Upregulation of Genes Involved in the Beta-Catenin Signaling Pathway in a Mouse Model of Chronic Lymphocytic Leukemia.

Binding of Wnt ligands to Frizzled (Fzd) family and LRP5/6 receptors results in stabilization of beta-catenin protein, its translocation to the nucleus, and in association with LEF/TCF family transcription factors, expression of target genes. This canonical Wnt signaling pathway plays a critical role in development and in the maintenance of tissue specific stem cell populations in the skin, gut, and bone marrow. Dysregulation of the beta-catenin signaling pathway has been described in numerous human malignancies, including chronic lymphocytic leukemia (CLL). Using the Eμ-TCL1 transgenic mouse model of CLL developed by C. Croce et al., we have examined expression of various components of the canonical Wnt pathway during lymphomagenesis by RT-PCR. The Eμ-TCL1 mouse spontaneously develops a hyperplasia of CD5+ B cells in the peripheral blood and peritoneum which progresses towards a monoclonal B cell leukemia/lymphoma with infiltration of spleen, bone marrow, and other organs. This provides the ability, largely lacking in human subjects, to analyze changes in gene expression during development of a lymphoid malignancy. We have FACS sorted normal CD5- B cells as well as CD5+ B cells from the hyperplastic, oligoclonal and malignant, monoclonal phases of leukemia development in Eμ-TCL1 mice and assessed their expression of various members of the Wnt, Fzd, LRP coreceptor, and LEF/TCF transcription factor families. Coreceptor LRP6 is expressed in all populations in approximately equal amounts as determined by quantitative RT-PCR. We find no expression of Fzd receptors 1, 2, 3, 4, or 9, nor Wnt4 or 5a in any population. We find mRNA for Fzd6 and LEF-1 are increased in hyperplastic and malignant CD5+ B cells in Eμ-TCL1 mice as compared to CD5- polyclonal B cells from the same animals. Of interest, both LEF-1 and Fzd6 expression also have been shown to be upregulated in human CLL B cells. To further assess Fzd6 expression during malignant transformation, we took advantage of the fact that only 3–5% of mouse B cells express lambda light chain. Therefore, large expansions of lambda expressing CD5+ B cells are most likely to be monoclonal in Eμ-TCL1 mice. In three separate mice, oligoclonal expansions of CD5+lambda- B cells coexisted with a clonal expansion of CD5+lambda+ cells, as confirmed by PCR analysis of germline immunoglobulin DNA. These two populations, as well as polyclonal CD5- B cells, were purified by FACS; and Fzd6 expression was quantified by real time RT-PCR. Fzd6 mRNA was increased in the oligoclonal CD5+ B cells by an average of 15.9 fold over the normal CD5- B cells (range 4.1-36.9). In the monoclonal lambda expressing subset, Fzd6 expression was increased 41.7 fold (range 23.7–68.3) over CD5- B cells, and increased over that seen in the oligoclonal CD5+ B cells of the same animal by 4.1 fold (range 1.9–5.7). These findings show that progressively increased expression of Fzd6 correlates with malignant transformation in B cell leukemogenesis in the Eμ-TCL1 mouse model and parallels the findings in fully transformed CLL B cells in humans. The functional consequences of enhanced Fzd6 and other components of the canonical Wnt signaling pathway in neoplastic B cells are currently being investigated.