Platelet-Derived Growth Factor Alpha Mediates the Proliferation of Peripheral T-Cell Lymphoma Cells Via an Autocrine Regulatory Pathway

Home , PDGFA, PDGFB, PDGFRA

ORIGINAL ARTICLE Platelet-derived growth factor alpha mediates the proliferation of peripheral T-cell lymphoma cells via an autocrine regulatory pathway

PP Piccaluga1,9, M Rossi1,9, C Agostinelli1, F Ricci2, A Gazzola1, S Righi1, F Fuligni1, MA Laginestra1, M Mancini3, MR Sapienza1, A De Renzo4, PL Tazzari2, D Gibellini5, P Went6, F Alviano3, PL Zinzani3, GP Bagnara3, G Inghirami7, C Tripodo8 and SA Pileri1 on behalf of the AIRC 5xMille consortium ‘Genetics-driven targeted management of lymphoid malignancies’ and the European T-cell Lymphoma Study Group10

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho- PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients’ plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.

Leukemia (2014) 28, 1687–1697; doi:10.1038/leu.2014.50

INTRODUCTION and others, with relevant clinical implications.13–15 In such Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) is instances, PDGFRA was found to have a pivotal pathogenic role, the most common type of T-cell-derived tumor.1,2 It is an with its function being altered by somatic mutations, aggressive disease for which current treatments are ineffective; amplification, genomic rearrangement and the activation of 9,13,16 novel, more rational targeted approaches are definitely autocrine/paracrine loops. Remarkably, as in these other warranted.3 malignancies, PDGFRs were demonstrated to be suitable 7,17,18 In this regard, the genetics of PTCL/NOS is quite heterogeneous, therapeutic targets in PTCLs, both ex vivo and in vivo. and no disease-specific genetic abnormalities have been identi- In this study, we aimed to identify the causes of PDGFRA fied to date.4,5 However, recent gene expression profile (GEP) activation in PTCL/NOS. studies demonstrated that some relevant genes are consistently deregulated.6–8 Interestingly, among them, the gene encoding platelet-derived growth factor receptor alpha (PDGFRA) was MATERIALS AND METHODS always found to be overexpressed, and the corresponding Gene expression analysis protein was constitutively phosphorylated, with potentially First, we analyzed GEP data for 78 cases of PTCL/NOS derived from relevant clinical consequences.6–8 Remarkably, PDGFRA expression cryo-preserved lymph nodes and 30 samples of normal T lymphocytes was later documented in other T-cell-derived malignancies, (CD4 þ , N ¼ 10; CD8 þ , N ¼ 10; HLA-DR þ , N ¼ 5; HLA-DR-, N ¼ 5) that were previously generated using the Affymetrix HG-U133 2.0 plus including angioimmunoblastic lymphoma, prolymphocytic 9,10 microarray (Affymetrix http://www.affymetrix.com/support/index.affx) and leukemia and T/NK-cell-derived neoplasms. In addition, PDGFRA are available at http://www.ncbi.nlm.nih.gov/projects/geo/ (GSE6338 and deregulation has been linked to several tumors, including chronic GSE19069). For technical details, see the references.7,8 In particular, we myeloproliferative neoplasms,11,12 gastrointestinal stromal tumors focused on the expression of PDGFRA, identified by four different probe

1Hematopathology Section, Department of Experimental, Diagnostic, and Specialty Medicine, S. Orsola-Malpighi Hospital, Bologna University Medical School, Bologna, Italy; 2Transfusion Medicine Service, S. Orsola-Malpighi Hospital, Bologna, Italy; 3Department of Experimental, Diagnostic, and Specialty Medicine, Bologna University Medical School, Bologna, Italy; 4Ematologia Universita` ‘Federico II’, Napoli, Italy; 5Microbiology Section, Department of Experimental, Diagnostic, and Specialty Medicine, S. Orsola-Malpighi Hospital, Bologna University Medical School, Bologna, Italy; 6Institute of Pathology, Basel, Switzerland; 7Department of Pathology and Center for Experimental Research and Medical Studies, University of Torino, Turin, Italy and 8Dipartimento di Scienze per la Promozione della Salute, Sezione di Anatomia Patologica, Universita` degli Studi di Palermo, Palermo, Italy. Correspondence: Professor PP Piccaluga, Molecular Pathology Laboratory, Hematopathology Section, Department of Experimental, Diagnostic, and Specialty, Bologna University Medical School, S. Orsola-Malpighi Hospital, Pavillon 8, Via Massarenti 9, Bologna 40138, Italy. E-mail: [email protected] 9These authors contributed equally to this work. 10See Appendix. Received 12 September 2013; revised 16 January 2014; accepted 17 January 2014; accepted article preview online 31 January 2014; advance online publication, 25 February 2014 Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1688 sets in the HG-U133 2.0 plus GeneChip (203131_at; 211533_at; 215305_at; USA), following the manufacturer’s protocol. Briefly, serially diluted 1554828_at), PDGFRB (202273_at), PDGFA (205463_s_at; 216867_s_ rhPDGF-AA protein at concentrations ranging from 0–1000 pg/ml was at; 229830_at) and PDGFB (204200_s_at; 216055_at; 216061_x_at; used to generate a standard curve for calculating the PDGF-AA 217112_at). The mean values obtained using different probes were used concentration in each sample. The PDGF-AA protein levels in the for the analyses.7,9,19–22 Notably, the tumor samples were selected platelet-poor plasma collected from two PTCL patients upon diagnosis according to the proportion of neoplastic elements, which was higher and five healthy subjects were also determined using the same assay. than 70% and generally higher than 90% in the PTCL/NOS samples.7 The gene expression analysis was conducted as previously reported.7 For details, see Supplementary Information. The study was approved by Treatment with neutralizing anti-PDGF antibody the local Ethical Committee (Protocol number 001–2011-U-Tess) and To test the hypothesis of an autocrine stimulation by PDGF, FePd and EOL- written consent was obtained from the involved patients. 1 cells were cultured with and without a neutralizing anti-PDGF antibody at different concentrations (0, 20, 30, 40 and 60 mg/ml) and evaluated at FISH analysis–massive parallel sequencing–direct sequencing of different times (0, 24, 48, 72 and 96 h). After treatment, the cells were the PDGF/PDGFRA loci–real-time quantitative PCR collected, fixed and permeabilized using an Intraprep kit (Beckman Coulter, Brea, CA, USA) for analysis of PDGFRA/p-PDGFRA expression by flow The details of the FISH, massive parallel sequencing, Sanger sequencing cytometry. In addition, cell counts and viability assessments (using trypan and real-time quantitative PCR analyses are reported in Supplementary blue) were performed. Furthermore, proliferation and apoptosis were Information and Supplementary Table 1. evaluated using bromodeoxyuridine and annexin-V assays, respectively. Subsequently, after 48 h of treatment with the anti-PDGF neutralizing Isolation of normal T lymphocytes antibody (20 mg/ml), human PDGF-AA peptide (PeproTech, Rocky Hill, NJ, Human primary naı¨ve CD4 þ and CD8 þ T lymphocytes were purified USA) were added for 6 h at a concentration of 10 ng/ml. After this from the peripheral blood mononuclear cells of healthy donors using a treatment, the cells were collected and fixed for analysis of PDGFRA/p- naı¨ve CD4 þ T-cell isolation kit II (Miltenyi, Auburn, CA, USA) and a naı¨ve PDGFRA expression using flow cytometry and for evaluation of the fraction CD8 þ T-cell isolation kit, respectively. The purity of the CD4 þ CD45RA þ , of mitotic cells using the bromodeoxyuridine assay. CD8 þ CD45RA þ CCR7 þ cells was higher than 97%, as determined by flow cytometry. Flow cytometric analysis of PDGFRA and p-PDGFRA expression Isolated CD4 þ or CD8 þ naı¨ve T cells were maintained in RPMI 1640 medium (Lonza, Basel, Switzerland) containing 10% FCS (Gibco, Paisley, FePd cells and normal lymphocytes isolated from the peripheral blood of UK) and were activated using PHA (5 m/ml, Sigma, St Louis, MO, USA) plus healthy donors (as control samples) were analyzed by flow cytometry to IL-2 (10 U/ml, Pierce, Rockford, IL, USA). The cells were harvested after 96 h detect the presence of PDGFRA (Santa Cruz, Santa Cruz, CA, USA), for RNA extraction. p-PDGFRA (Santa Cruz) (FC500, Beckman Coulter), STAT1 (Cell Signaling, Beverly, MA, USA), STAT3 (Santa Cruz), STAT5 (Santa Cruz), p-STAT1 Cell Signaling), psTAT3 (Cell Signaling), and psTAT5 (Cell Signaling) (NAVIOS, Immunohistochemical analysis Beckman Coulter). Briefly, the cells were fixed and then permeabilized Immunohistochemical analysis of tissue microarrays was conducted as using an Intra Prep kit (Beckman Coulter), following the manufacturer’s 23–26 previously reported. Briefly, tissue microarrays were constructed for instructions. The cells were then stained using standard procedures and 262 PTCL cases (Table 1). Seven reactive non-neoplastic lymph nodes and analyzed using a NAVIOS flow cytometer (Beckman Coulter). Cytograms one tonsil were used as controls. For details, see Supplementary were created using dedicated Kaluza software (for additional details see Information. Samples were considered positive if 30% or more of the Supplementary Information). tumor cells were stained with an antibody.23

Cell culture Western blot analyses Western blot (WB) analysis was performed on whole cell lysates according EOL-1 (a human eosinophilic leukemia cell line carrying the fusion FIP1L1- 29,30 PDGFRA),27,28 Jurkat (a T-cell leukemia cell line), CEM (a T-cell leukemia cell to published methods. FePd cell line treated with neutralizing PDGF line), SUP-M2 (an anaplastic large cell lymphoma carrying the typical t(2;5) antibody (20 mg/ml) and with PDGF-AA peptide (10 ng/ml) as above reported and treated with Imatinib 1 and 5 mM for 48 h was used. Brieﬂy, (p23;q35) leading to the NPM1-ALK (NPM-ALK) fusion gene and expressing 7 CD30) and FePd (a PTCL cell line expressing CD30) cell lines were cultured. 10 cells were lysed using RIPA buffer supplemented with 1 mM PMSF and For details, see Supplementary Information. protease inhibitor cocktail, 50 mg of cell lysates were loaded for SDS–PAGE. Anti-PDGFR-a, anti-phospho-PDGFR-a Tyr 754 and anti-beta actin were purchased from Santa Cruz Biotechnology. Anti-AKT, phospho-AKT ser473, PDGF-AA determination using an ELISA ERK, phosphor-ERK Thr202/Tyr204 were purchased by Cell Signaling The PDGF-AA protein levels in the supernatants of FePd and Jurkat cell Technology.31,32 Beta actin from Santa Cruz was used as controls for lines were measured. The levels were determined using the Quantikine protein loading. Signal intensities in single blots from two separate ELISA human/mouse PDGF-AA assay kit (R&D Systems, Minneapolis, MN, experiments were measured by means of ChemiDoc-It instrument

Table 1. Expression of PDGFR and PDGF ligands as evaluated using immunohistochemistry (samples were considered positive if 30% or more of the cells were stained with an antibody)

TCL subtype Number of analyzed cases Positive/evaluable cases

PDGFRA P-PDGFRA PDGF-A PDGFB

PTCL/NOS 156 128/141 105/110 74/86 85/92 AITL 45 36/36 21/22 20/23 23/24 ALCL ALK þ 16 16/16 10/12 13/13 9/12 ALCL ALK À 11 10/10 8/10 6/6 7/9 Mycosis fungoides 28 28/28 28/28 5/5 7/8 Enteropathy-associated T-cell lymphoma 5 5/5 NA 3/3 2/2 Subcutaneous panniculitis-like T-cell lymphoma 1 1/1 NA NA 0/1 Reactive lymph node 7 0/7 0/7 0/7 0/7 Tonsil 1 0/1 0/1 0/1 0/1 Abbreviations: AITL, angioimmunoblastic lymphoma; ALCL, anaplastic large-cell lymphoma; NA, not available; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PTCL/NOS, peripheral T-cell lymphomas, not otherwise speciﬁed; TCL, T-cell lymphoma.

Leukemia (2014) 1687 – 1697 & 2014 Macmillan Publishers Limited Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1689 equipped with a dedicated software (Launch VisionWorksLS, Euroclone, (FePd and CEM) and normal T lymphocytes. The results confirmed Milano, Italy). The statistical significance of differences among signal the upregulation of PDGFRA in the lymphoma samples (Figure 1b). intensities was assessed by means of ImageJ software. Subsequently, we studied PDGFRA expression in the 156 PTCL/NOS cases included in the tissue microarrays using immuno- Cytospin histochemistry, and we found that 128/141 (91%) evaluable FePd cells were washed and diluted to a concentration of 105/100 mlin PTCL/NOS cases exhibited positive staining. Notably, virtually all of PBS. An aliquot of B100–200 ml of PBS was loaded into each of the wells of the neoplastic cells were PDGFRA-positive (Figure 2; Table 1). a cytospin device, which was spun at 400 r.p.m. for 4 min. Then, each Interestingly, all of the other PTCL subtypes included in the sample (control FePd cells and FePd cells treated with the neutralizing analysis presented positive PDGFRA staining (Figure 2; Table 1). antibody anti-PDGF at concentration of 20 mg/ml) was aliquoted into the Conversely, the T-cell-rich zones in the reactive lymph nodes and appropriate wells of the cytospin device, which was spun at 400 r.p.m. for the tonsil appeared to be consistently negative, with the 4 min. The slides were dried and the cells fixed with acetone, and then exception of scattered CD30 þ activated lymphocytes. STAT1, STAT3, and STAT5 expression was assessed using immunohistochemistry (see Immunohistochemical analysis for Ab dilutions and details in Consistent with the PDGFRA expression data, the phosphory- Supplementary Information). lated form of PDGFRA was shown to be expressed in 105/110 (95%) evaluable PTCL/NOS cases. Similarly, among the other tested PTCLs, all but one case displayed positive p-PDGFRA Statistical analysis staining. In contrast, the reactive non-neoplastic samples were Statistical analyses were conducted using the StatView 5.0 software negative (Figure 2; Table 1). package (SAS Institute, Cary, NC, USA). ANOVA, unpaired t-tests and, when For control purposes, the expression of the other PDGFR, appropriate (specifically, when the sample size was less than ten cases in at least one group), non-parametric tests (Mann–Whitney) were utilized to PDGFRB, was evaluated in the same cases. No significant analyze the GEP data. The limit of significance for all of the analyses was differences were found between PTCL/NOS and normal cells at defined as Po0.05; two-sided tests were used in all of the calculations. the gene expression level. Immunohistochemistry (IHC) demonstrated the expression of the molecule in a scant minority of neoplastic cells and in a few mesenchymal cells, as well as in RESULTS scattered cells in the T-cell-rich zones of the reactive lymph nodes PDGFRA is overexpressed and activated in PTCL/NOS (data not shown). First, we studied PDGFRA RNA and protein expression in PTCL and Taken together, these results demonstrated that PDGFRA is non-neoplastic samples. In particular, we analyzed the level of selectively overexpressed and phosphorylated in PTCLs. PDGFRA gene expression in 78 PTCL/NOS cases and 30 samples of normal T lymphocytes for which DNA microarrays had been PTCL/NOS have an intact PDGFRA locus previously generated,7,8 and we found a significant overexpression We subsequently examined the PDGFRA genomic locus to identify in the tumors (0.205 vs À 2.986, Po0.0001; s.e. of 0.17 and 0.08, possible lesions that would sustain PDGFRA activation, as respectively; Figure 1a). Furthermore, to validate the microarray- described in other malignancies. based gene expression data, we used qPCR to evaluate PDGFRA FISH analysis performed using samples from 110 PTCL/NOS expression in PTCL/NOS primary samples, two PTCL cell lines cases revealed that there was no amplification/deletion in the

Box Plot Box Plot Split By: NOS vs Normal Split By: NOS vs Normal 4 2.5 P<0.0001 P=0.0003 3 2 2 1.5 1 1 0 Normal T-cells 0.5 Normal T-cells -1

value PTCL/NOS

value 0 PTCL/NOS -2 -0.5 -3 -1 -4 -1.5 Gene expression normalized Gene expression -5 normalized Gene expression -2 PDGFRA – microarray data PDGFA – microarray data Box Plot Split By: Cell type Box Plot 10 Split By: NOS vs Normal P<0.0001 2 9 P=0.02 8 1.5 7 CD4 6 CD8 1 5 CEM Normal T-cells 4 0.5

value PTCL/NOS 3 Fe-Pd 0 2 Primary PTCL/NOS 1 Gene expression value Gene expression -0.5 0 -1 normalized Gene expression -1 PDGFRA – qPCR data PDGFB – microarray data Figure 1. PDGFRA, PDGFA and PDGFB gene expression in PTCLs/NOS. Normalized gene expression values obtained by gene expression proﬁling are reported in a, c, and d. Real-time quantitative PCR values (relative to those of normal T lymphocytes, used as the reference control) are plotted in b. CD4: CD4 normal T cells (four samples), CD8: CD8 normal T cells (four samples), FePd: FePd cell line, CEM: CEM lymphoblastic cell line, primary PTCL/NOS: primary tumors with peripheral T-cell lymphomas, not otherwise speciﬁed (two patients).

& 2014 Macmillan Publishers Limited Leukemia (2014) 1687 – 1697 Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1690 showed no relevant conformational change. Moreover, also ab ExPasy Bioinformatic resource portal described the same no relevant conformational change (UniProtKB/Swiss-Prot P16234: Variant p.Ser478Pro: http://www.web.expasy.org/variant_pages/ VAR_034378.html). Such CNV could be definitely interpreted as germline SNPs as it was detected in normal matched DNAs. No differences in expression levels were recorded in cases carrying or not such polymorphism (data not shown), nor other specific clinicopathological feature was associated with it. Similarly PDGFA/PDGFB loci did not present with relevant abnormalities cd (Supplementary Table 2). Finally, consistent with the previous findings, high-density karyotyping of a series of PTCL/NOS using an Affymetrix 250k SNP array did not reveal any evidence of significant abnormalities in the PDGFRA locus.33

PDGF ligands are expressed and secreted by PTCL/NOS cells Because different methods actually excluded the possible ef existence of primary structural abnormalities able to justify intrinsic PDGFRA phosphorylation (that is, activation) in PTCLs, we explored the hypothesis of an autocrine stimulation. To this aim, we first analyzed the expression levels of two genes encoding PDGFRA ligands, PDGFA and PDGFB. We found that both of them were expressed in PTCLs/NOS (Figure 1); however, PDGFB expression was downregulated in the tumors compared with the normal samples (0.035 vs 0.247; standard errors of 0.05 and 0.05, respectively; P ¼ 0.02). Conversely, PDGFA expression was significantly higher in the neoplastic cells (0.111 vs À 0.598; gh standard errors of 0.10 and 0.14, respectively; P ¼ 0.0003). Subsequently, we investigated whether PTCL/NOS cells could secrete PDGFA, thus performing an autocrine stimulation. To this purpose, we cultured FePd cells, as well as Jurkat and SUP-M2 cells as positive and negative controls, respectively, and determined the PDGF-AA levels in the supernatants at 48 h. Significantly higher levels of PDGF-AA were detected in the Fepd samples (mean value: 3.345; s.d.: 0.261) than in the Jurkat (mean value: 1.054; s.d.: 0.174) or the SUP-M2 samples (mean value: 0.018; s.d.: Figure 2. Immunohistochemical analysis of PDGFRA PTCL subtypes. PDGFRA and p-PDGFRA in PTCL/NOS (a and b), respectively). 0.005) (Kruskal–Wallis test, P ¼ 0.0023) (Figure 3a). Following this Expression of PDGFRA in other PTCL subtypes: AITL (c), EATL (d), observation, we tested whether PDGF-AA was necessary for ALCL ALK þ (e) and ALK- (f) and MF (g). Please note in e and g, the PDGFRA activation by neutralizing PDGF in the cultures using a neoplastic elements (all PDGFRA þ ) indicated by black arrows. In specific antibody (Ab) and by evaluating PDGFRA phosphorylation the non-neoplastic reactive lymph nodes, only scattered activated using flow cytometry. Indeed, functional subtraction of PDGF elements exhibited PDGFRA expression (h); see black arrows). revealed a progressive dose- and time-dependent decrease in PDGFRA phosphorylation (Figures 3b and c; Supplementary Figure 2). At 48 h, with an Ab dosage of 40 mg/ml, a 1 Log PDGFRA locus. In two cases, trisomy of chromosome 4, which 10 decrease in phosphorylation (Figure 3b; Supplementary Figure 2) includes the PDGFRA gene, was detected. was observed. Moreover, treating neoplastic T cells with the We thus evaluated the PDGFRA locus by analyzing whole neutralizing antibody anti-PDGF restored PDGFRA phosphoryla- exome sequencing data generated using 10 PTCL/NOS samples tion upon re-challenge with exogenous recombinant human and relatively matched non-neoplastic DNA. Neither somatic non- PDGF-AA (Figure 3e; Supplementary Figure 2). To further verify the synonymous mutations nor known exonic short nucleotide specificity of the Ab-mediated PDGF depletion, the neutralizing polymorphisms were detected (Supplementary Figure 1; anti-PDGF Ab was added to cultures of the EOL-1 cell line, which is Supplementary Table 2). derived from a eosinophilic leukemia carrying the PDGFRA/FIP1L1 Direct sequencing of the PDGFRA coding and promoter regions rearrangement and exhibits constitutive PDGFRA activation. was then performed in 81 PTCL cases, including those studied As expected, anti-PDGF treatment had no effect on PDGFRA using GEP and IHC, to evaluate the possible existence of genomic phosphorylation in the EOL-1 cells (Figure 3d; Supplementary abnormalities that could affect protein viability. This approach did Figure 2). not reveal any significant alterations in the nucleotide sequence of These data demonstrated that PTCL/NOS cells produced and the receptor (coding and regulatory region) with the exception of secreted PDGF-AA, which is necessary and sufficient for PDGFRA exon 10 in 12 cases. In particular, in these 12 cases a transition phosphorylation in vitro, by sustaining an autocrine stimulation. from T to C in position 55 139 771 (forward strand) was detected. This alteration was recognized as a synonym polymorphism (rs35597368) and caused a change in the amino-acid sequence: Western Blot for PDGFRA/P-PDGFRA the wild-type serine in position 478, which is a neutral and polar To improve the robustness of the study, we confirmed the results amino acid, was replaced with a proline, which is a neutral and also by Western blot analysis. Neutralizing antibody anti-PDGF hydrophobic amino acid. Despite this change, the prediction of (20 mg/ml) induced the complete de-phosphorylation of PDGFRA the protein status of PDGFRA, performed using the bioinformatics at Tyr 754 on FePd cell line. The exposure to PDGF-AA restored program PolyPhen (http://www.genetics.bwh.harvard.edu/pph/), phosphorylation. These results were confirmed in two separate

Leukemia (2014) 1687 – 1697 & 2014 Macmillan Publishers Limited Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1691 Box Plot P=0.0023 Split By: Cell type 4 3.5 3 2.5 FE-PD 2 Jurkat (pg/ml)

10 1.5 NEG. Ctrl SUP-M2

Log 1 0.5 0 -0.5 PDGF-AA

100 100 80 80 60 PDGFRA 60 40 pPDGFRA 40

flow cytometry flow 20 Expression (%) at 20 at flow cytometry at flow 0 0

CTRL20 30 40 pPDGFRA Expression (%) 0 h24 h 48 h 72 h 96 h Treatment dose (microg/ml) Time CTRL + anti-PDGF Ab (20 microg/ml)

100 100

80 80

60 60 CTRL CTRL treated cells 40 40 treated cells flow cytometry flow flow cytometry flow 20 20 pPDGFRA Expression (%) at pPDGFRA Expression (%) at 0 0 +anti PDGF Ab +anti PDGF Ab Fe Pd EOL1 + PDGF-AA Figure 3. PDGFRA activation is mediated by an autocrine stimulation. PDGF-AA is secreted by surnatant of neoplastic cell lines (Jurkat as positive control, SUP-M2 as negative control, FePd cell line) when examined by ELISA assay. Cells CTRL negative is a negative control supplied by ELISA assay (R&D System) (a). The removal of PDGF using a neutralizing antibody anti-PDGF demonstrated the progressive dose (0, 20, 30 and 40 mg/ml)- (b) and time (0, 24, 48, 72 and 96 h with a concentration of 20 mg/ml)-(c) dependent de-phosphorylation of PDGFRA in FePd cell line. PDGFRA phosphorylation was restored by adding PDGF-AA peptide at dose of 10 ng/ml exceeding those of the antibody in Fepd cell line. (d). The phenomenon was not observed, as expected, in cells carrying primary genetic lesions affecting PDGFRA phosphorylation, such as the FIP1L1/PDGFRA rearrangement in EOL-1 eosinophilic leukemia cells (e). For all the panels, results were plotted as histograms (percentage of expression on basal values). experiments. The statistical significance of differences in signal obtained from PTCL patients (2 patients) proved to be significantly intensities (measured by means of ChemiDoc-It instrument) was higher than those of healthy individuals (5 subjects) (mean: confirmed by densitometric analysis performed with a dedicated 725.720; s.d.: 199.243 vs mean: 442.465; s.d.: 77.206) (Figure 4b). software (ImageJ) (Supplementary Figure 3). Based on these findings, we investigated whether PDGFRA signaling was active in primary tumors. We generated a GEP of cells exhibiting PDGFRA phosphorylation (that is, activation) and The PDGFA–PDGFRA axis is active in primary tumors and is treated or not treated with imatinib mesylate (1 mM) for 6 h. Both mediated by STAT1 and STAT5 PTCL cells and EOL-1 cells were profiled to minimize the possible To further test the hypothesis of autocrine PDGFA/PDGFRA off-target effects of the drug. We then performed a supervised stimulation, we used IHC to study PDGFA expression in a series analysis comparing treated vs untreated cells and identified a of 262 PTCLs, including all of the most common subtypes. Notably, molecular signature (24 probe sets corresponding to 21 annotated the majority of neoplastic cells displayed diffuse, intense staining genes; Supplementary Table 3) representative of PDGRA activa- (Figure 4a; Table 1) in 74/86 (86%) of the cases. Consistent with tion. We then tracked this ‘PDGFRA activation signature’ in primary this result, the PDGF-AA levels (pg/ml) in the plasma samples PTCL and normal T-cell samples. Interestingly, by clustering these

& 2014 Macmillan Publishers Limited Leukemia (2014) 1687 – 1697 Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1692

PDGFA TCEB2 BOLA2 /// 8... SON SON CBFB IRAK1 GLRX5 NDUF56

GTF3A NOMO1 /// ... SFR52 N/A ODC1 HSPA5 PTPRO

HNRNPA3 BHLHE40 Box Plot Split By: Sample type p=0.022 110 PTCL/NOS T-cells 100 Fischer exact test, p<0.0001 90 80 -2 -0 2 Ctrl 70 Patients

(pg/ml) 60 50 40 PDGF-AA plasma level 30 Figure 4. PDGFA expression and PDGFRA activation in primary tumors. PDGFA expression in primary tumors was demonstrated using immunohistochemistry (a): PTCL/NOS case expressing PDGFA; in the inset an interfollicular area of a reactive lymph node is represented, where small lymphocytes are PDGFA negative and an activated lymphoid blast was weakly positive), whereas its levels in PTCLs patients’ and healthy subjects’ (ctrl) platelet-poor plasma were assessed (units: pg/ml) using an ELISA assay (b). A molecular signature representative of PDGFRA signaling was found to be enriched in primary tumors, indicating the activity of the pathway (c). Speciﬁcally, the molecular signature obtained inhibiting PDGFRA signaling by imatinib (Supplementary Table 3) was tested in 28 PTCL/NOS and 15 normal T lymphocytes samples. The hierarchical clustering showed a clear-cut distinction of normal and neoplastic cells.

samples according to the PDGFRA activation signature, we found immunocytochemistry of the cytospin samples (Figure 5a). Finally, that normal cells and tumors were clearly distinct (Fischer exact immunohistochemical analysis of 28 primary cases confirmed that test, Po0.0001; Figure 4c), with the latter presenting a significant pSTAT5 was diffusely localized in nuclei of the neoplastic clone in enrichment of the expression of genes representative of PDGFRA the majority of cases, whereas pSTAT1 and pSTAT3 nuclear activation, as evaluated using GSEA (q-value p0.3). expression was variable, with a considerable fraction of negative Furthermore, because PDGFRA activity was predicted to be cases (Figures 5c and d; Supplementary Figure 3). These data based on signal transducer and activator of transcription proteins supported the hypothesis of an autocrine PDGFA-mediated according to a bioinformatic pathway analysis (Ingenuity, USA), PDGFRA stimulation in primary PTCL cases and demonstrated we investigated whether the PDGFRA signaling in PTCLs was that PDGFRA is active and most likely acts through STAT5. indeed mediated by these molecules. Specifically, the expression levels of STAT1, STAT3 and STAT5 genes, which are known to be downstream effectors of PDGFR signaling, were evaluated. We AKT/P-AKT and ERK1–2/P-ERK1–2 evaluation found that STAT1 (mean: 9.511; s.d.: 0.933 vs mean: 7.354; s.d.: As PDGFRA signaling might be mediated not only by signal 0.450; Po0.0001) and STAT5 (mean: 8.292; s.d.: 0.651 vs mean: transducer and activator of transcription, but also by AKT and 7.454; s.d.: 0.599; Po0.0001) were significantly overexpressed in ERK1–2/ we evaluated them by Western Blot in FePd cell line PTCLs compared with normal T lymphocytes, whereas STAT3 treated or not with neutralizing antibody anti-PDGF. Neutralizing expression was downregulated in the tumor samples (mean: antibody anti-PDGF induced a partial but significant de-phosphor- 8.041; s.d.: 0.787 vs mean: 8.967; s.d.: 0.486; Po0.0001), which is in ylation of ERK1/2 at Thr202/Tyr204 in Fepd cell line. Conversely, it accord with PDGFRB signaling being overruled by that of PDGFRA did not affect the phosphorylation of AKT at Ser473, which did in these tumors (Figure 5a). We observed consistently high not result phosphorylated in our in vitro model. Moreover, expression of STAT1, STAT5 and the corresponding phosphory- we observed a complete de-phosphorylation of ERK1/2 at lated forms in PTCL cell cultures using flow cytometry (Figure 5a). Thr202/Tyr204 in FePd cell line treated with Imatinib 1 and 5 mM Interestingly, when tumor cells were treated with the neutralizing (Figure 5b). These results were confirmed in two separate antibody anti-PDGF, STAT5 and, to a lesser extent, STAT1 showed experiments. The statistical significance of differences in decreased phosphorylation (Figure 5a). Accordingly, PDGFRA signal intensities (measured by means of ChemiDoc-It instrument) inhibition resulted in the blockage of the nuclear translocation was confirmed by densitometric analysis performed using a of both STAT1 and STAT5 in PTCL cells, as evaluated by dedicated software (ImageJ). Based on that, we studied by

Leukemia (2014) 1687 – 1697 & 2014 Macmillan Publishers Limited Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1693 P-STAT + STAT + Baseline STATs Baseline p-STATs anti-PDGF Ab STAT anti-PDGF Ab STAT1 STAT3 STAT5

STAT genes STAT proteins

P-AKT S473

AKT

P-ERK 1/2

ERK 1/2

BETA ACTIN CTRL + antiPDGF + PDGF-AA + ima1microM + ima5 microM PBMC K562

pSTAT1 pSTAT3 pSTAT5

Figure 5. PDGFRA signaling is mediated by signal transducer and activator of transcription proteins. Signal transducer and activator of transcription gene levels in PTCL/NOS and normal lymphocytes were determined (normalized gene expression values are plotted). Signal transducer and activator of transcription phosphorylation was assessed using flow cytometry at the baseline and in the presence of neutralizing antibody anti-PDGF, whereas signal transducer and activator of transcription protein localization was determined using cytochemistry (a). In panel 3A on the right, overlap of P-STAT1/3/5 expression peaks was showed (baseline cells vs treated cells); black peaks represent untreated cells; gray peaks represent treated cells with neutralizing antibody anti-PDGF. (b) Neutralizing Ab anti-PDGF induces a partial but significant de-phosphorylation of ERK1/2 at Thr202/Tyr204 in Fepd cell line. Conversely, it did not affect the phosphorylation of AKT at Ser473 which did not result phosphorylated in our in vitro model. To confirm our results, we treated our cell line with Imatinib 1 and 5 mM, and we observed a complete de-phosphorylation of ERK1/2 at Thr202/Tyr204. K562 cell lines were used as positive control for ERK1/2 and AKT expression and phosphorylation and PBMC was used as negative control. Beta actin was used as controls for protein loading. SDS–PAGE was used to investigate the protein expression levels. Beta actin was used as controls for protein loading. (c) Immunohistochemical profile of the 28 PTCL/NOS case studied for pSTAT1, pSTAT3, pSTAT3: a red box indicates pSTAT expression, whereas a green box indicates for pSTAT negativity (samples were considered positive if 30% or more of the tumor cells were stained with an antibody). pSTAT5 nuclear expression was most commonly observed in PTCLs/NOS lymphocytes, although a fraction of the case samples also expressed STAT1 and/or STAT3. (d) Examples of pSTAT1, pSTAT3 and pSTAT5 nuclear positivity in PTCL/NOS, using immunohistochemistry. immunohistochemistry 28 cases for P-ERK1/2 expression, but none the apoptosis rate of PTCL cells (FePd cells) treated or not with a of them was positive. neutralizing antibody anti-PDGF (20 mg/ml) using the annexin-V assay and their proliferation rate using the BrdU assay. After 24, 48, 72 and 96 h, a progressive decrease in cellular proliferation of up PDGFRA autocrine stimulation sustains PTCL/NOS cell proliferation to 80% (Figure 6a; Supplementary Figure 5) was observed in the We then investigated whether PDGFRA activation could foster treated cells. The growth inhibition was proportional to the PTCL cell proliferation and viability. For this purpose, we evaluated decrease in PDGFRA phosphorylation, and both of these events

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100 100 80 80 60 60 Ctrl 40 40 Treated

20 BrDU Incorporation

BrDU Incorporation 20 (Percentage on basal values) (Percentage (Percentage on basal values) (Percentage 0 0 0h 24h 48h 72h 96h + anti PDGF + anti PDGF + PDGF-AA Time Ctrl + anti-PDGF Ab (20 microg/ml)

7000 Peripheral blood PTCL 100 cells/microL 90 6000 80 70 5000 60 50 4000 40 30 3000 on basal values) 20 10

Cell Viability (Percentage 2000 0 0h 6h 12h 24h 48h 1000 Time 0 CTRL Imatinib 1microM Imatinib 5 microM Imatinib 10 microM

10/0824/08 07/0921/0905/1019/10 02/11 16/1130/1114/1228/12 11/0125/0108/0222/0207/0321/0304/04 18/04 Time Figure 6. PDGFRA signaling sustains PTCL/NOS cell proliferation and is a suitable therapeutic target. The addition of an anti-PDGF (for 48 h) antibody revealed the time-dependent reduction in the cell proliferation index, consistent with progressive cell cycle modification detected by BrDU assay (a). The effect in the cell proliferation was reversed by adding PDGF-AA peptide (b). The top panels shows BrDU incorporation detected by flow cytometry; the bottom panels shows the results plotted as histogram (percentage of expression on basal values). The administration of imatinib mesylate at escalating doses (0, 1, 5 and 10 mM) induced a significant reduction in cell viability in a time-dependent manner in primary PTCL cells refractory to daunorubicin and gemcytabine treatment (c). We consistently observed anti-lymphoma activity in a patient with progressive leukemic disease that was refractory to conventional therapies and autologous stem cell transplantation who exhibited a stable disease with the reduction of the tumor burden (peripheral blood lymphocyte count is plotted) for approximately 9 months (d). Please note the decrease in peripheral blood (PB) PTCL cells as assessed by flow cytometry during imatinib treatment.

were reversed by supplying the treated cells with recombinant disease remained stable for approximately 8 months with human PDGF-AA (Figure 6b; Supplementary Figure 5). Treatment evidence of a reduction in the tumor burden, which, however, with anti-PDGF, however, did not affect the apoptotic cell death did not match the criteria for disease remission. Notably, a gradual rate (data not shown). In contrast, treating PTCL/NOS cells with and progressive reduction in the leukemic component was escalating doses of imatinib mesylate, a prototypical PDGF observed, which led to the complete disappearance of leukemic tyrosine kinase inhibitor, achieved signiﬁcant induction of cells within 4 months of beginning the imatinib treatment apoptosis in a time- and dose-dependent fashion (Figure 6c). In (Figure 6d). Ten months after beginning the imatinib treatment, particular, after 48 h of imatinib treatment, the fraction of viable the disease had spread to the central nervous system, and cells ranged from 25–50% with a dosage range of 1–10 mM. imatinib treatment was consequently halted. Despite additional chemotherapy (6 courses of gemcitabine), the patient died in 3 months. Evidence of anti-lymphoma activity due to PDGFRA inhibition We obtained preliminary evidence of anti-lymphoma activity exerted by PDGFRA inhibition in the case of a 55-year-old male DISCUSSION affected by refractory PTCL/NOS, who received imatinib mesylate In this study, we investigated the determinants and biological as compassionate therapy. The disease originally presented in consequences of PDGFRA activation in PTCLs. The hypothesis that clinical stage IIA. First-line treatment with MACOP-B (12 courses) PDGFRA could be intimately involved in the biology of PTCL/NOS led to complete remission, which was followed, 8 years later, by clones was substantiated by expression analyses, which we disease relapse with diffuse nodal and extranodal (lung and bone) performed using GEP and IHC. Indeed, PDGFRA was found to be involvement. At the time of this relapse, the patient received overexpressed in PTCL/NOSs compared with normal T cells. This CHOP (6 courses), followed by high-dose cyclophosphamide (with evidence extends our original observation that PDGFRA is peripheral blood stem cell collection) and autologous stem cell overexpressed and consistently activated in PTCL subtypes, transplantation. However, despite an initial complete remission, including anaplastic large-cell lymphoma (ALCL), angioimmuno- the disease rapidly relapsed and became disseminated leukemia 2 blastic lymphoma, MF, cutaneous PTCL/NOS, panniculitis-like PTCL months after the autologous stem cell transplantation. After and enteropathy-type PTCL. This pattern of overexpression was testing the pathological material (the initial lymph node biopsy) not observed for the other member of the PDGF receptor family, for PDGFRA expression with informed consent, the patient PDGFRB, in keeping with the predominant PDGFRA involvement received imatinib at a daily dosage of 800 mg. Fortunately, the in the pathogenesis of mature T-cell neoplasms. The common

Leukemia (2014) 1687 – 1697 & 2014 Macmillan Publishers Limited Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1695 element of deregulated PDGFRA expression in the PTCLs of different hematological malignancies.45 STAT5 activation was prompted the idea that targeting tyrosine kinase inhibitors could confirmed in situ in a large series of primary PTCL cases. In contrast be a novel and promising treatment strategy.7,17,34,35 In this to STAT5, STAT3 protein, which is constitutively activated in ALK þ regard, we recently demonstrated the efficacy of TKI for the ALCLs through ALK signaling,40,46 appeared to be associated with treatment of both an ALCL mouse model and ALCL patients.18 the PTCL/NOS model. The finding that STAT3 and STAT5 activation In this type of tumor, remarkably, we also observed PDGFRB are disjointed is not surprising because this occurs in other expression, directly mediated by ALK þ signaling through AP-1.18 neoplastic settings characterized by constitutive PDGFRA In this study, we add additional information by demonstrating signaling, such as GIST.47 that PDGFRA activation demonstrated in PTCLs is not due to Finally, we provided insight into the outcome of PDGFRA genetic abnormalities, including gene amplification, translocations activation in PTCL cells by demonstrating that inhibiting the and point mutations. This finding is noteworthy because it autocrine stimulation did not induce cell death while achieving a makes PTCL a unique condition among the hematological significant reduction of cell proliferation and that selective malignancies characterized by PDGFRA overexpression and PDGFRA stimulation restored ‘full’ proliferative capability. Indeed, activation. In fact, in other diseases, such as hypereosinophilic because the high proliferation rate of these tumors appears to syndrome, aggressive mastocytosis and chronic myeloproliferative have a significant clinical role, conferring high aggressiveness to neoplasms, primary genomic lesions involving the PDGFRA the disease,23,48 it is conceivable that interfering with PDGFRA gene locus have been identified.13 Herein, we challenged the signaling may be beneficial for PTCL patients. Importantly, hypothesis that PDGFRA deregulation in PTCLs underlies the imatinib induced significant primary PTCL cell death ex vivo. mechanism of the autocrine stimulation. We demonstrated, in a However, we could not rule out that this effect, which could be large set of cases, that PTCL neoplastic cells actively produce ascribed to PDGFRA inhibition, would be magnified by quenching PDGFA in situ and that its synthesis and secretion is associated off-target signals. Moreover, we demonstrated that the other with increased platelet-poor plasma levels of this growth factor. known imatinib targets (KIT, ABL and PDGFRB) were not In addition, we demonstrated that PDGFA secretion is necessary overexpressed in PTCL cells. As a proof of principle, we treated and sufficient to induce PDGFRA phosphorylation in PTCL cells a PTCL/NOS patient with a refractory progressive disease with in vitro. Autocrine stimulation of tyrosine kinase receptors is a imatinib mesylate as a compassionate therapy. Remarkably, relatively common mechanism in human cancers,13,36 which has although the disease was advanced, imatinib exerted anti- been only rarely described in lymphomas. In particular, the lymphoma activity. In particular, although the criteria for bioinformatic predictions indicated that diffuse large B-cell complete or partial remission were not fulfilled, the patient’s lymphomas and certain leukemias can activate several pathways disease was stable for 8 months (a considerable period for a PTCL/ involved in proliferation and survival via autocrine loops.37 NOS in progression), with neoplastic cells cleared from the More recently, it was shown that PDGFRA activation is sustained peripheral blood. Consistent with our observation, two complete by an autocrine stimulation in T-prolymphocytic leukemia, a rare remissions were obtained in PTCL patients using dasatinib T-cell-derived leukemia.16 This finding is consistent with our monotherapy.34 In addition, we very recently documented a observations and fits the hypothesis that several different T-cell complete remission in an ALCL patient with relapsed/refractory neoplasms share common pathogenic features. In this regard, disease who was treated with imatinib,18 which constituted the although the genetics of PTCLs are quite complex5,38 we rationale for designing ad hoc clinical trials in this disease. previously showed that different PTCL types present with In conclusion, our study clarified the mechanism through which common altered pathways,9,39 an observation later confirmed by PDGFRA is activated in PTCL/NOS and reinforced the rational basis others.8,40 Taken together, these results have potentially relevant for targeting TKI in this disease. implications because they suggest that neoplastic cells would be able to induce local and systemic conditions favoring their fitness via PDGFRA synthesis and release. CONFLICT OF INTEREST Importantly, in addition to an autocrine stimulation, an in vivo The authors declare no conflict of interest. paracrine loop cannot be excluded because many reactive components of the tumor microenvironment may also produce PDGF ligands. In this regard, it should be noted that the reactive ACKNOWLEDGEMENTS milieu has a significant role in different lymphoid neoplasms.41 We are grateful to Dr Aurora Esposito for her technical assistance. This work was Recently, it was shown that non-neoplastic cells can support supported by the Centro Interdipartimentale per la Ricerca sul Cancro ‘G. Prodi’, tumor proliferation in Hodgkin lymphoma through the PD1-PD1 BolognAIL, AIRC (IG4987; IG10519; 10007 5xMille; IG 2013N.14355), RFO (Prof Pileri ligand loop, which can determine the prognostic features of and Prof Piccaluga), Fondazione Cassa di Risparmio in Bologna, Fondazione della follicular lymphoma42 and Hodgkin lymphoma.43 Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006 (Prof Pileri and Prof Piccaluga), and FIRB Futura 2011 RBFR12D1CB (Prof Piccaluga). We further characterized the signaling that is sustained by PDGFRA activation in PTCLs. Specifically, we generated a GEP signature representative of PDGFRA activity by inhibiting the þ REFERENCES receptor in different PDGFRA cell types and tracking it in primary 1 Jaffe ES, Harris NL, Stein H, Campo E, Pileri SA, Swerdlow SH. Introduction and PTCL samples, where it proved to be enriched compared with overview of the classification of the lymphoid neoplasms. In: Swerdlow S, Campo E, normal T cells, which suggested that a tyrosine kinase is activated Harris NL, Jaffe ES, Pileri SA, Stein H et al. (eds) WHO Classification of Tumors of in the primary cases. Notably, a similar approach was previously Hematopoietic and Lymphoid Tissues, 4th edn. Lyon: IARC, 2008, pp 158–166. adopted to identify the CD40 signaling GEP signature to track in 2 Dyer MJ, Siebert R. Peripheral T-cell non-Hodgkin’s lymphoma NOS: naming of normal and neoplastic B cells.44 parts. Leukemia 2006; 20: 208–209. The signature of PDGFRA activation in PTCL was further 3 Vose J, Armitage J, Weisenburger D. 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Leukemia (2014) 1687 – 1697 & 2014 Macmillan Publishers Limited Tyrosine kinase autocrine loop in T-cell lymphoma PP Piccaluga et al 1697 APPENDIX Claudio Doglioni, Andre´s Ferreri and Maurilio Ponzoni (San AIRC 5xMille consortium ‘Genetics-driven targeted management Raffaele Institute, Milano); Claudio Agostinelli, Pier Paolo of lymphoid malignancies’: Robin Foa`, Sabina Chiaretti, Filippo Piccaluga and Stefano Pileri (University of Bologna); Brunangelo Berardelli, Brunangelo Falini, Enrico Tiacci, Giorgio Inghirami, Falini, Stefano Ascani, and Enrico Tiacci (University of Perugia); Roberto Piva, Gianluca Gaidano, Davide Rossi, Stefano Pileri and Belgium: Peter Van Loo, Thomas Tousseyn, and Christiane De Pier Paolo Piccaluga. Wolf-Peeters (University of Leuven); Germany: Eva Geissinger The European T-Cell Lymphoma Study Group: Italy: Giorgio and Andreas Rosenwald, (University of Wuerzburg); Martin Leo Inghirami and Roberto Piva, (Azienda Ospedaliera Citta` Hansmann and Sylvia Hartmann (University of Frankfurt); della Salute e della Scienza di Torino, University of Torino); Spain: Miguel Angel Piris and Maria E. Rodriguez (Hospital Marco Chilosi and Alberto Zamo´ (University of Verona); Fabio Universitario Marque´s de Valdecilla, IFIMAV, Santander and Facchetti and Silvia Lonardi (University of Brescia); Anna De Instituto de Investigaciones Biome´dicas Alberto Sols, CSIC-UAM, Chiara and Franco Fulciniti (National Cancer Institute, Napoli); Madrid).

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