradation 6/51 Toll-like receptor signaling pathway 11/90 Long-term depression 4/77 N-Glycan biosynthesis 7/41 Neuroactive ligand-receptor interaction 6/299 Gap junction 3/92 GnRH signaling pathway 7/97 Tight junction 8/119 Tryptophan metabolism 9/84 Colorectal cancer 9/85 Pancreatic cancer 9/73 mTOR signaling pathway 8/50 Melanoma 7/70 Neurodegenerative Disorders 5/39 Propanoate metabolism 3/34 Selenoamino acid metabolism 3/34 Thyroid cancer 3/31 SNARE interactions in vesicular transport 3/36 Starch and sucrose metabolism 5/81 Prion disease 3/14 #1 #2 #6 #10 #5 #9 #3 #7 #4 #8 #1 #2 #6 #10 #5 #9 #3 #7 #4 #8 #1 #2 #6 #10 #5 #9 #3 #7 #4 #8

: adhesion-related pathways : lymphocyte signaling-related pathways

-1.50 +1.5

Figure 2. Representation of the expression pattern of 85 pathways differentially expressed as a consequence of CD31/CD38 interactions. Hierarchical clustering was applied to the matrix of the activity levels of the 85 KEGG pathways found by PLAGE analysis to be differen- tially expressed in the L-CD31+ samples compared with the basal and L-mock ones.

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+ basal L-mock L-CD31 Gene combined with clinical research: CD38 as both marker and key component of the pathogenetic Mcl-1 network underlying chronic lymphocytic leuke- PUMA mia. Blood. 108:1135–44. Harakiri 7. Jaksic O, et al. (2004) CD38 on B-cell chronic lym- Bim phocytic leukemia cells has higher expression in BAX lymph nodes than in peripheral blood or bone BAX Bcl-RAMBO marrow. Blood. 103:1968–9. P19 8. Damle RN, et al. (2007) CD38 expression labels an activated subset within chronic lymphocytic #9 #3 #2 #6 #1 #4 #8 #5 #7 #9 #3 #2 #6 #1 #4 #8 #5 #7 #9 #3 #2 #6 #1 #4 #8 #5 #7 #10 #10 #10 leukemia clones enriched in proliferating B cells. Blood. 110:3352–9. -1.50 +1.5 9. Pepper C, et al. (2007) Highly purified CD38(+) and CD38(-) sub-clones derived from the same Figure 3. Representation of the seven apoptosis-related genes differentially expressed chronic lymphocytic leukemia patient have dis- upon CD31/CD38 engagement. Expression pattern of 7 genes of the 1645 gene signatures tinct gene expression signatures despite their that are also part of the 30 apoptosis-regulating genes reported in (14). Red squares refer monoclonal origin. Leukemia. 21:687-96. 10. Deaglio S, et al. (2003) CD38 is a signaling mole- to upregulation in the sample with respect to the Universal Reference, and green squares cule in B-cell chronic lymphocytic leukemia cells. to downregulation. Expression ratios are represented in a log 10 scale. All the genes except Blood. 102:2146–2155. Mcl-1 increased their expression as a consequence of CD31/CD38 interactions. Two differ- 11. Deaglio S, et al. (2007) CD38/CD19: a lipid raft + ent Agilent probes specific for Bax are overexpressed in the L-CD31 condition. dependent signaling complex in human B cells. Blood. 109:5390–8. 12. Deaglio S, et al. (2005) CD38 and CD100 lead a tions. Independent immunohistochemical Berlucchi” (F Malavasi) and the Regione network of surface receptors relaying positive studies on lymph node sections revealed Piemonte (S Deaglio, T Vaisitti and signals for B-CLL growth and survival. Blood. a direct association between the number F Malavasi). The Fondazione Inter- 105:3042–50. 13. Patten PE, et al. (2008) CD38 expression in of endothelial cells (CD31+) and the level nazionale Ricerche Medicina Sperimen- chronic lymphocytic leukemia is regulated by the of CD38 expression by CLL cells (13), in- tale provided financial and administra- tumor microenvironment. Blood. 111:5173–81. directly confirming that CD31/CD38 tive assistance. S Aydin is a student of 14. Tonino SH, Spijker R, Luijks DM, van Oers MH, static interactions occur in vivo. Moreover, the Ph.D. program in Advanced Tech- Kater AP. (2008) No convincing evidence for a role the percentage of CD38+ CLL cells (8,18) niques in the Localization of Human Tu- of CD31-CD38 interactions in the pathogenesis of chronic lymphocytic leukemia. Blood. 112:840–3. and the density of tumor vessels (19,20) mors (University of Turin). 15. Deaglio S, et al. (2007) CD38 and ZAP-70 are correlate with neoplastic proliferation functionally linked and mark CLL cells with high and disease aggressiveness. DISCLOSURE migratory potential. Blood. 110:4012–21. In conclusion, these results provide The authors declare that they have no 16. Deaglio S, et al. (1998) Human CD38 (ADP-ribosyl further support to the hypothesis that competing interests as defined by Molecu- cyclase) is a counter-receptor of CD31, an Ig superfamily member. J. Immunol. 160:395–402. CLL maintenance and progression derive lar Medicine, or other interests that might 17. Tomfohr J, Lu J, Kepler TB. (2005) Pathway level not only from the essential drive pro- be perceived to influence the results and analysis of gene expression using singular value vided by the antigen but also from a discussion reported in this paper. decomposition. BMC Bioinformatics. 6:225. number of accessory signals that modify 18. Lin TT, et al. (2008) Highly purified CD38 sub- the microenvironment to favor localiza- REFERENCES populations show no evidence of preferential clonal evolution despite having increased prolif- tion of neoplastic cells to growth- 1. Malavasi F, et al. (2008) Evolution and function of erative activity when compared with CD38 sub- permissive sites. The CD31/CD38 axis is the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. Physiol. Rev. 88:841–86. populations derived from the same chronic lym- part of this network. 2. Deaglio S, et al. (2000) CD38/CD31, a receptor/ phocytic leukaemia patient. Br J Haematol ligand system ruling adhesion and signaling in 142:595–605. 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