Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic-targeting approach in acute myeloid leukemia

Abstract

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1

AR-42 treatment inhibits HDAC activity in AML. (a) HDAC activity in Kasumi-1 and NB4 cells at 24 hours after treatment with vehicle, AR-42 or TSA. (b) Increased histone acetylation in Kasumi-1 and NB4 and cells 48 hours after AR-42 treatment. (c) HDAC activity in primary AML patient blasts 24 hours after treatment with AR-42 (n=2; patients no.1 and no.2). (d) Increased histone acetylation in primary patient blasts 48 hours after AR-42 treatment (patients no.1 and no.2 as indicated).

Figure 2

miR-29b expression increases following AR-42 treatment. (a) in Kasumi-1, NB4, and FDC-P1-KITmut cells, 24 hours after treatment with 1 μM AR-42. (b) in eight primary AML patient samples (patients nos. 3–10) at 24 hours after treatment with 1 μM AR-42 and 2400 μM VPA.

Figure 3

Expression of the miR-29b targets DNMT1, DNMT3A, DNMT3B and SP1 decreases following AR-42 treatment. (a,b) Kasumi-1, NB4, and FDC-P1-KITmut cells treated with 1 μM AR-42 for 24 hours DNMT1, DNMT3A, DNMT3B and SP1, decreases on both mRNA and protein level. (c,d) Primary patient blasts were treated with 1 μM AR-42 for 24 hours with decrease of DNMT1, DNMT3A, DNMT3B and SP1 on both mRNA (n=3) and protein level (patients as indicated).

Figure 4

AR-42 followed by decitabine has the strongest activity on cell viability. Kasumi-1, NB4, and FDC-P1-KITmut cells treated with vehicle, decitabine 0.5 μM for 72 hours, decitabine 0.5 μM for 72 hours with AR-42 0.3 μM added at 24 hours, AR-42 0.3 μM for 72 hours, or AR-42 0.3 μM for 72 hours with decitabine 0.5 μM added at 24 hours. Cells treated with AR-42 followed by decitabine showed lowest cell viability.

Figure 5

Priming with AR-42 upregulates miR-29b and increases survival in murine models. (a) miR-29b levels were upregulated 20-fold in AR-42 treatment versus vehicle treatment group. (b) Dnmt1, Dnmt3a, Dnmt3b, and Sp1 were downregulated in AR-42 versus vehicle treatment group. (c) Overall survival. FDC-P1-KITmut cells injected into NOD/SCID mice showed 10 of 17 mice with survival at 60 days in AR-42 followed by decitabine group compared with no survival in mouse groups with decitabine treatment alone, decitabine followed by AR-42, or AR-42 treatment alone.