IL21R expressing CD14+CD16+ monocytes expand

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SMM7. 7662.433. 43.14486. SMM8. 5344.555. 46.07221. SMM9. 8406.226. 36.69782. SMM10. 11324.39. 60.34423. SMM11. 9027.734. 48.58276. SMM12.
Plasma Cell Disorders

SUPPLEMENTARY APPENDIX

IL21R expressing CD14+CD16+ monocytes expand in multiple myeloma patients leading to increased osteoclasts

Marina Bolzoni,1 Domenica Ronchetti,2,3 Paola Storti,1,4 Gaetano Donofrio,5 Valentina Marchica,1,4 Federica Costa,1 Luca Agnelli,2,3 Denise Toscani,1 Rosanna Vescovini,1 Katia Todoerti,6 Sabrina Bonomini,7 Gabriella Sammarelli,1,7 Andrea Vecchi,8 Daniela Guasco,1 Fabrizio Accardi,1,7 Benedetta Dalla Palma,1,7 Barbara Gamberi,9 Carlo Ferrari,8 Antonino Neri,2,3 Franco Aversa1,4,7 and Nicola Giuliani1,4,7

Myeloma Unit, Dept. of Medicine and Surgery, University of Parma; 2Dept. of Oncology and Hemato-Oncology, University of Milan; Hematology Unit, “Fondazione IRCCS Ca’ Granda”, Ospedale Maggiore Policlinico, Milan; 4CoreLab, University Hospital of Parma; 5Dept. of Medical-Veterinary Science, University of Parma; 6Laboratory of Pre-clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture; 7Hematology and BMT Center, University Hospital of Parma; 8Infectious Disease Unit, University Hospital of Parma and 9“Dip. Oncologico e Tecnologie Avanzate”, IRCCS Arcispedale Santa Maria Nuova, Reggio Emilia, Italy 1 3

©2017 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2016.153841 Received: August 5, 2016. Accepted: December 23, 2016. Pre-published: January 5, 2017. Correspondence: [email protected]

SUPPLEMENTAL METHODS Immunophenotype of BM CD14+ in patients with monoclonal gammopathies. Briefly, 100 μl of total BM aspirate was incubated in the dark with anti-human HLA-DR-PE (clone L243; BD), anti-human CD14-PerCP-Cy 5.5, anti-human CD16-PE-Cy7 (clone B73.1; BD) and anti-human CD45-APC-H 7 (clone 2D1; BD) for 20 min. Flow cytometry analysis was performed as 6-color experiment by using BD FACS Canto II with Diva software. First, a gate was set around monocytes based on their light-scatter properties. Then, two monocyte populations were defined in that gate: the classical CD14brightCD16- monocytes and CD14dimCD16bright monocytes. In some experiments, 106 mononuclear cells (MNCs), obtained from BM aspirates by density gradient centrifugation (Lympholite-H; Burlington, NC), were stained with anti-human CD14-PerCP-Cy 5.5, anti-human CD16-PE-Cy7 and anti-human CD360/IL-21R APC (Clone 17A12; BD), using the gating strategy described above. Fluorescence in situ hybridization (FISH) analysis. FISH analysis has been performed on purified CD138+ cells to test the presence of: hyperdiploidy (ON9RED/15GREEN; Kreatech Diagnostics; Durham, NC); del(13q) (D13 S319SO/CEP 12SG; Metasystems; Altlussheim, Germany); del(17p) (LSI ATM SG/p53SO; Metasystems); chromosome 14 translocation (14 BREAK-APART; Metasystems). If samples were positive for chromosome 14 translocation, the presence of t(4;14) (FGFR3SO/IGHSG; Abbott Laboratories; Abbott Park, IL), t(11;14) (LSI IGH/CCND1XT; Abbott Laboratories) and t(14;16) (IGH/MAF; Abbott Laboratories) were checked. Microarrays analysis Total RNA was extracted from BM CD14+ cells using the RNeasy total RNA isolation kit (Qiagen; Hilden, Germany). Gene expression profiles of BM CD14+ cells previously isolated were generated on GeneChip® HG-U133 Plus 2.0 arrays (Affymetrix; Santa Clara, CA). The biotin-labeled cRNA was prepared according to the Affymetrix GeneChip Expression Analysis Technical Manual

protocol. Log2-transformed expression values were extracted from CEL files and normalized using RMA procedure in the affy package for Bioconductor and the annotations included in the cdf definition

files

version

20

available

at

the

Brainarray

portal

(http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/20.0.0/version.html). Unsupervised and supervised analyses of gene expression data were carried out using the Significant Analysis of Microarrays (SAM) software version 4.00, as previously reported. 13 Data were deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE70345. Correlation between IL21R expression and that of CCR5, CD40 and SLAMF7 by BM monocytes arisen from gene expression analysis were evaluated by Spearman rho correlation test. Real Time PCR. RNA (0,5 µg) was reverse-transcribed with 400 U Moloney murine leukemia reverse transcriptase (Applied Biosystems, Life Technologies) in accordance with the manufacturer’s protocol. Real Time PCR was performed by adding complementary DNA to a universal master Mix primers and TaqMan probes (Applied Biosystems) for the following genes: IL21R: Hs00222310_m1, SLAMF7: Hs00221793_m1 and ABL: Hs01104728_m1. The Real Time ready assays for CCR5 (Assay ID: 104069) and CD40 (Assay ID: 100254) genes were purchased from Roche Diagnostics (Mannheim, Germany). The expression of selected genes was checked by Real Time PCR by Light Cycler 480 (Roche Diagnostics). To normalize the differences in RNA quality and reverse transcription efficiency, we applied the comparative Ct method using the endogenous reference gene ABL. In the experiments where treated cells were compared to untreated controls, the fold change in mRNA expression (n-fold) of each analyzed gene was calculated as 2-ΔΔCt, as previously described.30 However, when the aim of the analysis was to check the differences between groups of patients, mRNA expression levels were expressed as –ΔCt. STAT3 activity assay.

STAT3 activity was evaluated by means of an ELISA-based commercially available kit (Trans AM STAT Family, Active Motif, Vinci Biochem; Vinci, Florence, Italy), used in accordance with the manufacturers’ procedures. We tested duplicates of nuclear extracts (each containing 5 µg of proteins) of CD14+ cells purified from 3 MGUS, 3 SMM and 3 MM patients and nuclear extracts of HepG2 treated with rhIL-6 (100 ng/ml) were used as positive controls, as suggested in the manufacturers’ procedures. Cell nuclear extracts were obtained and total amount of proteins quantified as previously described.30 BM IL-21 levels in patients with monoclonal gammopathies. The levels of IL-21 in BM plasma were measured in a total cohort of 76 newly diagnosed MM, 42 SMM and 41 MGUS patients. Plasma (5 ml) was obtained after centrifugation from BM aspirates, aliquoted and stored at -20°C until analysis. BM IL-21 plasma levels were detected by Human Interleukin-21 ELISA kit (BioVendor; Brno, Czech Republic), following the manufacturers’ protocol.

SUPPLEMENTAL TABLES Supplemental Table 1: mRNA expression levels of CD14 (929_at) and SDC1 (6382_at). Monocyte samples reported in italic style were excluded from gene expression analysis due to high PC contamination according to 2-groups stratification based on K-means clustering of SDC1 expression. Monocyte sample

CD14

SDC1

MGUS1 MGUS2 MGUS3 MGUS4 MGUS5 MGUS6 MGUS7 MGUS8 MGUS9 SMM1 SMM2 SMM3 SMM4 SMM5 SMM6 SMM7 SMM8 SMM9 SMM10 SMM11 SMM12 SMM13 SMM14 SMM15 MM1 MM2 MM3 MM4 MM5 MM6 MM7 MM8 MM9 MM10 MM11 MM12

8789.818 9006.552 6403.874 9964.512 8146.314 8589.581 7803.366 9120.798 8895.179 8114.306 8029.939 7796.826 9010.986 8058.92 9088.744 7662.433 5344.555 8406.226 11324.39 9027.734 7547.065 7526.294 8964.173 8211.26 7492.444 6873.478 6079.989 8406.226 6458.982 5512.166 10195.97 8460.027 7921.082 7397.48 11284.27 7538.09

40.304 38.80391 35.66097 43.99736 59.22493 57.34123 44.29881 35.85622 32.07205 37.218 39.68942 28.66408 43.27907 43.83396 52.35158 43.14486 46.07221 36.69782 60.34423 48.58276 37.88982 65.80806 28.70343 45.07396 32.61221 163.3324 42.57115 70.01866 49.30689 55.35385 37.92994 33.74889 80.19244 47.13206 30.44155 64.00715

MM13 MM14 MM15 MM16 MM17 MM18 MM19 MM20 MM21 MM22 MM23 MM24 MM25 MM26 MM27 MM28 MM29 MM30 MM31 MM32

8060.529 9567.787 8702.203 8484.997 8436.607 8612.851 5049.836 8848.109 7571.708 9838.354 9886.636 4579.45 5069.29 7199.53 1372.92 7325.70 8592.04 7947.35 5844.00 5309.73

100.2527 37.23853 166.6147 99.10607 35.66015 35.29721 56.44912 74.04942 61.97922 104.0483 45.53263 655.08 1464.04 1209.62 7674.54 702.92 1117.68 2153.92 555.92 2219.51

Abbreviations: MGUS, Monoclonal Gammopathy of Undetermined Significance; SMM, Smoldering Multiple Myeloma; MM, Multiple Myeloma.

Supplemental Table 2: List of genes significantly modulated in BM monocytes across the three groups of patients (MM, SMM and MGUS) by multiclass analysis of gene expression data.

Gene ID FCER1A SLCO4C1 PTPN22 PADI4 SLAMF7 PGM1 RAB37 RPS17

Gene Name

Fc fragment of IgE receptor Ia solute carrier organic anion transporter family member 4C1 protein tyrosine phosphatase, non-receptor type 22 peptidyl arginine deiminase 4 SLAM family member 7 phosphoglucomutase 1 RAB37, member RAS oncogene family ribosomal protein S17 feline leukemia virus subgroup C cellular receptor family member FLVCR2 2 ANXA6 annexin A6 SERPINB10 serpin family B member 10 WLS wntless Wnt ligand secretion mediator CLTCL1 clathrin heavy chain like 1 TMEM55A transmembrane protein 55A SSBP4 single stranded DNA binding protein 4 GNGT2 G protein subunit gamma transducin 2 LOC100506844 uncharacterized LOC100506844 CPNE2 copine 2 CCR5 C-C motif chemokine receptor 5 UBR4 ubiquitin protein ligase E3 component n-recognin 4 SQLE squalene epoxidase CTSL cathepsin L SLC9A3R1 SLC9A3 regulator 1 PNMA1 paraneoplastic Ma antigen 1 LGALS12 galectin 12 CHST7 carbohydrate sulfotransferase 7 LHFPL2 lipoma HMGIC fusion partner-like 2 MYB MYB proto-oncogene, transcription factor ADA adenosine deaminase SRPK1 SRSF protein kinase 1 CENPK centromere protein K RUNX3 runt related transcription factor 3 VAMP5 vesicle associated membrane protein 5 IL21R interleukin 21 receptor hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoylHADHA CoA hydratase , alpha subunit TMEM170B transmembrane protein 170B FBN2 fibrillin 2 NCAPD2 non-SMC condensin I complex subunit D2

SAM Score(d)* 1.49 1.46 1.45 1.45 1.44 1.38 1.34 1.33 1.33 1.32 1.32 1.31 1.29 1.26 1.25 1.22 1.22 1.22 1.21 1.21 1.20 1.20 1.20 1.19 1.19 1.19 1.19 1.19 1.18 1.17 1.17 1.17 1.16 1.16 1.15 1.14 1.14 1.14

MTSS1 CCL18 EIF2D TCN2 DGKZ AZU1 SLC2A9 CDCA7 TUBB SPNS3 TMEM51 KBTBD11 IPCEF1 ECRP SCCPDH ORM1 GAS2L1 ANP32E ADAMTS5 GOT2 MIR205 NECTIN2 KIF11 NRM CTSV SIPA1L2 SLC26A8 EMB FAM101B MKI67 CDCA7L HEG1 B4GALT6 IGF1R DNASE2 IFNGR2 SELM EIF3L TRAF5 TMC8 E2F8 PRRG2 CIT PTCH1 PSRC1 INSIG1 MRPL37

MTSS1, I-BAR domain containing C-C motif chemokine ligand 18 eukaryotic translation initiation factor 2D transcobalamin 2 diacylglycerol kinase zeta azurocidin 1 solute carrier family 2 member 9 cell division cycle associated 7 tubulin beta class I sphingolipid transporter 3 transmembrane protein 51 kelch repeat and BTB domain containing 11 interaction protein for cytohesin exchange factors 1 ribonuclease A family member 2 pseudogene saccharopine dehydrogenase orosomucoid 1 growth arrest specific 2 like 1 acidic nuclear phosphoprotein 32 family member E ADAM metallopeptidase with thrombospondin type 1 motif 5 glutamic-oxaloacetic transaminase 2 microRNA 205 nectin cell adhesion molecule 2 kinesin family member 11 nurim cathepsin V signal induced proliferation associated 1 like 2 solute carrier family 26 member 8 embigin refilin B marker of proliferation Ki-67 cell division cycle associated 7 like heart development protein with EGF like domains 1 beta-1,4-galactosyltransferase 6 insulin like growth factor 1 receptor deoxyribonuclease 2, lysosomal interferon gamma receptor 2 selenoprotein M eukaryotic translation initiation factor 3 subunit L TNF receptor associated factor 5 transmembrane channel like 8 E2F transcription factor 8 proline rich and Gla domain 2 citron rho-interacting serine/threonine kinase patched 1 proline and serine rich coiled-coil 1 insulin induced gene 1 mitochondrial ribosomal protein L37

1.13 1.13 1.13 1.12 1.12 1.12 1.11 1.11 1.11 1.10 1.10 1.10 1.10 1.10 1.09 1.08 1.08 1.08 1.08 1.08 1.07 1.07 1.07 1.07 1.07 1.07 1.07 1.06 1.06 1.06 1.06 1.06 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.04 1.04 1.04 1.04 1.04

RRAS MAP4K4 PIM1 IFITM1 HIP1 ANLN PGD SPOCK1

related RAS viral oncogene homolog mitogen-activated protein kinase kinase kinase kinase 4 Pim-1 proto-oncogene, serine/threonine kinase interferon induced transmembrane protein 1 huntingtin interacting protein 1 anillin actin binding protein phosphogluconate dehydrogenase SPARC/osteonectin, cwcv and kazal like domains proteoglycan 1

1.04 1.04 1.03 1.03 1.03 1.03 1.03 1.03

ISG20

interferon stimulated exonuclease gene 20

1.03

LOC152225 BPI CKAP4 ZBED3 PKM

uncharacterized LOC152225 bactericidal/permeability-increasing protein cytoskeleton associated protein 4 zinc finger BED-type containing 3 pyruvate kinase, muscle

1.03 1.03 1.03 1.02 1.02

*Linear discriminant score defined by the Significance Analysis of Microarrays (SAM) analysis.

Supplemental Table 3: List of genes differentially expressed by BM monocytes obtained from supervised analysis of MM patients as compared to SMM.

DOWN Gene ID PTPN22 PADI4 FCER1A

Gene Name

protein tyrosine phosphatase, non-receptor type 22 peptidyl arginine deiminase 4 Fc fragment of IgE receptor Ia solute carrier organic anion transporter family member SLCO4C1 4C1 RAB37 RAB37, member RAS oncogene family ECRP ribonuclease A family member 2 pseudogene SERPINB10 serpin family B member 10 PGM1 phosphoglucomutase 1 RPS17 ribosomal protein S17 FBN2 fibrillin 2 LGALS12 galectin 12 ANXA6 annexin A6 MIR205 microRNA 205 SPNS3 sphingolipid transporter 3 TMEM55A transmembrane protein 55A SCCPDH saccharopine dehydrogenase ADAM metallopeptidase with thrombospondin type 1 ADAMTS5 motif 5 SLC2A9 solute carrier family 2 member 9 WLS wntless Wnt ligand secretion mediator KBTBD11 kelch repeat and BTB domain containing 11 SRPK1 SRSF protein kinase 1 PGD phosphogluconate dehydrogenase SLC26A8 solute carrier family 26 member 8 UBR4 ubiquitin protein ligase E3 component n-recognin 4 LOC100506844 uncharacterized LOC100506844 SLC9A3R1 SLC9A3 regulator 1 CLTCL1 clathrin heavy chain like 1 CENPK centromere protein K MYB MYB proto-oncogene, transcription factor EMB embigin TMC8 transmembrane channel like 8 EIF2D eukaryotic translation initiation factor 2D MTMR11 myotubularin related protein 11 BPI bactericidal/permeability-increasing protein MRPL33 mitochondrial ribosomal protein L33 NRM nurim FMO5 flavin containing monooxygenase 5

SAM Fold score(d)* Change -4.76 -4.65 -4.62

0.59 0.44 0.42

-4.57

0.45

-4.54 -4.46 -4.38 -4.29 -4.29 -4.18 -4.16 -4.08 -4.02 -3.99 -3.98 -3.97

0.58 0.47 0.29 0.64 0.79 0.54 0.60 0.65 0.76 0.64 0.67 0.72

-3.97

0.73

-3.96 -3.92 -3.88 -3.86 -3.85 -3.85 -3.82 -3.78 -3.78 -3.76 -3.73 -3.71 -3.70 -3.68 -3.61 -3.60 -3.55 -3.53 -3.52 -3.52

0.74 0.64 0.66 0.69 0.72 0.66 0.74 0.62 0.70 0.70 0.43 0.51 0.55 0.74 0.78 0.63 0.42 0.81 0.79 0.72

HIP1 KIF21B B4GALT6 DGKZ CDA HDDC2 MARC1 MAP4K4 TMEM170B CPNE2 ADGRA2 IPCEF1 CACNA2D3 MAP4K1 CDCA7 PKM RNASE2 PSRC1 CKAP4 FAM101B SPOCK1 NME8 CYTL1

huntingtin interacting protein 1 kinesin family member 21B beta-1,4-galactosyltransferase 6 diacylglycerol kinase zeta cytidine deaminase HD domain containing 2 mitochondrial amidoxime reducing component 1 mitogen-activated protein kinase kinase kinase kinase 4 transmembrane protein 170B copine 2 adhesion G protein-coupled receptor A2 interaction protein for cytohesin exchange factors 1 calcium voltage-gated channel auxiliary subunit alpha2delta 3 mitogen-activated protein kinase kinase kinase kinase 1 cell division cycle associated 7 pyruvate kinase, muscle ribonuclease A family member 2 proline and serine rich coiled-coil 1 cytoskeleton associated protein 4 refilin B SPARC/osteonectin, cwcv and kazal like domains proteoglycan 1 NME/NM23 family member 8 cytokine like 1

-3.52 -3.51 -3.49 -3.49 -3.48 -3.46 -3.46 -3.46 -3.43 -3.43 -3.43 -3.42

0.75 0.78 0.77 0.74 0.65 0.78 0.61 0.79 0.62 0.73 0.70 0.61

-3.41

0.60

-3.41 -3.41 -3.40 -3.40 -3.40 -3.39 -3.39

0.74 0.50 0.69 0.58 0.73 0.65 0.62

-3.38

0.65

-3.38 -3.37

0.74 0.62

UP Gene ID

Gene Name

SLAMF7 CCR5 CTSL CHST7 LHFPL2 PNMA1 SSBP4 PIM1 GAS2L1 GNGT2 TCN2

SLAM family member 7 C-C motif chemokine receptor 5 cathepsin L carbohydrate sulfotransferase 7 lipoma HMGIC fusion partner-like 2 paraneoplastic Ma antigen 1 single stranded DNA binding protein 4 Pim-1 proto-oncogene, serine/threonine kinase growth arrest specific 2 like 1 G protein subunit gamma transducin 2 transcobalamin 2 feline leukemia virus subgroup C cellular receptor family member 2 YES proto-oncogene 1, Src family tyrosine kinase squalene epoxidase vesicle associated membrane protein 5 adenosine deaminase cathepsin V

FLVCR2 YES1 SQLE VAMP5 ADA CTSV

SAM Fold score(d)* Change 4.74 2.44 4.55 2.26 4.46 2.13 4.33 1.87 4.15 2.20 4.07 1.55 4.06 1.28 4.03 1.63 3.98 1.37 3.97 1.40 3.91 1.50 3.90

1.51

3.87 3.86 3.85 3.83 3.81

1.22 1.84 1.62 1.79 1.43

IFNGR2

interferon gamma receptor 2

*Ratio of change in gene expression to standard deviation.

3.75

1.38

Supplemental Table 4: List of differentially expressed genes by BM monocytes arisen from supervised analysis of MM versus (MGUS plus SMM) patients.

DOWN Gene ID FCER1A

Gene Name

Fc fragment of IgE receptor Ia solute carrier organic anion transporter family member SLCO4C1 4C1 PADI4 peptidyl arginine deiminase 4 PTPN22 protein tyrosine phosphatase, non-receptor type 22 PGM1 phosphoglucomutase 1 RPS17 ribosomal protein S17 ANXA6 annexin A6 RAB37 RAB37, member RAS oncogene family WLS wntless Wnt ligand secretion mediator SERPINB10 serpin family B member 10 CLTCL1 clathrin heavy chain like 1 TMEM55A transmembrane protein 55A LOC100506844 uncharacterized LOC100506844 UBR4 ubiquitin protein ligase E3 component n-recognin 4 SLC9A3R1 SLC9A3 regulator 1 CPNE2 copine 2 MYB MYB proto-oncogene, transcription factor CENPK centromere protein K SRPK1 SRSF protein kinase 1 LGALS12 galectin 12 TMEM170B transmembrane protein 170B hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA HADHA thiolase/enoyl-CoA hydratase , alpha subunit EIF2D eukaryotic translation initiation factor 2D DGKZ diacylglycerol kinase zeta CDCA7 cell division cycle associated 7 FBN2 fibrillin 2 AZU1 azurocidin 1 IPCEF1 interaction protein for cytohesin exchange factors 1 TUBB tubulin beta class I SPNS3 sphingolipid transporter 3 NCAPD2 non-SMC condensin I complex subunit D2 ANP32E acidic nuclear phosphoprotein 32 family member E ADAM metallopeptidase with thrombospondin type 1 ADAMTS5 motif 5 ORM1 orosomucoid 1 KIF11 kinesin family member 11 NRM nurim

SAM Fold Score(d)* Change -5.70

0.40

-5.65

0.45

-5.58 -5.57 -5.35 -5.15 -5.12 -5.12 -5.03 -5.01 -4.92 -4.89 -4.71 -4.66 -4.63 -4.59 -4.58 -4.54 -4.53 -4.52 -4.40

0.46 0.60 0.64 0.79 0.64 0.60 0.63 0.32 0.68 0.67 0.62 0.74 0.70 0.71 0.51 0.43 0.70 0.63 0.61

-4.38

0.83

-4.37 -4.33 -4.29 -4.26 -4.25 -4.25 -4.23 -4.23 -4.22 -4.17

0.78 0.74 0.50 0.58 0.45 0.61 0.68 0.65 0.59 0.72

-4.17

0.72

-4.17 -4.14 -4.14

0.49 0.40 0.79

KBTBD11 GOT2 SLC2A9 FAM101B EMB CDCA7L SLC26A8 B4GALT6 E2F8 MKI67 EIF3L PSRC1 MRPL37 ANLN

kelch repeat and BTB domain containing 11 glutamic-oxaloacetic transaminase 2 solute carrier family 2 member 9 refilin B embigin cell division cycle associated 7 like solute carrier family 26 member 8 beta-1,4-galactosyltransferase 6 E2F transcription factor 8 marker of proliferation Ki-67 eukaryotic translation initiation factor 3 subunit L proline and serine rich coiled-coil 1 mitochondrial ribosomal protein L37 anillin actin binding protein SPARC/osteonectin, cwcv and kazal like domains SPOCK1 proteoglycan 1 MAP4K4 mitogen-activated protein kinase kinase kinase kinase 4 ZBED3 zinc finger BED-type containing 3 HIP1 huntingtin interacting protein 1 CKAP4 cytoskeleton associated protein 4 CIT citron rho-interacting serine/threonine kinase PKM pyruvate kinase, muscle RAB27A RAB27A, member RAS oncogene family ZWINT ZW10 interacting kinetochore protein FOXM1 forkhead box M1 S100A12 S100 calcium binding protein A12 CENPH centromere protein H MS4A3 membrane spanning 4-domains A3 PECR peroxisomal trans-2-enoyl-CoA reductase TOP2A topoisomerase II alpha CDA cytidine deaminase CENPF centromere protein F LOC100288637 OTU deubiquitinase 7A pseudogene BPI bactericidal/permeability-increasing protein SCCPDH saccharopine dehydrogenase GPSM2 G-protein signaling modulator 2 TMC8 transmembrane channel like 8 ZNF281 zinc finger protein 281 MRPL33 mitochondrial ribosomal protein L33 MIR205 microRNA 205 MARC1 mitochondrial amidoxime reducing component 1 HIPK2 homeodomain interacting protein kinase 2 KIF18B kinesin family member 18B NCAPG non-SMC condensin I complex subunit G RETN resistin TPCN1 two pore segment channel 1 TYMS thymidylate synthetase

-4.13 -4.13 -4.12 -4.11 -4.10 -4.08 -4.07 -4.07 -4.05 -4.04 -4.04 -4.00 -3.99 -3.99

0.69 0.75 0.76 0.63 0.56 0.54 0.68 0.77 0.44 0.54 0.81 0.74 0.84 0.44

-3.97

0.66

-3.96 -3.95 -3.94 -3.93 -3.92 -3.92 -3.90 -3.90 -3.89 -3.89 -3.87 -3.87 -3.86 -3.86 -3.84 -3.83 -3.82 -3.82 -3.82 -3.82 -3.81 -3.81 -3.80 -3.80 -3.79 -3.79 -3.78 -3.78 -3.78 -3.77 -3.77

0.80 0.70 0.76 0.67 0.65 0.71 0.71 0.49 0.61 0.69 0.59 0.43 0.67 0.40 0.67 0.55 0.59 0.46 0.76 0.63 0.77 0.73 0.81 0.79 0.65 0.75 0.50 0.47 0.58 0.81 0.44

RACGAP1 SHCBP1 GINS1 CACNA2D3 PTDSS1 ECRP PGD KIAA0101 MSL1 TPX2 TK1 FAM217B NUSAP1 NCAPG2 KIF20A BMX KIF15 HMGB2 IGF1R NME8 KIFC1 MEGF9 OIP5 MAP4K1 ADGRA2 CEP55 TACC3 NUF2 RASSF1 GPI CENPM ZNF367 EFCAB2 CTH KIF21B HIST1H4C PCNA MTMR11 TMC4 C20orf27 PLD1 UBE2C TTK FAM110B CBX4 FARSB

Rac GTPase activating protein 1 SHC binding and spindle associated 1 GINS complex subunit 1 calcium voltage-gated channel auxiliary subunit alpha2delta 3 phosphatidylserine synthase 1 ribonuclease A family member 2 pseudogene phosphogluconate dehydrogenase KIAA0101 male specific lethal 1 homolog TPX2, microtubule nucleation factor thymidine kinase 1 family with sequence similarity 217 member B nucleolar and spindle associated protein 1 non-SMC condensin II complex subunit G2 kinesin family member 20A BMX non-receptor tyrosine kinase kinesin family member 15 high mobility group box 2 insulin like growth factor 1 receptor NME/NM23 family member 8 kinesin family member C1 multiple EGF like domains 9 Opa interacting protein 5 mitogen-activated protein kinase kinase kinase kinase 1 adhesion G protein-coupled receptor A2 centrosomal protein 55 transforming acidic coiled-coil containing protein 3 NUF2, NDC80 kinetochore complex component Ras association domain family member 1 glucose-6-phosphate isomerase centromere protein M zinc finger protein 367 EF-hand calcium binding domain 2 cystathionine gamma-lyase kinesin family member 21B histone cluster 1 H4 family member c proliferating cell nuclear antigen myotubularin related protein 11 transmembrane channel like 4 chromosome 20 open reading frame 27 phospholipase D1 ubiquitin conjugating enzyme E2 C TTK protein kinase family with sequence similarity 110 member B chromobox 4 phenylalanyl-tRNA synthetase beta subunit

-3.77 -3.77 -3.76

0.56 0.56 0.51

-3.76

0.63

-3.76 -3.75 -3.75 -3.74 -3.74 -3.73 -3.73 -3.73 -3.73 -3.72 -3.72 -3.71 -3.70 -3.69 -3.69 -3.68 -3.68 -3.68 -3.68 -3.67 -3.67 -3.67 -3.65 -3.64 -3.64 -3.64 -3.63 -3.62 -3.61 -3.61 -3.60 -3.60 -3.59 -3.59 -3.58 -3.58 -3.58 -3.57 -3.57 -3.56 -3.56 -3.55

0.82 0.54 0.76 0.46 0.81 0.48 0.66 0.67 0.51 0.62 0.47 0.72 0.50 0.78 0.73 0.76 0.72 0.62 0.54 0.76 0.71 0.47 0.75 0.50 0.82 0.79 0.62 0.51 0.67 0.53 0.80 0.57 0.62 0.68 0.72 0.78 0.81 0.54 0.50 0.76 0.78 0.80

MAD2L1 FMO5 ERG ANAPC15 FAM64A KIF14 SLC25A40 THBS4 SSR1 POLR3B PCMT1 SIDT1 ITGAM CAT NCL LPGAT1 CCNE2 HAL MCM7 STMN1 BIRC5 SDHD CDC20 PHKA2 MPO BUB1B DTL RRM2 TFF3 VRK1 PTTG1 TSPAN2 SLC40A1 GYS1 KIF4A NPIPB15 PLCL2 TOP1MT TCF19 SFXN5 MELK CYTL1 CSF3R MCM4 USP48 CDK1

MAD2 mitotic arrest deficient-like 1 flavin containing monooxygenase 5 ERG, ETS transcription factor anaphase promoting complex subunit 15 family with sequence similarity 64 member A kinesin family member 14 solute carrier family 25 member 40 thrombospondin 4 signal sequence receptor subunit 1 RNA polymerase III subunit B protein-L-isoaspartate O-methyltransferase SID1 transmembrane family member 1 integrin subunit alpha M catalase nucleolin lysophosphatidylglycerol acyltransferase 1 cyclin E2 histidine ammonia-lyase minichromosome maintenance complex component 7 stathmin 1 baculoviral IAP repeat containing 5 succinate dehydrogenase complex subunit D cell division cycle 20 phosphorylase kinase regulatory subunit alpha 2 myeloperoxidase BUB1 mitotic checkpoint serine/threonine kinase B denticleless E3 ubiquitin protein ligase homolog ribonucleotide reductase regulatory subunit M2 trefoil factor 3 vaccinia related kinase 1 pituitary tumor-transforming 1 tetraspanin 2 solute carrier family 40 member 1 glycogen synthase 1 kinesin family member 4A nuclear pore complex interacting protein family member B15 phospholipase C like 2 topoisomerase I, mitochondrial transcription factor 19 sideroflexin 5 maternal embryonic leucine zipper kinase cytokine like 1 colony stimulating factor 3 receptor minichromosome maintenance complex component 4 ubiquitin specific peptidase 48 cyclin dependent kinase 1

-3.54 -3.54 -3.53 -3.53 -3.52 -3.51 -3.51 -3.50 -3.50 -3.50 -3.49 -3.48 -3.47 -3.47 -3.46 -3.46 -3.45 -3.45 -3.45 -3.44 -3.44 -3.44 -3.43 -3.43 -3.43 -3.43 -3.43 -3.43 -3.43 -3.42 -3.42 -3.42 -3.42 -3.42 -3.42

0.54 0.76 0.85 0.82 0.70 0.55 0.75 0.79 0.85 0.79 0.78 0.71 0.76 0.72 0.87 0.76 0.62 0.73 0.72 0.79 0.64 0.82 0.50 0.83 0.49 0.55 0.51 0.46 0.80 0.74 0.64 0.69 0.45 0.79 0.56

-3.41

0.73

-3.41 -3.40 -3.40 -3.40 -3.39 -3.39 -3.38 -3.36 -3.36 -3.35

0.77 0.75 0.67 0.87 0.56 0.65 0.82 0.73 0.78 0.54

CIDEB CARS2 IVNS1ABP DLGAP5 TMEM106C RCC1 ASPM GTSE1 BZW2 RNASE2 EP300-AS1 SMC2 RALGAPA2 CCNA2 VILL SSRP1 GGH SMYD2

cell death-inducing DFFA-like effector b cysteinyl-tRNA synthetase 2, mitochondrial influenza virus NS1A binding protein DLG associated protein 5 transmembrane protein 106C regulator of chromosome condensation 1 abnormal spindle microtubule assembly G2 and S-phase expressed 1 basic leucine zipper and W2 domains 2 ribonuclease A family member 2 EP300 antisense RNA 1 structural maintenance of chromosomes 2 Ral GTPase activating protein catalytic alpha subunit 2 cyclin A2 villin like structure specific recognition protein 1 gamma-glutamyl hydrolase SET and MYND domain containing 2

-3.35 -3.35 -3.34 -3.34 -3.34 -3.33 -3.33 -3.32 -3.32 -3.32 -3.32 -3.31 -3.31 -3.31 -3.30 -3.30 -3.30 -3.30

0.76 0.80 0.74 0.43 0.71 0.85 0.54 0.73 0.83 0.63 0.85 0.65 0.84 0.55 0.83 0.82 0.65 0.81

UP Gene ID

Gene Name

SLAMF7

SLAM family member 7 feline leukemia virus subgroup C cellular receptor family member 2 single stranded DNA binding protein 4 G protein subunit gamma transducin 2 squalene epoxidase paraneoplastic Ma antigen 1 adenosine deaminase vesicle associated membrane protein 5 interleukin 21 receptor runt related transcription factor 3 lipoma HMGIC fusion partner-like 2 MTSS1, I-BAR domain containing transcobalamin 2 cathepsin L transmembrane protein 51 carbohydrate sulfotransferase 7 C-C motif chemokine receptor 5 nectin cell adhesion molecule 2 heart development protein with EGF like domains 1 signal induced proliferation associated 1 like 2 TNF receptor associated factor 5 deoxyribonuclease 2, lysosomal insulin induced gene 1

FLVCR2 SSBP4 GNGT2 SQLE PNMA1 ADA VAMP5 IL21R RUNX3 LHFPL2 MTSS1 TCN2 CTSL TMEM51 CHST7 CCR5 NECTIN2 HEG1 SIPA1L2 TRAF5 DNASE2 INSIG1

SAM Fold Score(d)* Change 5.56

2.35

5.01

1.58

4.81 4.71 4.64 4.58 4.55 4.49 4.48 4.47 4.44 4.38 4.25 4.22 4.22 4.20 4.20 4.13 4.08 4.07 4.05 4.05 4.01

1.29 1.41 1.81 1.52 1.75 1.61 1.49 1.62 2.03 1.43 1.45 1.87 1.71 1.68 1.95 1.25 1.51 1.34 1.36 1.60 1.69

RRAS IFITM1 PTCH1 ISG20 LOC152225 PIM1 IFNGR2 CD9 PRRG2 LFNG GAS2L1 DHRS9 PAPSS2 CTSV MAP3K7CL EPSTI1 STAT4 AMPD1 CASP5 MARCO CD40 SELM CACNA1A ZDHHC23 HIVEP2 LINC00847 IDI1 PRORSD1P GJA3 ADGRE2 TESK1 LAP3 MLLT11 SLC16A6 EIF5A2 PIK3IP1 IFI27 ERGIC1 CXCL11

related RAS viral oncogene homolog interferon induced transmembrane protein 1 patched 1 interferon stimulated exonuclease gene 20 uncharacterized LOC152225 Pim-1 proto-oncogene, serine/threonine kinase interferon gamma receptor 2 CD9 molecule proline rich and Gla domain 2 LFNG O-fucosylpeptide 3-beta-Nacetylglucosaminyltransferase growth arrest specific 2 like 1 dehydrogenase/reductase 9 3'-phosphoadenosine 5'-phosphosulfate synthase 2 cathepsin V MAP3K7 C-terminal like epithelial stromal interaction 1 signal transducer and activator of transcription 4 adenosine monophosphate deaminase 1 caspase 5 macrophage receptor with collagenous structure CD40 molecule selenoprotein M calcium voltage-gated channel subunit alpha1 A zinc finger DHHC-type containing 23 human immunodeficiency virus type I enhancer binding protein 2 long intergenic non-protein coding RNA 847 isopentenyl-diphosphate delta isomerase 1 prolyl-tRNA synthetase associated domain containing 1, pseudogene gap junction protein alpha 3 adhesion G protein-coupled receptor E2 testis-specific kinase 1 leucine aminopeptidase 3 myeloid/lymphoid or mixed-lineage leukemia; translocated to, 11 solute carrier family 16 member 6 eukaryotic translation initiation factor 5A2 phosphoinositide-3-kinase interacting protein 1 interferon alpha inducible protein 27 endoplasmic reticulum-golgi intermediate compartment 1 C-X-C motif chemokine ligand 11

*Ratio of change in gene expression to standard deviation.

4.01 4.00 3.99 3.98 3.97 3.96 3.95 3.94 3.94

1.48 2.52 1.12 1.68 1.43 1.58 1.34 2.26 1.20

3.92

1.37

3.91 3.91 3.91 3.88 3.87 3.87 3.83 3.82 3.79 3.79 3.78 3.78 3.77 3.76

1.31 1.77 1.49 1.36 1.94 2.05 1.40 1.72 1.64 1.47 1.29 1.55 1.25 1.24

3.75

1.34

3.74 3.72

1.42 1.27

3.72

1.22

3.71 3.71 3.70 3.69

1.16 1.50 1.21 1.44

3.68

1.31

3.67 3.66 3.64 3.62 3.60 3.60

1.50 1.29 1.21 3.13 1.25 2.01

Supplemental Table 5: IL-21 expression by BM microenvironment cells. -ΔCt BM MSCs

ND

+

ND

CD14 cells +

CD138 cells

ND

+

-8,3

CD3 cells +

Activated CD3 cells

-1

+

CD8 cells

ND

+

-8,6

CD4 cells

Abbreviation: ND=not detectable

LEGENDS OF SUPPLEMENTAL FIGURES Supplemental Figure 1: BM monocytes purity. Representative flow cytometry plots to assess the purity of BM monocytes after purification with anti-CD14 Ab conjugated microbeads. Monocytes were identified as CD14+CD45+CD138- cells.

Supplemental Figure 2: Monocyte sub-population sorting. Example of flow cytometry analysis with anti-human CD16 and anti-human CD14, showing the two sub-population of monocytes before (A) and after (B, C) sorting. Purity of CD14+CD16+ (B) and CD14+CD16- (C) cells.

Supplemental Figure 3: Gene expression profiling of purified BM CD14+ cells from patients with different monoclonal gammopathies. (A) Heatmap of the differentially expressed genes resulted from the multiclass Significant Analysis of Microarrays (SAM) of the monocytes samples clustered according to the diagnosis of the patients. Heatmaps of the differentially expressed genes arisen from the supervised SAM analyses comparing BM CD14 + cells from MM versus SMM (B) or active MM versus asymptomatic forms (MGUS plus SMM) (C).

SUPPLEMENTAL FIGURES Supplemental Figure 1

Supplemental Figure 2

Supplemental Figure 3