Valproic Acid Induces Cell Cycle Arrest and Differentiation of t(15;17) Positive Leukemic Cells Independent from ATRA Sensitivity and Has No Effect on CD34pos Cells.

Abstract

The translocation t(15;17) in acute promyelocytic leukemia results in the PML/RARα fusion protein. PML/RARα recruits histone deacetylases (HDAC) and represses target genes of wild-type RARα. Recently, the anticonvulsant drug valproic acid (VPA) has been described as an HDAC inhibitor. In order to evaluate the role of VPA in the treatment of acute promyelocytic leukemia, the effect of VPA on the t(15;17) positive cell line NB4 was examined. To exclude toxicity on non malignant stem cells, CD34pos cells were analyzed. In NB4 cells, VPA led to an increase of acetylated histone H4. Histone acetylation was associated with a dose dependent inhibition of proliferation. Furthermore, inhibition of NB4 clonogenic growth was observed. In contrast, clonogenic growth of CD34pos cells was not affected by the presence of VPA. The VPA induced inhibition of cell growth in malignant cells was not caused by altered apoptosis. Rather an arrest at the G1/G0 phase was observed in the presence of VPA. In agreement with p21 expression of leukemic cell lines not harboring the t(15;17) translocation, VPA induced p21 mRNA expression also in t(15;17) positive cells. However, no induction of p21 was observed in normal CD34pos cells. VPA triggered the differentiation of NB4 cells measured by NBT test and surface expression of CD11b and CD11c. Flow cytometry of colonies from CD34pos cells showed an increased fraction of CD34pos cells in the presence of VPA. Thus, VPA does not induce differentiation in non malignant hematopoiesis. To study whether VPA and ATRA induced differentiation are mediated via identical pathways, the process of differentiation was studied in NB4 cells and ATRA resistant NB4-R2 cells. While differentiation in the presence of ATRA led to induction of C/EBPβ and C/EBPε, no change in the expression of these transcription factors was observed after VPA treatment. Downregulation of c-myc was detected upon ATRA as well as VPA treatment, an additive effect was seen after the combination. Resistance to ATRA did not interfere with effects of VPA on cell differentiation. However, upon ATRA treatment no c-myc downregulation was observed in the NB4-R2 cell line. Taken together, VPA acts as an HDAC inhibitor in t(15;17) positive cells and induces myeloid differentiation by mechanisms distinct from ATRA and independent of ATRA-resistance. Moreover, VPA does not interfere with normal hematopoiesis. Therefore, this substance might be helpful in patients with APL - especially in ATRA-resistant disease.