High affinity neurotrophin receptors and ligands ...

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Prepublished online December 4, 2008; doi:10.1182/blood-2008-05-155200

High affinity neurotrophin receptors and ligands promote leukemogenesis Zhixiong Li, Gernot Beutel, Mathias Rhein, Johann Meyer, Christian Koenecke, Thomas Neumann, Min Yang, Jurgen Krauter, Nils von Neuhoff, Michael Heuser, Helmut Diedrich, Gudrun Gohring, Ludwig Wilkens, Brigitte Schlegelberger, Arnold Ganser and Christopher Baum

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Blood First Edition Paper, prepublished online December 4, 2008; DOI 10.1182/blood-2008-05-155200

High affinity neurotrophin receptors and ligands promote leukemogenesis 1

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Zhixiong Li , Gernot Beutel , Mathias Rhein , Johann Meyer , Christian Koenecke , Thomas Neumann1, Min Yang1, Jürgen Krauter2, Nils von Neuhoff3, Michael Heuser2, Helmut Diedrich2, Gudrun Göhring3, Ludwig Wilkens3, Brigitte Schlegelberger3, Arnold Ganser2, Christopher Baum 1,4

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Department of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, 30625 Hannover, Germany 3 Institute of Cell and Molecular Pathology, Hannover Medical School, 30625 Hannover, Germany 4 Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039, U.S.A. 2

ZL, GB, and MR contributed equally to this work.

Corresponding author: Zhixiong Li, MD or Christopher Baum, MD Department of Experimental Hematology, OE6960 Hannover Medical School Carl-Neuberg-Straße 1 30625 Hannover Germany Phone: +49 511-532-5148 Fax: +49 511-532-5105 E-mail: [email protected] [email protected]

Running title: leukemogenesis of TRK signaling Category: neoplasia

Part of the data was presented in an oral session at the 2007 Annual Meeting of the American Society of Hematology in Atlanta.

1 Copyright © 2008 American Society of Hematology


Abstract

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high affinity NT-receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients (mean age 54.3 years), we demonstrate for the first time cell surface expression of the three TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. While only few point mutations were found in TRK +

sequences by RT-PCR, we observed co-expression of BDNF (ligand for TRKB) in >50% of TRKB

cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Co-expression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target.

Keyword: acute leukemia, TRKs, BDNF, autocrine loop, protein-tyrosine kinase

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Introduction

Current concepts of leukemogenesis postulate a collaboration of ‘class I’ mutations that result in for example constitutively activated protein-tyrosine kinases (PTKs) with ‘class II’ mutations of transcription factors such as AML/Runx or ETS proteins. In this scenario, class I mutations (such as BCR/ABL and FLT3-ITD) promote proliferation but generally do not inhibit differentiation, while the reverse is true for class II mutations.1 While the molecular analysis of patient samples has supported this concept in human acute myeloid leukemia (AML), there still remain a substantial proportion of patients in whom both types of mutations have not yet been demonstrated. There is growing evidence for involvement of multiple PTK oncogenes, their immediate downstream targets (e.g. phosphatidylinositol 3-kinase=PI3K), or of proteins regulating their function in hematological malignancies.2-5 The fact that cytogenetic remissions can be achieved in the majority of patients with chronic myeloid leukemia (CML) demonstrates a causal role of the BCR-ABL oncoprotein 6

in this disease. Analysis of activated PTK is also of clinical relevance.

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At least one third of AML

patients carry mutated FLT3 alleles and have unfavorable prognosis.8,9 It is thus important to identify other PTKs that are activated in the remaining patients. Moreover, co-activation of receptor PTKs has been suggested to be important for tumor development and to affect the tumor cell response to targeted therapy.10 Oncogenic transformation by PTKs occurs in different ways,11 e.g. by genomic rearrangements, such as chromosomal translocations, gain-of function (GOF) mutations, PTK overexpression or small deletions in receptor PTKs and cytoplasmic PTKs. Autocrine and/or paracrine loops have been suggested as important mechanisms for aberrant kinase activation in human solid tumors12 and leukemia,13 and may have therapeutic potential.14 However, few experimental studies convincingly demonstrate the oncogenic potential of autocrine/paracrine circuits of PTKs in animal models,12,15 and a prognostic role of autocrine loops in human leukemia has not been demonstrated. The neurotrophins (NTs), which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4, and NT-6, play a major role in neuronal survival. NTs are unique in that they utilize two different classes of receptors: the TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) and the low affinity NGF receptor (LNGFR=p75NTR),16 a member of the tumor necrosis factor cytokine receptor family. The biologically active receptors for NGF, BDNF and NT-3 are TRKA, TRKB, and TRKC, respectively. NT-3 can bind to all of the TRK

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receptors, and NT-4 binds preferentially to TRKB. NT binding to TRK receptors leads to dimerization of receptors and kinase activation. LNGFR may modulate the activity of this signaling complex but is not required for its function.16 Of note, human embryonic stem cell survival has recently been shown to be mediated through activation of TRK receptors by NTs.17 Members of the TRK family have been found in several non-neural cell types,

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and may also play a crucial role in initiation, progression, and

metastasis of many tumors in humans, e.g. neuroblastoma, medullary thyroid carcinoma and breast cancer.20-23 In addition, some data indicate relevance of TRK receptors as prognostic factors.20 For 24

example, TRKB is associated with bad prognosis in Wilms´ tumor.

However, relatively little is known

about the mechanisms of oncogenesis mediated by altered TRK signaling.25,26 Many PTK oncogenes are derived from genes (e.g. Abl, FLT3, c-Kit and PDGFR-ß) normally involved 2

in the regulation of hematopoiesis or hematopoietic cell function. TRK receptors and their respective ligands are also expressed at various stages of hematopoiesis.27,28 A role for neurotrophins in hematopoiesis has yet to be confirmed using conditional knockouts. Nevertheless, recent data suggested important functions of TRK signaling in hematopoiesis. TRKs promote proliferation and survival of lymphocytes and monocytes/macrophages. 29 TRKB expression is greatest in precursor CD4-CD8- thymocytes and progressively declines throughout the T cell differentiation pathway.30 Importantly, there is increasing evidence for involvement of TRK receptors in leukemogenesis. A cryptic translocation t(12;15) (p13;q25), which resulted in the chimeric transcript TEL-TrkC, was found in an AML patient.25,31 Furthermore, a deleted form of TRKA (ΔTrkA), in which 75 amino acids are lacking in the extracellular domain, was identified in another AML patient. 32 In mouse models, we found that ΔTrkA is a very potent oncogene that transforms cells mainly via PI3K and mTOR.33 Another study revealed the induction of TRKA and a contribution of NGF to survival signaling in human cord blood cells transduced with retroviral vectors encoding the AML1-ETO oncogene.

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addition, we had evidence to suggest that a cytoplasmically deleted form of LNGFR may contribute to leukemia in mice.35 Furthermore, we observed expression of LNGFR in patients with acute leukemia 36

(AL), preferentially in common ALL. Taken together, these data suggest a previously underestimated role of NT signaling in leukemogenesis. However, with the exception of one report showing expression of TRKA mRNA in primary leukemic cells in 44% of AML patients, 37 the expression pattern of other TRKs and NTs and their potential prognostic relevance have not been reported in primary leukemic cells.

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Here, we screened AL patients for expression of TRKA, TRKB and TRKC. A distinct expression pattern of these receptors in different leukemia subtypes as well as expression of NTs was observed. Co-expression of TRKB and BDNF was associated with poor outcome and induced leukemia in a murine model. Moreover, TRK signaling was important for maintenance of leukemic cells in vitro. This study expands current concepts of leukemogenesis and encourages further evaluation of NT receptor signaling as a drug target in leukemia therapy.

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Methods

TRK and NTs expression in leukemic blasts of AL patients We studied tumor specimens from AL patients who had been diagnosed in the Hannover Medical School from 2004 to 2006. Blood and/or bone marrow (BM) samples from AL patients were collected at diagnosis after informed consent was obtained in accordance with the Declaration of Helsinki. Mononuclear cells from all samples studied were immediately isolated by centrifugation over Ficoll 0

gradient and freshly used or stored at –180 C until further use. The following monoclonal antibodies were used: anti-TRKA (clone H10, Biodesign), anti-TRKB (clone75133, R&D), anti-TRKC (clone 75219, R&D), anti-BDNF (Cat# GF35L, CALBIOCHEM), anti-NT-3 (clone 41512, R&D). A polyclonal antibody against human NGF (cat# BAF256, R&D) was used for detection of NGF. Antibodies were validated on cell lines that expressed retrovirally encoded TRK receptors and NTs. The study was approved by the ethics committee of the Hannover Medical School. The majority of patients were treated according to previously described protocols.

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For surviving patients, the median follow-up

period after diagnosis was 30 months. In all cases, cytomorphologic classification according to FAB criteria was made on bone marrow and/or peripheral blood smears.

Retroviral vectors, vector production, retroviral transductions, in vivo tumorigenesis assays, and tumor phenotyping A retroviral vector encoding full-length human TRKA was kindly provided by Dr. Gary Reuther (University of South Florida, Tampa, Florida).32 Plasmid SF91.IRES-EGFP.WPRE, which mediates efficient transgene expression in hematopoietic cells, has been described.33 The retroviral vector is referred to as SF91-IE. A PCR fragment containing the cDNA of human TRKB or BDNF was generated and cloned into the NotI site before the IRES-EGFP cassette of SF91-IE. Resulting vectors encoding TRKB or BDNF were named SF91-TRKB and SF91-BDNF, respectively. Cell free high-titer supernatants containing the ecotropic envelope protein were generated as described.

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Retroviral

transductions, in vivo tumorigenesis assays, and tumor phenotyping were performed as previously described (supplementary information).33

Radiation-induced apoptosis assay and leukemic cell growth assays

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2.5×105 32D cells were starved from IL-3 and serum for 3 hours, placed in 24-well plates, and exposed to 5 Gy -irradiation. Immediately after irradiation, cells were supplemented with NGF, BDNF (each 100 ng/mL), IL-3 (2 ng/mL), or no factor. Cell viability was analyzed using the Annexin-V assay. Cells staining negative for both Annexin-V and propidium iodide were counted as viable cells. To 2

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analyze clonal growth, 2x10 or 2x10 murine cells were plated per dish in M3234 media (StemCell Technologies, Vancouver, Canada) in the presence of signal transduction inhibitors. The assays were plated as duplicates or quadruplicates, and colonies were counted on day 6. Mononuclear cells from patients were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and exposed to inhibitors and/or idarubicin.39 Inhibitors K252a, AG879 (Calbiochem, Merck, Bad Soden, Germany) and anti-human BDNF antibody (Promega, Mannheim, Germany) were used.

Western blot analysis For signal transduction analysis, leukemic cell extracts were prepared following established protocols.

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Cell lysates were used as indicated in results. Antibodies were purchased from Santa

Cruz Biotechnology (Santa Cruz, California, U.S.A.).

RNA extraction and reverse transcriptase– polymerase chain reaction (RT-PCR), small interfering RNA (siRNA)-mediated knockdown, BDNF-ELISA, and statistical analysis Please refer to supplementary information.

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Results

Frequent expression of TRK receptors in leukemic blasts of AL patients We analyzed expression of TRKs in AL patients. 94 adult patients (42 female, 52 male) with a mean age of 54.3 years and diagnosis of primary or secondary AML (87%), ALL (12%), or AUL (1%) were enrolled after informed consent. The patients´ clinical characteristics are summarized in Table 1. Expression of TRKA, TRKB and TRKC was detected by flow cytometry using monoclonal antibodies. If +

>20% of leukemic blasts expressed at least one of the TRK receptors, cases were considered TRK

(Figure 1A, B, S1).40 Thus, 55% of the analyzed cases expressed at least one TRK receptor, without statistically meaningful differences in expression rates between AML (43/82) and ALL (8/11). We observed expression of TRKA on AML blasts, which is in agreement with a previous study that demonstrated expression of TRKA on the RNA level.37 For the first time, we found expression of TRKB and TRKC in human leukemia. Interestingly, while TRKB can be expressed alone in blasts, TRKA or TRKC expression always occurred concomitantly with TRKB. About 18% of leukemia cases co-expressed all TRK receptors. Co-expression of two or more TRK receptors (i.e. TRKA+TRKB, TRKB+TRKC and TRKA+TRKB+TRKC) was observed in AML, while ALL blasts exclusively expressed only TRKB. Although TRK mRNA was found in hematopoietic cells from healthy volunteers,27,37 we did not detect TRK receptors on the surface of normal mononuclear cells by flow cytometry (data not shown), which is in agreement with previous studies.

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Correlation of TRK expression and French-American-British (FAB) leukemia classification Next, we analyzed the relationship between patient age, FAB subtype, white blood cell (WBC) counts, ECOG status, cytogenetics, FLT3 mutation and TRK expression. In agreement with recent publications,8 we found internal tandem duplications of the juxtamembrane region of the FLT3 receptor (FLT3-ITD) in 25% (17/67) of AML patients. There was no significant correlation of patient age, WBC counts, ECOG status, FLT3-ITD or cytogenetics with TRK expression (Table 1). However, in contrast to a previous study,37 we established a clear correlation of TRK expression pattern and the FAB classification (Table 1). In particular, TRKA was expressed in 21 of 34 myelo-monocytic/monocytic leukemias (AML M4 and M5) (62%) whereas only 5 of 48 non-myelo-monocytic/monocytic leukemias

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(10%) were positive (p