3089 – ERK INHIBITORS SUPPRESS THE GROWTH OF LEUKEMIA CELLS

RAS gene mutations is the most frequent oncogenic mutations in various cancers. MAPK signaling is thought to be the important pathway in RAS signal and crucial of cell survival. Inhibition of activated ERK1/2 induced by RAS mutation is considered a promising target for treatment of various cancers. Therefore, we investigated the effects of direct ERK inhibitors on in vitro growth of leukemia cell lines. We used 4 AML cell lines (OCI/AML3, HL-60, THP-1 and U-937), 2 T-ALL cell lines with activated NOTCH1 mutation (Jurkat and KOPT-K1), and 2 normal lymphocyte samples. OCI/AML3, HL-60 and THP-1 possess NRAS gene mutation. Three ERK inhibitors, SCH772984, LY3214996 and Ulixertinib were used. Cells were cultured with the inhibitors and cell growth was assessed with a colorimetric assay. Induction of apoptosis and analysis of cell cycle were evaluated by flow cytometry. Expression and activation of signaling proteins in lysates from the cultured cells were examined by immunoblotting. The specificity of the effects of inhibitors was confirmed by ERK1/2 knockdown experiment with siRNA induction. The three inhibitors suppressed the growth of only 3 cell lines with NRAS mutation in a dose-dependent manner. Flow cytometric analysis revealed that the growth suppression was mainly caused by G0/G1 arrest in cell cycle and partially by induction of apoptosis. Suppressive effects on normal lymphocytes were milder than those on leukemia cell lines. Immunoblotting revealed that these inhibitors generally induced the suppression of phosphorylation of ERK1/2, RSK, down-regulation of DUSP4, c-MYC, cyclin D3, although the effects was different depending on the inhibitors and cell lines. siRNA transfection experiment showed that the suppressive effects were specific to ERK1/2. Our findings indicate that ERK inhibitors can be novel molecular-targeted drugs for leukemia with RAS mutation.