Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin's lymphoma found in children and young adults. ALCLs frequently carry a chromosomal translocation that results in expression of the oncoprotein nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). The key molecular downstream events required for NPM-ALK–triggered lymphoma growth have been only partly unveiled. Here we show that the activator protein 1 family members JUN and JUNB promote lymphoma development and tumor dissemination through transcriptional regulation of platelet-derived growth factor receptor-β (PDGFRB) in a mouse model of NPM-ALK–triggered lymphomagenesis. Therapeutic inhibition of PDGFRB markedly prolonged survival of NPM-ALK transgenic mice and increased the efficacy of an ALK-specific inhibitor in transplanted NPM-ALK tumors. Notably, inhibition of PDGFRA and PDGFRB in a patient with refractory late-stage NPM-ALK+ ALCL resulted in rapid, complete and sustained remission. Together, our data identify PDGFRB as a previously unknown JUN and JUNB target that could be a highly effective therapy for ALCL.
ALCLs are T cell lymphomas1,2 comprising 10–20% of all non-Hodgkin's lymphoma cases in children and 3% in adults3. About half of ALCL cases are positive for the NPM-ALK fusion chimera caused by the t(2;5)(p23;q35) translocation4. ALK translocations or point mutations have also been described in diffuse large B cell lymphomas and in several nonlymphoid neoplasms5,6,7,8. Inhibition of ALK fusion proteins by specific compounds such as crizotinib showed promising clinical responses in ALCL and non–small cell lung cancer9,10. However, ALK mutations conferring resistance to crizotinib have also been reported11.
Recent studies have linked NPM-ALK expression to induction of the activator protein 1 (AP-1) transcription factors JUNB and JUN12,13. To investigate their role in NPM-ALK–driven T cell lymphomas, we conditionally deleted JUN and/or JUNB in T cells of transgenic mice carrying the human NPM-ALK fusion protein under the control of the mouse CD4 promoter14 (CD4-NPM-ALK) (Fig. 1a). We confirmed gene deletion by DNA genotyping (data not shown), real-time PCR, western blotting and immunohistochemistry (Supplementary Fig. 1a–c). JUN deletion did not affect expression of oncogenic NPM-ALK in T cells (Supplementary Fig. 1a–c), and all genetic cohorts showed normal T cell development before onset of lymphoma (Supplementary Fig. 1d,e and data not shown). CD4-NPM-ALK mice developed T cell lymphomas around 8 weeks after birth. The overall survival was not affected by deletion of JUN or JUNB alone (Fig. 1b), but survival was substantially prolonged in mice in which both JUN and JUNB were deleted (CD4-NPM-ALK-CD4ΔΔJUN) (Fig. 1b). Lymphomas from CD4-NPM-ALK-CD4ΔΔJUN mice showed markedly reduced proliferation and significantly (P = 0.014) increased apoptosis compared to lymphomas from CD4-NPM-ALK mice (Fig. 1c and Supplementary Fig. 2a,b). Consistently, CD4-NPM-ALK-CD4ΔΔJUN but not CD4-NPM-ALK lymphoma cells failed to grow in vitro (data not shown), and Cre-mediated deletion of loxP-flanked Jun and Junb in established CD4-NPM-ALK cell lines (CD4-NPM-ALK-JUNflox/flox;JUNBflox/flox) induced cell death (Supplementary Fig. 2c). Freshly isolated CD4-NPM-ALK-CD4ΔΔJUN lymphoma cells formed palpable tumors when engrafted into severe combined immunodeficient (SCID) mice, albeit after a long latency (Supplementary Fig. 2d). These results show that JUN and JUNB are important regulators of NPM-ALK–driven T cell lymphoma development.
T cell lymphomas spread to many organs in CD4-NPM-ALK transgenic mice14. Although CD4-NPM-ALK-CD4ΔΔJUN mice eventually developed lymphomas, we did not detect dissemination of tumor cells (Fig. 1d,e and Supplementary Fig. 2e). Notably, blood vessel numbers were significantly decreased in primary lymphomas of CD4-NPM-ALK-CD4ΔΔJUN mice compared to CD4-NPM-ALK controls (Fig. 2a). Expression and activation of the pericyte marker PDGFRB was readily detectable in lymphoma cells of CD4-NPM-ALK mice but essentially absent in T cell lymphomas of CD4-NPM-ALK-CD4ΔΔJUN mice (Fig. 2b,c). Tumor cells from single mice lacking only JUNB or JUN (designated CD4-NPM-ALK-CD4ΔJUNB and CD4-NPM-ALK-CD4ΔJUN, respectively) showed intermediate PDGFRB protein expression (Supplementary Fig. 3a). PDGFRB mRNA expression was also decreased in lymphomas of CD4-NPM-ALK-CD4ΔΔJUN mice when compared to CD4-NPM-ALK lymphomas (Fig. 2d), suggesting transcriptional regulation of PDGFRB by JUN proteins. Consistently, AP-1 consensus sequences were identified within the mouse PDGFRB promoter and the first intron and were conserved among other species15,16 (Supplementary Fig. 3b,c). Analysis of ENCODE transcription factor binding tracks revealed binding of JUN and JUNB to the PDGFRB intronic AP-1 site in the human K562 leukemia cell line15,17 (Supplementary Fig. 3d). Electrophoretic mobility shift assay analysis of nuclear extracts from NPM-ALK lymphomas demonstrated AP-1 DNA binding to the consensus sequence, which was reduced after antibody-mediated JUN and JUNB depletion (Fig. 2e). Moreover, binding of JUN and JUNB to the PDGFRB promoter was confirmed by chromatin immunoprecipitation assays of human dermal fibroblasts (Fig. 2f), mouse NPM-ALK cell lines and tumor tissues (Supplementary Fig. 3e,f) and luciferase reporter assays in Jurkat cells (Fig. 2g) and HeLa cells using PDGFRB promoter constructs with and without an AP-1 site (Supplementary Fig. 3g). These data demonstrate transcriptional regulation of PDGFRB by JUN and JUNB. PDGFRs have not been implicated in ALCL but are known physiologic regulators of tumor growth18,19,20. We therefore investigated the effects of the PDGFR kinase inhibitor imatinib on established mouse A333, MEL406 and CD4-417 lymphoma cell lines. Imatinib also blocks activity of the BCR-ABL kinase, the receptor tyrosine kinase KIT and PDGFRA with high affinity21, but it has no effect on ALK22,23. The NPM-ALK lymphoma cell lines did not express BCR-ABL (data not shown) or PDGFRA, but they expressed KIT at variable levels (Fig. 3a and Supplementary Fig. 4a,b). PDGFRB expression ranged from high to non-detectable (Fig. 3a and Supplementary Fig. 4a). We then used tumor cell transplantation experiments to evaluate the effects of imatinib on tumor growth. Notably, imatinib treatment of tumor-bearing mice resulted in markedly reduced tumor growth (Fig. 3b and Supplementary Fig. 4c). The response rate of individual transplanted cell lines correlated with the level of PDGFRB expression, as A333-derived lymphomas (lacking PDGFRB but expressing high levels of KIT) were not affected by imatinib treatment (Fig. 3b). We obtained similar results using nilotinib (Supplementary Fig. 4d), another PDGFRB kinase inhibitor24,25. Imatinib treatment did not affect expression of ALCL-associated cytokines and their receptors (such as IL-9 and IL-22 (refs. 26,27; Supplementary Fig. 4e), peripheral and splenic T cell populations (Supplementary Fig. 5a–c) or tumor-infiltrating T cell subsets (Supplementary Fig. 5d,e). These results indicate that inhibition of PDGFRB markedly impairs growth of transplanted ALCL tumor cells.
We next explored the effects of imatinib on primary lymphomas in CD4-NPM-ALK mice. Imatinib treatment substantially increased overall survival (Fig. 3c), reduced in vivo tumor cell proliferation and enhanced apoptosis (Fig. 3d). Imatinib also blocked proliferation and enhanced cell death of mouse PDGFRB+ lymphoma cell lines in vitro (Supplementary Fig. 6a,b). As expected, imatinib reduced PDGFRB phosphorylation in CD4-NPM-ALK cell lines (Supplementary Fig. 6c) and in tumor tissues from CD4-NPM-ALK mice (Fig. 3e), resulting in reduced phosphorylation of protein kinase B (PKB, also known as AKT) and signal transducer and activator of transcription 3 (STAT3) but not of extracellular signal–regulated kinase (ERK) (Supplementary Fig. 6d,e). Furthermore, PDGFRB phosphorylation in PDGF-β (PDGFB)-stimulated lymphoma cells was impaired by imatinib in a dose-dependent manner (Supplementary Fig. 6f,g), resulting in reduced proliferation (Supplementary Fig. 6h). Imatinib also reduced PDGFB mRNA levels (Supplementary Fig. 6i) in CD4-NPM-ALK lymphomas, indicating an autoregulatory PDGF secretion loop. Notably, in imatinib-treated CD4-NPM-ALK-CD4ΔΔJUN mice, no tumors developed during the 30-week observation period (Fig. 3c), and tumor cell dissemination to distant organs was completely blocked (Supplementary Fig. 7a). This is probably owing to an additional effect of imatinib on the tumor stroma, which has been reported for other cancers28,29 (Supplementary Fig. 7b–f).
We next tested imatinib treatment in combination with the ALK inhibitor crizotinib9. Imatinib and crizotinib synergistically reduced tumor growth of grafted mouse NPM-ALK+ lymphoma cells (Fig. 3f). Similar results were obtained with the ALK inhibitor CEP28122 (ref. 30). Imatinib reduced lymphoma growth and the percentage of lymphomas that relapsed after CEP28122 treatment (Fig. 3g,h). These results show that therapeutic blockade of PDGFRs can markedly alleviate relapse of ALCLs after ALK inhibition.
The vast majority of human NPM-ALK+ and NPM-ALK− ALCLs showed high expression of JUNB, JUN, PDGFRA and PDGFRB mRNA31,32,33,34 (Fig. 4a) and protein (Fig. 4b and Supplementary Fig. 8a,b). In contrast to peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) and angioimmunoblastic T cell lymphoma (AITL), most ALCL samples showed high expression of both PDGFRA and PDGFRB. Consistently, two highly conserved AP-1 binding sites were identified in the human PDGFRA promoter (Supplementary Fig. 8c). We did not detect wild-type or mutated forms of KIT or BCR-ABL in ALK+ lymphomas (Supplementary Fig. 8d and data not shown). Established human ALK+ ALCL cell lines were negative for PDGFRA and PDGFRB (Supplementary Fig. 8e), but ALK expression was required to maintain JUNB mRNA expression (Supplementary Fig. 8f). These results demonstrate that primary human ALK+ ALCLs express high levels of PDGFRA, PDGFRB and JUNB.
On the basis of our results, we tried imatinib treatment in a patient with an ALCL of poor prognosis. This NPM-ALK+, JUNB+, JUN+, PDGFRA+, PDGFRB+, KIT− ALCL (Fig. 4c) was refractory to conventional chemotherapy and relapsed after autologous stem cell transplantation. The patient entered complete clinical remission with reduced tumor markers and diminished PDGFB levels after 10 d of imatinib therapy (Fig. 4d–g). The patient is now free of ALCL 22 months after initiation of imatinib therapy (Supplementary Case Report). We also observed high PDGFB levels in the serum of four additional patients with ALCL (Supplementary Fig. 8g).
JUNB and JUN are thus crucial for the growth and metastasis of NPM-ALK+ ALCL, exerting this effect through the direct regulation of PDGFR expression. ALK induces the expression of JUNB and JUN12,13, which subsequently bind to and activate the PDGFRB promoter. We show that PDGFR expression and activation is a key driver of ALCL proliferation, lymphoma cell survival and tumor cell dissemination. PDGFRs might promote ALCL formation through a combined effect on lymphoma cells and tumor stroma. The mechanism by which PDGFRs promote ALCL formation in the tumor stroma and the qualitative contribution of stromal effects to drug responses in the mouse models are unclear at present, and further experiments will be required to address these issues. Although the precise mechanism is not fully understood, inhibition of PDGFRB by imatinib resulted in an excellent therapeutic response in the CD4-NPM-ALK ALCL mouse model and in a terminally sick patient with NPM-ALK+ ALCL.
Our patient data show that PDGFRs are present in NPM-ALK+ T cell lymphomas but also in highly malignant NPM-ALK− ALCL. This would imply that, in the absence of ALK, other kinases drive the expression of PDGFRs, through AP-1 or other transcription factors. It should be noted that we have not proven that PDGFRs functions within the context of the signaling networks in ALK− tumor cells. Future experiments will be required to investigate whether PDGFR expression is a crucial common driver for lymphomagenesis.
The major clinical challenge in treating NPM-ALK+ ALCL is its high rate of relapse. Notably, we have shown here that the dual inhibition of ALK and PDGFR reduces lymphoma growth and alleviates relapse rates. Our findings suggest that imatinib is a potential therapeutic option for patients with crizotinib-resistant lymphomas11.
In summary, we provide evidence that PDGFR inhibition is a highly effective treatment for NPM-ALK+ ALCL, and these findings might also be relevant for ALK− lymphomas. We are now designing a clinical trial on the basis of PDGFR expression in tumors, for which we will enroll patients with second-stage ALCL.
Cell lines CD-4-4, CD4-417, MEL406, VAC, bT02, A943 and A333 were isolated from CD4-NPM-ALK mice and seeded at 1 × 106 cells/ml in RPMI 1640 Medium supplemented with 10% FBS and 100 IU/ml penicillin, 50 mg/ml streptomycin sulfate. Cell numbers were determined with an electronic cell counter (CASY-1, Schärfe-System).
Mice carrying the human NPM-ALK cDNA expressed under the CD4 promoter4,14 were crossed with CD4-Cre mice35, as well as mice carrying loxP-flanked JunB and/or Jun36,37. The genetic background of these intercrosses was C57BL/6 × BALB/c. For the Kaplan-Meier curve, only isogenic littermates of the following genotypes were used: wild-type control, CD4-NPM-ALK-JunBflox/flox;Junflox/flox (CD4-NPM-ALK), CD4-NPM-ALK-CD4-JunB−/−;Jun−/+ (CD4-NPM-ALK-CD4ΔJUNB), CD4-NPM-ALK-CD4-JunB−/+;Jun−/− (CD4-NPM-ALK-CD4ΔJUN) and CD4-NPM-ALK-CD4-JunB−/−;Jun−/− (CD4-NPM-ALK-CD4ΔΔJUN) mice. Mice were kept in a pathogen-free facility and all animal experiments were done in agreement with the ethical guidelines of the Medical University of Vienna.
Twelve-week-old female SCID mice were subcutaneously implanted with 1–5 × 106 cells. After two weeks, tumors had grown to the size of <1.0 cm in diameter and mice received imatinib or nilotinib (100–200 mg per kilogram of body weight per day), crizotinib (33 mg per kilogram of body weight per day) + imatinib (200 mg per kilogram of body weight per day), or PBS for 7 d by oral gavage.
1 × 106 BALB/c ALK+ ALCL cells (VAC and bT02) were subcutaneously implanted into 4- to 6-week-old syngeneic BALB/c recipients. Mice with tumor masses <1.0 cm were treated with CEP28122 (100 mg per kilogram of body weight twice a day (b.i.d.)), CEP28122 (100 mg per kilogram of body weight b.i.d.) + imatinib (200 mg per kilogram of body weight b.i.d.), or CEP28122 (100 mg per kilogram of body weight b.i.d.) for 18–21 d, followed by CEP28122 (100 mg per kilogram of body weight b.i.d.) + imatinib (200 mg per kilogram of body weight b.i.d.) for 18 d.
Imatinib treatment of CD4-NPM-ALK tumor-bearing mice.
CD4-NPM-ALK–positive mice were treated with imatinib at 6 weeks of age and mice were sacrificed at 30 weeks of age. All animal experiments were approved by the ethical committee for animal experiments of the Medical University of Vienna and the Federal Ministry of Science and Research of Austria (animal license numbers: BMWF-66.009/0139-C/GT/2007, BMWF-66.009/0092-II/10b/2009, BMWF-66.009/0137-II/10b/2010 and BMWF-66.009/0001-II/3b/2011).
Patient samples, treatment and blood testing.
The patient with ALCL was treated under supervision of the Department of Internal Medicine I and in agreement with the ethics committee of the Medical University of Vienna with 400 mg imatinib per day after receipt of informed consent. Plasma samples were analyzed for CRP and haptoglobin concentrations (nephelometric assays). PDGFB, soluble CD30 (sTNFRSF8) and β2 microglobulin (B2M) plasma levels were quantified by commercially available ELISAs (eBioscience). Patient samples shown in Figure 4b and Supplementary Figure 8 were collected at the Clinical Institute of Pathology, Medical University Vienna, the Pathology and Hematopathology Unit, Department of Hematology and Oncology 'L. and A. Seràgnoli,' S. Orsola-Malpighi Hospital, University of Bologna and the Department of Molecular Biotechnology and Health Sciences, Center for Experimental Research and Medical Studies, University of Turin with approval from the responsible ethics committees.
Immunohistochemistry and immunofluorescence.
Immunohistochemistry and immunofluorescence staining were performed with formalin-fixed, paraffin-embedded tissues after receipt of informed patient consent and in accordance with the Declaration of Helsinki. Antibodies used were: antibody to Ki67 (1:1,000; Novocastra, NCL-KI67-P), anti-PDGFRA (1:100; Neomarkers, RB-9027), anti-PDGFRB (1:80; Cell Signaling, #3169), anti-pPDGFRB (1:150; SCBT, sc-12909), anti–cleaved caspase 3 (1:100; Cell Signaling, #9661), anti-STAT3 (1:200; SCBT, sc-7179), anti–p-STAT3 (1:80; Cell Signaling, #9145), anti–p-AKT (1:50; Cell Signaling, #3787), anti-JUNB (1:300; SCBT, sc-46), anti-JUN (1:100; SCBT, sc-1694), anti-JUN (1:100; Cell Signaling, #9165), anti-ALK (1:50; Zymed, 51-3900), anti-CD30 (1:50; DAKO, M0751), anti-CD31 (1:50; Dianova, DIA 310), anti-S100A/B (1:500; Dako, Z0311), anti–collagen IV (1:50; Chemicon, AB756P), anti–smooth muscle actin (1:200; Neomarkers, MS-113) and anti-vimentin (1:80; Abcam, AB28028). TUNEL staining was performed with the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. Images were captured with a Zeiss AxioImager Z1 microscope and quantified using HistoQuest and TissueQuest software (TissueGnostics GmbH, Vienna, Austria, http://www.tissuegnostics.com/).
Chromatin Immunoprecipitation (ChIP).
Conserved AP-1 binding sites within the PDGFRB promoter were identified using the ECR Browser (http://ecrbrowser.dcode.org/)38. ChIP was performed as described39. Cells (107 CD4-417 or BJ-1) or tumor tissue (100 μg, CD4-NPM-ALK or CD4-NPM-ALKΔ/ΔJUN lymphomas) were used with 10 μg each anti-JUNB (sc-73X, Santa Cruz), anti-JUN (sc-1694X, Santa Cruz) and control IgG (normal rabbit IgG, sc-2027, Santa Cruz). Primer sequences amplifying a conserved AP-1 binding site in the proximal mouse PDGFRB (muPDGFRB) promoter were: muPDGFRB-FW: 5′-CTCCATTTGACAGGCATCAG-3′; muPDGFRB-Rev: 5′-CTTCCTCCTTTCCCTCTGCT-3′, muPDGFRBneg-FW: 5′-TAGGCTGAGCAGGTCAACT-3′ and muPDGFRBneg-Rev: 5′-TGTGCTCAGGGAGATGACAG-3′ for negative control and muVEGF-FW: 5′-AATGGGATCCTCTGGGAAGT-3′ and muVEGF-Rev: 5′-CACAGTGCATACGTGGGTTT-3′ for positive control. For the human PDGFRB (huPDGFRB) promoter, primer sequences were: huPDGFRB-FW: 5′-CAGGTCATCTGCTCCAAGTG-3′ and huPDGFRB-Rev: 5′-TTGCACTGTCCTGTCTGTCC-3′ as well as huPDGFRBneg-FW: 5′-GGGTATATGGCCTTGCTTCA-3′ and huPDGFRBneg-Rev: 5′-GAGGAATCCCTCACCCTCTC-3′ for negative control.
Promoter analysis and reporter gene assays.
The UCSC Genome Browser on Mouse (July 2007; ref. 15), and MathInspector16 were used for in silico promoter analysis. Luciferase assays were performed as previously described40. The wild-type PDGFR promoter was cloned by PCR in front of the firefly luciferase in the pGL3 (Promega) vector (PDGFRB-luc) as well as a PDGFRB promoter mutant lacking the AP-1 site (PDGFRB ΔAP-1-luc). Human JUN and JUNB pCDNA3.1 (Invitrogen) expression vectors were used together with a β-galactosidase control vector (pMIR-REPORT; Ambion) for relative luciferase quantification. Jurkat lymphoma cells were transfected with the indicated vectors and subjected to OneGlo luciferase assay after 36 h.
Electromobility shift assay.
Oligonucleotides were annealed at equimolar concentrations (40 μM) in 200 μl annealing buffer (0.0625× PCR buffer II (Roche) and 0.94 M MgCl2). For supershift reactions, 2 μg of antibodies specific for JUNB and JUN (anti-JUNB sc-73X and anti-JUN sc-1694X) were used.
Gene expression profiling.
The gene expression data were obtained from three publicly available data sets (GSE6338, GSE14879 and GSE19069 at the NCBI GEO repository http://www.ncbi.nlm.nih.gov/geo/)31,32,33 and proprietary34 and unpublished data sets including T cells from ALCL and PTCL. Expression values were extracted from CEL files and normalized with RMA. Selected probe lists were visualized in a heat map format using Heat Map Viewer, available as a GenePattern module.
Detailed methodology is described in Supplementary Methods.
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We thank E.F. Wagner for providing the JUNBflox/flox and JUNflox/flox mice, A. Ostman for helpful discussions and technical help with PDGFRB analysis, and S. Reichmann for technical help with soluble-CD30 and PDGFB assays. L.K. is supported by the Fonds zur Förderung der wissenschaftlichen Forschung (FWF, P-18478-B12) and the Genome Research in Austria InflammoBiota project. R.M., V.S. and R.E. are supported by the FWF (SFB-F28) and V.S. is supported by FWF project 19723. G.E. is supported by an Elise Richter Fellowship (FWF V102-B12) and an FP7 Marie Curie International Reintegration Grant (IRG 230984). H.D. is supported by the Herzfelder Family Foundation and the Niederösterreichische Forschungs- und Bildungsges.m.b.H. P.W.V. and P.B.S. were supported by Jubiläumsfonds of the Österreichische Nationalbank (P-12147), and P.W.V. was awarded an E. Schroedinger Grant (J 2922). T.W. is supported by the Else-Kröner Fresenius Stiftung. S.P. and G.I. are supported by Italian Association for Cancer Research (AIRC) Special Program in Clinical Molecular Oncology, Milan (5x1000 No. 10007). G.I. is supported by Regione Piemonte (ONCOPROT, CIPE 25/2005); ImmOnc ('Innovative approaches to bust the immune responses', Programma Operativo Regionale, Piattaforme Innovative BIO F.E.S.R. 2007/13, Asse 1 'Ricerca e innovazione' della LR 34/2004) and the Oncology Program of Compagnia di San Paolo, Torino. R.P. is supported by AIRC grant IG-8675. R.E. is supported by the FWF Doktoratskolleg-plus Inflammation and Immunity grant, the Comprehensive Cancer Center Vienna Research Grant and the Austrian Federal Ministry of Science and Research GENAU Austromouse grant. J.M.P. is supported by the Austrian Academy of Sciences and an Advanced Grant from the European Research Council. This study was performed on behalf of the European Research Initiative on ALCL (ERIA).
The authors declare no competing financial interests.
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Laimer, D., Dolznig, H., Kollmann, K. et al. PDGFR blockade is a rational and effective therapy for NPM-ALK–driven lymphomas. Nat Med 18, 1699–1704 (2012). https://doi.org/10.1038/nm.2966
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