Modulation of Anthracycline-Induced Cytotoxicity by Targeting the Prenylated Prenylome in Myeloid Leukemia Cells

Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development.

Myeloid leukemia cell lines (HL60, KG1a, THP-1, K562) were screened for idarubicin-induced apoptosis, cell cycle progression, cell-cycle dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Analysis of synergy was done by the method of Chou and Talalay using the CompuSyn program. Analysis of the prenylated proteome was performed using a tagging-via-substrate approach. Knock-down of K-Ras and Cdc42 protein levels was done by RNA interference (RNAi) using lentiviral shRNA constructs.

Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2a expression levels. Although cells were most sensitive to idarubicin, DPI L-778,123 and FTI BMS-214,662 also potently inhibited leukemia cell growth with IC₅₀ values in the micromolar and sub-micromolar range. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling, Top2 expression and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines (KG1a, THP-1 and K562), but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Both BMS-214,662 and L-778,123 effectively inhibited prenylation of the majority of larger molecular weight proteins (e.g. Lamins, NAP1L1, HDJ-2), and some smaller molecular weight proteins (e.g. H-Ras, N-Ras). Additionally, only DPI L-778,123 blocked prenylation of smaller molecular weight proteins such as K-Ras and Cdc42. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation in all cell lines tested. However, RhoB and RhoC remained prenylated even upon combined FTI/DPI treatment. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis.

Our results suggest that nearly complete inhibition of protein prenylation using an FTI+DPI combination is the most effective method to induce apoptosis and to block anthracycline induced activation of ERK signaling.

Reuter:Sanofi: Honoraria; Amgen: Honoraria.