Enzasataurin Enhances ATRA-induced Differentiation of Acute Myeloid Leukemia Cells

Abstract Background All-trans retinoic acid (ATRA) is considered to be the sole clinically useful differentiating agent in the treatment of acute myeloid leukemia (AML). However, it has been effective only in acute promyelocytic leukemia (APL) but not other subtypes of AML. Therefore, finding strategies to sensitize cells to ATRA may develop ATRA-based therapy in the treatment of non-APL AML patients. Methods Cell proliferation was assessed by cell growth. Cell death was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11b expression and morphology. To explore the underlying mechanisms, we studied the role of PKCβ, MEK, ERK, AKT, PU.1, C/EBPβ and C/EBPε by Western-blotting analysis. Results In this study, a clinically achievable concentration of enzastaurin enhanced ATRA-induced differentiation of AML cell lines, HL-60 and U937 as well as non-APL AML primary cells, while it also restored ATRA sensitivity in ATRA-resistant cell line, HL-60Res. Mechanistically, in all these cell lines, enzastaurin-ATRA (enz-ATRA) enhanced the protein levels of PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε. The activity of protein kinase C β (PKCβ) was suppressed by enz-ATRA treatment in HL-60 and HL-60Res cells. However, another PKCβ-selective inhibitor mimicked the cellular and molecular effects of enzastaurin only in HL-60 cells. Only in U937 cells, enz-ATRA activated MEK and ERK, and a MEK specific inhibitor suppressed enz-ATRA-triggered differentiation and reduced the protein levels of PU.1, C/EBPβ and C/EBPε. Enz-ATRA activated Akt in HL-60 and HL-60Res cells. However, an Akt inhibitor blocked enz-ATRA-triggered differentiation and restored the protein levels of PU.1, C/EBPβ and C/EBPε only in HL-60Res cells. Therefore, PKCβ inhibition, MEK/ERK and Akt activation are involved in enz-ATRA-induced differentiation in HL-60, U937 and HL-60Res cells, respectively by modulation of the protein levels of C/EBPβ, C/EBPε and PU.1. Conclusions Enzastaurin, at the clinically achievable concentration, enhances ATRA-induced differentiation of AML cells by PKCβ inhibition, MEK/ERK and Akt activation. This study may provide a potential therapeutic strategy for AML patients.