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 MSCVbased 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.

Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma

“…These data suggest that the formation of t(2;5) might not be the first step leading to malignant transformation, and might not be required for formation of systemic ALCL. This model fits well with the facts that (i) in a significant fraction of ALCL cases, t(2;5) is not found (4), (ii) although being transforming, the NPM-ALK fusion protein is not sufficient to induce ALCL-like tumors in animal models (4,25), and (iii) a significant number of patients with t(2;5)-positive, CD30 ϩ lymphoproliferations restricted to the skin without systemic dissemination has been reported (26,27). We focused in our work on the most common ALK rearrangement t(2;5)/NPM1-ALK, therefore the impact of chromosomal spatial reorganization on the formation of the rare other ALK-involving rearrangements (3) remains to be investigated.…”

The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib

“…For the methylcellulose assays, colonies were picked after 14 days and analyzed by flow cytometry. On average, 20 colonies were analyzed from each plate (range [15][16][17][18][19][20][21][22][23][24][25]. Generally, the colony number decreased with higher imatinib concentrations (data not shown) and revealed a strong selection of EGFP þ /Bcr-Abl mutant expressing cells upon addition of imatinib, whereas without imatinib there was no significant difference in the percentage of EGFP þ /Bcr-Abl mutant cells compared to the initially plated ratio (Figure 3c and d).…”

“…21 Briefly, after staining with Fc-block and CD 45-Cy5 cells were incubated with PE-conjugated antibodies against CD11b, CD45R/B220 and CD90.2(Thy1.2) (BD Pharmingen, Heidelberg, Germany). Propidium iodide was used to exclude dead cells and samples were analysed on a Coulter XL2 cytometer.…”

“…Cells were suspended in BBMM (IMDM, 20% FCS, 1% BSA) containing growth factors (mIL-3 10 ng/ml, IL-6 10 ng/ml, SCF 50 ng/ml (R&D Systems, Wiesbaden, Germany)) and prestimulated for 24 h. BM cells were transduced with viral supernatants by spin infection four times every 12 h as described previously. 21 The cells were harvested 12 h after the last spin infection, washed with PBS, resuspended in Hanks balanced salt solution (Sigma-Aldrich, Irvine, UK) and MIG-Bcr-Abl mutant infected cells were tested for EGFP expression by FACS analysis. Lethally irradiated (800 rad) female Balb/c recipient mice were transplanted with 2.5 Â 10 6 cells of a 3:1 mixture of Bcr-Abl wt and either Bcr-Abl T315I or Bcr-Abl Y253H mutant infected BM cells via tail vein injection.…”

Activation of -diacylglycerol kinase is critical for the mitogenic properties of anaplastic lymphoma kinase

“…4,5 The tumorigenic properties of NPM/ALK have been demonstrated in vitro in different cell systems [6][7][8][9] and confirmed in vivo by the generation of NPM/ALK-mediated tumor models. [10][11][12][13] Despite several studies, the pathogenic mechanisms leading to ALK-mediated transformation remain still poorly defined.Thus far, several signaling molecules have been identified that associate and/or are activated by ALK, including growth factor receptor-bound protein 2 (Grb2), Src homology and collagen (Shc), insulin receptor substrate-1 (IRS-1), phospholipase C-␥ (PLC-␥), p60 src , and phosphatidylinositol 3-kinase (PI3-K), although only a few have been shown to be strictly specific for ALK's transforming potential. 3 In particular, several lines of investigation have indicated that both PLC-␥ and PI3-K are critical transducers of NPM/ALK-mediated oncogenesis through activation of mitogenic and/or survival signals, 8,[14][15] while the exact role of the ras/mitogen activated protein kinase (MAPK) cascade needs further evaluation.…”