Gene Expression Profiling Identifies Oncogenic Molecular Pathways in T-Lymphoma Induction by SL3-3 Retrovirus Insertional Mutagenesis in Inbred NMRI Mice.

Abstract

The non-oncogene bearing retrovirus SL3-3 murine leukemia virus (MLV) induces T-cell lymphomas with a remarkably consistent immunophenotype when injected into newborn inbred NMRI (NMRI-i) mice, making it, at present, the most homogeneous murine model of MLV induced T-lymphomas1. The oncogenic effects of SL3-3 are caused by proviral insertional mutagenesis of the host genome in or near genes of major importance for lymphoma/leukemia development. Determination of SL3-3 integration sites in the NMRI-i genome therefore provides an efficient whole genome screening method for identifying genes involved in murine and potentially also human T-cell lymphomagenic processes. Here, the impact of integration sites on latency, tumor immunophenotype, clonality, and gene expression profiles reflecting the affected molecular pathways involved in the carcinogenesis was analyzed. So far, a total of 22 retroviral integration sites were identified in thymic (14) and mesenteric lymph node tumors (8) from 10 SL3-3-infected NMRI-i mice. The majority of tumors were CD3+CD4+CD8− (2 thymus tumors were CD3+CD4+CD8+) and TCRβ clonality was demonstrated for all tumors by Southern analysis. Gene expression profiling using 60-mer oligonucleotide microarrays representing 10.000 different genes was performed in triplicate for 3 thymic tumors and a pool of 3 normal thymuses from uninfected NMRI-i mice. Unsupervised clustering of microarray data, two tailed student t-tests and significance analysis of microarrays (SAM) showed the T-cell thymus tumors to be more closely related to each other than to normal thymus tissues. The most common integration sites identified in thymic T-cell tumors were in or close to growth factor independence-1 (Gfi-1) and G1/S-specific cyclin D3 (CCND3), both involved in regulation of cell cycle progression in T cells. The two thymic tumors with CCND3 integrations contained homogenous CD3+CD4+CD8− single positive populations. The transcript levels determined by microarray of CCND3 were for both tumors 2-fold higher that what was observed in normal thymuses. D cyclins and cyclin-dependent kinases (CDKs) regulate the G1/S checkpoint and the interaction partner of CCND3, CDK6 was also up-regulated as compared to the normal thymus tissue. Similar expression features for CCND3 and CDK6 were observed for the third thymus tumor with, at present, unknown integration sites. Thus, for tumors with CCND3 integrations a promoted S phase entry leading irreversibly to cell division in the tumor cells can be suggested as further supported by the observation of decreased expression levels of the negative regulator of CDK2, p27KIP1 and increased expression of mRNA encoding G1 to S phase transition 2 (Gspt2). Notably, CCND3 is required for T-cell receptor (TCR) dependent expansion of transformed murine T-lymphocytes. Among other genes with a differential up-regulated expression in T-cell derived thymus tumors with SL3-3 integration in CCND3 were members of the TCR signaling cascade (Zap70, Fyn, VAV2, VAV3 and RAC2) and oncognes (Rel and Ect2) illustrating some of the transformation processes occurring in the malignant T-cells. In summary, this murine model of T-lymphomas enables a specific coupling of common integrations sites, like CCND3 or Gfi-1 to their effects on downstream molecular pathways.