Significance of BAALC (brain and acute leukemia, cytoplasmic) as a poor prognostic factor for patients with cytogenetically normal acute myeloid leukemia (CN-AML) and acute lymphoblastic leukemia (ALL) has now been widely established, however, its molecular mechanisms are still mostly unknown. Available evidence suggests that BAALC is a cytoplasmic protein. Based on this finding, we carried out phosphoprotein array of cytoplasmic signal transduction-related proteins and sought to find out the deregulated signaling underlying BAALC-mediated leukemogenesis. Using BAALC-overexpressed HEK293T cells, we found enhanced phosphorylation of Extracellular signal-regulated kinase (ERK), and its downstream target p90RSK. Besides, Jurkat cells, a human ALL cell line, showed sustained phosphorylation of ERK when BAALC is overexpressed. These results suggest that BAALC acts as a scaffold-like molecule for the cytoplasmic mitogen-activated protein kinase kinase (MEK), upstream ERK activator and activates ERK signaling pathway. Immunoprecipitation assay revealed that BAALC binds to MEK in HEK293T cells. Co-localization of BAALC and MEK in the cytoplasm of HEK293T was also detected by immunofluorescence experiment. The proportion of the phosphorylated form in the MEK-bound ERK was increased compared to its control, indicating that BAALC binds to MEK and directly up-regulates its kinase activity to phosphorylate ERK. BAALC accelerated cell growth and increased the phosphorylation of ERK when overexpressed in Jurkat or HEL cells. Those cells showed accelerated cell cycling with no change of apoptotic status. On the other hand, BAALC knockdown decreased cellular proliferation as well as phosphorylation of ERK in the human leukemia cell line expressing a high level of BAALC, such as Kasumi-1 cells. Decelerated cell cycling was also observed in the BAALC-knocked down cells. Notably, the proportion of the phosphorylated ERK bound to MEK was decreased when BAALC was knocked down, which is consistent with BAALC-overexpressed experiments. Interestingly, in AML cell lines without active Ras mutations, the expression of BAALC was positively correlated to its sensitivity to MEK inhibitor (U0126). Moreover, growth advantage of BAALC overexpression in leukemic cell lines was canceled by the treatment with U0126, implying that BAALC-induced growth advantage depends on MEK-ERK signaling activity. Analysis of deletion mutants of BAALC revealed that binding ability of BAALC to MEK resides in its N-terminal region, which is highly conserved among mammals. This N-terminal region of BAALC was necessary for phosphorylation of ERK and the mutant lacking this region failed to promote proliferation of the leukemic cell lines. Taken together, BAALC acts as a scaffold-like protein that binds to MEK in the cytoplasm and directly up-regulates MEK-ERK signal transduction pathway. The fact that overexpression of BAALC significantly promotes proliferation of leukemic cells is consistent with the clinical observation that BAALC-high leukemia has a poor prognosis. The novel function of BAALC as a scaffold-like protein that activates MEK-ERK signal transduction pathway may account for the underlying mechanism for the generation of aggressive form of leukemia and would be a promising therapeutic target of BAALC-high leukemia.

Disclosures:

Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.

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