Histone deacetylase inhibitors: potential targets responsible for their anti-cancer effect

HDACs are classified by their homology to yeast HDACs. Eighteen are known, of which the 11 zinc-dependent enzymes belonging to class I, II, and IV constitute the focus of research, and of this review. HDACs, usually in conjunction with other corepressors, deacetylate lysine moieties in amino-termini of histones [6]. The acetylation status of the histone depends on the balance between deacetylase activity and histone acetyl transferase (HAT) activity. Deacetylation results in a relatively closed chromatin conformation that often leads to repressed transcription [6]. Thus, HDAC inhibitors are generally considered to be transcriptional activators [7]. However, gene expression profiling shows that as many genes may be repressed as de-repressed after exposure to an HDACi. This is likely to be a consequence of the direct and indirect effects of these drugs on other transcriptional regulators and cell signaling pathways and/or due to the dynamic and complex interrelations between chromatin remodeling and regulated gene transcription [8, 9].

HDACi are currently classified according to their chemical structure, and each agent varies in its ability to inhibit individual HDACs (Table 1). HDACi share a common pharmacophore containing a cap, connecting unit, linker and a zinc binding group that chelates the cation in the catalytic domain of the target HDAC [10]. The pan-deacetylase inhibitors include vorinostat (suberoylanlide hydroxamic acid, SAHA), panobinostat (LBH589) and trichostatin A which inhibit class I, II and IV HDACs, while valproate, entinostat (MS-275) and romidepsin (depsipeptide, FK228) are considered class-I—specific, and tubacin, HDAC6-specific. (Table 1)

HDAC6 warrants special attention as a HDAC predominantly, [11, 12] but not exclusively [9] localized to the cytoplasm. HDAC6-specific effects, particularly those on cell motility and the proteasome and aggresome pathways (discussed below) are considered by some investigators to be responsible for much of the cytotoxicity of the HDACi. This is one example of how HDACi vary in their targets—the pan-HDACi include HDAC6 amongst their targets, while the class1-selective HDACi (such as romidepsin) do not. Such differences provide the rationale for the development of novel, highly HDAC-specific agents. For now, it is easiest to group the HDACi in commercial development into the pan-HDACi versus those that are class 1-specific, and it is probably not unreasonable to make generalizations about HDACi targets on that basis.

HDACi can induce high rates of apoptosis at sub-micromolar concentrations in many cell-line models of hematological malignancy. Precisely which of the effects discussed below is most important remains a matter of conjecture and may well be cell-type and agent-specific. The two major apoptotic pathways are the death receptor (direct) and mitochondrial (indirect) pathways. HDACi have been shown to induce apoptosis through effects on mediators within either pathway or by inducing other signals within other cellular pathways that activate apoptosis.

Another potential trigger of HDACi-induced cell death arises through the potential effect of these agents on the misfolded protein response (MPR). The MPR is comprised of a number of cellular processes which protect the cell from toxicity arising from the accumulation of misfolded proteins. Misfolded proteins may arise as a consequence of defective protein synthesis, or due to other cellular derangements that result in a change in conformation of pre-formed protein [58]. Folding of proteins occurs in the endoplasmic reticulum (ER) and is reliant on the chaperone function of HSP90 [59, 60]. In this way, HSP90 prevents degradation of client proteins.

The ER responds to increased transcriptional activity in the cell by activation of the ER stress response. Through signaling from the ER three responses to increased ER stress can be initiated: 1. Decreased protein transcription, 2. Increased transcription of genes of the ER to increased long term processing capacity, or 3. apoptosis [60]. Apoptosis may be initiated by a number of trans-membrane receptors in the ER that then activate the intrinsic apoptotic pathway via c-Jun terminal kinase (JNK) [61]. Misfolded proteins may also be targeted for destruction through the proteasome. Targeting to the proteasome occurs through a number of protein modifications most importantly, ubiquitinylation.

Aggregates of misfolded protein are relatively resistant to destruction by the proteasome, and form in the context of proteasome inhibition, insufficiency or dysfunction [62]. Misfolded proteins accumulate focally in into an aggresome via a microtubule—an HDAC-6-dependent mechanism. The aggresome is then targeted for destruction by the autophagosome [12, 62‐64]. Overall, the aggresome pathway is a homeostatic and cytoprotective mechanism which may rescue the cell in the context of proteosomal overload, inhibition or dysfunction. The ubiquitin-proteasome-aggresome pathways are thought to be particularly relevant targets for anti-cancer therapy of myeloma, where production of immunoglobulin requires a properly functioning endoplasmic reticulum and proteasome.

HDAC inhibitors affect functioning of the proteasome / aggresome pathways in three key ways (which make their combination with proteasome inhibitors particularly appealing) [65]. Firstly, inhibition HDAC6 results in hyperacetylation of HSP90 and HSP70 [66] which subsequently promotes misfolding and depletion of client proteins, including c-RAF, AKT and CDK4 and induces ER stress [67‐70]. Recent evidence suggests that in a model of mantle cell lymphoma, induction of the ER stress-response gene CHOP is critical to panobinostat-induced cytotoxcity [68]. Secondly, HDAC hyperacetylation of tubulin leads to defective function of the dynein motor complex required for aggresome formation [7, 62, 63, 65, 68]. Inability to compensate for additional ER stress through a functioning aggresome pathway primes the cell for, and potentially initiates, apoptosis. Finally, a loss of function screen, discovered that proteasome deregulation through a pathway involving HR23B (RAD23B) was in part responsible for HDACi-induced apoptosis [72]. HR23B possesses ubiquitin-like domains and shuttles proteins to the proteasome. It also has a role in nucleotide excision repair, which was not shown to be critical in the effects of the HDACi. In this study, CTCL cells possessing higher levels of HR23B were more sensitive to HDACi induced death, and HDACi were shown to decrease proteasome function in treated cells in an indirect manner. Experimental depletion of the HR23B restored proteasomal function and reduced HDACi sensitivity. These observations were expanded in a subsequent study [73] in which an association between reduced HR23B expression in CTCL tissue biopsies and clinical response was observed. The authors concluded that HR23B expression may prove to be a useful biomarker to predict responsiveness to HDACi.

The evidence pointing to aggresome and proteasome dysfunction after HDACi therapy and the importance of HDAC6 in the maintenance of ubiquitin-proteasome-aggresome function [74] has been the basis for combinations of HDACi with proteasome inhibitors [71, 75]. However HDAC6 inhibition and tubulin acetylation may not be required, either for HDACi efficacy as a mono therapy or for synergy from the combination of HDACi with the proteasome inhibitors [76]. Buglio et al, hypothesized that the HDAC-selective mocetinostat (which has no effect on HDAC6) would make a more attractive agent for combination with bortezomib due to a perceived lower risk of thrombocytopenia compared to the pan-HDACi. Their preclinical experiments showed that the combination of mocetinostat and bortezomib was synergistic in Hodgkin lymphoma cell lines through reduction of the NfKb levels typically associated with HDAC inhibition. This synergy was HDAC6-independent, and brings into question the necessity for HDAC6 inhibition for combination therapies with the proteasome inhibitors. In a recent clinical study, the combination of bortezomib with the HDAC 1 and 2-specific romidepsin was able to rescue patients with bortezomib-refractory myeloma, adding weight to the concept that inhibition of HDAC6 is not required for this type of drug combination to be of benefit to patients [77]. Indeed, although not compared directly in a trial, romidepsin appears to be at least as effective as the pan-HDACi, vorinostat for cutaneous T-cell lymphoma, the only indication for which HDACi have earned FDA approval [78].

p53 is one of the most commonly altered transcription factors in cancer, and is found to be inactivated or mutated in various acute leukemias, CLL, myeloma and lymphoma. It is a promiscuous transcription factor with interactions with many key cellular pathways, including, but not limited to, those of Rb-E2F, MAP-kinase, IGF-1/AKT, Wnt-beta-catenin and cyclin-CDK via p21 [79]. Wild-type p53 is activated and accumulates in the nucleus in response to stress signals such as DNA damage, hypoxia, spindle damage and heat shock, amongst others. This response is modified by kinases (ATM, AT, CHK2, CHK1), acetyltransferases (CBP/ p300, pCAF, TRAF), PML, SUMO-1 and HMG1 and also deacetylases including the HDAC1/mSin3 complex [79‐85]. Ubiquitin-mediated proteasomal degradation contributes to the control of p53 levels. The overall effect of p53 activation is cell cycle arrest (predominantly by activation of p21^Waf1/Cip1) and apoptosis (through increased expression of pro-apoptotic genes of the intrinsic pathway).

Many of the pathways discussed in this review influence p53, and thus the HDACi have several means of modulating p53. The importance of the acetylation status of p53 (and thus the role of direct HDAC-p53 interactions) is controversial [86], however there is evidence that acetylation of p53 is enhanced in the setting of cellular stress [81], is required to interrupt Mdm-2 mediated repression of p53, [84, 87] increases the affinity of p53 for DNA [88], reduces ubiquitin-mediated degradation of the transcription factor [89], and can enhance expression of p21^Waf1/Cip1 [89]. A number of studies demonstrate activation of p53 after HDAC inhibition [89, 90]. However in most reports the apoptosis and p21 induction following HDAC inhibition can be induced in a p53 independent manner—an observation that may be clinically relevant for the treatment of tumors harboring mutated p53 [32, 48, 91‐93].

It is postulated that the HDACi-mediated effects on the cell cycle may be a key reason for the differential toxicity and responses between normal and transformed cells. Cell cycle arrest at G1 associated with induction of CDKN1A/p21^WAF1/CIP1 is a key response to almost all of the currently available HDACi [1, 7, 94, 95]. Down-regulation of CCND1/cyclin D may also contribute [96, 97]. However, induction of cell cycle arrest may protect cells against cytotoxic agents that require cell cycling for efficacy. Cell cycle arrest may also partly explain the tumor selectivity of HDACi [98, 99]. HDACi can also induce cell cycle arrest at G2/M. Tumor cells lacking a functional G2 checkpoint and that proceed into mitosis after HDACi therapy, undergo apoptosis. By contrast, normal cells (with an intact G2 checkpoint) are able to maintain arrest G2/M following withdrawal of HDACi treatment [98, 99]. This difference may go some way towards explaining the tumor selectivity of the HDACi.

Hematological malignancies are frequently associated with altered cytokine dependency, with perturbation of cytokine expression, receptor abnormalities, or with dysfunction of the post-receptor signaling cascades. Generally, binding of a cytokine to its receptor results in receptor dimerization. The cytoplasmic domains of cytokine receptors bind JAKs (Janus kinases) which phosphorylate the receptor and activate each other. In turn, the signal transducer and activators of transcription (STATs) are activated, also by phosphorylation, and STAT dimers enter the nucleus to initiate transcription at specific promoter regions. Chromatin remodeling is required for maximal transcriptional effect, and this is achieved through recruitment of HATs, [100, 101] as well as HDACs [102‐105].

Activation of the STAT3 signaling pathway is associated with multiple cellular effects including increased proliferation and cell survival (through induction of pro-survival Bcl-2 family members), induction of angiogenesis, inhibition of p53, and with activation of Rel/NFκβ [106]. STAT3 hyper activation is described in multiple myeloma (where IL-6 dependence is of particular significance), [107‐109] Hodgkin lymphoma, [110‐114] c-myc dependent lymphoma, [115] diffuse large B cell lymphoma, [116] and the T-cell lymphomas [42, 106]. Mycosis fungoides and Sézary syndrome are associated with constitutive activation of STAT3 and probably induced over activity of STAT5 [100, 117, 118]. STAT5 hyper activation is also described in Hodgkin lymphoma, [119, 120] as is IL-4/STAT6 activation (with production of the cytokine thymus and activation-regulated chemokine- TARC) [121‐124]. For a review of the role STATs in cancer, the reader is referred to a review by Yu and Jove [125]. The aberrancy of STAT activation in many hematological malignancies makes the STATs a rational target for anticancer agents.

The STATs are among the non-histone proteins hyper acetylated in response to HDACi. In addition to phosphorylation, STAT1 activity is partially regulated by CBP-induced acetylation or HDAC1-influenced deacetylation [101, 105, 126]. Acetylated STAT1 binds to the RelA subunit of NFκβ and prevents its nuclear translocation and anti-apoptotic effects [127]. Furthermore, STAT1- and STAT2 - mediated transcription of genes is reduced after HDAC inhibition [86, 102, 103, 128, 129]. HDAC inhibition prevents the transcription of the targets of STAT5, by preventing the recruitment of SMRT, rather than by alterations of histone acetylation [104].

In a murine xenograft model of CTCL, panobinostat reduced levels of (activated) pSTAT3 and pSTAT5 in biopsies, but not the overall quantity of either STAT protein [49]. An early study suggested that STAT6 expression was reduced after treatment with vorinostat in skin lymphoma biopsies without changes to expression to STAT3 [130]. Subsequently, an important study by Fantin and colleagues demonstrated that clinical response to vorinostat was associated with a change in localization of pSTAT3 from predominantly nuclear to predominantly cytoplasmic, presumably reflecting functional inactivation of pSTAT3 by vorinostat in these responding patients. A lack of in vitro and clinical response of CTCL to vorinostat appears to be associated with persistent accumulation of nuclear pSTAT3 [42, 118].

In Hodgkin lymphoma, mocetinostat downregulates the expression of STAT6 and its target cytokines TARC and IL-5, with paradoxical increases in IL-13 [121]. It is postulated that altered cytokine profile results in a shift to a T_H1-type cellular response to the Reed-Sternberg cell.

Together, these observations suggest that effects on the JAK/STAT pathways and altered cytokine signaling are putatively important therapeutic targets of the HDAC inhibitors that warrant further clarification. Indeed, STAT-dependency may explain why it is the hematological malignancies that show the most promising responses to these agents.

Levels of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hypoxia-induced factor 1-α (HIF1α) are increased in the in a number of hematological malignancies, especially in the bone marrow microenvironment [176]. Targeting tumor angiogenesis has proven to be a valuable strategy in the therapy of solid tumors using VEGF/EGFR inhibitors, as well as myeloma and myelodysplasia through the use of immunomodulatory agents (lenalidomide and thalidomide) and for the former, the proteasome inhibitors [176] Hypoxia-inducible factor 1-α (HIF1α) is considered a master regulator of the cell’s response to normoxic and hypoxic conditions. Levels of HIF1α are modulated through ubiquitination and proteasomal degradation, with complex interactions between p300/CBP, pVHL and HDACs 1,3, 4, 6, and 7 influencing this process (see review by Ellis et al.) [177]. Increased levels of these HDACs appear promote angiogenesis, conversely HDAC5 appears to be an inhibitor of angiogenesis [178].

HDACi have been shown to suppress angiogenesis in a number of cell types across a range of experimental conditions [177, 179‐186]. Down regulation of genes associated with angiogenesis (GUCY1A3, ANGPT1, COUP-TFII) has been documented in clinical CTCL samples after treatment with panobinostat [50] and TSP1 after treatment with vorinostat [118]. Similarly, reduction in VEGF, sVEGFR1, and bFGF were seen in samples from patients with myeloma who had been treated with panobinostat [187] and skin biopsies of cutaneous lymphoma from patients treated with vorinostat show reduced microvessel density [118].

Combination strategies with specific small-molecules inhibitors of angiogenesis are being explored in the solid tumor setting, and should be considered for haematalogical malignancies thought to depend on angiogenesis [181, 182, 188].

Given that tumor-regrowth occurs after clinical remission implies the presence of subpopulation of cancer cells that are relatively resistant to primary treatment [189]. Such resistance may be present in a subset of cells prior to treatment or may develop as a consequence of exposure to drugs, through a process of natural selection. The cancer stem cell hypothesis somewhat controversially proposes that within a cancer there is a phenotypically distinct subpopulation of cells responsible for the clonogenic potential of the tumor [190‐195]. The putative cancer stem cells are said to form the minority of overall cancer cell population, have the capacity for self-renewal, and importantly are potentially more resistant to anti-cancer agents. A host of mechanisms of resistance to a variety of anti-cancer treatments have been demonstrated in various putative cancer stem cell models, including hedgehog signaling in multiple myeloma [191, 194, 196], increased drug efflux, and changes in Notch and Wnt signaling in AML, CML and T-ALL. (See reviews by Lin et al. and references therein) [190, 193].

Sharma et al. recently demonstrated the ability to detect a subpopulation of PC9 lung cancer cells that were resistant to erlotonib, termed “drug-tolerant persisters” (DTPs) [197]. These DTPs all possessed the putative cancer stem cell marker CD133 that was present on only 2% of the original, untreated PC9 population. When grown in drug-free media, the cells re-acquired a drug-sensitive phenotype, this ‘elasticity’ implying an epigenetic mechanism of drug resistance. Supporting this was data from gene expression profiling of the two cell lines (drug-sensitive and drug-resistant) which was consistent with a global epigenetic modification. The authors identified that the retinoblastoma protein and HDAC-demethylating protein KDM5A was unregulated in the DTPs and found that histone H3 was consistently hypoacetylated in the DTPs. Trichostatin A was lethal to DTPs but not to the drug-sensitive cells, supporting the theory that the drug-resistance state was dependent on global chromatin changes and HDAC-dependence. Application of four different HDAC inhibitors to PC9 cells prior to exposure to erlotonib and a number of other anti-cancer drugs including cisplatin, prevented the development or expansion of DTPs without effect on the proliferation or survival of the PC9 cells. These observations offer the tantalizing possibility that HDACi can target the putative cancer stem cell or circumvent acquired drug resistance, and clearly offer a direction for further research.

The last decade of the research into HDACi has been one where dogma around their effects and targets are continually being challenged and refined. No longer can the HDACi be regarded as simple activators of transcription, or agents that achieve their activity predominantly through direct effects on the pathways of apoptosis. Apart from induction of cell death, these agents have complex effects on p53 and on cytokine signaling pathways, and must now be considered immune-modifiers as well as anti-angiogenic agents. Alteration of transcription is only one mechanism but non-histone targets are clearly critically important and we need more information on the effects on the host environment (Fig. 1).

One example of the challenges we face in developing these compounds is the developing story of HDAC6; tantalizing evidence that specific HDACs (such as HDAC6) make rational targets for drug development must be tempered by evidence that the targets of HDAC6 may not actually be necessary for clinical synergy with the drugs such as the proteasome inhibitors. Another challenge is to determine if HDACi can contribute to the therapy of AML with recurrent cytogenetic abnormalities.

The work now is to better dissect which on-target effects are most critical for HDACi efficacy, in which specific clinical situations, and how we may overcome the relatively modest single agent response rates using rational combination therapies [218, 219].

In vitro models do not replicate the tumor microenvironment or the immune milieu and therefore probably do not, alone, provide sufficient basis for clinical studies. A greater emphasis on immune-competent in vivo models and biomarker studies are essential to establish which targets are critical in patients, and which combinations will be the most promising.