The Farnesyltransferase Inhibitor (FTI) L-778,123 Displays Promising Anti-Leukemia Activity

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.