Interaction of the Leukemogenic CALM/AF10 Fusion Protein with the Hematopoietic Key Regulator Ikaros.

Abstract

The focus of our research group is the study of the t(10;11)(p13;q14) translocation that leads to the fusion of the proteins CALM and AF10. This translocation can be found in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ALL) and also in malignant lymphomas. In some patients the t(10;11) is the only cytogenetic abnormality which indicates that the CALM/AF10 fusion is a causal event during leukemogenesis. Previous studies of our group have shown that the expression of CALM/AF10 in hematopoietic stem cells triggers the development of an aggressive leukemia in a murine bone marrow transplantation model. CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) has a function in Clathrin mediated endocytosis. AF10, a putative transcription factor with a PHD-Motive (Plant Homeo Domain) and a Leucine Zipper domain, was initially identified as fusion partner of MLL. The underlying mechanism of CALM/AF10 dependent leukemogenesis, however, remains mostly unknown. Recently we could show that AF10 interacts with the transcription factor Ikaros (ZNFN1A1) in yeast-two-hybrid assays. Interestingly, Ikaros is a key regulator of hematopoesis, required for normal differentiation and proliferation of B- and T-lymphocytes. The structure of the protein is characterized by a DNA-binding and an oligomerisation domain. Through interaction with many factors in the cell nucleus, Ikaros can act both as activator and repressor of transcription. In various forms of ALL as well as chronic myeloid leukemia (CML) an aberrant expression pattern of Ikaros has been found. In a murine model the expression of a dominant negative isoform of Ikaros causes leukemias and lymphomas. Using various AF10 deletion mutants in the yeast, the Ikaros interaction domain of AF10 was mapped to the Leucine Zipper domain of AF10 which has also been shown to be required for malignant transformation by the MLL/AF10 fusion protein. Overexpression of fluorescently labelled proteins reveals a similar distribution pattern of AF10 and Ikaros in the nucleus, whereas in the presence of CALM/AF10 Ikaros appears to be localized predominately in the cytoplasm. The interaction between AF10 and Ikaros has been confirmed by GST-pull-down assays. In order to further study this interaction and its role in leukemogenesis we have raised monoclonal antibodies against the C-terminus of AF10. These antibodies are currently established for Western Blot analysis and Immunoprecipitation experiments. Reporter gene assays are carried out to measure the impact of CALM/AF10 on Ikaros' function as repressor or activator of transcription. These studies may provide new insights into the mechanism of CALM/AF10 induced leukemia and thereby facilitate the development of new therapies.