Deregulated RAS signaling plays an important role in the molecular pathogenesis of myeloid leukemias, and strategies which target RAS signaling pathways are therapeutically promising. Inhibition of RAS post-translational modification (e.g. by farnesyltransferase inhibitors = FTIs) is one strategy to impede oncogenic RAS function in vivo and FTIs are currently being tested in clinical trials in acute myeloid leukemia (AML). In this study, we tested the anti-leukemia effects of FTI L-778,123 in myeloid leukemia cell lines (HL-60, Kasumi-1, K562) and primary myeloid leukemia cells (n=6). FTI L-778,123 substantially inhibited myeloid leukemia cell proliferation as quantified by MTS assays with IC50 values ranging from 0.2 μM–1.8 μM for cell lines and 0.1 μM–161.8 μM in primary samples. Inhibition of HL-60 (FAB M2) cell proliferation was found to be caused by cell cycle arrest at the G1/S interface as demonstrated by FACS analysis of propidium iodide stained cells. We observed no induction of apoptosis in L-778,123-treated HL-60 cells as quantified by FACS analysis following annexin-V incubation. In contrast, L-778,123 induced a G2M blockade followed by apoptosis in NB-4 (FAB M3) cells. Western blotting demonstrated that blockade of RAS protein prenylation by L-778,123 was both time- and concentration-dependent. H-RAS prenylation in HL-60 cells was almost completely inhibited within 12 hours of treatment with 0.1, 0.5 or 1 μM L-778,123, and by 6 hours with 5 μM of the drug. N-RAS prenylation was strongly inhibited within 3 hours with all four FTI concentrations. Interestingly, an accumulation of unprocessed N-RAS protein was observed after 6 hours of FTI treatment. K-RAS prenylation was only partially blocked after 48 hours incubation with 10 to 20 μM of the drug. This finding probably reflects the 10–20-fold higher affinity of K-RAS for farnesyltransferase, as compared to the other RAS isoforms. Western blotting and FACS analysis showed that L-778,123 also inhibited phosphorylation of MEK-1/2 and MAPK-1/2, down-stream components of the RAS-to-MAPK signaling cascade. Treatment of HL-60 cells with L-778,123 (5 μM, 24 hours) led to approximately 40–50% lower levels of intracellular phosphorylated MEK-1/2. Similarly, L-778,123 treatment caused both time- and concentration-dependent reduction of activated, diphosphorylated MAPK-1/2 levels. Higher L-778,123 concentrations (0.5, 1 and 5 μM) potently decreased diphosphorylated MAPK-1/2 levels within 6 to 12 hours, while a lower drug concentration (0.1 μM) elicited similar effects after 36 to 48 hours. In summary, our data demonstrate that L-778,123 inhibits multiple components of the RASto-MAPK signaling cascade and exhibits impressive activity against myeloid leukemia cell proliferation. Interestingly, L-778,123 displayed differential cell cycle effects in two different myeloid leukemia cell lines (namely, G1 arrest in HL-60 and G2M arrest in NB-4), indicating that this drug may employ leukemia-sub-type-specific activities.

Disclosures: No relevant conflicts of interest to declare.

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