NPM-ALK Induces Tumour Development in a Murine Model of Anaplastic Large Cell Lymphoma (ALCL) Independently of Bcl-3 Expression.

Anaplastic large cell lymphomas (ALCLs) define a subgroup of aggressive non-Hodgkin’s lymphomas. In 40–60% of systemic ALCLs, a t(2;5) (p23;q35) translocation is found, which generates a fusion gene between nucleophosmin (NPM) and the receptor tyrosine kinase gene ALK (anaplastic lymphoma kinase). The NPM-ALK chimeric gene encodes a constitutively activated tyrosine kinase and is believed to initiate the process of lymphomagenesis. Since NPM-ALK has been shown to activate PI3-kinase and STAT3, proteins that are involved in apoptosis regulation, altered apoptosis might contribute to ALCL development. Bcl-3, which exerts an anti-apoptotic effect in B and T lymphocytes, has been reported to be up regulated in a subgroup of ALCLs. This raised the question whether Bcl-3 is required for tumour development induced by NPM-ALK. In a first set of experiments, the expression levels of Bcl-3 in NPM-ALK negative and NPM-ALK positive tumour cells were analysed. Bcl-3 expression was enhanced in NPM-ALK expressing cells compared to NPM-ALK negative cells or cells expressing a kinase dead NPM-ALK mutant. Next we used primary murine bone marrow cells from wt versus Bcl-3 −/− mice in order to clarify the contribution of Bcl-3 to NPM-ALK induced transformation. We employed a retroviral infection system utilizing a MCSV-based vector co-expressing NPM-ALK together with the enhanced green fluorescent protein (EGFP) via an internal ribosomal entry site to infect Bcl-3−/− and wild type (wt) bone marrow cells. Transformation of bone marrow cells was analysed by methylcellulose assay without cytokines. Transformation of NPM-ALK infected Bcl-3−/− bone marrow cells was comparable to NPM-ALK infected BL6 wt bone marrow cells. No colony formation was detectable after transfection of Bcl-3−/− bone marrow with empty vector as control. Finally, we utilized a murine transplantation model of ALCL. Lethally irradiated BL6 wt mice were transplanted with retrovirally NPM-ALK infected wild type or Bcl-3−/− bone marrow cells. As a control, BL6 wt mice received a transplant of Bcl-3−/− bone marrow cells infected with supernatant from viral producer cells transfected with empty vector. Mice transplanted with NPM-ALK infected wild type or Bcl-3−/− bone marrow cells rapidly died within a median survival time of 16 and 17 days respectively, whereas mice transplanted with Bcl-3 −/− bone marrow cells transfected with empty vector survived healthy for more than 300 days. Diseased mice macroscopically showed involvement of the spleen, predominantly. Histologically, spleens of diseased mice showed an extensive infiltration of ALK-positive tumour cells with proliferation of large histiocytic cells both in mice transplanted with NPM-ALK infected Bcl-3−/− and wild type bone marrow. In both groups, FACS-analysis revealed a high percentage of EGFP positive and thus NPM-ALK positive cells in bone marrow and spleen. In conclusion, NPM-ALK is able to transform bone marrow cells and to induce a lymphoma-like disease in the absence of Bcl-3. Bcl-3 is thus dispensable for ALCL development in a murine mouse model. Lack of Bcl-3 in the knockout mice may be compensated by the expression of other proteins. Therefore, Bcl-3 upregulation in a subgroup of human ALCLs may be not critical for lymphoma development.