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The oncogenic fusion protein nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) induces two distinct malignant phenotypes in a murine retroviral transplantation model

Abstract

A t(2;5) (p23;q35) chromosomal translocation can be found in a high percentage of anaplastic large-cell lymphomas (ALCL). This genetic abnormality leads to the expression of the NPM–ALK fusion protein, which encodes a constitutively active tyrosine kinase that plays a causative role in lymphomagenesis. Employing a modified infection/transplantation protocol utilizing an MSCV-based vector, we were able to reproducibly induce two phenotypically different lymphoma-like diseases dependent on the retroviral titers used. The first phenotype presented as a polyclonal histiocytic malignancy of myeloid/macrophage origin with a short latency period of 3–4 weeks. Clinically, the diseased mice showed rapidly progressive wasting, lymphadenopathy and pancytopenia. Mice displaying the second phenotype developed monoclonal B-lymphoid tumors with a longer latency of approximately 12–16 weeks, primarily involving the spleen and the bone marrow, with less extensive lymph node but also histologically evident extranodal organ infiltration by large immature plasmoblastic cells. The described retroviral mouse model will be useful to analyse the role of NPM–ALK in lymphomagenesis in vivo and may contribute to the development of new treatment options for NPM–ALK induced malignancies.

Main

The anaplastic large-cell lymphomas (ALCL) were first described in 1985 by Stein et al. (1985) as a defined subgroup of high-grade NHL. These lymphomas consist mostly of large CD30 (Ki-1 antigen)-positive cells with variable morphologic patterns comprising a common form, a small cell variant and a lymphohistiocytic form, as well as a variety of other more rare morphological appearances (Stein et al., 2000). In more than 50% of systemic ALCL, a t(2;5)(p23;q35) translocation can be found (Falini et al., 1999; Morris et al., 2001). The corresponding genes have been cloned and represent a fusion between the NPM and the ALK genes (Morris et al., 1994). The translocation of the first four exons of the NPM gene to the exons encoding the intracellular domain of the ALK receptor tyrosine kinase leads to a constitutive activation of the ALK tyrosine kinase, and is believed to initiate the process of lymphomagenesis (Drexler et al., 2000; Duyster et al., 2001). The oncogenic potential of NPM–ALK has been demonstrated in vitro by transformation assays with rodent fibroblasts or cytokine-dependent murine hematopoietic cell lines such as Ba/F3, 32D and in primary murine bone marrow (Bischof et al., 1997; Bai et al., 1998,2000; Mason et al., 1998). The role of NPM–ALK for transformation in vivo was underlined by demonstrating that mice develop lymphoid tumors after transplantation with bone marrow retrovirally infected with NPM–ALK (Kuefer et al., 1997). However, in this transplantation model the efficiency of lymphoma induction was low, rendering reliable analysis of factors contributing to lymphoma development difficult.

We aimed to establish a murine model of ALCL with high reproducibility and accuracy by employing a modified retroviral transduction/transplantation system allowing for higher expression of the NPM–ALK cDNA. We used a bicistronic retroviral vector coexpressing NPM–ALK together with the enhanced green fluorescent protein (EGFP) via an internal ribosomal entry site (Figure 1a). Since the phenotype in the previously published model may have arisen from low infection efficiencies, we decided to compare infection conditions of high versus low multiplicity of infection (MOI ranging from 0.05 to 5.0). This led to infection efficiencies ranging from <1% up to 50% infected cells measured by FACS analysis for EGFP fluorescence (Figure 1b). The mice transplanted with high-titer virus (MOI 5.0)-infected cells showed first signs of illness within 3 weeks after transplantation and rapidly succumbed to their disease, with a median survival of less than 1 month (Figure 1c). In contrast, mice transplanted with bone marrow cells infected with a low-titer virus (MOI of 0.05) had a disease latency of more than 8 weeks (Figure 1c). In these mice, disease progression was considerably slower, with some mice surviving several weeks after their first signs of illness. The median survival was approximately 4.1 months. All mice transplanted with BM cells infected with retrovirus stock prepared using empty control vector were alive and well after up to 14 months following transplantation. Thus, disease onset and overall survival strongly correlate with the MOI used.

Figure 1
figure1

(a) Schematic of the retroviral vector construct used to generate retrovirus stock for murine bone marrow infections. The NPM–ALK cDNA was cloned into the HpaI site of the MigRI vector (Hawley et al., 1994; Pear et al., 1998) and transfected into Phoenix E viral producer cells (G Nolan, Stanford, USA). The EGFP is coexpressed via an internal ribosomal entry site (IRES). LTR, viral long-terminal repeat. (b) Two representative FACS analyses of murine bone marrow after retroviral infection with titers of either approximately 106 viral particles per milliliter (left) or 104 particles per milliliter (right) are shown. The percentage of infected cells is determined by plotting the FITC versus the PE channels to exclude autofluorescent cells from the EGFP-positive fraction. (c) Survival curves of mice transplanted with NPM–ALK -infected bone marrow (BM). Whereas all mice infected with control vector generated retrovirus stock (•) were alive after 12 months, mice transplanted with a high-titer virus-infected BM (▪) had a median survival of only 0.8 months and mice transplanted with a low-titer virus-infected BM () had a median survival of 4.13 months

Mice with short-latency disease macroscopically showed involvement of the spleen, multiple enlarged lymph nodes and macroscopic tumor growth in extranodal organs, with a tumor infiltration pattern in the liver and kidneys similar to the spleen (data not shown). In contrast, mice with long-latency disease showed spleens infiltrated with multiple bulky tumors and less macroscopic tumor infiltration in extranodal organs, with liver and kidney being macroscopically infiltrated in only two of 12 mice (data not shown). Also, only a few enlarged lymph nodes were found in these mice, limiting the prominent organ manifestations to the spleen and bone marrow (Table 1). In all mice, the percentage of EGFP-positive tumor cells in the analysed organs varied and was, considering the tumor size, surprisingly low, reflecting high reactive tissue load. Variable fractions of EGFP-positive cells between below 1% and up to 50% of all cells could be detected in the spleen, bone marrow and lymph nodes of the diseased animals (data not shown). Strikingly, in the peripheral blood, no EGFP-positive cells could be found in any of the analysed mice, indicating that the transformed cells were retained in the bone marrow and lymphoid organs.

Table 1 Summary of NPM–ALK retroviral infection/transplantation experiments

In an attempt to determine the immunophenotype of the different tumor cells, a panel of different PE-labelled antibodies against a variety of surface molecules was used (Figure 2). Most of the EGFP-positive cells from mice with short-latency disease reacted with CD11b (Figure 2a). The myeloid/macrophage cell fraction of both EGFP-positive as well as EGFP-negative cells from these mice was highly expanded and seemed to contain a substantial percentage of neutrophils, as determined by staining with Gr-1 (Figure 2 and data not shown). Interestingly, although the spleen and bone marrow were significantly infiltrated by Gr-1-positive cells, the peripheral blood in these mice was devoid of leukocytes at the end stage of their disease. The immunophenotype of EGFP-positive cells from mice with long latency disease could not be determined by FACS analysis, since these cells reacted only with CD44 and CD45 antibodies, which are expressed on virtually all leukocytes.

Figure 2
figure2

(a) Representative FACS analysis of spleen cells from diseased mice with short (upper row) or long (lower row) disease latency with markers for macrophages (CD11b), B (B220) or T cells (Thy1.2). (b) Table of cell surface molecules examined by FACS. In the short-latency disease, most EGFP-positive cells were CD11b-positive, and the EGFP-negative cell fraction also mostly stained for CD11b. In contrast, the majority of the EGFP-positive cells from the spleens of mice with long-latency disease failed to react with any of the indicated antibodies. PE-conjugated antibodies were purchased from Pharmingen, staining and FACS analysis were performed according to standard laboratory methods

Histologically, an extensive infiltration of ALK-positive tumor cells in the spleen, lymph nodes and other extranodal organs could be confirmed in all diseased mice. Also, in macroscopically unaffected organs such as liver and kidney, microscopically ALK-positive, distinctive and mostly perivascular infiltrates could be found in both disease phenotypes (Figure 3e, h). All the tissues analysed for ALK expression showed a cytoplasmic and nuclear staining of the ALK-positive cells in accordance with the cellular distribution expected for NPM–ALK (Figure 3). Tissues from mice with short-latency disease presented histologically with a proliferation of large histiocytic cells with ample, in part finely vacuolated, cytoplasm and vesicular nuclei (Figure 3a, b). Many of these cells expressed ALK and the histiocyte marker BM8 on histochemical staining in sections from the spleen (Figure 3c, d) and liver (Figure 3e), in accordance with the results obtained from the FACS analysis. The second phenotype presenting in mice with long-latency disease showed tissues predominantly infiltrated by large basophilic cells with a perinuclear halo and plasmoblastic appearance (Figure 3f, g). These cells were ALK-positive (Figure 3h) and reacted with anti-IgM and syndecan (CD138) antibodies, which established their plasma-cell origin (Figure 3i, j).

Figure 3
figure3

Sections from mice with histiocytic (ae) or plasmacytoid (fj) disease. Analysis of H+E stained sections of spleens from mice with short-latency disease revealed the proliferation of large histiocytic cells with ample, in part finely vacuolated, cytoplasm and vesicular nuclei among nests of normal lymphocytes (a, b). ALK staining was strong in both the cytoplasmic and nuclear compartments (c) and could also be found in histiocytic perivascular infiltrates in the liver (e). Many of the cells with histiocytic appearance in the spleen sections reacted with an antibody against the histiocyte marker BM8 (d). H+E stained liver tissue from long-latency mice with the second disease phenotype shows a monotonous infiltration by plasmacytoid/plasmoblastic cells with basophilic cytoplasm and perinuclear halo surrounding a bile duct (f, g). ALK staining of this tissue shows strong nuclear and cytoplasmic reactivity (h). The plasma cells also stain intensively with anti-IgM (i) and anti-CD138 (syndecan-1) antibody (j)

Southern blotting for the detection of clonality displayed a polyclonal malignancy in the mice with short-latency disease and a mono- or oligoclonal insertion pattern for the mice with long-latency disease (data not shown). The long-latency disease showed a clonal rearrangement of the immunoglobulin heavy-chain genes, which further corroborated its B-cell origin. A T-cell-receptor rearrangement could not be detected in either disease phenotype (data not shown).

After serial transplantation of 2 × 106 spleen cells from mice with short-latency disease, two out of four sublethally irradiated mice succumbed to a similar disease within 4 weeks, whereas the other two mice developed a long-latency disease phenotype. Four out of six mice transplanted with 2 × 106 spleen cells from mice with long-latency disease became ill after a mean time of 5.4 months with a disease phenotype identical to the primary tumors in regard to histology and immunophenotype.

It has recently been shown that NPM–ALK induces a lymphoma-like disease in mice transplanted with NPM–ALK-infected bone marrow (Kuefer et al., 1997). The utility of this mouse model was compromised by the relatively low frequency and the long latency of lymphoma induction. Similar to mouse models described for Bcr-Abl (Pear et al., 1998; Zhang and Ren, 1998; Li et al., 1999), we tried to improve the current limitations by using a different retroviral vector, higher retroviral titers and a modified infection protocol. The MSCV-derived retroviral vector employed in our study has been shown to be active in a broad range of hematopoietic cells including early progenitors and T cells, thus also supporting expression in the putative target cells where the t(2;5) translocation is believed to arise (Hawley et al., 1994; Falini et al., 1998). Furthermore, a modified transfection protocol utilizing the 293T-cell-based Phoenix retroviral producer line and infecting the bone marrow cells by spin infection may also have contributed to the more efficient and reproducible induction of a lymphoma-like disease in our experiments (Pear et al., 1993).

Employing high-titer virus, a clinically aggressive histiocytic malignancy with a short latency of 2–3 weeks could be induced. A histiocyte-rich form of ALK-positive ALCL has been well described, and in many ALK-positive ALCLs, an extensive inflammatory reaction can be observed, which is also associated with a massive neutrophilic infiltration in some cases (Cheuk et al., 2000; Parker et al., 2001). It is in part because of the strong reactive tissue load that the morphological diagnosis of ALCL can be a difficult task. In fact, many of these malignancies were catalogued under the term ‘malignant histiocytosis’ before the advent of more refined immunohistochemical staining. Since ALK-positive lymphomas have been described to produce proinflammatory cytokines, a cytokine-induced reactive expansion might explain the neutrophilic infiltration found in the spleen and bone marrow of the diseased mice. Studies determining the cytokine expression profiles in these animals are currently being performed in our laboratory.

The second NPM–ALK-induced disease phenotype was reproducibly generated by using diluted retroviral titers. The diseased mice showed macroscopic tumor infiltration in the spleen and extranodal organs, the malignant cells reacted with anti-IgM and CD138, but not with CD30 antibodies, and had rearranged immunoglobulin heavy-chain genes in Southern blot analysis, suggesting that NPM–ALK is able to transform B-lymphoid cells at a particular stage of development without blocking subsequent differentiation. Certain substrains of Balb/c mice have been described as being especially susceptible to the induction of B-cell lymphoma (Beecham et al., 1991; Potter et al., 1999), and mice infected with a retrovirus coexpressing the v-abl tyrosine kinase and c-myc, or double transgenics, develop plasma cell tumors (Potter et al., 1986; Troppmair et al., 1989; Largaespada et al., 1992). NPM–ALK itself has been shown to activate a variety of downstream signalling molecules, among them PLCγ, PI3-kinase, STAT3 and STAT5 (Bai et al., 1998,2000; Nieborowska-Skorska et al., 2001; Slupianek et al., 2001; Zhang et al., 2002). Since activation of PI3-kinase and STAT3 has also recently been demonstrated to be an important mechanism for prevention of apoptosis and growth regulation in plasma cells (Hyun et al., 2000; Rawat et al., 2000; Tu et al., 2000; Hideshima et al., 2001), NPM–ALK might promote B-cell lymphoma development by inducing these pathways in a permissive cellular context.

The recent development of an efficient and reproducible mouse model for CML has led to a plethora of new insights into the pathogenesis of Bcr–Abl-induced leukemia in vivo (Pear et al., 1998; Zhang and Ren, 1998; Li et al., 1999). By retrovirally introducing specific Bcr–Abl mutants into murine hematopoietic cells (Million and Van Etten, 2000; Roumiantsev et al., 2001; Zhang et al., 2001), or by infecting bone marrow from mice deficient for genes believed to be important for transformation (Sexl et al., 2000; Di Cristofano et al., 2001; Li et al., 2001), it is possible to examine specific signalling pathways related to Bcr–Abl in vivo. Thus, mouse models represent a powerful tool to dissect molecular pathways involved in malignant transfor-mation by specific oncogenes. The mouse model described here will contribute to the molecular characterization of NPM–ALK-induced lymphomagenesis and to the identification and development of new therapeutic options for NPM–ALK-related malignancies.

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Acknowledgements

JD is supported by a grant from the Wilhelm-Sander Stiftung. CM is supported by a fellowship from the Deutsche Jose Carreras Leukämie Stiftung (DJCLS 2001/NAT-2). The work was also supported by National Cancer Institute (NCI) Grant CA69129 (SWM), NCI Cancer Center Core Grant CA21765 and by the American Lebanese Syrian Associated Charities (ALSAC), St Jude Children's Research Hospital. We thank W Pear for supplying the MigRI retroviral vector.

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Correspondence to Justus Duyster.

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Miething, C., Grundler, R., Fend, F. et al. The oncogenic fusion protein nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) induces two distinct malignant phenotypes in a murine retroviral transplantation model. Oncogene 22, 4642–4647 (2003). https://doi.org/10.1038/sj.onc.1206575

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Keywords

  • NPM–ALK
  • mouse model
  • tyrosine kinase
  • lymphoma

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