Lysine acetylation at the ε-amino group is one of the most abundant posttranslational modifications (PTM) in eukaryotic cells. Due to the neutralization of positive charge on lysine residues, acetylated proteins can interact with different molecules and adopt different folds. Thus, reversible lysine acetylation plays a crucial role in many biological processes, including gene expression, chromatin remodeling, cell cycle control, cell signaling and protein quality control (PQC) (Kouzarides, 2000). Protein acetylation is a classic example of a reversible PTM which can accommodate the needs of cells and reflect responses to environmental changes. Traditionally, enzymes which “write” the acetylation mark on lysine residues are termed histone acetyltransferases (HAT), enzymes which remove the acetylation mark from lysine residues in proteins, the erasers, are named histone deacetylases (HDAC) (New et al., 2012). The name “histone” deacetylases reflects the fact that many lysine residues in histones are acetylated and explains to some extent the epigenetic aspect of histone deacetylases and therefore histone deacetylase inhibitors (HDACi). However, it has been demonstrated that thousands of other proteins can be acetylated and deacetylated both in the cytoplasm and in the nucleus (Choudhary et al., 2009). Consequently, most HDACs act on proteins which occur in the nucleus and in the cytoplasm, implying that HDACi can affect not only histones, but all other proteins, depending on their class specificity. Bromodomain (BRD)-containing proteins act as “readers” and recognize acetylation marks. They translate acetylation signals to downstream signaling cascades, leading for example to further histone modifications or chromatin remodeling, finally shaping the cell into diverse phenotypes (Filippakopoulos and Knapp, 2014; Figure 1).

Histones are proteins, which organize the DNA into a compact form called nucleosome (Olzscha et al., 2015), and the bond strength between histone units and DNA can be determined by the acetylation of lysine residues of histones (Luger and Richmond, 1998). The acetylation of distinct lysine residues of histones (H2A, H2AX, H2B, H3, and H4) has different functions. Generally, histone acetylation is associated with transcriptional activation: If the histones are acetylated at many lysine residues, the nucleosome is present in its open form and genes can be transcribed by RNA-polymerases. It is assumed that the acetylation neutralizes the positive charge of the amino terminus and therefore the binding between histones and DNA is weakened (Annunziato and Hansen, 2018). Consequently, lysine residues regain their positive charge upon deacetylation and the affinity of the negatively charged DNA phosphate backbone to the amino terminus of histones is increased (Shahbazian and Grunstein, 2007). Other functions of lysine acetylation are DNA repair (H2AX on Lys5 and Lys36, H2A on Lys5, H3 on Lys9, 14, 18, 23, 27, 36, 56, and H4 on Lys5, 8, 12, 16, 91), histone deposition (H3 on Lys9,14 and H4 on Lys5, 12), transcriptional elongation (H3 on Lys14 and H4 on Lys8), chromatin assembly (H3 on Lys56), telomeric silencing (H4 on Lys12), chromatin decondensation (H4 on Lys16) and DNA replication (H4 on Lys91) (Koprinarova et al., 2016). Acetylation of H2A and H2B are mostly taking part in transcriptional activation, while acetylation on H2AX, H3 and H4 lysine residues can have different effects. In conclusion, deacetylation of histone proteins with HDACs is required for chromatin remodeling, many downstream processes and regulatory pathways (Wade, 2001; Figure 1).

Eighteen human HDACs have been described and classified into four groups. We provide only a short introduction about the different HDAC classes, as there are many comprehensive reviews which give overviews about the HDAC classes and also chemical classes of HDACi, for example (New et al., 2012) or (Seto and Yoshida, 2014). The classification in Homo sapiens is based on the HDAC’s homology to yeast proteins (Dokmanovic et al., 2007). HDAC1, 2, 3, and 8 belonging to class I are homolog to the yeast RPD3 protein and are localized in the nucleus; they are involved in cell survival and proliferation. The class II HDACs (HDAC4, 5, 6, 7, 9, and 10) are supposed to play a tissue-specific role (Lagger et al., 2002). They are homolog to the yeast HDAC HDA1 (histone deacetylase 1) and can be found in the nucleus or cytoplasm. HDAC4, 5, 7, and 9 belong to class IIa and contain only one catalytic domain, while class IIb HDACs (6 and 10) have two catalytic domains and can only be detected in the cytoplasm. HDACs of class I and II contain Zn²⁺ in their catalytic sites, and thus are known as Zn²⁺-dependent HDACs. The HDACs from class III (SIRT1-7) are homolog to the Sir2 yeast protein. They do not contain Zn²⁺ in their catalytic sites, but require NAD⁺ for their enzymatic activity (Bolden et al., 2006). Class IV consists of only one protein, HDAC11. Regions in its catalytic center are similar to both class I and II sequences; hence, it is also classified as Zn²⁺-dependent HDAC (Gao et al., 2002).

The abundance and enzymatic activity of HDACs in cells is regulated on various levels e.g., by changes in gene expression, protein complex formation, PTMs, subcellular localization and by the availability of metabolic cofactors (Sengupta and Seto, 2004).

Histone deacetylase inhibitors suppress HDAC activity. There are six structurally defined classes of HDACi: small molecular weight carboxylates, hydroxamic acids, benzamides, epoxyketones, cyclic peptides and hybrid molecules. They mainly act on HDACs of the classes I, II and IV by binding the Zn²⁺-containing catalytic domain (Drummond et al., 2005). The first discovered HDACi, the natural antifungal antibiotic trichostatin A (TSA), belongs to hydroxamic acid-type chelators (Yoshida et al., 1990), and the TSA structural analog vorinostat, also known as suberoylanilide hydroxamic acid (SAHA) was the first HDACi being approved by the U.S. Food and Drug Administration (FDA). The other three HDACi approved by the FDA so far are romidepsin, belinostat and panobinostat (Yoon and Eom, 2016). NAD⁺-dependent class III HDACs are inhibited by NAD⁺ and its derivates, dehydrocoumarin, splitomycin, 2-OH-naphtaldehyde, sirtinol and M15 (Porcu and Chiarugi, 2005). However, in this review, we focus on the “classic” HDACs belonging to the classes I, II and IV and their respective HDACi.

Vorinostat (Zolinza^®) was approved in October 2006 for treatment of advanced primary cutaneous T-cell lymphoma (CTCL) (Mann et al., 2007). Romidepsin (Istodax^®) was licensed for CTCL treatment in 2009 (Whittaker et al., 2010), and later, in 2011 for peripheral T-cell lymphoma (PTCL) (Coiffier et al., 2012). Belinostat (Beleodaq^®) was approved by the FDA in 2014 for the treatment of PTCL. The fourth approved HDACi panobinostat (Farydak^®) was licensed in 2015 for the treatment of multiple myeloma (MM).

As already mentioned, HDACi have a profound effect on the structure of chromatin and therefore on the transcriptional activity of the affected gene chromatin regions. This is why HDACi can be seen as established epigenetic modulators, since they affect the read-out of genes without changing the DNA sequence (Olzscha et al., 2015).

Epigenetics can be defined as inherited changes in phenotypes or entities, which are not encoded in the nucleotide sequence of the organism, but are passed on to daughter cells (Olzscha et al., 2015). Exogenous influences and altered environmental conditions can change epigenetic signatures and may give a hint about the origin of different malignancies, such as cancer or neurological disorders (Tsankova et al., 2007; John and Rougeulle, 2018). One appearance of epigenetics can be biochemical post-replicative modifications of the DNA-sequence, either through alteration of single bases or as described above in proteins (Handy et al., 2011).

Traditionally, cancer has been defined as a group of diseases leading to uncontrolled cell proliferation caused by genetic mutations in tumor-suppressor genes and oncogenes or chromosomal abnormalities (Hanahan and Weinberg, 2000). However, cancer may also be driven by epigenetic changes (Baylin and Ohm, 2006). According to its definition, epigenetic changes can be heritable and also known as epimutations, equivalent to mutations; however, some changes, in particular, histone deacetylation that repress gene expression by wrapping DNA more tightly, are not heritable, but have been also described as “epigenetic” (Berger et al., 2009). Thus, acetylation can influence transcriptional regulation, cell cycle control, apoptosis and autophagy, but also the activity of further proteins that maintain protein homeostasis, which will be described below (Nihira et al., 2017).

Since thousands of proteins can be acetylated by HATs and deacetylated by HDACs, HDACi will not only act on an epigenetic level, but will also influence crucial protein functions, especially in PQC systems. These systems and the underlying effects will be described in this review and how this knowledge is utilized to develop combination therapies of HDACi and modulators of PQC processes. Clinical trials with HDACi alone or in combination are systematically evaluated for their potential to identify novel targets of PQC systems and their effect on epigenetic modulation. We also exemplify the development of novel HDACi which are in clinical trials, provide evidence that PQC systems are involved and how the underlying proteins can be used as biomarkers. Finally, we give an outlook on current and future HDACi development, its impact on proteostasis and how this knowledge can be utilized to improve precision medicine for cancer patients.

Recent studies suggest that cancer cells have increased concentrations of HDACs. For instance, according to clinical and preclinical studies, class I HDACs may stimulate cell proliferation and survival (Yoon and Eom, 2016). It has been shown that HDAC1 is overexpressed in prostate (Halkidou et al., 2004), gastric (Choi et al., 2001), colon (Wilson et al., 2006), and breast (Zhang et al., 2005) carcinomas. HDAC2 is reported to be responsible for the loss of adenomatous polyposis coli (APC) expression in colorectal cancer (CRC) (Zhu et al., 2004) and displays increased expression in cervical (Huang et al., 2005) and gastric (Song et al., 2005) carcinomas. HDAC3 and HDAC6 are also reported to show increased concentration in colon and breast carcinoma cells (Zhang et al., 2004; Wilson et al., 2006).

One of the problems in cancer is the heterogeneity which can occur from different mutations and/or different epigenetic patterns. Genomic variability occurs sometimes even if the cells display similar phenotypes or when there are differences in the phenotype, even though the cells originate from one tumor population (Cantor and Sabatini, 2012). These observations strengthen the hypothesis that epigenetics plays an important role in cancer development. Tumor suppressor genes might be silenced and oncogenes activated upon epigenetic changes without any influence on the genotype (Llinàs-Arias and Esteller, 2017). It is hypothesized that molecular chaperones such as the heat shock protein 90 (HSP90) act as regulators of the genotype-to-phenotype interplay and offer an evolutionary buffer to protect cells from malignant transformation (Whitesell and Lindquist, 2005; Jarosz, 2016).

The first discovered non-histone target of HATs and HDACs was p53 (Sakaguchi et al., 1998; Liu et al., 1999), a tumor suppressor protein that is able to bind DNA. Therefore, it can affect chromatin structure and can epigenetically change gene expression, whereby the binding is regulated by acetylation. Furthermore, acetylated p53 is able to induce apoptosis and autophagy (Fridman and Lowe, 2003; Mrakovcic and Fröhlich, 2018). Apoptosis is a form of highly controlled, energy-dependent programmed cell death, and malignant cells often depend on inherent or acquired mechanisms to resist cell death, which is seen as a hallmark of cancer (Hanahan and Weinberg, 2000). It is still under investigation, whether acetylation alters the interaction with other proteins or whether it results in a conformational change of p53 (Mrakovcic et al., 2018).

Another example for a silenced tumor suppressor in cancer cells is p21, which acts as a cyclin-dependent kinase (CDK) inhibitor. As p21 plays a crucial role in the regulation of CDKs, its expression has a big impact on cancer growth. Silencing of p21 occurs as a result of hypoacetylation of its promotor and consequently, it has been shown that HDAC1 inhibits the promotor by binding at the SP1-site (specificity protein 1) and competes with p53 activating the promoter of p21 (Gui et al., 2004). Using HDACi, there are two different mechanisms known leading to an enhanced expression of p21, with one of them p53-independent and the other p53-dependent. In the p53-independent mechanism, treatment with HDACi results in a release of HDAC1. Therefore, the promotor loses its repression and the gene of p21 is transcribed. The p53-dependent mechanism displays an enhanced expression of p21, as the HDACi induces acetylation of p53 resulting in a higher binding affinity to the p21 promotor (Ocker and Schneider-Stock, 2007).

HDAC1 is also known to bind ETO (eight-twenty-one), which can be fused with AML1 (acute myeloid leukemia 1). This fusion protein AML1-ETO arises as a result of a t(8;21) translocation and it has been shown that HDACi is efficient as a treatment against AML, suggesting a non-epigenetic effect. A study using valproic acid (VPA) as an HDACi reported a dissociation of the AML1-ETO/HDAC1 complex from the AML1-ETO promotor. Other studies reported a proteasomal degradation of AML-ETO after HDACi treatment, again demonstrating the importance of the PQC systems in cancer cells (Hug and Lazar, 2004).

In order to ensure the correct protein folding and protection of the proteome, eukaryotic cells developed a complex PQC system. Molecular chaperones, the ubiquitin proteasome system (UPS) and autophagy form a complex network which maintains the integrity of the proteome (Mogk and Bukau, 2006; Chen et al., 2011; Olzscha, 2019; Figure 2).

As mentioned in section “The Role of HDACs in Epigenetics and Cancer Cells,” HDACi cause hyperacetylation of histones which is one reason for the induction of apoptosis. As autophagy and apoptosis functionally counteract each other in tumor cells (see section “The Role of Protein Quality Control Systems in Cancer Cells”), one could assume that autophagy is inhibited during treatment with HDACi (Gump and Thorburn, 2011). However, it has been demonstrated that administration of HDACi can also induce autophagy, leading to the paradox situation that autophagy has beneficial effects in the treatment of cancer cells and even facilitates tumor suppression (Zhang et al., 2015). The effect of promoting cell survival or cell death is dependent on the cell type and genetic predisposition of the tumor, as well as the duration and dose of the HDACi. There are many examples described in the literature, one of them is the pan-HDACi panobinostat. On the one hand, panobinostat can inhibit autophagy by increasing the level of acetylation of autophagy-related gene products, for instance ATG7. The acetylation causes a repression of ATG7, which leads to a promotion of apoptosis and decreased autophagy in myeloid leukemia cells (Stankov et al., 2014). On the other hand, it has been reported that autophagy is induced in panobinostat-treated Eμ-myc lymphoma cells, the c-myc gene is here driven by the IgH enhancer. Thereby, an apoptotic protease activating factor 1 (Apaf-1) or caspase-9 deletion has been reported. This deletion causes an apoptosome inactivation and thus a suppression of apoptosis (Mrakovcic et al., 2017, 2018). Furthermore, it has been demonstrated that HDACs themselves can induce autophagy. For example, HDAC6 can induce autophagy as a result of an impaired UPS (Kaliszczak et al., 2018). It binds polyubiquitinated proteins and plays an essential role for the fusion of autophagosomes with lysosomes.

It seems to be a drawback that pan-HDACi not only inhibit histone deacetylation in the nucleus, but also a variety of proteins which can be found in virtually all cellular compartments. However, a greater understanding of the control and homeostasis mechanisms of HDACs is required to enable more effective application of HDACi for the treatment of specific tumor cell types.

As existing HDACi are mostly pan-HDACi, they do not entail satisfactory specificity. Accordingly, it is of great interest to develop new and more specific inhibitors (see also section “Novel Strategies of HDAC Inhibitors Affecting Protein Quality Control Pathways”). HDAC6 is a potential selective target due to its unique molecular structure with two catalytic domains and its localization in the cytoplasm (Li et al., 2018). Critical substrates with a role in PQC include p300 and HSP90 (Cosenza and Pozzi, 2018). Both are known to play a crucial role in tumorigenesis, showing the importance for the development of specific HDAC6 inhibitors. Tubastatin A is an example for an established HDAC6 inhibitor, often used in pre-clinical studies (Wang et al., 2016). However, a more specific HDAC6 inhibitor (marbostat-100) has been developed and was published in 2018 with a K_i-value of 0.7 nM (Sellmer et al., 2018). In comparison, the value of the most common used selective HDAC6 inhibitor tubastatin A is 10-times higher. A preferred substrate of HDAC6 is α-tubulin; the reverse reaction is catalyzed by the α-tubulin acetyltransferase ATAT1. The deacetylated α-tubulin polymerizes with β-tubulin to form microtubules, components of the cytoskeleton, which play an important role in DNA segregation during mitosis. Inhibition of HDAC6 with marbostat-100 results in hyperacetylated α-tubulin. The specificity of this HDAC6 inhibitor was determined by comparing the enrichment of acetylated histone H3 in marbostat-100 treated cells with entinostat (MS 275) treated cells. It is an HDAC1 and HDAC3 specific inhibitor in phase II clinical studies. This enrichment of acetylated histone H3 could also be detected using the FDA-approved pan-HDACi panobinostat (LBH589) (Grünstein, 2018). It has been demonstrated in different human cell lines and in mice that marbostat-100 is considerably more specific than panobinostat, led more efficiently to hyperacetylation of α-tubulin and displayed only minor proteolytic effects on the target enzyme HDAC6 (Grünstein et al., 2019).

In analogy to marbostat-100, the application of the established HDAC6i tubastatin A led to hyperacetylation of α-tubulin. However, upon oxidative stress, tubastatin A activated the heat shock transcription factor 1 (HSF1) leading to the upregulation of the molecular chaperones HSP70 and HSP25 and increased cell survival (Leyk et al., 2017). Upon proteasomal stress, HDAC6 could initiate autophagy, as it is involved in the transport of ubiquitinated proteins along microtubules (Leyk et al., 2015). This influence of the PQC system has been observed in some preclinical studies of HDACi, which are described below in section “HDAC Inhibitors in Combination With Proteostatic Drugs.”

Two further examples for inhibitors that influence molecular chaperones as well as autophagy are trichostatin A and sodium butyrate. Both can affect the chromatin structure at the site where the gene for HSP70 is located (Chen et al., 2002). HSP70 is a molecular chaperone supporting the folding of many newly synthesized proteins and can recognize the KFERQ motif in proteins resulting in CMA.

In addition to its impact on molecular chaperones and consequently on CMA, another mechanism of action of trichostatin A is known. It has been shown that trichostatin A enhances the ubiquitination of the HAT p300, resulting in its proteasomal degradation. Since it is a co-activator of the expression of the NADPH oxidase 4 (Nox4) (Hakami et al., 2016), an important factor in angiogenesis, cancer cells suffer from oxygen and nutrient deficiency due to its reduced expression in trichostatin A treated cells. Thus, trichostatin A constitutes a paradigm in its ability to impact on molecular chaperones, the UPS and autophagy.

Another example for an inhibitor affecting the ubiquitin-proteasome system is MC1568 (Table 1). It is a class IIa selective HDACi that increases the specific sumoylation of HDAC4. Sumoylation is a PTM using “small ubiquitin-related modifier” (SUMO) to label the target protein and can direct it to different pathways. MC1568 induced HDAC4 down-regulation by increasing its specific sumoylation followed by activation of proteasomal degradation pathways. MC1568 alters not only the pattern of PTMs and activates the degradation of substrates via the UPS, but also changes epigenetic pathways that may be affected by HDAC4 (Scognamiglio et al., 2008).

Besides trichostatin A, there are other HDACi known to induce autophagy. For instance, OSU-HDAC42 led to downregulation of Akt/mTOR signaling and the induction of the ER stress response to induce autophagy (Liu et al., 2010). On the contrary, triple negative breast cancer cells treated with the HDACi YCW1 showed a downregulation of BNIP3 (Bcl-2/adenovirus E1B 19 kDa protein interacting protein 3) resulting in autophagic cell death. Similarly, treatment of mice with YCW1 led to a decline in lung tumor growth (Huang et al., 2014).

The fact that HDACi can influence PQC systems led to a novel strategy where HDACi are used in combination with drugs modulating PQC systems, i.e., PI or modulators of autophagy.

In a study conducted in 2017, trichostatin A was tested in combination with the PI bortezomib for the treatment of ovarian cancer cells and displayed an inhibition of the proliferation of A2780 cells inducing apoptosis. Furthermore, similar results were shown in A2780T cells that are resistant to cytostatic taxanes (Jin et al., 2017). In another attempt, a combination of sodium butyrate was tested with the PIs MG115, MG132, PSI-1, PSI-2, or epoxomicin in human CRC cells (SW48, SW1116, and SW837). In these studies, additive and synergistic anticancer effects, namely growth inhibition and apoptosis, were observed in combination with all tested PIs (Abaza, 2010).

Cell death induced by accelerated autophagy in cancer cells has been shown to be effective in a combined treatment with HDACi and an inducer of autophagy. In Burkitt lymphoma and lymphocyte cell lines, VPA induced autophagosome formation and increased autophagy led to an autophagy-mediated cell death in combination with an mTOR inhibitor (Dong et al., 2013). This experiment has been carried out with the mTOR-specific inhibitor temsirolismus, but as OSU-HDAC42 is known to act as an mTOR inhibitor, it is suspected that OSU-HDAC42 would show similar results (Liu et al., 2010). Furthermore, it has been demonstrated that the protein levels of HDACs, especially HDAC6, are reduced by autophagy after treatment with the HDACi AR42 (OSU-HDAC42). The combination of this HDACi with the kinase inhibitor pazopanib in melanoma cells demonstrated an inhibition of the ATPase activity of the molecular chaperones HSP90 and HSP70. In this setting, HDAC6 could activate HSP90 by deacetylation and the inhibition was enhanced by combined treatment (Booth et al., 2017). Using YCW1 (Table 1) in combination with radiation also demonstrated increased cell death in cancer cells due to ER stress and the induction of autophagy (Chiu et al., 2016). In addition, preclinical studies revealed an enhanced cisplatin effect against YCW1-treated non-small cell lung cancer (NSCLC), whereas cisplatin causes mitochondria-mediated apoptosis (Huang et al., 2014). This highlights again the discrepancy that cancer cells sometimes utilize autophagy to their advantage and sometimes inhibit the autophagic pathway.

Protein ubiquitination can be also mediated by cullin-ring E3 ligases (CRLs), which have to be activated by neddylation with NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), another protein similar to ubiquitin acting as PTM (Enchev et al., 2015). Neddylation with NEDD8 is achieved by NEDD8-activating enzymes (NAEs), which are druggable enzymes. It has been shown in several studies that NAE-inhibitors, for instance pevonedistat (MLN4924), can act with other anti-cancer agents including bortezomib in a synergistic manner in MM (Gu et al., 2014). In a different pre-clinical study, it has been demonstrated that the NAE inhibitor pevonedistat acts synergistically with the HDACi belinostat in various AML cell types, especially those with reciprocal effects on homologous recombination (HR) and non-homologous end-joining (NHEJ) mechanisms (Zhou et al., 2016).

There are 61 bromodomains known in the human proteome, integrated in 46 proteins. All of them have a conserved left-handed bundle of four α-helices linked by flexible and variable loops. The best-known proteins containing a bromodomain are part of the bromodomain and extra terminal family (BET). This BRD family is characterized by the presence of two tandem bromodomains (BD1 and BD2) at the N-terminus, an extra terminal domain (ET), and a C-terminal domain (CTD). They play a crucial role in cancer cells, especially in cell proliferation by regulating the expression of oncogenes, for instance c-MYC or nuclear factor κ light chain enhancer of activated B cells (NF-κβ)-dependent genes (Pérez-Salvia and Esteller, 2017).

It has been demonstrated that hyperacetylation of proteins induced by HDACi can result in amyloid-like protein aggregation. This can lead to a reduction of the proteolytic capacity of the UPS, increased autophagy and downregulated translation, summarized as proteostatic failure (Olzscha et al., 2017). Similarly, it has been observed that trichostatin A induced a dramatic increase of the acetylation of tau proteins, which aggregation can be seen under pathological conditions as one of the underlying causes for Alzheimer’s disease (AD) (Cohen et al., 2011). The increase of acetylated tau levels and resulting aggregation is also shown in tubastatin A treated oligodendrocytes, and an alteration of the cell morphology was observed containing a reduced microtubule binding activity of tau (Noack et al., 2014). As described above, tubastatin A is an HDAC6-specific inhibitor; consequently, HDAC6 plays an important role in the cytotoxic accumulation of protein aggregates which may explain the higher level of aggregated tau proteins (Boyault et al., 2007b). Marbostat-100 also inhibits HDAC6, the treatment may have similar effects on the acetylation of the tau protein; however, it remains unclear whether it has an effect on aggregation.

The potential of HDACi-induced aggregation raises the question, how this knowledge can be utilized for benefits in cancer therapy, at the same time preventing adverse side effects and suppressing aggregation. It has been demonstrated that the bromodomain-containing proteins CBP and p300 are involved in the formation of protein aggregates after treatment with HDACi and their depletion results in a reduction of aggregation. This opens a new strategy for cancer treatment without the formation of aggregates. Bromodomain inhibitors are small proteins which can block the binding of bromodomain-containing proteins to acetylated residues and therefore have the potential to reverse the aggregation-induced cytotoxicity and restore proteostasis (Olzscha et al., 2017).

The first published BET inhibitor was (+)-JQ1 tested in NUT (nuclear protein in testis) midline carcinoma cells. NUT can fuse with the bromodomain BRD4 forming the oncoprotein BRD4-NUT, which plays an important role in the differentiation and proliferation of cancer cells. (+)-JQ1 acts as a competitor binding at the acetyl-lysine binding motif and prevents the formation of the fusion protein and the resulting proliferation (Filippakopoulos et al., 2010). It was then tested in many other cancer types, for instance glioblastoma (Cheng et al., 2013), colon cancer (McCleland et al., 2016), lung cancer (Lockwood et al., 2012), Burkitt’s lymphoma (Mertz et al., 2011), and MM (Soodgupta et al., 2015). It led to downregulation of c-MYC, an oncogene responsible for altered transcription and proliferation and showed synergistic effects with the HDACi mocetinostat (Borbely et al., 2015). However, it has never reached a clinical trial, due to its short half-life; therefore, analogs of (+)-JQ1 with a longer half-life were synthesized, one of them is called CPI203 (Alqahtani et al., 2019). It has shown some success in bortezomib-resistant mantle cell lymphoma (Moros et al., 2014) and MM cells (Díaz et al., 2017) in combination with the immunomodulator lenalidomide, whereby reduced c-MYC-levels leading to a downregulation of IRF4 (interferon regulatory factor 4). IRF4 is a transcription factor, which is necessary for the survival of lymphoma and myeloma cells, leading to an induction of apoptosis in these cells. I-BET151 is another BET-inhibitor, which also represses c-MYC in myeloma cells (Chaidos et al., 2014). As a pan-BET inhibitor, it also displayed anti-cancer effects in other types of cancers, for example in medulloblastoma cells by suppressing the Hedgehog-activity or in NUT midline carcinoma (Long et al., 2014).

Other examples where bromodomain inhibitors can influence proteostasis in combination with HDACi are CBP and p300. These transcription modulators are not only bromodomain-containing proteins, they also act as histone acetyltransferases. Thus, they recognize lysine acetylation and may cause further acetylation in histones leading to a relaxation of DNA and an activation of transcription. Two p300/CBP-specific bromodomain inhibitors, I-CBP112 and SGC-CBP30, were investigated in preclinical studies, I-CBP112 for leukemia and prostate cancer (Picaud et al., 2015) and SGC-CBP30 in MM (Hay et al., 2014). I-CBP112 activates the HATs CBP and p300 resulting in a repression of the proliferation in cancer cells (Zucconi et al., 2016). SGC-CBP30 can suppress IRF4 in myeloma cells (Conery et al., 2016). However, currently it is not brought into clinical trial, as it displays a short half-life. Another inhibitor which targets non-BET bromodomains as well as BET-bromodomains is bromosporine (Theodoulou et al., 2016). This pan-BDi reduced the formation of protein aggregates only slightly after HDACi treatment (Olzscha et al., 2017).

There is still an interest in developing more specific and more efficient HDACi for cancer treatment. Most of the current HDACi influence several pathways in the cells, including the PQC system. This can be used by combining HDACi with other cancer treatments, like radiation, PI, bromodomain inhibitors, autophagy- and chaperone-modulating agents. On the other hand, there are also new HDACi broadening the spectrum of molecular actions and therefore their monotherapeutic use has to be reconsidered as a treatment option.

Various novel HDACi have undergone pre-clinical and clinical studies over the past 5 years, both HDACi which target specific HDAC classes and HDACs which can be considered as pan-HDACi. CXD101 is a novel class 1-selective HDACi and has shown effects in some hematological malignancies (Eyre et al., 2019). The observed high levels of the proteasomal shuttling factor HR23B indicate a positive outcome, resembling the results of pan-HDACi (Khan et al., 2010; New et al., 2013). In fact, it has been demonstrated that also class I HDACi are able to induce protein aggregation in human cells (Olzscha et al., 2017). As described above, the induced protein aggregation may contribute to the overall cytotoxicity exerted by HDACi and their success in hematological malignancies. Since CBP/p300-specific bromodomain inhibitors are able to partially abrogate this effect (Olzscha et al., 2017), it is likely that the aggregation indirectly affects nuclear proteins and therefore modulates epigenetic regulation in cells. Interestingly, a clinical trial of CXD101 in combination with the tissue-agnostic drug pembrolizumab for relapsed or refractory diffuse large B-cell lymphoma (PLACARD, NCT03873025) is underway, taking the levels of PD-L1 (programmed death-ligand 1) in PD-1 positive cells into account (see also section “Evaluating Alterations of PQC Systems: Precision Medicine Upon HDAC Inhibitor Treatment”).

As mentioned above (section “HDAC Inhibitors in Combination With Proteostatic Drugs”), a common strategy is to apply HDACi in combination therapy with other anti-cancer drugs. However, this strategy faces several problems, including incompatibilities, pharmacokinetic problems when reaching different compartments and unexpected interactions, which may alleviate the activities, but also lead to an increased possibility to generate undesired cytotoxic effects (de Lera and Ganesan, 2016). To overcome some of the problems, several chimeric HDACi have been developed. They consist of hybridized functional groups of an HDACi structure and the respective different group to inhibit or bind to a second target (Nepali et al., 2014). For instance, the inhibitor fimepinostat (CUDC-907), which is a dual HDACi and phosphatidylinositol-3-kinases (PI3K) (Gunst et al., 2019) was tested in a trial in patients with lymphoma (NCT01742988). A striking example of this strategy, which affects epigenetic outcome and PQC pathways, is the generation of chimeras between HDACi and bromodomain inhibitors (BDi). Bromodomains cannot only “read” acetylation marks on proteins, they can also act synergistically with HDACs to guide them to the respective protein and remove the acetylation mark (Olzscha et al., 2015). Many promising examples of BDi have been generated, targeting several bromodomain-containing proteins, including CBP/p300, affecting both chromatin and interacting proteins, including p53 (Picaud et al., 2015). Several chimeric compounds consist of the functional groups of pan-HDACi such as SAHA and different BDi including the BET inhibitors JQ1 and I-BET295 (Atkinson et al., 2014), as well as BRD-4 specific inhibitors (Amemiya et al., 2017). Besides the established functions as epigenetic modulators, some HDACs and bromodomain-containing proteins (BRDs) exert their activities also in the cytoplasm, affecting crucial PQC pathways. Effects of HDACs and BRDs on the protein degradation machinery demonstrate that these mechanisms contribute to the overall cytotoxicity of the single substances or their chimeras (New et al., 2013; Olzscha et al., 2017).

A novel HDACi which affects proteostasis is MPT0G413, a selective HDAC6 inhibitor. This inhibitor did not only inhibit the growth in MM cells, the combination of MPT0G413 and bortezomib enhanced also polyubiquitinated protein accumulation and synergistically reduced MM viability, showing increased caspase-3, caspase-8, and caspase-9 levels (Huang et al., 2019). Since it is an HDAC6 inhibitor, the effects are likely to reflect disturbances in PQC pathways and therefore only indirectly affect epigenetic features in the nucleus.

Cancer cells are highly proliferative and show an extensive protein turnover and thus rely heavily on proteasomal degradation of abnormal or mutant proteins (Adams, 2004). Therefore, it can be argued that cancer cells are more dependent on functioning PQC systems such as the UPS, and autophagy than non-transformed cells (Goldberg, 2007). Indeed, several preclinical studies established that proteasome inhibition has a more severe effect on malignant cells than on normal cells (An et al., 1998; Masdehors et al., 2000; Hideshima et al., 2001; LeBlanc et al., 2002). Thus, proteasome inhibition would overload the cancer cell with protein material and accumulation of ubiquitinated proteins, finally causing cell death (Adams, 2004).

First attempts on using bortezomib, as a single agent PI, showed success in the treatment of relapsed and refractory MM. Various preclinical and clinical trials provided data on significant benefit in patients’ respond and outcome (Orlowski et al., 1998; Hideshima et al., 2001; LeBlanc et al., 2002; Richardson et al., 2005a). The Assessment of Proteasome inhibition for EXtending remissions (APEX) trial confirmed significant benefit in the bortezomib group over the patients treated with dexamethasone in patients with relapsed MM (Richardson et al., 2005b). These findings laid the foundation for the approval of bortezomib by the FDA in 2003. Although thrombocytopenia and peripheral neuropathy were the most frequently associated dose limiting toxicities, the FDA authorized bortezomib for the use in relapsed and refractory myeloma patients who showed no response to two or more prior therapies (Field-Smith et al., 2006). Accordingly, two other substantial drug discoveries were found to have a beneficial effect on MM patients: the immunomodulatory drugs thalidomide and lenalidomide (Dimopoulos et al., 2007; Rajkumar et al., 2008). However, despite all the promising new drug developments and outcomes, a number of patients refractory to prior use of bortezomib, thalidomide, or lenalidomide still only showed poor responses (Kumar et al., 2017). As MM cells have been described to possess an abnormal acetylome, another approach to this group of patients was the implementation of HDACi (Mithraprabhu et al., 2014). In fact, data of preclinical studies demonstrated an anti-proliferative effect of vorinostat, romidepsin, dacinostat and panobinostat resulting in apoptosis of MM cells (Catley et al., 2003; Khan et al., 2004; Campbell et al., 2010; Ocio et al., 2010; Sanchez et al., 2011; Holkova et al., 2012). However, when transferring single agent use of HDACi into clinical trials, only limited effect on MM cells was noted (Richardson et al., 2008; Niesvizky et al., 2011).

Eventually, several preclinical studies postulated synergistic effects of HDACi and proteasomal inhibition, paving the way of combinational therapy of these two agents (Pei et al., 2004; Campbell et al., 2010). The best characterized and coherent explanation of the synergy between PI and HDACi is the dual inhibition of the proteasome and aggresome pathway (Hideshima et al., 2005; Catley et al., 2006). Targeting both of the degradation pathways with bortezomib and HDACi in tumor cells would exponentiate their effect and result in greater accumulation of polyubiquitinated proteins, increased cellular stress and apoptosis. More specifically, despite the fact that proteasome inhibition results in accumulation of ubiquitinated proteins and cell death, malignant cells have shown to evade this life-threatening end by an alternative pathway. Here, malignant cells form aggresomes, engulfing the polyubiquitinated proteins to transport them with the help of HDAC6 via microtubules (Ouyang et al., 2012). Disruption of this alternative pathway was reported using both nonselective and selective HDACi through HDAC6 inhibition thus synergizing with bortezomib and inducing cells to undergo apoptosis in multiple hematologic and epithelial malignancies (Catley et al., 2006; Nawrocki et al., 2006; Heider et al., 2008). In the same vein, the beforementioned observation that pan-HDACi treatment in clinical concentration of human cells led to the formation of amyloid fibrils gave a further proof that HDACi may act synergistically on PQC pathways (Olzscha et al., 2017). On account of the described synergy, dual inhibition could exploit full therapeutic potential of both proteasome and HDAC inhibition.

A hallmark of MM cells is the production of abundant amounts of immunoglobulin which must either be properly folded or degraded. Accordingly, dual disruption in protein degradation seemed to be especially effective in these types of cancer cells (Lee et al., 2003; Obeng et al., 2006). This assumption was substantiated by preclinical data, indicating a combination of PI and HDACi to be an attractive and novel strategy for the treatment of MM (Table 2).

Preliminary data from phase I, II and III studies evaluated success in the treatment regime of panobinostat or vorinostat as HDACi plus bortezomib in patients with relapsed or refractory MM (Badros et al., 2009; San-Miguel et al., 2013). Subsequently, combinational therapy was implemented into clinical settings. Patients with relapsed or refractory MM were examined on the effect of HDACi combined with proteasome inhibition. On the one hand, the phase II VANTAGE trial analyzed vorinostat plus bortezomib (Dimopoulos et al., 2013), whereas on the other hand, the phase II PANORAMA 1 trial tested the combination of panobinostat plus bortezomib and dexamethasone in patients with relapsed and refractory MM (San-Miguel et al., 2011). Despite the fact that VANTAGE displayed prolonged progression-free survival (median PFS of 7.6 vs. 6.8 month) when combining vorinostat and bortezomib, clinical relevance needed to be further examined. The PANORAMA 1 trial was able to show modest overall survival benefit when combinational therapy of panobinostat, bortezomib and dexamethasone was applied (median PFS of 12 vs. 8 month). This led to the approval of panobinostat in 2015 by the FDA. The results of this therapeutic approach were evaluated on bortezomib-refractory patients in the PANORAMA 2 trial (Richardson et al., 2013). According to this trial, it has been proposed that combinational treatment of panobinostat, bortezomib and dexamethasone recaptures response in 34.5% of pre-treated, bortezomib-refractory MM patients. In summary, both results of PANORAMA 1 and 2 are partly coherent with preclinical studies and elucidate considerably the role of panobinostat in combination with bortezomib and dexamethasone, especially in patients with relapsed or bortezomib refractory MM. Furthermore, the results hypothesize HDACi to sensitize patients with bortezomib-resistant MM. Despite the clinical benefit of this combined agent regime, it harbors the danger of poor side effects. Especially the overlapping toxicity profiles make this regime rather toxic. A grade 3 – 4 thrombocytopenia (67%) and gastrointestinal toxicity (diarrhea 25%) indicate a poor safety profile. In order to optimize the safety profile, combinations of panobinostat with second-generation PI were tested at different doses and schedules. Carfilzomib is one example of a second-generation PI and obtained approval by the FDA for the treatment of relapsed and refractory MM, in patients who were given at least two prior therapies (Groen et al., 2019). Combination of panobinostat and carfilzomib was tested in a phase I/II clinical trial in patients with relapsed or refractory MM with promising response rates and an acceptable safety profile (ClinicalTrials.gov identifier: NCT01496118) (Berdeja et al., 2015).

Another idea to address the rather toxic combination of pan-HDACi with PI was to substitute pan-HDACi with class-selective HDACi. In contrast to pan-HDACi, selective class I HDACi rarely induce thrombocytopenia, thus seeming to be more suitable agents. As the key mechanism underlying the synergistic effect of HDACi and PI was mainly explained by HDAC6-dependent aggresome function, it can be argued that class-specific HDACi may still synergize with PI without having a poor safety profile. Therefore, an isoform selective HDAC6 inhibitor, ricolinostat, was introduced in the combinational treatment regime with bortezomib and dexamethasone in relapsed or refractory MM patients (Vogl et al., 2017). This phase I/II study demonstrated that combinational therapy with an isoform selective HDACi shows less severe gastrointestinal, hematologic, and constitutional toxicities in comparison to non-selective HDACi. This raised the idea to test the novel combinational regime in different malignancies. It has been demonstrated that class I HDACi mocetinostat/MGCD0103 has a potent antiproliferative activity in Hodgkin lymphoma (HL) cell lines in an HDAC6-independent manner (Buglio et al., 2010). Regarding these results, mocetinostat was especially interesting to investigate further, as it is a class I HDACi with no effects on HDAC6 (Fournel et al., 2008). The generated data demonstrated that inhibition of class I HDAC by mocetinostat results in an adequate induction of cell death in HL cell lines. On account to that, a broader inhibition of HDACs, including HDAC6, is not needed for a sufficient antiproliferative effect in vitro. Furthermore, they were able to show a synergistic effect of mocetinostat with PI. Mocetinostat induced the expression of various inflammatory cytokines resulting in the activation of NF-κB, which in turn mitigated the killing effect of mocetinostat on tumor cells. As PIs inhibit NF-κB activation, this novel combination would explain how PIs enhance mocetinostat activity, independent of HDAC6. A following phase II trial tested mocetinostat, a class I/IV HDACi for relapsed HL, whereby 85 mg were administered three times per week. 14 of 51 patients (27%) treated with mocetinostat had a complete or partial response whereas only one patient out of 25 (4%) had a partial response on pan-HDACi vorinostat. Single agent use of mocetinostat also induced a reduction in tumor size in more than 4/5 of patients (Younes et al., 2011). Collectively, these data suggest the potential and clinical value of class-specific HDACi in patients with HL. Combination of panobinostat and bortezomib in patients with relapsed or refractory PTCL shows encouraging activity, however displaying a relatively high number of adverse events with 10 out of 25 patients (40%) (Tan et al., 2015).

Noticeably, HDACi and PI have been tested and analyzed in a variety of hematological malignancies. However, their effects are not well investigated in solid tumor malignancies. First attempts in investigating the safety and efficacy of vorinostat and bortezomib were tested 2012 in NSCLC. Here, they examined the two-agent use as induction therapy with an adjacent surgery in patients with NSCLC (Jones et al., 2012). The obtained results showed a decrease in metabolic activity in the tumors. However, due to the short duration of induction treatment, no significant change in tumor size has been observed. A following phase II study testing vorinostat and bortezomib as third-line therapy in patients with advanced NSCLC was terminated at its first temporary analyses due to a lack of anti-tumor activity (Hoang et al., 2014). Nonetheless, they highlighted the relevance of potential biomarkers predicting drug activity and thus driving clinical development.

In another setting, HDACi and PI have been tested in glioblastoma multiforme (GBM) cells, as promising preclinical studies proposed activity against GBM cell lines and glioma models (Eyüpoglu et al., 2005; Ugur et al., 2007; Yin et al., 2007). However, this result was not confirmed in clinical trials. A phase II trial of bortezomib in combination with vorinostat in recurrent glioblastoma had disappointing results and was clinically ineffective (Friday et al., 2012). It should be considered that unlike vorinostat, bortezomib cannot pass an intact blood-brain barrier (BBB) and thus may be the reason for an unsatisfactory result. Since other PIs such as marizomib can cross the BBB, it would eventually be a more beneficial combination with an HDACi in treating tumors beyond the BBB (Gozzetti and Cerase, 2014).

Overall, HDACi in combination with PI showed synergistic effects, which could be validated in several phase I trials in different tumor entities. However, there is still an unmet need of further investigation on molecular mechanisms underlying the combinational treatment regime and especially of the development of predictive biomarkers. These biomarkers (see also section “Evaluating Alterations of PQC Systems: Precision Medicine Upon HDAC Inhibitor Treatment”) would allow clinicians to stratify patients who would benefit from the treatments (Table 2).

Another approach, going for the same train of thought as seen in the combinational regime of HDACi and PI is the substitution of PI with autophagy inhibition (Table 2). As outlined in section “The Role of Protein Quality Control Systems in Cancer Cells,” autophagy represents a hallmark of the PQC as the proteasome does. Preclinical studies showed a context-dependent effect of autophagy in different states of malignant pathogenesis. As outlined in the section about pre-clinical studies, autophagy has a protective function in premalignant cells. While preventing defective cells from proliferating, it hampers the acquisition of additional mutations that would even promote tumor development. However, looking at advanced cancer cells, autophagy can promote mechanism for oncogenesis. Here, it enhances cell survival under stressful conditions in the tumor microenvironment, such as hypoxia and nutrient deprivation (White, 2012). Besides the already existing endogenous stress, autophagy is even further promoted by anti-cancer treatment, leading to additional protection of tumor development (Janku et al., 2011). This could give a rationale for the poor therapeutic efficacy, as the survival of malignant cells is maintained through autophagy (Carew et al., 2007a, 2012; Amaravadi et al., 2011). Recent investigations confirm the diminished effect of therapies due to autophagy, promoting cancer cell survival (Strait et al., 2002; Duan et al., 2005). Thus, inhibition of autophagy represents a novel strategy to augment cancer treatment efficacy.

HDACi have been described to induce autophagy in many clinical trials, but its full therapeutic potential is hampered due to the protective action of autophagy. Subsequently, disruption of autophagy would boost the pro-apoptotic and cytostatic effects of HDACi. In fact, this mode of action was confirmed by preclinical studies in models of imatinib-resistant chronic myeloid leukemia and colon cancer (Carew et al., 2007b, 2010). In 2014, Mahalingam et al. reported about a phase I clinical trial of the autophagy inhibitor hydroxychloroquine (HCQ) in combination with vorinostat in adult patients with advanced refractory solid malignancies (Mahalingam et al., 2014). In the majority of patients, no significant benefit was observed; only renal cell carcinoma patients had a dramatic and durable response to the novel combination of vorinostat plus hydroxychloroquine (HCQ). Besides the deficient results, the authors accentuated the need of predictive biomarker for assessing clinical sensitivity to autophagy inhibitors in order to optimize drug development. In 2016, Patel et al. revived the scheme and performed a single-arm expansion cohort to assess the efficacy, safety and effects on immunity of vorinostat and HCQ in patients with refractory metastatic CRC (Patel et al., 2016). Results implied no substantial benefit over other oral drug as survival showed to be comparable to other oral drugs for refractory CRC including regorafenib (Grothey et al., 2013). Despite this outcome, vorinostat plus HCQ had a favorable toxicity profile and can be discussed as an alternative treatment for refractory CRC. Subsequently, a randomized phase II trial of vorinostat and HCQ versus regorafenib (a tyrosinkinase inhibitor) is now open to enrollment. Alongside, another phase II trial exhibits the therapeutic benefit on vorinostat plus HCQ over regorafenib in chemo-refractory metastatic colorectal cancer. Results implied that survival of the two treatment regimens showed comparable survival (Arora et al., 2019).

Another interesting new idea was to combine HDACi with modulators of molecular chaperones. As outlined in section “The Role of Protein Quality Control Systems in Cancer Cells,” molecular chaperones and particularly HSP90 and HSP70 play not only a role in protein folding, but also in signal transduction and interact in several pathways to maintain cellular protein homeostasis and cell survival (Wiech et al., 1992). HSP90 has been described as a key player to stabilize proteins that are particularly important in cancer cells including BCR-ABL (BCR, breakpoint cluster region; ABL, Abelson), ERB-B2 (erythroblastic oncogene B), proto-oncogene B-Raf (BRAF), AKT Serine/Threonine Kinase (AKT), vascular endothelial growth factor receptor (VEGFR), FMS like tyrosine kinase 3 (FLT3), androgen and estrogen receptors, hypoxia-inducible factor (HIF-1α) and a constantly growing list, affecting a variety of cancer-related functions (Welch and Feramisco, 1982; Powers and Workman, 2006). In some cancerous cell lines, HSP90 has been found in much higher levels compared to normal cells (Kamal et al., 2003) and shows beneficial effects on many oncoproteins (da Rocha Dias et al., 2005; Shimamura et al., 2005). Preclinical studies demonstrated the anti-cancer effect of HSP90 inhibition and suggested its ability could affect several oncogenic signaling pathways simultaneously. Thus, it reduces the likelihood of the tumor acquiring resistance to any single therapeutic pathway and is a major advantage upon other agents (Banerji, 2009). However, HSP90 inhibitors such as 17-AAG have not reached clinical trials beyond phase III, due to minimal effects and toxicity, especially liver toxicity (Hyun et al., 2018). Therefore, the combination with other agents, for instance HDACi, could be a promising alternative, in order to reduce the effective concentration of HSP90 inhibitors. Following this hypothesis, HDAC6 and HSP90 are interactors as HDAC6 can deacetylate HSP90 (Boyault et al., 2007a; Figure 3). Adversely, when inhibiting HDAC6, HSP90 is present in a hyper-acetylated state, losing the bond with the co-chaperone p23 and finally its overall chaperone activity (Kovacs et al., 2005; Aldana-Masangkay and Sakamoto, 2011). This makes the synergistic use a promising anti-cancer treatment strategy (Table 2). Lung cancer has been described to be particularly susceptible to HSP90 inhibition. Overexpressed or mutant ERB-B2 and BRAF are often the driving force in lung cancer development. Interestingly, all of them are degraded with the assistance of HSP90, giving a rationale for the beneficial use of HSP90 inhibitors in lung cancer (Shimamura and Shapiro, 2008; Zismanov et al., 2014). This is now implemented into an ongoing clinical trial with 20 participants aiming to investigate the combined effect of HSP90 inhibitor and PI or HDACi on lung cancer cell fate and ER/Golgi homeostasis (NCT01270399).

This novel combination treatment has not been well investigated in a clinical set-up to this point. Preclinical studies proposed synergistic effects in breast cancer (Rahmani et al., 2003). One phase I trial tested GSK525762C (molibresib besylate) and entinostat in patients with advanced or refractory solid tumors or lymphomas. Results are yet to come.

Overall, epigenetics has become an inevitable part of cancer research. Until now, HDACi have been shown and tested to synergize with a wide range of very different agents. Of particular interest is the synergistic use of HDACi with the inhibition of PQC systems. Herein, progress has been made, implicating that HDACi exhibit their anticancer activity through a multitude of pathways. However, there is still an unmet need for further investigations on detailed mechanistic action of HDACi. The therapeutic effect of HDACi not only depends on the cancer type, but on the stage of cancer, treatment dosage, the individual patient’s biological signature, and other factors. In order to boost the development of HDACi and PQC modulating agents these factors need to be considered. To achieve significant improvement in HDACi therapeutic outcomes, better patient selection and monitoring of biomarkers are strongly required.