Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia

Abstract

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations, invariably involving the retinoic acid receptor alpha (RAR alpha) gene fused to one of several distinct loci, including the PML or PLZF genes, involved in t(15;17) or t(11;17), respectively. Patients with t(15;17) APL respond well to retinoic acid (RA) and other treatments, whereas those with t(11;17) APL do not. The PML-RAR alpha and PLZF-RAR alpha fusion oncoproteins function as aberrant transcriptional repressors, in part by recruiting nuclear receptor-transcriptional corepressors and histone deacetylases (HDACs). Transgenic mice harboring the RAR alpha fusion genes develop forms of leukemia that faithfully recapitulate both the clinical features and the response to RA observed in humans with the corresponding translocations. Here, we investigated the effects of HDAC inhibitors (HDACIs) in vitro and in these animal models. In cells from PLZF-RAR alpha/RAR alpha-PLZF transgenic mice and cells harboring t(15;17), HDACIs induced apoptosis and dramatic growth inhibition, effects that could be potentiated by RA. HDACIs also increased RA-induced differentiation. HDACIs, but not RA, induced accumulation of acetylated histones. Using microarray analysis, we identified genes induced by RA, HDACIs, or both together. In combination with RA, all HDACIs tested overcame the transcriptional repression exerted by the RAR alpha fusion oncoproteins. In vivo, HDACIs induced accumulation of acetylated histones in target organs. Strikingly, this combination of agents induced leukemia remission and prolonged survival, without apparent toxic side effects.

Figures

Figure 1

The effects of HDACIs alone and in combination with RA on NB4 cell growth, apoptosis, and differentiation. (a) Structures of the HDACIs utilized in this study. (b) NB4 cells cultured at an initial cell density of 105/ml in the presence of PB, TSA, or SAHA alone and in combination with RA (10–6 M). Growth curves were performed on NB4 cells cultured with 1 mM PB, 0.12 μM TSA, or 0.6 μM SAHA alone or in combination with RA. (c) 3H-Thymidine incorporation was assayed after 20 hours’ culture with PB, TSA, or SAHA at the indicated concentrations and in combination with RA (10–6 M) and a further 24 hours with 3H-Thymidine. Each point represents results from three independent experiments performed in triplicate for b and c (mean ± SD). (d) The percentage of apoptotic cells was measured after 48 hours’ culture by using flow cytometry analysis of Annexin V–labeled cells. Each point represents results from three independent experiments performed in duplicate (mean ± SD). (e) The NBT reduction assay was performed after 4 days of culture and measured as OD540nm/106 cells. Each point represents results from three independent experiments performed in duplicate (mean ± SD). The OD540nm value induced by various HDACIs with or without 10–6 M RA normalized to untreated control (untreated = 1).

Figure 2

Northern blot analysis of potential RA and SAHA target genes after culture of NB4 cells with RA (1 μM), SAHA (0.9 μM), and the combination of RA and SAHA for the indicated times. Total RNA (10 μg) was isolated and fractionated on 1.0% agarose/formaldehyde and transferred to a nylon membrane. The membrane was probed by using 32P-random labeled cDNA probes as indicated. As a loading control, the same membranes were also probed with a 50-mer oligonucleotide specific for 18S rRNA.

Figure 3

HDACIs reverse the transcriptional repression caused by PML-RARα and PLZF-RARα. Cos-1 cells were transfected with an 0.8 μg DR5-Luc reporter; 0.3 μg of Tk-β-galactosidase internal standard; and 0.4 μg of pSG5, pSG5-RARα, pSG5-PML-RARα, or pSG5-PLZF-RARα expression vectors and then incubated with various HDACIs in combination with 10–7 M RA. (–), 10–7 M RA alone. Luciferase activity was measured in a Luminometer (Turner Designs, Sunnyvale, California, USA) and normalized with the internal β-galactosidase standard. Values represent the mean ± SD from one of three independent transfections performed in triplicate.

Figure 4

Effects of SAHA alone or in combination with RA on leukemic cells from PLZF-RARα/RARα-PLZF double TM. BM cells collected from leukemic double TM were cultured at an initial cell density of 2 × 106/ml. Each sample from the leukemic transgenic mice was divided equally and incubated in parallel with or without SAHA (0.6 μM), RA (10–6 M), or the combination, as indicated. Results shown here are from one of three independent experiments performed in triplicate (mean ± SD). (a) SAHA alone causes growth inhibition and promotes RA-induced growth inhibition. 3H-Thymidine incorporation rate was measured after 44 hours’ culture as indicated above, with [3H]thymidine added for the final 24 hours. These cultures were maintained in the presence of 2% pokeweed mitogen–stimulated SCCM containing IL3 and GM-CSF). Results are expressed as percentage of inhibition upon treatment compared with untreated control. (b) SAHA alone causes apoptosis. TUNEL-positive cells were stained and scored after 48 hours of culture in the presence of SCCM and expressed as the induction of apoptosis upon treatment (untreated =1). (c) SAHA in combination with RA displays an additive effect on differentiation induction. NBT reduction assay was performed after 4 days of incubation as indicated and in the absence of SCCM. Results are expressed as the fold increase in OD540/106 cells (untreated =1).

Figure 5

In vivo effects of SAHA, RA, and the combination of both on PLZF-RARα/RARα-PLZF double leukemic TM. (a) Western blot analysis of acetylated histone H4 in murine cells upon in vivo administration of SAHA. Wild-type and leukemic TM were given SAHA (20 μg/gbw) by intraperitoneal injection. Histones were acid extracted from murine peripheral blood, BM, and spleen cells in untreated mice and 2 hours after SAHA administration. (b) SAHA in combination with RA treatment prolongs survival in PLZF-RARα/RARα-PLZF double TM with leukemia. Upon presentation of leukemia as monitored by automatic and differentiated counts on peripheral blood samples from PLZF-RARα/RARα-PLZF double TM, RA was administered daily at a dose of 1.5 μg/gbw for 2 weeks. The RA-treated mice were then randomly assigned into three groups: RA alone (1.5 μg/gbw), SAHA (50 μg/gbw) alone, and SAHA (50 μg/gbw) in combination with RA (1.5 μg/gbw). The treatment was continued for 4 weeks. During and after the treatment, the mice were bled weekly, and automatic and morphological differential counts were performed on each sample to evaluate response to treatment until each animal died. Kaplan Meier analysis was used to compare the cumulative survival period between SAHA in combination with RA (n = 11) and RA (n = 9) or SAHA (n = 6) alone. The black bar on the abscissa represents the 28-day period of treatment. Survival time reflects the days from the initiation of therapies until death for each mouse. (c) Complete remission was induced by SAHA + RA treatment in six of 11 leukemic PLZF-RARα/RARα-PLZF double transgenic mice. Analyses were performed weekly as described above. SAHA + RA causes the same duration of disease-free survival (time in remission) in PLZF-RARα/RARα-PLZF leukemic mice as that achieved in PML-RARα transgenic leukemic mice treated with RA (1.5 μg/gbw).