By studying the drug sensitivity profiles of several HDAC inhibiors across panels of lung cancer, breast cancer and melanoma cells we have identified unique patterns of drug response specific for each inhibitor. Interestingly, the pan-inhibitor TSA showed greater specificity for cancer vs normal cells than the class 1 selective inhibitors depsipeptide and apicidin. TSA inhibited HDAC activity in purified systems in vitro, while depsipeptide's inhibition of HDACs at doses that blocked cancer cell viability required components of cell extracts. Consistent with this, we found that although both drugs increased global histone acetylation levels, only depsipeptide caused a concomitant decrease in global levels of the repressive histone 3 lysine 9 trimethylation mark, indicating that depsipeptide may indirectly affect other factors in addition to its HDAC inhibitory activity. Microarray gene expression analysis of an isogenic cell line pair, which showed identical sensitivity to TSA but discrepant sensitivity to depsipeptide, revealed that resistance to the cyclic peptide inhibitor may be mediated at least in part by chemotherapy-induced increases in multidrug resistance gene expression. Thus, in spite of targeting the same enzyme family, the various HDAC inhibitors studied here each demonstrated unique phenotypes that may impact the current clinical development of this drug class.
The divergent patterns of drug sensitivity of human cancer cells across HDAC inhibitors we observed allowed us to conclude that each inhibitor, including those that have selectivity for class 1 HDACs, must have activities beyond just HDAC inhibition, which contribute to overall drug effects such as cytotoxicity. In the case of depsipeptide, one such activity may be its ability to lower global levels of histone methylation as we observed at concentrations that blocked the viability of cancer cells. Consistent with this, Wu et al (2008)
have reported decreased H3K9 methylation at promoters of genes upregulated by depsipeptide. The global changes in H3K9 methylation we see in response to depsipeptide but not TSA suggest that depsipeptide may potentially affect the activity or level of histone methylases or demethylases directly, as was shown in H719 cells for G9a and SUV39H1 (Wu et al, 2008
). We did not observe any significant differences, however, in the basal levels of these methylases across cells with a range of depsipeptide sensitivities (data not shown).
In our analysis of drug sensitivity profiles, we found that cancer cell responsiveness to the HDAC inhibitors tested in this study was independent of the histological characteristics and the stage of the tumour from which the cell lines were derived. Likewise, we found no general correlation between drug sensitivity and the available mutational status of cells. Interestingly, however, one of the cell lines that showed resistance to TSA but was sensitive to depsipeptide, H1437, has been recently found to harbour a mutation in HDAC9 (Forbes et al, 2010
), a class 2 HDAC. Whether this may be a feature that contributes to TSA resistance generally, remains open to investigation. It is tempting to speculate that molecular determinant of responsiveness may be uncovered by a more thorough analysis of the molecular features of the cell line panels used here, analogous to the discovery of the correlation between EGFR mutations and hypersensitivity to tyrosine kinase inhibitors (Lynch et al, 2004
Surprisingly, we did see a large shift in responsiveness to depsipeptide, but not other HDAC inhibitors, in two cell lines, one derived from a lymph node lesion before chemotherapy treatment, the other derived from the primary tumour after exposure to etoposide and cisplatin. Comparison of this isogenic pair by microarray gene expression profiling indicated that higher levels of enzymes in the glutathione pathway corresponded to resistance to depsipeptide seen in the line derived after chemotherapy. Xiao et al (2003)
reported the presence of various glutathione-depsipeptide conjugates in serum, and proposed that these may represent metabolites with altered activity. In addition, depsipeptide's disulphide bond is subject to reduction and this appears to require cytosolic activities and to increase the potency of depsipeptide and its analogues (Xiao et al, 2003
; Crabb et al, 2008
), yet decreasing the active drug's stability. The glutathione pathway enzymes upregulated in H2073 may potentially affect this reduction and/or increase depsipeptide conjugation to reduced glutathione (through the action of GSTP1, GSTA4 and GSTM3, for example), keeping the cyclic peptide in a less stable form. The connection between drug potency and the status of the disulphide bond within depsipeptide may also help explain the lack of inhibition of HDACs in purified systems at concentrations that had clear biological effects in cells and lysates. Whether sensitivity to depsipeptide may be compromised by prior exposure to chemotherapy in clinical settings remains an open possibility, supported by our data, which must be taken into consideration for future trials.
Despite the greater general potency of depsipeptide compared with TSA or apicidin, the specificity profile of this drug was inferior to that of TSA. Thus, although less selective as an HDAC inhibitor, TSA was the most specific anticancer agent, preferentially targeting cancer vs
normal cells. A TSA analogue, SAHA/vorinostat, has been approved for clinical use by the FDA against cutaneous T-cell lymphoma (Duvic and Vu, 2007
) with a toxicology and side effect profile that meets standards. To date, therapeutic use of depsipeptide or apicidin analogues or other HDAC inhibitors has not been approved, although clinical trials are ongoing (Sandor et al, 2002
; Ryan et al, 2005
; Arce et al, 2006
; Candelaria et al, 2007
; Prince et al, 2009
; Rocca et al, 2009
). Our study suggests that at least in the preclinical setting, TSA-related compounds may offer the better blend of effectiveness and specificity for lung and breast cancer and melanomas. This may potentially also hold in the setting of combination therapy. A recent study by Frenkel, Gazdar and colleagues concluded that depsipeptide co-administered with EGFR inhibitors gave a substantial advantage over EGFR inhibitors alone in xenograft models of lung cancer (Zhang et al, 2009
). Parallel studies comparing several HDAC inhibitors in combination with standard chemotherapies or targeted therapies will be needed to identify the most effective and safe combinations. Since vorinostat already has approval status, this would be a rational first choice of HDAC inhibitor, as several cancer centres with currently open trials have already realised.