Disease Models and Transformation Mechanisms Mediated by MLL-AF4 Family Oncoproteins in Human Leukemia.

The Mixed Lineage Leukemia (MLL) gene codes for a histone methyltransferase that is required for hematopoietic development. As a consequence of chromosomal translocations, MLL is fused with over 40 different genes to yield in-frame fusion proteins in acute leukemias. AF4, the most common fusion partner, accounts for 40% of MLL leukemias. The AF4-related proteins, LAF4 and AF5q31, are also fusion partners for MLL in rare cases of leukemia, whereas a fourth family member, FMR2, is a candidate protein for fragile X mental retardation syndrome. MLL fusions with AF4 family proteins manifest as acute biphenotypic or lymphoblastic leukemias that are associated with a poor prognosis, but the lack of appropriate disease models has hampered progress in understanding their underlying molecular mechanisms. Here we report that each of the MLL-AF4 family fusion proteins (the leukemia associated MLL-AF4, MLL-LAF4, and MLL-AF5q31 as well as an artificial MLL-FMR2) is capable of altering the growth and self-renewal properties of primary murine hematopoietic stem/progenitor cells in a serial methylcellulose culture myeloid replating assay. However, significant differences were observed in their respective oncogenic potentials. Cells transduced with MLL-LAF4, MLL-AF5q31 and MLL-FMR2 rapidly expanded during serial replatings, adapted to growth in liquid culture, and induced leukemias in syngeneic recipient mice within 6 months. Conversely, bone marrow cells transduced with MLL-AF4 yielded modest numbers of blast-like colonies in the third round of plating, only transiently expanded in vitro, and quickly underwent terminal differentiation. MLL-LAF4 and MLL-AF5q31 were also capable of enhancing the self-renewal of transduced cells with B lymphoid progenitor phenotypes. A structure/function analysis demonstrated that the previously reported transcriptional transactivation domains of AF4 family proteins were neither necessary nor sufficient for in vitro transformation. Furthermore, the recently reported AF9 interaction motif that is conserved in all AF4 family proteins was also shown to be dispensable for in vitro transformation. These data indicate that transactivation per se, and interaction with AF9 in particular, are not absolutely required for the oncogenic actions of MLL-AF4 family proteins. Conversely, a minimal transformation domain was mapped to the highly conserved carboxy-terminal homology domain shared among all AF4 family proteins, including the Drosophila homolog Lilliputian, and shown to be necessary and sufficient for transformation. Taken together, our studies establish transformation models for MLL-AF4 family fusion proteins and provide critical mechanistic insights into their underlying molecular mechanisms.