Decitabine and Vitamin D3 Differentially Increase C-Jun, PU.1, and Sp1 Hematopoietic Transcription Factors To Induce Monocytic Differentiation.

Abstract

Standard chemotherapy is not curative for many patients with acute myeloid leukemia (AML). New treatment strategies combining demethylating agents, such as decitabine, and drugs that induce myelomonocytic differentiation (i.e. Vitamin D3) may reestablish functional hematopoiesis in these patients.We studied the effects of decitabine alone or in combination with Vitamin D3 (VD3) on promonocytic U937 cells and Ficoll-enriched blast cells from patients with AML and analyzed their impact on cell growth and viability (assessed by trypan blue exclusion), surface marker expression (FACS analysis of CD11b, C14), and the expression and function of target molecules (Northern blot, Western blot, EMSA). Doses ranging from 0.1 to 1 uM of decitabine induced CD11b and CD14 surface expression 3- to 18-fold and 3- to 4-fold, respectively. 3 to 100nM of VD3 induced CD11b and CD14 expression 1.5- to 10-fold and CD14 1.5- to 8-fold, resp. Interestingly, decitabine synergized with VD3 to increase CD11b and CD14 surface marker expression up to 73-fold and 11-fold, resp., and to induce morphologic differentiation of U937 cells along the monocytic lineage. The mechanisms of decitabine- and VD3-induced monocytic differentiation are currently unclear. Therefore, we investigated the effects of the two drugs on several transcription factors that have been implicated in monocytic differentiation. Northern and Western blotting showed that decitabine induced transcription of c-jun but not PU.1, while VD3 increased PU.1, IRF8, and C/EBPbeta but not c-jun. Monocytic target promoters such as the CD11b promoter have been shown to be activated by a complex containing PU.1 and c-jun. Using electromobility shift assays, we now demonstrate increased DNA binding of nuclear proteins from decitabine- and VD3-induced U937 cells to the CD11b promoter. In addition to the effects seen with the CD11b promoter, we investigated whether the myeloid transcription factor Sp1 played a role in decitabine- and VD3-induced CD14 expression. Indeed, we found that mithramycin A, a specific inhibitor of Sp1, inhibited both VD3- and decitabine-induced upregulation of CD14, which is in line with previous data showing that Sp1 is critical for CD14 promoter activity. Induction of CD11b and/or CD14 by decitabine and/or VD3 was confirmed in primary AML patient samples at the time of diagnosis.In conclusion, decitabine synergizes with Vitamin D3 to induce CD11b and CD14 expression, likely by enhancing PU.1/c-jun and Sp1 transcriptional activity.