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The enzymatic activities of CD38 enhance CLL growth and trafficking:
implications for therapeutic targeting
Authors: Tiziana Vaisitti1,2, Valentina Audrito1,2, Sara Serra1,2, Roberta Buonincontri1,2, Giovanna
Sociali3, Elena Mannino3, Andrea Pagnani2, Antonella Zucchetto4, Erika Tissino4, Candida Vitale5,
Marta Coscia5, Cesare Usai6, Chris Pepper7, Valter Gattei4, Santina Bruzzone3, Silvia Deaglio1,2*
Affiliations: 1Department of Medical Sciences, University of Torino, Torino, Italy; 2Human Genetics
Foundation (HuGeF), Torino, Italy; 3DIMES-Section of Biochemistry AND CEBR, University of
Genova, Genova, Italy; 4Centro di Riferimento Oncologico (CRO) IRCCS, Aviano, Italy; 5Division of
Hematology, University of Torino, Azienda Universitaria Ospedaliera Città della Salute e della
Scienza di Torino, Torino, Italy; 6Institute of Biophysics, CNR, Genova, Italy; 7Institute of Cancer &
Genetics, Cardiff University, Heath Park, Cardiff, UK.
Running title: Role of the enzymatic activities of CD38 in CLL
Correspondence: Silvia Deaglio, MD, PhD, Department of Medical Sciences, University of Torino
School of Medicine & Human Genetics Foundation, via Nizza, 52, 10126 Torino, Italy. Email:
[email protected].
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Supplemental Materials and Methods
Cells
Peripheral blood mononuclear cells (PBMC if purity > 95%) or purified B lymphocytes were used
for in vitro and in vivo assays. Mec-1 cells were cultured in RPMI-1640 with 10% fetal calf serum
(Sigma Aldrich, Milan, Italy).
Reagents
Chemokines used were CCL19, CXCL10 and CXCL12 (all at 200 ng/ml R&D Systems, Milan, Italy).
Antibodies
Antibodies used for immunofluorescence were: anti-CD38 Alexa488-conjugated (AbD Serotec,
Milan, Italy), -CD19-PE, -CD38-PE, -CD19-FITC, -CD45-PerCP (all from eBioscience, Milan, Italy).
Anti-CD38 antibodies used were: IB4, AT-1, OKT10 and SUN-4B7 (from Prof. F. Malavasi, University
of Turin, Italy).
Co-capping experiments were carried using anti-CD19 (BU12), anti-CD81, anti-CD29 (Moon-4, from
Prof. F. Malavasi, University of Turin, Italy) and anti-CD49d (clone HP2/1, Abd Serotec) antibodies.
Alexa633-labeled GMIg (Dako, Glostrup, Denmark) was used to induce capping, anti-CD38Alexa488 (AbD Serotec) to counterstain (15).
Production of lentiviruses
Lentiviral particles containing the genetic material for WT CD38 were generated as described (47).
The green fluorescent protein (GFP) virus, driven by the same promoter, was used as the control
(19). A lentiviral plasmid encoding for the CD38 mutant was generated using the Site Directed
Mutagenesis Kit (Agilent, Milan, Italy) introducing a single base pair change (glutamic acid at
position 226 into aspartic acid, E226D).
Intracellular staining for Vav-1
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When indicated, cells were permeabilized (0.1% Tween-20), blocked (10% goat serum) and stained
with anti-phospho-Vav-1 (Tyr-174, Abcam, Cambridge, UK), followed by TRITC-conjugated
secondary antibody (Millipore, Billerica, MA). Alexa633 phalloidin (Life Technologies) was used to
counterstain. Data from at least 100 cells were recorded. Results were reported as:
phosphoprotein mean fluorescence intensity in the presence of VCAM-1 / mean fluorescence
intensity obtained in the presence of polylysine.
Antibodies used for immunohistochemistry
Anti-CD20 (1:50, clone L26) and anti-CD38 () were from Leica Microsystems. Anti-mouse HRPconjugated antibodies and 3,3′-diaminobenzidine (EnVision™ System, Dako) were used to visualize
the reaction.
Gene expression profiling analysis
The analysis of the scanned images and the determination of the detection call for each probe set
of the array were obtained with the GenomeStudio Gene Expression Module Software (Illumina)
applying a standard quantile normalization. Expression values were background subtracted and
filtered against a rather stringent p-value threshold of 0.01. All probes were selected if
simultaneously passed the p-value threshold test and if their expression level was higher than the
background.
The P value threshold adopted for the analysis was P<0.01, considering only sequences with a
minimum fold change higher than one in absolute value (|logFc|>1).
Unsupervised clustergram analysis of the whole probe-set was performed using the clustergram
tool of the of MATLAB bioinformatics package (http://www.mathworks.it/) using a Pearson
correlation coefficient as metric. Differentially expressed lists were constructed on probes which:
(i) simultaneously passed the the p-value threshold an all considered experiments, and (ii) whose
minimum fold change was larger than one in absolute value (|logFc|>1).
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Venn diagrams were then created to visualize intersections between class comparison results and
to
select
the
sequences
of
interest
using
the
Venny
software
(http://bioinfogp.cnb.csic.es/tools/venny/). For enrichment analysis, differentially expressed
genes were classified according to their gene ontology (GO) annotations and cellular pathway
association according to Kyoto Encyclopedia of Genes and Genomes (KEGG) database, using online
tools: Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics
Resources (http://david.abcc.ncifcrf.gov/), and GeneCodis (http://genecodis.cnb.csic.es/), applying
cumulative hypergeometric distribution and a cut-off on Bonferroni-corrected P-value (p=0.05).
All microarray data have been deposited with Gene Expression Omnibus. The submission number
is GSE56369 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56369).
The expression of selected genes derived from the microarray data in the CD38 clone were
calculated using the expression value (fluorescence signals) obtained by chip and normalized over
the GFP clone. The comparison between microarray and qPCR data was indicated as the
expression values are expressed as log2 of the fold change (FC).
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Supplemental figure legends
Supplementary Figure 1. Characterization of Mec-1 clones expressing a WT (CD38WT) or an
enzymatically inactive (CD38M) form of CD38. (A) qRT-PCR data indicate comparable expression
levels of CD38WT and CD38M molecules in Mec-1 clones. (B) Western blot analysis confirming
protein expression in both Mec-1 variants. AT-1 anti-CD38 mAb was used to visualize the protein.
(C) Surface expression of CD38 as determined by using 5 different monoclonal antibodies. AT-13/5
was directly conjugated to Alexa-488, while the other 4 were used in indirect immunofluorescence
using a FITC-conjugated goat anti-mouse IgG as secondary reagent. (D) HPLC analyses to measure
NAD glycohydrolase and cADPR hydrolase activities in the different Mec-1 clones.
Supplementary Figure 2. Ligation of CD38WT and CD38M molecules activates a similar signaling
pathway. (A) Western blot analysis of the activation of ERK1/2 or p38/JNK following ligation of
CD38 using agonistic antibodies. Results obtained using Mec-1/CD38WT or /CD38M clones are
shown. (B) Cumulative data from 3 independent experiments. Results are expressed as fold
increase over untreated cells kept under the same conditions.
Supplementary Figure 3. Comparable expression of chemokine receptors in the different Mec-1
variants. Representative profiles of CCR7 (A), CXCR4 and CXCR3 (B) in Mec-1/CD38WT, /CD38M or
/GFP. Dotted lines indicate expression levels of cells in culture, while full line levels of cells
immediately after in vivo growth. (C) MFI values of CXCR4 and CXCR3 in the different Mec-1
variants, in vitro (open bars) and in vivo (black bars) conditions. Data from at least 3 independent
experiments. (D) qRT-PCR data showing expression levels of CXCR4 and CXCR3 in vitro (open bars)
and in vivo (black bars).
Supplementary Figure 4. Mec-1/CD38WT cells show a more aggressive behavior in vivo than Mec1/CD38M or /GFP cells. Immunohistochemical analysis of CD20 staining in liver (A) or lung (B)
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sections from mice injected with the different Mec-1 variants and analyzed after 25 days. CD38
expression is maintained after in vivo growth in both Mec-1/CD38WT and /CD38M, as determined
by immunohistochemical analysis of kidney sections (C) or immunofluorescence using cells
purified from kidney metastases (D).
Supplementary Figure 5. Gene expression profile analysis and qRT-PCR validation of the
microarray data. (A) Unsupervised hierarchical clustering of Mec-1/CD38WT, /CD38M or /GFP cells
shows clustering of /CD38M and /GFP cells, while /CD38WT are clearly distinct both after in vitro
culture and in vivo xenograft. (B) Pie chart showing the most represented GO terms and KEGG
pathways obtained analyzing the 2,514 sequences representing the Mec-1/CD38WT signature.
Comparison between expression levels using microarray (open bars) or qRT-PCR (black bars) of
CXCL10 (C), IL18RAP (D) or IL-10 (E). The different conditions are indicated in the x axis. Data are
presented as relative expression calculated as Log2 of the signal intensity for microarray data,
while qRT-PCR expression levels were obtained using the CT method, standardizing over actin
levels. (F) Linear regression curve showing direct correlation between microarray and qRT-PCR
data (R2= 0.79, p<0.0001). Individuals dots expression of single genes selected for analysis
(CXCL10, IL-18RAP, CADM1, VCAM-1, CCL17, CCL22, CXCL13, CXCR3, CX3CL1, CXCR4 and IL-10). For
each gene in vitro and in vivo conditions were examined for each Mec-1 clone studied.
Supplementary Figure 6. Kuromanin inhibits endogenous CD38 enzymatic activities in
splenocytes from NSG mice. NSG male mice (age 8-12 weeks) were treated with kuromanin for
the indicated time points. At the end of the incubation time, animals were euthanized, splenocytes
recovered and the enzymatic activities of CD38 measured using the NGD assay. Results are
expressed as arbitrary units of fluorescence.
Supplementary Figure 7. Kuromanin inhibits CLL cell chemotaxis. PBMC from CLL patients were
exposed to kuromanin (100 M, 30 minutes, 37 °C) before plating in the upper chamber of a
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Boyden chamber. CXCL12 (100 ng/ml) was used as chemoattractant. The migration index was
calculated after staining for CD19 and CD5.
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