Histone deacetylases and their inhibitors in cancer ... - Nature

REVIEWS

Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders Katrina J. Falkenberg1 and Ricky W. Johnstone1,2

Abstract | Epigenetic aberrations, which are recognized as key drivers of several human diseases, are often caused by genetic defects that result in functional deregulation of epigenetic proteins, their altered expression and/or their atypical recruitment to certain gene promoters. Importantly, epigenetic changes are reversible, and epigenetic enzymes and regulatory proteins can be targeted using small molecules. This Review discusses the role of altered expression and/or function of one class of epigenetic regulators — histone deacetylases (HDACs) — and their role in cancer, neurological diseases and immune disorders. We highlight the development of small-molecule HDAC inhibitors and their use in the laboratory, in preclinical models and in the clinic. Histone deacetylases (HDACs). A family of 18 proteins in humans, consisting of class I proteins (HDAC1, HDAC2, HDAC3 and HDAC8), class IIa proteins (HDAC4, HDAC5, HDAC7 and HDAC9), class IIb proteins (HDAC6 and HDAC10), class III proteins (sirtuins 1–7) and class IV proteins (HDAC11). These enzymes remove acetyl groups from lysine on histones and other proteins.

Epigenetic modifications Reversible, heritable genetic changes that occur without changes in DNA sequence. Cancer Therapeutics Program, The Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne 3002, Victoria, Australia. 2 Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville 3052, Victoria, Australia. Correspondence to R.W.J. e-mail: ricky.johnstone@ petermac.org doi:10.1038/nrd4360 Published online 18 August 2014 1

Many links between genomic and genetic variations and human diseases have been identified and have provided a greater understanding of disease aetiology. Moreover, this information is now being used to derive new thera peutic regimens that are underpinned by a strong molec ular rationale, and has accelerated the development of ‘personalized medicines’ for the treatment of cancer, cardiac disease, neurological disorders, infections and inflammatory diseases. We also now recognize that epigenetic aberrations — which are often directly caused by genetic defects resulting in the loss or gain of function of epigenetic regulatory proteins — contribute substan tially to the onset and progression of human diseases1. In this article, we focus on cancer and other human diseases that are linked to abnormal expression and/or function of histone deacetylases (HDACs), the develop ment of HDAC inhibitors and our understanding of the mechanisms of action and the emerging clinical role of these agents. Epigenetic modifications lead to chromatin remodelling, altered gene expression and changes in the cellular pheno type. The most intensely studied epigenetic modification is DNA methylation2; however, the most diverse modifi cations are those that occur on histone proteins. Histone amino‑terminal regions can undergo acetylation, methyl ation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deimination, proline isomerization, crotonylation, propionylation, butyrylation, formyla tion, hydroxylation and O-GlcNAcylation3,4. In addition to DNA and histone modification, chromatin structure

and function are regulated by chromatin remodelling complexes (for example, SWI/SNF and NuRD (nucleo some remodelling and deacetylase) families), non-coding RNAs (for example, HOX transcript antisense RNA (HOTAIR) and HOXA distal transcript antisense RNA (HOTTIP)) and mutations in histone proteins themselves (reviewed in REF. 5). Epigenetic regulators can be divided into distinct groups based on broad functions: epigenetic writers lay down epigenetic marks on DNA or histones; these marks are removed by epigenetic erasers and recognized by epigenetic readers (FIG. 1). The enzymatic nature of epige netic writers and erasers has facilitated the generation of pharmacological inhibitors for many of these enzymes, some of which are clinically approved5. Inhibitors of epi genetic readers are also in development 6. These inhibi tors show potent anticancer efficacy as well as activity in a range of other disease states. The most successful and long-standing inhibitors of epigenetic processes are the US Food and Drug Administration (FDA)-approved DNA-demethylating agents azacitidine (also known as 5‑azacytidine (Vidaza; Celgene)) and decitabine (also known as 5‑aza‑2ʹ‑deoxycytidine (Dacogen; Eisai)), which are used to treat myelodysplastic syndromes (MDS) and a range of other malignancies7,8. Other inhib itors of epigenetic writers, such as the histone methyl transferases DOT1L9,10 and EZH2 (REFS 11,12), have exciting potential for cancer treatment, whereas protein arginine methyltransferase (PRMT) inhibitors show promise for cancer and immune-mediated diseases13,14.

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Epigenetic eraser Writers e.g., HATs, HMTs or PRMTs

Erasers e.g., HDACs and KDMs • Transcriptional activation or repression • Changes in DNA replication • Changes in DNA damage repair Epigenetic reader

Epigenetic writer P P

Me

Me

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Ac Readers e.g., bromodomains, chromodomains and Tudor domains

Figure 1 | Epigenetic writers, readers and erasers. Epigenetic regulation is a dynamic process. Epigenetic writers such as histone acetyltransferases (HATs), histone Nature Reviews | Drug Discovery methyltransferases (HMTs), protein arginine methyltransferases (PRMTs) and kinases lay down epigenetic marks on amino acid residues on histone tails. Epigenetic readers such as proteins containing bromodomains, chromodomains and Tudor domains bind to these epigenetic marks. Epigenetic erasers such as histone deacetylases (HDACs), lysine demethylases (KDMs) and phosphatases catalyse the removal of epigenetic marks. Addition and removal of these post-translational modifications of histone tails leads to the addition and/or removal of other marks in a highly complicated histone code. Together, histone modifications regulate various DNA-dependent processes, including transcription, DNA replication and DNA repair.

Chromatin remodelling An alteration in chromatin structure that affects the nuclease sensitivity of a region of chromatin. Accomplished by covalent modification of histones and/or the action of ATP-dependent remodelling complexes.

Epigenetic writers Enzymes such as histone acetyltransferases, methylases and kinases, which covalently modify the amino-terminal ‘tails’ of histone proteins, and DNA methyltransferases, which modify the DNA itself.

Targeting the epigenetic erasers, the HDAC inhibitor vorinostat (Zolinza; Merck) is approved by the FDA for the treatment of cutaneous T cell lymphoma (CTCL)15, romidepsin (Istodax; Celgene) is approved for the treat ment of CTCL and peripheral T cell lymphoma (PTCL), and belinostat (Beleodaq; Spectrum Pharmaceuticals) has recently been approved for the treatment of PTCL. Furthermore, inhibitors of histone lysine demethylases (KDMs) may be effective for the treatment of cancer 16,17, β-globinopathies18 and neurological disorders19,20. In contrast to inhibiting the catalytic domains of epigenetic writers and erasers, inhibition of epigenetic readers involves disrupting protein–protein interactions. The landmark example of this is the small-molecule inhi bition of bromodomain-containing BET (bromodomain and extraterminal) proteins21,22. A particularly exciting effect of BET inhibition is the ability to downregulate the previously undruggable MYC oncogene, resulting in downregulation of its transcriptional targets23,24 and anti tumour effects in MYC-driven models of acute myeloid

leukaemia (AML)25,26, Burkitt’s lymphoma24 and multiple myeloma23. Bromodomain inhibition is also promising for the treatment of immune-mediated diseases by inhibiting the interaction of bromodomain-containing protein 2 (BRD2) and/or BRD4 with cyclin-dependent kinase 9 (CDK9) and decreasing the expression of inflammatory cytokines22,27. In this Review we discuss the role of HDACs in vari ous human diseases and the rationale for therapeutically targeting them, primarily in cancer; however, the potential therapeutic applications of HDAC inhibitors in neuro logical diseases, infection and inflammation are also dis cussed. As a prelude to a more extensive review of HDACs and their pharmacological inhibitors (detailed below), it is important to clarify three points. First, we believe that HDACs should be more simply and correctly called lysine deacetylases or ‘KDACs’. Acetylation is a very common post-translational modification and over 1,750 proteins can be acetylated at lysine residues in human cells28. Therefore, HDACs are responsible for the removal of acetyl groups on a vast array of nuclear and cytoplasmic proteins. In this Review we retain the term ‘HDAC’ for simplicity and focus heavily on the epigenetic effects of HDAC inhibitors. However, HDAC inhibitors are likely to exhibit their biological effects through hyperacetylation of a large number of cellular proteins. Second, the rather naive initial view that there is always a direct relationship between transcriptional activation and histone hyperacetylation at a given gene promoter has been superseded by more sophisticated models that describe a complex interplay of different epi genetic modifications to regulate gene transcription29–31. Indeed, as many genes are repressed following treatment with HDAC inhibitors as are activated32,33. Finally, it is well understood that HDACs function as the catalytic subunits of large protein complexes34,35, and the field is — rightly — starting to consider HDAC inhibition in the context of these large multiprotein complexes rather than the inhibition of single or multiple enzymes functioning as independent entities.

Role of HDACs in cancer onset and progression The original development of HDAC inhibitors as anti cancer agents occurred through empirical screens for agents that induced tumour cell differentiation, and the molecular targets (HDACs) of agents such as butyrate, trichostatin A (TSA) and vorinostat were discovered after the biological effects of the compounds were well described36,37. Given the potent antitumour effects of HDAC inhibitors, one would predict that HDACs them selves are oncogenic, and this is supported by correlative data showing that HDAC function and/or expression is perturbed in a variety of cancers and is often associated with poor prognosis (see REF. 15 for details). In addi tion to expression or genetic changes, HDACs can be aberrantly recruited to specific gene promoters by onco genic fusion proteins to drive leukaemogenesis (FIG. 2). For example, the AML1–ETO fusion protein found in patients with t(8;21) AML recruits HDAC1, HDAC2 (REF. 38) and/or HDAC3 (REF. 39) to repress AML1 target genes, thereby preventing myeloid differentiation and

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REVIEWS resulting in cellular transformation40. Furthermore, the fusion proteins PML–RARα (promyelocytic leukaemia– retinoic acid-related receptor-α) and PLZF–RARα recruit HDACs and DNA methyltransferases (DNMTs) to silence RARα target genes41. In the case of PML–RARα, retinoic acid restores RARα target gene expression and leads to reversion of the transformed phenotype42,43; however, leukaemias driven by PLZF–RARα respond poorly to retinoid treatment 44. Identification of the recruitment of HDAC repressor complexes to these chi meric receptors provided a strong rationale for the treat ment of patients with acute PML with HDAC inhibitors (TABLE 1). There is minimal definitive experimental evidence demonstrating that overexpression of HDACs is onco genic. Overexpression of HDAC1 in tumour cells can induce proliferation and de-differentiation45; however, there are no data showing that aberrant expression of HDACs can be a primary oncogenic effect. By contrast, knockdown of HDACs can induce a range of antitumour effects such as cell cycle arrest and inhibition of prolif eration, induction of apoptosis, differentiation and senescence, and disruption of angiogenesis15. This pro vides some indication that HDAC expression is required

Epigenetic erasers Enzymes such as histone deacetylases, phosphatases, deubiquitylases and demethylases that reverse covalent modifications within the amino-terminal ‘tails’ of histone proteins.

Epigenetic readers Proteins containing chromodomains, bromodomains, Tudor domains and DNA methyl-binding domains that recognize specific histone marks and recruit other chromatin modifiers and remodelling proteins to alter chromatin architecture and function.

/D

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Figure 2 | Leukaemia-associated fusion proteins recruit HDACs. Acute myeloid leukaemia 1 (AML1)–ETO (part a) and PML–RARα (promyelocytic leukaemia–retinoic Nature Reviews | Drug Discovery acid-related receptor-α) (part b) are oncogenic fusion proteins that recruit a range of proteins, including histone deacetylases (HDACs), to form multiprotein complexes to alter transcription and drive leukaemogenesis. Recruitment of HDACs to these fusion protein complexes provides a rationale for targeting HDACs in leukaemias driven by these fusion proteins. The ETO portion of AML1–ETO (part a) binds directly and indirectly to HDAC1, HDAC2 and HDAC3 (REF. 208). Nuclear receptor co-receptor complex (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) bind HDAC3, and SIN3A binds HDAC1 and HDAC2. However, it is unlikely that NCoR, SMRT and SIN3A are members of the same complex (depicted by a dotted line around SIN3A)208. AML1–ETO recruits other epigenetic modifying enzymes, including DNA methyltransferase 1 (DNMT1)209 and protein arginine methyltransferase 1 (PRMT1) 210 as well as core binding factor (CBF)211. PML–RARα (part b) dimerizes with retinoid X receptor (RXR) and recruits repressors including NCoR and SMRT, as well as HDAC1, HDAC2 and HDAC3 (REF. 212). DNMT1, DNMT3A and the histone methyltransferase EZH2 have also been shown to form a complex with PML–RARα42,213.

to ensure the survival and growth of a tumour cell; however, given that HDAC-knockout experiments have demonstrated an essential role for individual HDACs in normal cellular and tissue development (Supplementary information S1 (table)), it remains unclear whether the effect of HDAC knockdown is tumour-cell-selective. The importance of wild-type HDAC2 in tumorigenesis has been suggested using transgenic mice that express a catalytically inactive mutant form of HDAC2; these mice are unable to integrate into co‑repressor complexes with the paired amphipathic helix protein SIN3B46. When these mice were crossed with ApcMin mice, which are prone to develop gastrointestinal polyps and tumours owing to a mutation within adenomatous polyposis coli (Apc), reduced tumour rates were observed compared to mice with functional HDAC2 (REF. 46), indicating for the first time in vivo that HDAC2 may have a functionally important role in colorectal tumorigenesis. Whereas inhibiting class I HDACs has had demonstr able anticancer success in the clinic, emerging evidence using genetically engineered mice paradoxically points to tumour-suppressor roles for HDAC1, HDAC2 and HDAC3. Liver-specific knockout of Hdac3 resulted in deregulated DNA replication and a loss of genome stability, resulting in hepatocellular carcinoma47. More recently, three studies48–50 demonstrated that knockout or knockdown of HDAC1 and/or HDAC2 resulted in the development of haematological malignancies. Deletion of Hdac1 and Hdac2 in T cells caused neop lastic transformation of immature T cells concomitant with elevated levels of MYC and decreased tumour suppres sor p53 activity 48,49. Consistent with a putative genetic interaction between MYC and HDAC1 or HDAC2, our laboratory showed that knockdown of these HDACs in haematopoietic progenitor cells accelerated MYCdriven lymphomagenesis50. Interestingly, knockdown of HDAC1 and, to a lesser extent, HDAC2 also accel erated tumorigenesis driven by PML–RARα or loss of p53, which indicates a more general tumour-suppressor role for these HDACs. In all cases, deletion or depletion of HDAC1, HDAC2 or HDAC3 appeared to impair genomic stability, perhaps pointing to an under-appreciated role for HDACs in maintaining genomic integrity on a more global scale. These data raise the intriguing and dis turbing possibility that inhibition of HDAC1, HDAC2 or HDAC3 may, in certain circumstances, be tumourpromoting, with experimental evidence demonstrating accelerated tumour development following treatment of pre-leukaemic PML–RARα transgenic mice with valproic acid (VPA)50.

HDACs in neurological disease Aberrant HDAC expression and function leads to neuro pathology. The role of HDACs in regulating brain func tion, neurological development and deterioration has been examined using experimental models. For example, HDAC2 overexpression negatively regulates synaptic plas ticity, synaptic number and dendritic spine density, leading to learning and memory dysfunction51. Consistent with this, HDAC2‑deficient mice or HDAC2‑overexpressing mice treated with HDAC inhibitors had improved



REVIEWS Table 1 | Mechanisms of action of HDAC inhibitors Biological effect

Key effects of HDAC inhibitors

Other comments

Direct effects on tumour cells Cell death

• Induction of apoptosis through the intrinsic and extrinsic apoptosis pathways • Enhanced ROS production and decreased production of free radical scavengers132,205,229–231 • Accumulation of DNA damage through transcriptional downregulation or impaired function of DNA repair proteins (KU70, KU80, RAD50, MRE11, DNA‑PK, BRCA1, EXO1 and CHK2)232; impaired recruitment of DNA repair proteins to sites of DNA damage (RAD51 and BRCA1)233; ROS production234; and inhibition of DNA replication107,235 • Immunogenic cell death

• Intrinsic pathway requires BH3‑only proteins (for example, BIM, BID and BMF), and BCL‑2 protein overexpression inhibits apoptosis196,229,129 • Extrinsic pathway is activated by increased expression of death receptors (for example, DR4, DR5 and FAS) and their cognate ligands (TRAIL and FASL), and/or downregulation of intracellular regulatory molecules such as CFLIP130,131 • Pre-exposure to antioxidants protects from HDAC inhibitor-induced cell death236 • HDAC inhibitor treatment sensitizes cells to DNA-damaging agents237 • Apoptosis is important for therapeutic efficacy in vivo

Cell cycle arrest

• Induction of cell cycle arrest, often in combination with other effects, such as cell death and differentiation • We propose a hierarchy whereby cells that are resistant to apoptosis undergo cell cycle arrest, where G1/S phase‑induced arrest is dominant over G2/M phase-induced arrest143,238

• HDAC inhibitor-induced G1/S phase arrest occurs primarily through transcriptional changes in cell cycle regulatory genes, such as induction of the CDK inhibitors p21 (also known as WAF1 and CIP1; encoded by CDKN1A), p15INK4B (encoded by CDKN2B), p19INK4D (encoded by CDKN2D) and p57 (also known as KIP2; encoded by CDKN1C)143,239,240 • HDAC inhibitor-induced G2/M phase arrest is less well understood and may involve transcriptional changes and inactivation of G2/M complexes241

Senescence

Induction of features of senescence in treated cells is commensurate with tumour growth suppression242–245

HDAC inhibitor-induced senescenceassociated secretory phenotype (SASP) may have tumour-promoting effects246

Differentiation

Reversal of differentiation block in fusion-protein-driven AMLs (PML–RARα, AML1–ETO) and NMC (BRD4–NUT) tumours mediates antitumour effects149,150,247–249

Strong molecular rationale to combine HDAC inhibitors with ATRA in PML–RARα-driven AML, with excellent effects observed in preclinical studies and clinical trials250–252

Autophagy

• Induction of autophagy • Limited evidence that autophagy is important for the antitumour effects of HDAC inhibitors

Autophagy has been shown to be required for, to have no effect on and to even suppress HDAC inhibitor-induced apoptosis253–258

Tumour immunogenicity

• Enhanced immunogenicity • Enhanced antigen-presenting capacity

Increased expression of putative tumour antigens, MHC class I and II, co‑stimulatory molecules and NK cell-activating ligands148

Effects on non-tumour cells Regulation of distinct immune cell subsets

• Inhibition of dendritic cell differentiation and function77,259 • Cytotoxicity to macrophages, neutrophils and eosinophils77 • Induction of apoptosis in proliferating B cells92,196 • Increased tumour killing by NK cells and cytotoxic T cells260,261 • Increased differentiation and function of CD8+ T cells262,263 • In TReg cells, broad-spectrum HDAC inhibitors increase production and suppressive function; class I HDAC inhibitors suppress TReg cell numbers88,264–266 • Suppression of inflammatory cytokine production267,268

• Activated NK and T cells are resistant to the immunosuppressive action of HDAC inhibitors269, which suggests that immune-activating therapies followed by treatment with HDAC inhibitors may be a viable anticancer approach • HDAC inhibitors may engage the host adaptive immune response to enhance and prolong antitumour responses147 • In TReg cells, broad-spectrum HDAC inhibitors are used for inflammatory disease, whereas class I HDAC inhibitors are used for inhibition of tumour growth266 • In the context of bone marrow transplantation for leukemia treatment, HDAC inhibitors suppress inflammatory cytokines, thereby ameliorating GVHD while preserving the T cell-mediated anti-leukaemia effect270,271 • HDAC inhibitors are effective in ameliorating the severity of arthritis in preclinical models and can prevent bone destruction188,189

Inhibition of angiogenesis

Suppressed expression of pro-angiogenic genes143,272–274

Hypoxia induces the expression of HDAC1, HDAC2 and HDAC3 (REF. 275)



REVIEWS Table 1 (cont.) | Mechanisms of action of HDAC inhibitors Biological effect

Key effects of HDAC inhibitors

Other comments

Neuroprotection

• Neuroprotection against oxidative stress induced by glutathione depletion276, BAX-induced apoptosis277, HTT neurotoxicity through hyperacetylation and autophagic degradation67, glutamate-induced cytotoxicity278 and tauopathies through reduced phosphorylation and/or stabilization of tau64,65,179 • Induction of the expression of neurotrophins — molecules that are important for neuron survival and function173

• p21 is sufficient but not necessary for HDAC inhibitor-induced neuroprotection against oxidative stress, independently of cell cycle effects277 • HDAC inhibitors reduce the expression of genes that are implicated in Huntington’s disease, thus preventing the neurodegeneration associated with Huntington’s disease174 • HDAC inhibitors alter phosphorylation179 and/or reduce the stability of tau64,65, resulting in decreased clinical features of tauopathies

Reactivation of latent virus

Reduction in resting cell populations latently infected with HIV through induced expression of integrated provirus and suppression of inhibitory factors for HIV expression95,191,279

HDAC inhibitors do not increase the susceptibility of peripheral blood mononuclear cells to HIV infection279

AML, acute myeloid leukaemia; ATRA, all-trans retinoic acid; BAX, BCL‑2‑associated X protein; BCL‑2, B cell lymphoma 2; BID, BH3‑interacting domain death agonist; BIM, BCL‑2‑interacting mediator of cell death; BMF, BCL‑2‑modifying factor; BRCA1, breast cancer susceptibility 1; BRD4, bromodomain-containing protein 4; CDK, cyclin-dependent kinase; CFLIP, cellular FLICE-like inhibitory protein; CHK2, checkpoint kinase 2; DNA-PK, DNA-dependent protein kinase; DR4, death receptor 4; DR5, death receptor 5; EXO1, exonuclease 1; FASL, FAS ligand; GVHD, graft-versus-host disease; HDAC, histone deacetylase; HTT, huntingtin; MHC, major histocompatibility complex; MRE11, meiotic recombination 11; NK, natural killer; NMC, NUT midline carcinoma; NUT, nuclear protein in testis; PML, promyelocytic leukaemia; RARα, retinoic acid-related receptor-α; ROS, reactive oxygen species; TRAIL, TNF-related apoptosis-inducing ligand; TReg, regulatory T.

Tauopathies A class of neurodegenerative diseases associated with the pathological aggregation of tau protein in the human brain.

memory facilitation 51. Furthermore, HDAC2 has a putative pathogenic role in neurodegenerative diseases such as Alzheimer’s disease; its levels are elevated in patients with Alzheimer’s disease as well as in mouse models52. This has been linked to increased occupancy of HDAC2 at the promoters of target genes involved in cognitive function and neuroplasticity 52. The functional significance of HDAC2 was demonstrated in mouse models of neurodegeneration in which HDAC2 knock down restored learning and memory function52. As no effect on neuron survival was observed52, this study provides evidence to suggest that the neuroprotective effects of HDAC inhibitors may be due to reinstating gene expression profiles of existing cells rather than pre venting further neuronal cell loss. By contrast, mice with brain-specific knockout of both Hdac1 and Hdac2 (but not single-knockout mice) had severely disrupted corti cal, hippocampal and cerebellar organization, and neu ronal precursors from these mice underwent apoptosis rather than differentiation53. This may be explained by the fact that HDAC1 and HDAC2 appear to have impor tant roles in synaptic transmission in immature neurons, stabilizing synaptic networks during early development of the central nervous system54. Therefore, HDAC2 expression may be important for neuronal development but altered expression may contribute to pathogenesis in mature neurons. In addition to their well-defined roles in skeletal muscle development and function (Supplementary information S1 (table)), the class IIa HDACs (HDAC4, HDAC5 and HDAC9) have an important role in brain development and neurological functions involved in long-term memory and depression, as well as in neuro logical diseases such as Parkinson’s disease55. HDAC4

is likely to be the most important class IIa HDAC in neurobiology, as Hdac4‑knockout mice showed delayed development of the cerebellum and HDAC4 appears to have a role in protecting neurons from cell death56. Selective deletion of Hdac4 in the brain resulted in loss of learning and memory function57, and haploinsufficiency of HDAC4 in humans is associated with brachydactyly mental retardation syndrome58. Early studies suggest a role for HDAC6 in mood regulation, as Hdac6‑deficient mice and those treated with an HDAC6 inhibitor exhibited antidepressant behaviour and less anxiety 59. Aberrant protein acetylation and accumulation in neuropathology. Aberrant accumulation of insoluble hyperphosphorylated tau (p‑tau) is a hallmark of neuro degenerative tauopathies, including Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‑17)60. Acetylation of tau at Lys280 can promote p-tau aggregation and prevent p-tau from being degraded, leading to impaired cognitive func tion61. Elevated levels of acetylated tau were identified in murine models62 and in patients61,63 with Alzheimer’s disease and related tauopathies, specifically in diseased tissues, thus implicating tau acetylation in the pathogen esis of these diseases. The histone acetyltransferase p300 is involved in acetylation of tau, as small-molecule inhi bition of p300 resulted in decreased tau acetylation and expression of pathogenic p-tau61. Remarkably, inhibition of HDAC activity or depletion of HDAC6 can reduce p-tau levels, and in preclinical models it can alleviate the clinical features of tau-driven neurological disorders64,65. HDAC-mediated regulation of tau expression may occur through direct changes in acetylation at multiple lysine residues within the substrate protein65 and/or through



REVIEWS effects on tau-associated proteins such as α‑tubulin or heat shock protein 90 (HSP90)64. Clearly, there is a com plex interplay linking tau acetylation at different sites, its phosphorylation, its interaction with binding partners (such as HSP90) and its stabilization, and understanding this requires greater experimental insight. However, the evidence that HDACs can directly or indirectly regulate the activity of this key microtubule-associated protein and therefore affect pathogenesis has indicated that these epigenetic regulators may be viable therapeutic targets for a range of tauopathies. As indicated above, HDAC6 may have an important deacetylating function in neurobiology and neuropathol ogy owing to its key role in regulating cellular responses to protein aggregation66. The accumulation of misfolded proteins is a major pathological event in Parkinson’s dis ease, Alzheimer’s disease and Huntington’s disease. For example, Huntington’s disease is caused by the neuronal accumulation of the mutant protein Huntingtin (HTT), and acetylation of HTT at Lys444 targets it for autophagic degradation, which leads to improved clearance of the mutant protein and reverses neurotoxicity 67. The ability of HDAC6 to regulate aggresome formation and HSP90 function — both of which are essential cellular response mechanisms to the accumulation of misfolded proteins — places it at the nexus of neurological disease onset and progression. Although there is a rationale for link ing HDAC6 function to neuropathologies, experimental evidence demonstrating a general role for HDAC6 in neurodegenerative diseases remains to be fully defined. On one hand, depletion of HDAC6 in a mouse model of Alzheimer’s disease suppressed the destructive effects associated with amyloid-β‑mediated impairment of mito chondrial trafficking and enhanced cognitive function68,69. On the other hand, in a mouse model of Huntington’s disease, knockout of Hdac6 did not affect disease onset or progression70. To add to the complexity in defining a clear role of HDAC6 in neuropathologies, studies using a fruitfly model of spinobulbar muscular atrophy driven by an impaired ubiquitin–proteasome system (UPS) demonstrated that HDAC6 had a neuroprotective effect71. Gaucher’s disease is caused by mutations in the lysosomal enzyme β‑glucocerebrosidase (also known as glucosylceramidase) that result in accumulation of β‑glucocerebroside in affected tissues72. As these mis sense mutations lead to premature degradation of β‑glucocerebrosidase through HSP90 chaperone function and the UPS, it was hypothesized and later demonstrated that treatment with HDAC inhibitors may increase the stability of β‑glucocerebrosidase by inhibiting proteasome degradation73. This effect may be a direct consequence of inhibiting HDAC6, which is consistent with studies show ing that HDAC6 inhibition leads to hyperacetylation of HSP90 and inhibition of chaperone function74; however, this remains to be formally demonstrated.

HDACs in inflammation and infection HDACs can have a role in innate immunity through the regulation of Toll-like receptor (TLR) pathways and interferon (IFN) signalling75, and in the adaptive immune system through the regulation of antigen presentation

and B and T lymphocyte development 76 and function77. Class I HDACs seem to be centrally involved in innate immunity through the regulation of inflammatory cytokine production, whereas class IIa HDACs seem to be crucial for adaptive immunity, particularly in T cell function (FIG. 3). HDACs as regulators of innate immunity. Negative regulation of TLR signalling appears to be predomi nantly controlled by class I HDACs. Promoter activity of inflammatory genes (for example, interleukin‑12 subunit p40 (IL‑12p40), cyclooxygenase 2 (COX2) and IFNβ) is directly repressed by HDAC1, whereas HDAC2 represses pro-inflammatory gene expression by sequestering the transcriptional activator metastasis-associated protein MTA1 in a co‑repressor complex, which can be relieved upon lipopolysaccharide (LPS)-induced S‑nitrosylation of HDAC2 (REF. 75). Nuclear factor-κB (NF‑κB) signal ling is activated by TLR ligation and is negatively regu lated by HDAC1, HDAC2 and HDAC3 (REF. 75). HDAC1 interacts directly with the REL domain of the p65 subunit of NF‑κB, resulting in the recruitment of HDAC2 and the repression of inflammatory genes targeted by p65. HDAC3 dampens NF‑κB signalling through deacetyl ation of the p65 NF‑κB subunit, allowing its association with the inhibitory subunit NF‑κB inhibitor-α (IκBα) and subsequent nuclear export 75. The role of HDAC2 in the negative regulation of inflammation is supported by observations that HDAC2 expression was reduced in alveolar macrophages from patients with chronic obstructive pulmonary disease, and correlated with dis ease severity and resistance to corticosteroid therapy 75. In contrast to the repressive activity of class I HDACs, HDAC6 is a co‑activator for the transcription of IFNβ, and cells lacking HDAC6 exhibited increased virus replication upon infection with an experimental green fluorescent protein (GFP)-encoding vesicular stomatitis virus (VSV-GFP), which demonstrates an important role for this HDAC in innate immunity 75. Despite the common association of HDACs with transcriptional repression, class I HDACs are required for the expression of IFN-stimulated genes (ISGs) down stream of both type I IFN (IFNα and IFNβ) signalling — which is regulated by signal transducer and activator of transcription (STAT) — and type II IFN (IFNγ) signal ling 75. STAT5 recruits HDAC1, which in turn deacetyl ates CCAAT/enhancer-binding protein-β (CEBPβ), thereby activating the transcription of ISGs78. A number of groups have demonstrated that class I HDACs, in particular HDAC1, are important for STAT1- and/or STAT2‑dependent IFN signalling. HDACs either directly regulate the acetylation and subsequent activity state of STATs, or they affect STAT-mediated transcrip tion once STAT proteins are bound to promoters of ISGs79,80. Although at present there is no unified mecha nism to describe how HDACs regulate STAT function and downstream ISG expression, it is clear that innate immune responses against viral infections are impaired in the absence of class I HDAC expression or activity 81,82, and mice treated with HDAC inhibitors can be more susceptible to bacterial and fungal pathogens75.



REVIEWS IFN TLR

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TLR signalling HDAC1 and cytokine production

IFN signalling and cytokine production





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TLR signalling TReg cell HDAC7 and cytokine T cell development function production HDAC9

T cell function

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Negative regulation

T cell function Effector T cell

TReg cell function

LFA1

Positive regulation

Figure 3 | The role of HDAC isoforms in regulating immunity. Histone deacetylases (HDACs) have pleiotropic effects within the immune system. In general, class I HDACs (including HDAC1, HDAC2 and HDAC3) predominantly regulate innate immunity, whereas class IIa HDACs (including HDAC4, HDAC7 and HDAC9) predominantly regulate adaptive | Drug immunity, although overlap exists. Immune processes within the innate and adaptive Nature immuneReviews responses areDiscovery both positively (green boxes) and negatively (red boxes) regulated by different HDAC isoforms. In Toll-like receptor (TLR) and interferon (IFN) signalling, HDACs regulate the expression of inflammatory cytokines through deacetylating chromatin and upstream signalling molecules, thereby directly and indirectly regulating the transcription of TLR-stimulated genes (TSGs) and IFN-stimulated genes (ISGs). HDAC6 is important for the correct formation of the immune synapse through regulating microtubule acetylation. Conventional T cell development and function is regulated through the expression of cytokines, cytokine receptors and adhesion molecules. HDACs are also essential for regulatory T (TReg) cell homeostasis. IL-2, interleukin-2; IL-6, interleukin-6; LFA1, leukocyte function-associated molecule 1; MHC, major histocompatibility complex; TCR, T cell receptor.

Studies using selective small-molecule inhibitors of class IIa HDACs indicate that these HDACs may be important for the regulation of chemokines and other immunoregulatory molecules specifically in monocytes and not in lymphocytes83. The potential importance of class IIa HDACs in regulating innate immune responses derived from myeloid cells was further demonstrated by studies indicating that HDAC7 positively regulates TLRinducible pro-inflammatory gene expression in inflamma tory macrophages84. HDAC7 was recently shown to have a

more global cell-lineage regulatory role, in particular in determining the lymphocyte or myeloid cell fate devel opment decision through the control of a transcriptional programme that is important for macrophage function and differentiation85. Deregulated HDAC7 expression affected the expression of pro-inflammatory cytokines and immune responses mediated by macrophages85. Taken together, these studies indicate the putative role of class IIa HDACs, and in particular HDAC7, in regulating myeloid-specific innate immune responses.



REVIEWS

Regulatory T cell (TReg cell). A T cell type that suppresses the immune responses of other cells to maintain tolerance to self-antigens and abrogate autoimmune disease.

HDACs as regulators of adaptive immunity. Class IIa HDACs appear to have crucial roles in T cell develop ment and function. HDAC7 is important for normal T cell development in the thymus, and nuclear export of HDAC7 regulates the expression of cytokines, cytokine receptors and adhesion molecules that are required for cytotoxic T lymphocyte (CTL) func tion77,86. Interestingly, nuclear export of HDAC4 is also important for induced expression of IL‑5 in activated T cells, implicating the regulated shuttling of class IIa HDACs as a general mechanism for controlled expres sion of cytokine production75. HDAC9 is essential for maintaining regulatory T cell (TReg cell) homeostasis, as Hdac9−/− mice have elevated numbers of TReg cells87. The effect of HDAC9 on TReg cell development is probably through its interaction (together with HDAC7) with forkhead box P3 (FOXP3); interestingly, treatment with HDAC inhibitors that target class II HDACs results in elevated numbers and function of TReg cells88. Studies of Hdac6‑knockout mice demonstrated enhanced TReg cell function in the absence of increased TReg cell numbers89. Unlike HDAC9, which appeared to affect TReg cell activity through FOXP3, HDAC6 regulated TReg cell functions through HSP90 and heat shock factor protein 1 (HSF1). The observation that an HSP90 inhibitor phenocopied the effects of the HDAC6‑selective inhibitor tubacin provided experimental support for this model89. HDAC6 can also regulate T cell function through its role in the immune synapse that forms between T cells and antigenpresenting cells (APCs) and through its ability to regu late microtubule function. HDAC6 overexpression led to disordered CD3 and lymphocyte function-associated antigen 1 (LFA1) at the immune synapse as well as impaired antigen-specific microtubule organizing centre (MTOC) translocation and IL‑12 secretion90. In addi tion, HDAC6 is present at the leading edge in migrating T cells, and HDAC6 overexpression led to increased T cell migration and chemotaxis91. Although class I HDACs predominantly regulate innate immune responses, they also have a role in adap tive immunity. HDAC1 and HDAC2 appear to negatively regulate antigen presentation through inhibition of the MHC class II transactivator (CIITA), which is the master transcriptional regulator of major histocompatibility complex (MHC) class II genes75. In addition, HDAC1 and HDAC2 are essential for B cell proliferation during devel opment and following antigen stimulation of mature cells, but are dispensable for resting B cells92. In an in vivo model of allergic airway inflammation, HDAC1 depletion in T cells led to enhanced T helper 2 (TH2) cytokine pro duction from T cells together with increased inflamma tory cell infiltrates, mucus hypersecretion and enhanced airway resistance93, which indicates a suppressive role for HDAC1 in airway inflammation. The class IV HDAC, HDAC11, is important for antigen presentation as forced expression of this protein in macrophages increased anti gen presentation and the expression of CD40 and CD86 (two APC co‑stimulatory molecules), as well as IL‑12 (a T cell stimulatory cytokine). Conversely, disruption of HDAC11 in APCs led to upregulation of IL‑10 and impairment of antigen-specific T cell responses94.

HDACs as regulators of viral latency. Viral proteins aberrantly recruit HDACs to induce the latent viral phase, which is a major barrier to successful antiviral therapy. Latent HIV infection as a result of viral silencing can occur as a result of class I HDAC-mediated deacetyl ation of the HIV long terminal repeat (LTR) promoters, leading to heterochromatin formation95. Accordingly, inhibition of HDAC1, HDAC2 and HDAC3 can effi ciently induce ex vivo viral outgrowth from the resting CD4+ T cells of HIV-infected individuals95. These data provide a rationale for treating patients with latent HIV infection using class I-specific or broad-spectrum HDAC inhibitors and, as discussed in more detail below, clini cal trials are underway. However, a recent study using a new ex vivo assay questioned the effectiveness of HDAC inhibitors and other putative latency-reversing agents in eliminating the latent HIV reservoir when used as single agents in vivo96,97. These studies should provide a more accurate interpretation of clinical results using HDAC inhibitors.

HDAC inhibitors Several HDAC inhibitors have been synthesized or isolated as natural products, and these agents have varying target specificity, pharmacokinetic proper ties and activity in laboratory 98 and clinical settings15 (FIG. 4) (Supplementary information S2 (table)). The most commonly used HDAC inhibitors target multiple HDACs (Supplementary information S2 (table)), which makes it difficult to determine whether the biological consequences of HDAC inhibition (including clinical toxicities) are due to inhibition of a specific HDAC, the combined effect of inhibiting multiple HDACs and/or effects on one or more multiprotein complexes that incorporate specific HDACs as key enzymatic compo nents — for example, the co-repressor of RE1‑silencing transcription factor (CoREST) complex, the NuRD complex, the SIN3 complex and the nuclear receptor co-receptor (NCoR) complex34. The physical or enzymatic effect of one HDAC on the activity of another HDAC or HDACs that are a part of a larger, multiprotein complex is an underappreciated factor that requires further investigation. For example, the catalytic domain of HDAC4 interacts with HDAC3 within a larger NCoR–SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) complex, yet HDAC4 contains minimal intrinsic deacetylase activity. However, HDAC4 is essential for HDAC activity and for transcriptional repression of target genes by the NCoR–SMRT–HDAC3 complex 99. In addition, heterodimerization between HDAC4 and HDAC5 is necessary for calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent gene regulation through the physical association of HDAC4 with CaMKII100. The dependence of one HDAC on another for the enzymatic activity of a higher-order epigenetic com plex indicates that small-molecule HDAC inhibitors that putatively have target selectivity may in fact have broader effects than previously recognized. Indeed, a landmark study by Bantscheff and colleagues34, who profiled the inhibitory activity of different HDAC



REVIEWS Hydroxamates (generally target several classes of HDAC) Cap

Linker

Cap

Linker

O

H N

HN

O

O

H N

NHOH

NHOH

Vorinostat

Panobinostat

Benzamides (generally target class I HDACs) Cap

Linker

Cap

Linker

O N

O

N N H

NH2

H N

N

H N

N

O

Etinostat

N H

Mocetinostat

NH2

O

Thiols O O NH

H O S

S

NH

Romidepsin

O H

NH NH

Zinc-binding region

O

O

Figure 4 | Structure of HDAC inhibitors. In general, the pharmacophore of histone deacetylase (HDAC) inhibitors is composed of three regions: a ‘cap’ region or Nature Reviews | Drug Discovery ‘surface recognition domain’, which occludes the entrance of the active site pocket; a ‘zinc-binding group’, which chelates the zinc ion in the active site and is required for catalytic function; and a ‘linker’ region, which connects the two. Historically, most HDAC inhibitors have been hydroxamates; however, other zinc-binding groups are now being used to increase specificity and selectivity, including benzamide derivatives, thiols, sulphamides, ketones and trithiocarbonates (reviewed in REF. 214). Hydroxamate derivatives generally inhibit HDACs from multiple classes (for example, vorinostat and panobinostat), whereas benzamide derivatives are generally restricted to inhibiting class I HDACs (for example, etinostat, mocetinostat and the HDAC3‑specific inhibitors RG2833 and RGFP966). The cap region is predominantly responsible for selectivity, and as the cap region can not only bind to the HDAC itself but also to other complex components near the active site215, it could be used to design inhibitors that target HDACs residing in specific complexes. Finally, linker modifications may also serve to direct specificity, such as the addition of an aromatic ring that can fit into the enlarged catalytic pocket of HDAC7 that is not present in class I or class IIb HDACs216.

inhibitors using isolated HDAC enzymes and native HDAC-containing complexes, demonstrated distinctly different effects of various HDAC inhibitors depending on the physical state of the HDAC. For example, using purified HDACs as substrates, the aminobenzamides BML‑120 and tacedinaline showed modest preference for HDAC3 over HDAC1 and HDAC2. However, in the context of repressor complexes, these agents inhibited the HDAC3–NCoR complex at low micromolar concen trations but failed to inhibit the HDAC1– and HDAC2– SIN3 complexes at all doses tested. In addition, HDAC inhibitors demonstrated varying affinities for different

complexes containing the same HDACs. When testing the affinity of BML‑120, romidepsin, tacedinaline and VPA for different HDAC1- or HDAC2‑containing com plexes, an affinity hierarchy was seen (CoREST had the highest affinity, followed by NuRD, followed by SIN3). This study provides the strongest evidence to date that the activity of HDAC inhibitors is dependent on the macromolecular complexes in which HDACs reside, and we therefore advocate screening for HDAC inhibi tor specificity using HDAC-containing complexes rather than isolated enzymes.

New and emerging HDAC inhibitors Class IIa-selective HDAC inhibitors. Until recently, specific inhibitors of class IIa HDACs have not been available (Supplementary information S2 (table)). Trifluoromethyloxadiazole (TFMO)-based inhibitors that specifically inhibit the residual catalytic activity and acetyl-lysine binding pocket of class IIa HDACs have recently been developed; these inhibitors leave the scaffolding function of these proteins intact 83. Perhaps not surprisingly, these inhibitors failed to induce apop tosis or the profound changes in gene expression that were seen with vorinostat treatment; however, they have been shown to modulate lymphocyte response to colony-stimulating factors83. YK‑4‑272 is an inhibitor of class II HDACs that inhibits their nuclear import 101. Interestingly, YK‑4‑272 restricted tumour cell growth in vivo101, thus supporting the notion that nuclear class II HDACs may be necessary for, or at least augment, the enzymatic activity of nuclear class I HDACs. Class IIa HDACs have much weaker deacetylase activity than class I HDACs when assessed with standard acetyl-lysine substrates102; however, this may indicate that the natural substrate or substrates for these enzymes have yet to be identified rather than suggesting that the class IIa HDACs have low catalytic activity. For example, class IIa HDACs, but not class I or class IIb HDACs, are active against trifluoroacetyl-lysine substrates102. Isoform-selective HDAC inhibitors. There are still relatively few isoform-specific HDAC inhibitors avail able, but compounds with proposed selectivity for HDAC3, HDAC6 and HDAC8 (based on assays using recombinant purified proteins) have been developed (TABLE 2). HDAC3 is strongly implicated in the pathogen esis of the neurodegenerative disease Friedreich’s ataxia, which is caused by aberrant heterochromatin formation at the frataxin (FXN) gene locus owing to triplet repeat expansion, ultimately resulting in FXN repression103,104. The HDAC3‑specific inhibitor RG2833 increased FXN expression in clinical samples and ameliorated the dis ease phenotype in a murine model105,106, and is currently in Phase I clinical trials for the same disease. A related HDAC3‑selective inhibitor, RGFP966, inhibited the growth of CTCL cells in vitro and inhibited DNA replica tion. Importantly, these molecular and biological effects were phenocopied by small interfering RNA (siRNA)mediated knockdown of HDAC3, providing supportive genetic evidence for the HDAC3‑selective effects of RGFP966 (REF. 107).



REVIEWS Table 2 | Isoform-selective HDAC inhibitors HDAC inhibitor

Specificity

Stage

Diseases

Refs

Compound 60

HDAC1, HDAC2

Preclinical

Neurology

112

MRLB‑223

HDAC1, HDAC2

Preclinical

Cancer

111

RG2833; 109

HDAC3

Phase I trial

Friedreich’s ataxia

105,106

RGFP966

HDAC3

Preclinical

Cancer, neurology

107,217

BG45

HDAC3

Preclinical

Cancer

Rocilinostat (ACY‑1215)

HDAC6

Phase IIa trial

Cancer

ACY‑738, ACY‑775

HDAC6

Preclinical

Neurology

Tubacin

HDAC6

Preclinical

Cancer

219,220

Tubastatin A (tubastatin)

HDAC6

Preclinical

Inflammation, neurodegeneration

178,221

C1A

HDAC6

Preclinical

Cancer

222

HPOB

HDAC6

Preclinical

Cancer

223

Quinazolin‑4‑one derivatives

HDAC6

Preclinical

Alzheimer’s disease

224

PCI‑34051

HDAC8

Preclinical

Cancer

225

C149

HDAC8

Preclinical

Cancer

226

Jδ

HDAC8

Preclinical

NA

227

BRD73954

HDAC6, HDAC8

Preclinical

NA

228

108 * 218

HDAC, histone deacetylase; NA, not applicable. *See ClinicalTrials.gov identifiers NCT01323751, NCT01997840 and NCT01583283 for further information.

Polypharmacological molecules Single drug molecules that bind to multiple targets.

BG45, a new HDAC3‑selective inhibitor was recently shown to induce the death of multiple myeloma cells concomitant with hyperacetylation and hypophospho rylation of STAT3, although it was not demonstrated that deregulated STAT3 activity was necessary for the observed effects against multiple myeloma. Combined treatment with BG45 and bortezomib was effective against multiple myeloma cells in vitro and in vivo, although the molecular events that underpinned this observed combination effect were not determined108. Of the HDAC6‑specific inhibitors available, rocilinostat (ACY‑1215) is being tested clinically for the treatment of multiple myeloma in combination with bortezomib, following promising preclinical results 109. Multiple myeloma cells produce a large number of misfolded pro teins that need to be processed and removed through the aggresome (regulated by HDAC6) and the protea some110. Dual inhibition of both pathways results in the accumulation of misfolded proteins and subsequent tumour cell death through a process coined proteotox icity, thus providing a mechanistic rationale for combi nation studies using HDAC6 inhibitors and proteasome inhibitors for the treatment of multiple myeloma. HDAC8‑specific inhibitors such as PCI‑34051 and C149 have been developed with only minimal biological or molecular analysis performed on cells treated with these compounds (TABLE 2). Decreased proliferation and induction of apoptosis was observed in T cell leukaemias and lymphomas; however, given that the physiological function of HDAC8 is poorly understood, the ration ale for the use of HDAC8‑selective inhibitors to treat human diseases such as cancer remains unclear. To date, selective inhibitors of HDAC1 or HDAC2 have not been

developed but agents that preferentially inhibit both of these class I HDACs have recently been produced (TABLE 2). MRLB‑223 and Compound 60 are new inhibi tors of HDAC1 and HDAC2; MRLB‑223 has been shown to induce histone hyperacetylation and has in vivo anti tumor effects111, whereas Compound 60 induces mood stabilization and antidepressant effects112. Interestingly, the antitumour effects of MRLB‑223 were similar to those observed using vorinostat in the same experimen tal systems111 but vorinostat did not mediate the same mood-related behavioural effects as Compound 60 did, and the changes in gene expression observed in the brains of mice following Compound 60 treatment were dissimilar to those seen following vorinostat treat ment 112. This again raises questions regarding the true specificity and selectivity of HDAC inhibitors and the differential effects of small molecules on HDACs in the recombinant, purified form compared to physiological conditions where HDACs almost certainly exist pre dominantly as part of large macromolecular complexes. Polypharmacological HDAC inhibitors. Various poly pharmacological molecules are being developed that exhibit dual inhibition of HDACs and other therapeutic targets (reviewed in REF. 113; see Supplementary information S2 (table)). Three such polypharmacological drugs have reached clinical trials. CUDC‑907 contains a hydroxamic acid and a morpholinopyrimidine to inhibit both HDACs and phosphoinositide 3‑kinase (PI3K)114, and a Phase I trial of this drug is underway for the treatment of lym phoma and multiple myeloma (ClinicalTrials.gov iden tifier: NCT01742988). CUDC‑101 inhibits HDACs, epidermal growth factor receptor (EGFR) and HER2



REVIEWS (also known as ERBB2) by the addition of a hydroxamic acid to the methoxyethoxy group of the phenylamino quinazoline backbone of receptor tyrosine kinase inhibi tors115,116 and is currently being evaluated in Phase I trials as a treatment for solid tumours (ClinicalTrials.gov iden tifiers: NCT01702285, NCT01171924, NCT01384799 and NCT00728793). Tefinostat contains an esterasesensitive chemical motif that is cleaved upon uptake into macrophages and monocytes, activating the drug specifically in cells of the myeloid lineage117. A recent Phase I trial showed specificity of the compound for mye loid but not lymphoid cells, partial responses in patients with myeloid malignancies and a lack of dose-limiting toxicities118. Promising results have been observed in preclinical studies of chemical conjugates combining hydroxamic-acid-based HDAC inhibitors, such as vorino stat, with daunorubicin (a topoisomerase II inhibitor)119, camptothecin (a topoisomerase I inhibitor)120, bexaro tene (a retinoid X receptor agonist)121, tamoxifen (an oestrogen receptor‑α and -β antagonist)122, lovastatin (a 3‑hydroxy‑3‑methylglutaryl CoA reductase (HMGR) inhibitor)123, 1α,25‑vitamin D (a vitamin D receptor agonist)124,125, a SRC inhibitor 126 or selective oestrogen receptor modulators127.

Mechanisms of action and preclinical studies Through hyperacetylation of histone and non-histone HDAC substrates, HDAC inhibitors can induce a range of cellular and molecular effects, any one or more of which may underpin the antitumour, immunological and neurological responses observed in experimental and clinical settings. The cellular response to HDAC inhi bition is likely to be determined by a combination of factors, including the cell of origin, genetic and epi genetic anomalies present within the cell, the specific HDACs and HDAC-containing complexes inhibited and the effect of stromal cells and soluble factors that might regulate the response. There has been substantial research into the mechanisms of action of HDAC inhibi tors in oncology, which are detailed below and in TABLE 1 and followed by a discussion on mechanistic insights and preclinical studies in immune and neurological disorders.

Oncogene addiction The reliance of tumours on a single dominant oncogene for growth and survival, so that inhibition of this specific oncogene is sufficient to halt the neoplastic phenotype.

How do HDAC inhibitors function as oncology drugs? A major unanswered question relates to how or why HDAC inhibitors can have tumour-cell-selective effects, especially with respect to non-reversible effects such as apoptosis and senescence. Dawson and Kouzarides5 suggested that this is due to multi-tiered and semiredundant epigenetic regulation in normal cells during homeostasis. They posited that in transformed cells epigenetic regulators may be required for maintaining the expression of a set of key genes, and disturbing this delicate balance may lead to cell catastrophe, thereby likening this ‘epigenetic vulnerability’ to the paradigm of ‘oncogene addiction’128. In this way, it is suggested that following an ‘epigenetic insult’, normal cells engage alter native compensatory pathways, whereas cancer cells — which are reliant on specific epigenetic pathways — are unable to adapt.

Alternatively, it is possible that owing to the epi genetic reprogramming that occurs during cellular transformation, the different epigenetic ‘wiring’ of normal and tumour cells results in diverse molecular and biological outputs to HDAC inhibition that result in tumour-cell-selective cell death. For example, using isogenic matched tumour and normal cells, we recently identified a tumour-cell-selective, pro-apoptotic tran scriptional signature in response to treatment with an HDAC inhibitor, which was heavily weighted towards the upregulation of pro-apoptotic B cell lymphoma 2 (BCL‑2) family genes and the downregulation of prosurvival BCL‑2 genes129. Moreover, the induction of death receptors and ligands was also demonstrated to be a tumour-cell-selective response to HDAC inhibi tors130,131. Furthermore, treatment with vorinostat or MS‑275 led to the accumulation of reactive oxygen species and caspase activation in transformed but not normal cells, and increased levels of a key reducing pro tein, thioredoxin, in normal but not transformed cells132. Collectively, these studies demonstrate that tumour-cellspecific transcriptional responses to HDAC inhibitors, resulting in the activation of intrinsic or extrinsic apop totic pathways or altered responses to reactive oxygen species, can underpin the selective lethality of HDAC inhibitors to transformed cells. Tumour cells can be ‘addicted’ to chaperoned onco genic proteins, and hyperacetylation of HSP90 through HDAC6 inhibition can lead to the release and subsequent degradation of these oncogenic client proteins, includ ing ERBB2 (REF. 133), BRAF, CRAF, AKT134, BCR–ABL135 and KIT136. For example, the oncoprotein BCR–ABL drives t(9:22) chronic myeloid leukaemia, and treatment of BCR–ABL-positive leukaemia cells with the HDAC inhibitors LAQ824 or vorinostat led to HSP90 hyper acetylation and decreased association with BCR–ABL, resulting in the degradation of BCR–ABL and subse quent tumour cell apoptosis135,137. The ability of HDAC inhibitors to induce the degradation of HSP90 client oncoproteins might prove to be more beneficial than first perceived, as tumours with acquired resistance to small-molecule inhibitors of kinases such as BCR–ABL following mutation of the target enzyme remain sensi tive to HDAC inhibitors138. In addition, treatment with HDAC inhibitors can repress MYC expression and oncogenic function, and this is an undervalued effect of HDAC inhibitor treatment, particularly in tumours driven by aberrant MYC activity. Treatment with HDAC inhibitors leads to downregulation of MYC expression in a broad range of tumour types139,140. Furthermore, MYC can deregulate gene expression through its inter action with HDAC2 (REF. 141) and HDAC3 (REF. 142), and HDAC inhibition resulted in reactivation of MYC target genes and suppression of tumour growth. Taken together, these data indicate that HDAC inhibitors may, in certain circumstances, function by indirectly targeting MYC expression and/or function in a manner not dissimilar to the proposed activity of BET-family bromodomain inhibitors that have recently gained prominence as puta tive anticancer drugs for the treatment of MYC-driven tumours23.



REVIEWS Clearly, HDAC inhibitors have effects that are both tumour-cell autonomous and tumour-cell non-auto nomous, and it remains unclear precisely how important this interplay is for the responses to HDAC inhibitors seen in vivo. The effects of HDAC inhibitors on tumour cells themselves include induction of tumour cell death, cell cycle arrest, senescence, differentiation, autophagy and increased tumour immunogenicity (TABLE 1). The most common and widely studied antitumour effect of HDAC inhibition is cell death, and there is experimental evidence demonstrating a direct correlation between antitumour efficacy and apoptosis induced by HDAC inhibitors143,144. HDAC inhibitors can also induce immunogenic tumour cell death, which is characterized by the expression of cell surface molecules (for example, calreticulin) and the release of soluble factors (for example, HMGB1 and ATP) that potentiate the presentation of tumour antigens by dendritic cells to T cells145. Immunogenic cell death can lead to enhanced tumour clearance by CTL killing 146 and dendritic cell phagocytosis147, and we have demonstr ated that immune-stimulating antibodies designed to enhance APC and CTL activity strongly enhanced the in vivo antitumour effects of HDAC inhibitors and resulted in sustained adaptive antitumour immunity 147. Moreover, we recently showed that an intact host immune system was essential for vorinostat and panobinostat to induce sustained anticancer responses against solid and haematological tumours148. HDAC inhibitors such as butyrate were first identified as potential anticancer therapies based on their ability to induce differentiation36, and there is convincing evidence that the ability of HDAC inhibitors to induce the differen tiation of transformed cells contributes to their anticancer effects149,150. HDAC inhibitors can also affect the immune system and tumour microenvironment by regulating the differentiation, function and survival of distinct immune cell subsets and inhibiting angiogenesis (TABLE 1). Several mechanisms of resistance to HDAC inhibitors have been identified, further contributing to our knowledge of the mechanisms of action of these agents (BOX 1).

Therapeutic potential of HDAC inhibitors HDAC inhibitors for the treatment of cancer. The HDAC inhibitor vorinostat has been approved by the FDA for the treatment of refractory CTCL151,152, romidepsin has been approved for the treatment of CTCL and PTCL, and belinostat has been approved for the treatment of PTCL. More than 350 clinical trials have been completed or are underway using HDAC inhibitors for the treatment of various human malignancies with a focus on haematologi cal tumours153, both as single agents154 and in combination with chemotherapies and other targeted therapeutics155 (Supplementary information S2 (table)). Most clinical trials involving HDAC inhibitors are carried out in heavily pretreated patients with late-stage refractory disease, and this may contribute to the poor efficacy seen in many trials156. Indeed, in preclinical studies we demonstrated that an intact host immune system was important for the efficacy of HDAC inhibitors148, providing circumstantial evidence that immunodeficient patients would probably be less responsive to HDAC inhibitor therapy.

Rationally designed drug combinations for cancer treat ment. HDAC inhibitors have been combined with a broad range of agents, both empirically and with and rational hypothesis-based approaches, with varying degrees of success157. The most prominent example of the empirical testing of HDAC inhibitors in combination is with DNA-damaging chemotherapeutics, which have led to many successful outcomes158. For instance, the com bination of vorinostat with idarubicin and cytarabine in previously untreated patients with AML and MDS resulted in a remarkable overall response rate of 85%156. HDAC inhibitors have also been successfully combined with DNMT inhibitors158, but it remains unclear whether the addition of HDAC inhibitors actually improves the efficacy of DNMT inhibitors159. To address this, a large Phase II trial is currently underway to assess the effi cacy of 5‑azacytidine with and without etinostat for the treatment of MDS, chronic myeloid leukaemia and AML (ClinicalTrials.gov identifier: NCT00313586). Arguably, the most successful combination therapy involving an HDAC inhibitor is the rationally designed combination of HDAC inhibition and proteasome inhi bition. Two Phase I trials have been carried out with vorinostat and bortezomib for the treatment of relapsing and/or refractory multiple myeloma (using different dosing schedules) with overall response rates of 42% and 27%, respectively 160,161. Another Phase I trial evaluated vorinostat in combination with the novel non-peptidebased proteasome inhibitor marizomib in patients with melanoma, pancreatic cancer or lung cancer, and the combination treatment achieved stable disease in 61% of patients, all of whom had melanoma162. Another novel proteasome inhibitor, carfilzomib, is currently being evaluated in combination with vorinostat for the treat ment of refractory multiple myeloma and lymphoma after showing positive preclinical results163,164. The combination of HDAC inhibition and hormone therapy is a promising approach given the ability of HDAC inhibitors to repress the transcription of oestrogen receptors and androgen receptors, as well as the involve ment of acetylation in regulating the function of oestrogen receptors and androgen receptors165. Following positive preclinical results158, a Phase II clinical trial of vorinostat and tamoxifen was carried out in patients with hormone therapy-resistant breast cancer, and an objective response rate of 19% was achieved165. The anti-androgen receptor agent bicalutamide is currently being evaluated in com bination with vorinostat and panobinostat for the treat ment of prostate cancer (ClinicalTrials.gov identifiers: NCT00589472 and NCT00878436), with positive results anticipated based on preclinical studies158. Another example of the sensitization of HDAC inhibition to a current therapy is in Epstein–Barr virus (EBV)-driven lymphomas that are unresponsive to ganciclovir. Resistance to ganciclovir arises when EBV fails to express EBV thymidine kinase, the molecular target of ganciclovir. It was found that butyrate could induce EBV thymidine kinase expression in vitro166, and a subsequent Phase I/II trial showed that the butyrate plus ganciclovir combination was well tolerated and resulted in clinical responses in 10 out of 15 patients



REVIEWS Box 1 | Mechanisms of tumour cell resistance to HDAC inhibitors Studying the processes underlying acquired and intrinsic resistance to histone deacetylase (HDAC) inhibitors provides scope to understand the functionally important mechanism or mechanisms of action of these agents; it can also lead to the development of rational combination therapies using HDAC inhibitors and highlight biomarkers for responsiveness to HDAC inhibitors. Drug efflux. Romidepsin is a substrate for the ATP-binding cassette transporters ABCB1 and ABCC1, and inhibition of their efflux activity can restore sensitivity to romidepsin-induced apoptosis193,194. Cell lines with acquired resistance to romidepsin as a result of prolonged drug exposure showed increased expression of ABCB1 but not ABCC1, which correlated with enhanced histone hyperacetylation at the ABCB1 promoter194. In contrast to romidepsin, vorinostat is not a substrate of these efflux pumps, resulting in equally potent vorinostat-induced cell death in T cell leukaemia cells both with and without ABCB1 expression195. Overexpression of pro-survival BCL‑2 family proteins. Overexpression of B cell lymphoma 2 (BCL‑2) or knockout of the genes encoding the pro-apoptotic proteins BCL‑2‑interacting mediator of cell death (BIM) or BH3‑interacting domain death agonist (BID) conferred relative resistance to vorinostat-induced apoptosis in vitro and in vivo, as well as loss of therapeutic efficacy196. All five pro-survival BCL‑2 family proteins (BCL‑2, BCL‑XL, myeloid cell leukaemia 1 (MCL1), BCL‑W and BCL‑A1) suppressed vorinostat- and valproic acid (VPA)-induced apoptosis172; however, interestingly, romidepsin retained efficacy in the presence of BCL‑2 overexpression197 through an undefined mechanism. Combination studies using vorinostat and ABT‑737 were efficacious for tumours overexpressing BCL‑2 or BCL‑XL but not MCL1, BCL‑A1 or BCL-W, reflecting the specificity of ABT‑737 for these pro-survival proteins. Although these preclinical studies indicate that high levels of pro-survival BCL‑2 proteins may be a negative predictive marker for clinical responses to HDAC inhibitors, there is little formal evidence to support this. JAK–STAT signalling. Elevated levels and constitutive activation of signal transducer and activator of transcription 1 (STAT1), STAT3 and STAT5 in cutaneous T cell lymphoma are putative predictive biomarkers for resistance to vorinostat198. Inhibition of Janus kinase (JAK)–STAT signalling in vorinostat-resistant cells decreased the expression of BCL‑2, BCL-XL and MCL1, and resensitized cells to vorinostat-induced apoptosis198. These preclinical findings were supported by histological analysis of skin biopsy samples taken from patients enrolled in Phase II and Phase IIb clinical trials of vorinostat. Patients with low nuclear staining for phosphorylated STAT3 had a significantly higher probability of responding to vorinostat treatment than patients with high-level nuclear staining151,198. Additionally, increased STAT1 staining and nuclear localization was identified in non-responders compared to partial responders198. RAD23B. A genome-wide gene knockdown screen identified the UV excision repair protein RAD23 homolog B (RAD23B; also known as HR23B) as a sensitivity determinant for apoptosis induced by HDAC inhibitors199. RAD23B expression correlated positively with vorinostat sensitivity in tumour cell lines199, and the response to HDAC inhibitors in clinical trials could be predicted based on RAD23B expression200,201. RAD23B shuttles ubiquitylated cargo proteins to the proteasome for degradation and regulates the ability of HDAC inhibitors to initiate tumour cell apoptosis (when RAD23B levels are high) or tumour cell survival through the induction of autophagy (when RAD23B levels are low)202,203. High levels of RAD23B result in decreased proteasome function and sensitivity to apoptosis induced by HDAC inhibitors in a manner that phenocopies the effect of small-molecule proteasome inhibition by agents such as bortezomib. Recent studies showed that HDAC6 physically associated with RAD23B, resulting in decreased RAD23B expression204. Remarkably, the negative regulatory effect of HDAC6 occurred in the absence of its catalytic activity but required the chaperone-active form of heat shock protein 90 (HSP90)204. This provocative and complex mechanistic model does shed light on how RAD23B levels are predictive of patient response to class I‑specific HDAC inhibitors such as romidepsin, which induce apoptosis without inhibiting HDAC6 (REF. 200). Antioxidants. Levels of the antioxidant thioredoxin, as well as its reactive oxygen species (ROS)-scavenging activity, are induced in normal fibroblasts upon vorinostat treatment, resulting in resistance to vorinostat-induced death132. In transformed cells, however, thioredoxin levels and activity remained at the baseline, or were reduced upon vorinostat treatment, resulting in the accumulation of ROS and caspase-independent cell death132,205. Expression analysis of a panel of 17 antioxidant genes in patients with advanced leukaemia and myelodysplastic syndromes who were enrolled in a Phase I trial of vorinostat revealed that elevated expression of the antioxidant signature correlated with resistance, with 76% of non-responders having elevated antioxidant gene expression206. In vitro treatment of primary human leukaemia cells with vorinostat and β‑phenylethyl isothiocyanate, a compound that reduces cellular levels of the antioxidant glutathione, resulted in increased vorinostat cytotoxicity207, further supporting the role of ROS scavengers in sensitivity and resistance to HDAC inhibitors.

(four complete responses, six partial responses) 167. Panobinostat, MS275 and largazole are more potent than butyrate in combination with ganciclovir in vitro, potentially leading to greater clinical efficacy 168. Finally, combining HDAC inhibitors with agents that target apoptosis proteins has shown very promising results in preclinical models. Given the ability of HDAC

inhibitors to modulate the expression of key apoptosis proteins (TABLE 1), there is a strong rationale to combine HDAC inhibitors with agonists of death receptors and antagonists of pro-survival proteins. Indeed, sublethal doses of HDAC inhibitors sensitize tumour cells, but not normal cells, to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis169. We observed



REVIEWS highly synergistic antitumour activity between vorinostat or panobinostat and the agonistic TRAIL receptor (TRAILR)-specific antibody MD5‑1 in syngeneic murine models of breast cancer and adenocarcinomas170,171. Furthermore, antagonism of the intrinsic apoptosis pathway with ABT‑737 is effective in combination with HDAC inhibitor treatment in BCL‑2- or BCL-XLoverexpressing lymphoma-bearing mice172.

is being delineated in a range of neuropsychiatric disorders, and a rational, targeted approach has been taken to investigate HDAC inhibitors in these diseases. As previ ously discussed, the HDAC3‑specific inhibitor RG2833 is currently in Phase I clinical trials for Friedreich’s ataxia. Spinal muscular atrophy is caused by loss of the sur vival of motor neuron 1 (SMN1) gene, which results in neuromuscular degeneration182. A promising therapeutic approach is the activation of SMN2, which is a nearly identical gene that is formed by gene duplication and is abnormally spliced. Successful transcriptional activa tion and alternative splicing of SMN2 has been achieved following treatment with a range of HDAC inhibitors183. A Phase III trial is currently assessing VPA in combina tion with levocarnitine in children with spinal muscular atrophy (ClinicalTrials.gov identifier: NCT01671384). Specific HDACs are important in SMN2 expression and splicing in the context of spinal muscular atrophy, although suppressing HDAC2 and HDAC6 can enhance appropri ate splicing and expression of SMN2 (REF. 184). Recent preclinical studies have demonstrated that combining proteasome inhibitors such as bortezomib with HDAC inhibitors is more effective than single-agent treatment, providing a rationale for this approach in the clinic185.

HDAC inhibitors in preclinical models of neurological disease. HDAC inhibitors are effective in several ani mal models of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, ischaemic stroke, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (reviewed in REF. 173) (TABLE 1). In most cases, broad-spectrum HDAC inhibi tors have been studied, but isoform-specific inhibitors may prove to be more efficacious with less toxicity. For example, inhibition of HDAC3 alone was sufficient to pre vent Huntington’s disease-associated eye neurodegenera tion in a Drosophila melanogaster model and reduced the expression of Huntington’s disease-associated genes174. In the same study, HDAC1 inhibition also showed positive results in Huntington’s disease174, as did pan-HDAC inhi bition with vorinostat and butyrate in a D. melanogaster model of Huntington’s disease175. However, selective inhi bition of HDAC1 in neuronal tissue may prove to be prob lematic as HDAC1 inactivation results in double-stranded DNA breaks induced by cyclin-dependent kinase 5 (CDK5)–p25 (also known as CDK5R1), which leads to neuronal cell death176. Upon induction of oxidative stress by glutathione depletion, broad-spectrum hydroxamate-based HDAC inhibitors exhibit neurotoxicity 177, whereas the HDAC6‑specific inhibitor tubastatin A is neuro protective178. In addition, depletion of HDAC6 and spe cific inhibition of HDAC6 can reduce levels of pathogenic tau and decrease clinical features of tauopathies64,179. Although this may be a successful approach in some neurodegenerative diseases, HDAC6 inhibition requires careful evaluation in polyQ-induced neurodegenerative diseases, as HDAC6 may have both neurodegenerative and neuroprotective effects180. Therefore, owing to the diverse roles of different HDACs in neurobiology and neurological disease, as well as the neurological adverse events seen with HDAC inhibitors in the clinic181, the clinical development of pan-HDAC inhibitors to treat neurological diseases should proceed with caution.

HDAC inhibitors in preclinical models of inflammatory disease. HDAC inhibitors have shown positive results in rodent models of inflammatory diseases, including arthritis, inflammatory bowel disease, hypertension, septic shock, colitis and graft-versus-host-disease (GVHD)76, and in many cases this is due to enhanced TReg cell number and function186. Class II-, class IIa- or isoform-specific inhibi tors may be best suited for the treatment of inflammatory diseases. Broad-spectrum HDAC inhibitors have been shown to compromise host defence and exacerbate athero sclerosis and chronic obstructive pulmonary disease187, and this may be due to inhibition of class I HDACs, which are key repressors of pro-inflammatory cytokines. The use of class II-specific HDAC inhibitors is further supported by studies on TReg cell function in the context of inflamma tory disease186. Broad-spectrum HDAC inhibitors (TSA and vorinostat) but not class I-specific HDAC inhibitors (MS275) prevented development and promoted the reso lution of colitis in murine models, and this was dependent on FOXP3 TReg cells. Recent studies have demonstrated the effectiveness of HDAC inhibitors in preclinical models of inflammatory arthritis, highlighting the potential for the clinical development of these agents in this setting188,189.

HDAC inhibitors for the treatment of neurological disease. HDAC inhibitors have been used in neurology for many years, particularly in the context of psychiatry and neuro degeneration, well before the molecular targets of these drugs were identified. VPA was licensed for use in 1978 as an anticonvulsant drug for the treatment of absence seizures as well as partial and generalized seizure disor ders, and was marketed in the USA in 1983. VPA is still widely used as an anticonvulsant and mood-stabilizer drug in the treatment of epilepsy and bipolar disorder as well as major depression, schizophrenia and migraines. Years later, the role of acetylation and individual HDACs

HDAC inhibitors as an antiviral therapy. HDAC inhib ition has recently gained prominence as an antiviral therapeutic strategy. A challenge in HIV treatment is the eradication of latent virus reservoirs, as cells infected with integrated latent provirus are able to evade the immune system and pharmacological attack. HIV production can be enhanced in vitro by either class I-selective or broadspectrum HDAC inhibitors95. Reactivation of latent virus induced by HDAC inhibitors was seen in infected cell lines and in resting CD4+ T cells from patients receiving antiretroviral therapy 190. Recently, these preclinical results were reproduced in a clinical study in patients with latent



REVIEWS HIV infection191. Treatment with vorinostat resulted in increased cellular acetylation and HIV RNA produc tion in CD4+ T cells in patients whose viraemia was fully suppressed with antiretroviral therapy. Vorinostat, pan obinostat and VPA are currently being tested in combina tion with various antiretroviral therapies (ClinicalTrials. gov identifiers: NCT01680094, NCT01319383 and NCT01365065). Treatment of patients with herpes simplex virus (HSV) infection may be another applica tion for HDAC inhibitor-mediated eradication of latent virus reservoirs owing to role of HDAC1 and HDAC2 in maintaining the latent phase of HSV97. Although the use of HDAC inhibitors may prove to be a successful approach for eradicating viruses such as HIV, this also poses a potential major risk for patients who are latently infected with a virus when receiving HDAC inhibitors for another indication. Indeed, activation of latent hepatitis B virus and EBV has been observed in patients with cancer following treatment with romidepsin192. In the context of immune modulation and inflam mation, current clinical trials of HDAC inhibitors are focused on sickle cell disease (ClinicalTrials.gov identifi ers: NCT01245179 and NCT01000155), in which inflam mation is a major contributor to disease pathology, and in the treatment and/or prevention of GVHD in the context of stem cell transplantation (ClinicalTrials.gov identifiers: NCT00810602, NCT01789255 and NCT01111526).

Conclusions It is now widely recognized that alterations to the epigenome can initiate and prolong human disease. Fortunately, owing to the dynamic nature of epigenetic

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programming, in many instances these effects may be reversible through the targeting of distinct epigenetic enzymes such as HDACs. As our understanding of specific HDACs in disease aetiology grows and as we continue to define the molecular and biological conse quences of HDAC inhibition, it stands to reason that these agents will be more effectively and precisely used in the clinic to treat diseases such as cancer, neurological disorders and autoimmunity. Although HDAC inhibi tors with broad target specificity have been successfully used in the clinic, especially for the treatment of cer tain haematological malignancies, the development of agents that can selectively target single HDACs offers the possibility of greater efficacy and less toxicity. Clearly, a greater understanding of the mechanisms of action of HDAC inhibitors is required, and the realization that the inhibition of one HDAC may have considerable conse quences on the activity of another presents another layer of complexity to this already daunting task. However, the genetic and biochemical tools required to more defini tively determine the molecular and biological roles of distinct HDACs and HDAC-containing complexes are now at hand, which will no doubt hasten these impor tant mechanistic studies. This deeper understanding will also enhance the more strategic use of HDAC inhibitors in combination with other agents. Preclinical combina tion studies with HDAC inhibitors are already proving to be effective, and their rational clinical application in concert with other agents and companion predictive bio markers for response and resistance to HDAC inhibitor treatment will undoubtedly take prominence in the years to come.

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Acknowledgements

R.W.J. is a Principal Research Fellow of the National Health and Medical Research Council (NHMRC) of Australia and his research is supported by the NHMRC Program and Project Grants, Cancer Council Victoria, the Leukaemia Foundation of Australia and the Victorian Cancer Agency. The research of K.J.F. was supported by a postdoctoral fellowship from Cancer Council Victoria. The authors thank S. Mincci for helpful comments and advice.

Competing interests statement

The authors declare competing interests: see Web version for details.

FURTHER INFORMATION ClinicalTrials.gov: http://www.clinicaltrials.gov

SUPPLEMENTARY INFORMATION See online article: S1 (table) | S2 (table) ALL LINKS ARE ACTIVE IN THE ONLINE PDF


ADDENDUM

Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders Katrina J. Falkenberg & Ricky W. Johnstone Nature Reviews Drug Discovery 13, 673–691 (2014); doi:10.1038/nrd4360

On page 678, it was noted that it has been reported that the acetylation of signal transducers and activators of transcription (STATs) is regulated by histone deacetylase (HDAC) inhibitors, based on reference 79 in the reference list. The authors would like to note that another paper (Mol. Cell. Biol. 31, 3029–3037; 2011) has reported that STAT1 signalling is not regulated by a phosphorylation–acetylation switch.

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