Abstract
The hydroxamic acid analogue (HA) class of histone deacetylase (HDAC) inhibitors (HDIs), e.g., SAHA, LAQ824 and LBH589, are active in inducing growth arrest and apoptosis of human acute and chronic leukemia cells. These agents have been shown to inhibit class I, IIA and IIB HDACs, induce histone H3 and H4 acetylation, increase p21 levels, as well as induce pro-death molecules Bax, Bak and Bim, while attenuating the levels of the antiapoptotic Bcl-xL, Bcl-2 and XIAP. Additionally, treatment with HA-HDIs have also been shown to induce acetylation of the heat shock protein (hsp) 90, thereby inhibiting its ATP binding and chaperone function, which directs the client proteins of hsp90 (e.g., AKT, c-Raf, FLT-3 and Bcr-Abl) to polyubiquitylation and proteasomal degradation. Collectively, these mechanisms explain the anti-leukemia activity of HA-HDIs. In the present studies, we cultured human AML HL-60 cells in the continuous presence of increasing levels of the cinnamic acid hydroxamate LAQ824, and isolated the HL-60/LR cells that are capable of growth in the continuous presence of 200 nM of LAQ824. HL-60/LR cells were 40 fold more resistant to the cell cycle growth inhibitory and apoptotic effects of LAQ824 than the parental control HL-60 cells, and showed variable degree of cross resistance to the other HA-HDIs, e.g., LBH589 (100 nM) and trichostatin A (250 nM), as well as to phenylbutyrate (3 mM). As compared to the control HL-60, HL-60/LR cells expressed markedly higher levels of Bcl-xL, XIAP, AKT and c-Raf, but lower levels of Bak and Bim. Exposure to 200 to 500 nM of LAQ824 for 24 hours induced histone acetylation in both HL-60 and HL-60/LR cells, suggesting that LAQ824 was able to inhibit HDAC activity in both cell types. However, as compared to HL-60, HL-60/LR cells expressed markedly lower HDAC6 but higher HDAC4 & 10 levels, while HDAC2 & 3 levels were similar in the two cell types. LAQ824-induced apoptosis was accompanied with the induction of p21, Bax and Bak and attenuation of Bcl-2, Bcl-xL and XIAP levels in the control HL-60 cells. This was not observed in HL-60/LR cells, which also demonstrated cross-resistance to apoptosis induced by Ara-C (0.5 to 2.0 μM for 48 hours) and TRAIL (100 to 500 ng/ml for 48 hours) but not to the topoisomerase II inhibitor etoposide (up to 2.0 μM). In contrast, HL-60/LR cells were collaterally more sensitive to the hsp90 inhibitor 17-allylamino-demothoxy geldanamycin (17-AAG) (Kosan Biosciences, Hayward, CA) (0.5 to 5.0 μM for 48 hours). These studies demonstrate that the in vitro resistance of HL-60/LR cells to HA-HDI LAQ824 is associated with perturbations in the levels of specific pro-survival and pro-death proteins. The cross resistance profile and the collateral sensitivity pattern of HL-60/LR cells points to 17-AAG and topoisomerase II inhibitor-based anti-leukemia combinations to override the de-novo or acquired resistance of AML cells to HA-HDIs.
Design of Dual Inhibitors of Histone Deacetylase 6 and Heat Shock Protein 90
