Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins.
OBJECTIVE
Mixed lineage leukemia (MLL) gene rearrangement is commonly observed in human leukemias. Many of the resultant MLL fusion proteins are found correlated with Ras signaling. Nevertheless, Ras mutations have only been reported in a small subset of MLL-rearranged leukemia. With the potential of developing new therapeutic regimens targeting Ras signaling pathway, we studied the role of MLL-AF4 family fusions and MLL-septin family fusions in the activation of Ras signaling in leukemogenesis.


METHODS
Elk-1-driven luciferase reporter system was used to study the role of MLL-AF4, MLL-AF5q31, MLL-LAF4, MLL-CDCrel, MLL-MSF, and MLL-Septin 6 in the activation of Ras signaling. Dominant negative Ras S17N mutant and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 were employed to demonstrate the involvement of Ras and MEK in this transactivation event. The activation of endogenous Ras/MEK signaling pathway by MLL fusion proteins in leukemia cell lines was also addressed by immunoblot analysis and small interfering RNA knockdown approach.


RESULTS
We demonstrated that MLL-AF4, MLL-AF5q31, and MLL-LAF4 activated Elk-1 transcription factor, one of the major downstream effectors of Ras. This activation was abolished in the presence of dominant negative Ras or MEK inhibitor U0126, indicating the requirements of Ras and MEK. We further showed that endogenous MEK is phosphorylated in a MLL-AF4-expressing leukemia cell line, whereas depletion of MLL-AF4 by small interfering RNA reduced the phospho-MEK level.


CONCLUSIONS
Our findings suggest that MLL-AF4 family fusion oncoproteins can activate Elk-1 through Ras/MEK/extracellular signal-regulated kinase (ERK) pathway and strongly support the role of Ras signaling in the pathogenesis of MLL-rearranged leukemia.
Ng
Ming-Him James
MH
Division of Haematology, The SH Ho Foundation Research Laboratories in Pathology, Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR China.
Ng
Ray Kit
RK
Kong
Cheuk-Ting
CT
Jin
Dong-Yan
DY
Chan
Li-Chong
LC
D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D051788:Myeloid-Lymphoid Leukemia Protein / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D050807:ets-Domain Protein Elk-1 / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
C113855:MLL-AF4 fusion protein, human; D015514:Oncogene Proteins, Fusion; D034741:RNA, Small Interfering; D050807:ets-Domain Protein Elk-1; D051788:Myeloid-Lymphoid Leukemia Protein; D011494:Protein Kinases; D018631:ras Proteins