The oncoprotein MLL-ENL disturbs hematopoietic lineage determination and transforms a biphenotypic lymphoid/myeloid cell.

Mixed-lineage leukemia (MLL) fusion proteins are associated with a unique class of leukemia that is characterized by the simultaneous expression of lymphoid-specific as well as myeloid-specific genes. Here we report the first experimental model of MLL. Murine bone marrow cells were retrovirally transduced to express the MLL-eleven nineteen leukemia (MLL-ENL) fusion protein. When cultivated in flt-3 ligand, stem cell factor and interleukin-7 (IL-7) in a stroma-free culture system MLL-ENL-transduced as well as control cells showed a wave of B-lymphopoiesis. Whereas the controls exhausted their proliferative capacity in a CD19+/B220+ state, a continuously proliferating CD19-/B220+ cell population emerged in the MLL-ENL-transduced cultures. Despite the lymphoid surface marker, these cells were of monocytoid morphology. The immortalized cells contained unrearranged retrovirus, expressed MLL-ENL mRNA and were able to grow in syngenic recipients. From the diseased animals an MLL-ENL positive, B220+/CD19- cell type could be reisolated and cultivated in vitro. In analogy to human MLL, MLL-ENL-transformed cells not only coexpressed lymphocyte-specific (rag1, rag2, pax5, Tdt) and monocyte-specific genes (lysozyme, c-fms), but also showed rearrangements of the genomic immunoglobulin locus. This model shows that MLL-ENL influences events of early lineage determination and it will enable the investigation of the underlying molecular processes.

Transcriptional activation is a key function encoded by MLL fusion partners.

Chromosomal translocations that fuse the mixed lineage leukemia gene (MLL) to a variety of unrelated partner genes are frequent in pediatric leukemias. The novel combination of genetic material leads to the production of active oncoproteins that depend on the contributions of both constituents. In a search for a common function amongst the diverse group of MLL fusion partners we constructed artificial fusions joining MLL with generic transactivator and repressor domains (acidic blob, GAL4 transactivator domain, Herpes simplex VP16 activation domain, KRAB repressor domain). Of all constructs tested, only MLL-VP16 was able to transform primary bone marrow cells and to induce a block of early myeloid differentiation like an authentic MLL fusion. Interestingly, the transformation capability of the artificial MLL fusions was correlated with the transcriptional potential of the resulting chimeric protein but it was not related to the strength of the isolated transactivation domain that was joined to MLL. These results prove for the first time that a general biological function - transactivation - might be the common denominator of many MLL fusion partners.

Critical role of retinoid/rexinoid signaling in mediating transformation and therapeutic response of NUP98-RARG leukemia.

While the nucleoporin 98-retinoic acid receptor gamma (NUP98-RARG) is the first RARG fusion protein found in acute leukemia, its roles and the molecular basis in oncogenic transformation are currently unknown. Here, we showed that homodimeric NUP98-RARG not only acquired unique nuclear localization pattern and ability of recruiting both RXRA and wild-type NUP98, but also exhibited similar transcriptional properties as RARA fusions found in acute promyelocytic leukemia (APL). Using murine bone marrow retroviral transduction/transformation assay, we further demonstrated that NUP98-RARG fusion protein had gained transformation ability of primary hematopoietic stem/progenitor cells, which was critically dependent on the C-terminal GLFG domain of NUP98 and the DNA binding domain (DBD) of RARG. In contrast to other NUP98 fusions, cells transformed by the NUP98-RARG fusion were extremely sensitive to all-trans retinoic acid (ATRA) treatment. Interestingly, while pan-RXR agonists, SR11237 and LGD1069 could specifically inhibit NUP98-RARG transformed cells, mutation of the RXR interaction domain in NUP98-RARG had little effect on its transformation, revealing that therapeutic functions of rexinoid can be independent of the direct biochemical interaction between RXR and the fusion. Together, these results indicate that deregulation of the retinoid/rexinoid signaling pathway has a major role and may represent a potential therapeutic target for NUP98-RARG-mediated transformation.