The Complexity of the Epigenome in Cancer and Recent Clinical Advances

Published OnlineFirst August 17, 2012; DOI: 10.1158/1078-0432.CCR-12-2037

Clinical Cancer Molecular Pathways Research

Molecular Pathways: The Complexity of the Epigenome in Cancer and Recent Clinical Advances

Mariarosaria Conte1 and Lucia Altucci1,2

Abstract Human cancer is causally linked to genomic and epigenomic deregulations. Epigenetic abnormalities occurring within signaling pathways regulating proliferation, migration, growth, differentiation, transcription, and death signals may be critical in the progression of malignancies. Consequently, identification of epigenetic marks and their bioimplications in tumors represents a crucial step toward defining new therapeutic strategies both in cancer treatment and prevention. Alterations of writers, readers, and erasers in cancer may affect, for example, the methylation and acetylation state of huge areas of chromatin, suggesting that epi-based treatments may require "distinct" therapeutic strategies compared with "canonical" targeted treatments. Whereas anticancer treatments targeting histone deacetylase and DNA methylation have entered the clinic, additional chromatin modification enzymes have not yet been pharmacologically targeted for clinical use in patients. Thus, a greater insight into alterations occurring on chromatin modifiers and their impact in tumorigenesis represents a crucial advance- ment in exploiting epigenetic targeting in cancer prevention and treatment. Here, the interplay of the best known epi-mutations and how their targeting might be optimized are addressed. Clin Cancer Res; 18 (20); 1–9. 2012 AACR.

Background strategies both in cancer treatment and prevention (2). A number of epigenetic deregulations, such as DNA Alterations of writers, readers, and erasers in cancer may methylation, histone modifications, and microRNA-based affect the status of chromatin in vast areas of the epigenome, modulation, have been progressively reported as causally thus suggesting that epi-based treatments may require "dis- involved in tumorigenesis and progression. In the past tinct" therapeutic strategies compared with "canonical" decade, several chromatin-modulating enzymes have been targeted treatments (3). discovered and classified, and their aberrations linked to cancer. The state of chromatin is widely controlled by Chromatin modifiers and cancer specific DNA- and protein-related modifications. Under- Chromatin structure can be remodeled by covalent mod- standing the histone code and its crucial role in biology led ifications of DNA involving methylation of cytosine within to the study of enzymes that "write" these modifications, the CpG islands and a multitude of histone modifications in so-called writers, those that recognize modified chromatin, both their N-terminal and C-terminal tails, or even in their termed readers, and erasers, which are able to remove a globular domains. Histone modifications dynamically specific modification (1). The interpretation of epigenetic change chromatin structure (so-called "epigenetic plastici- changes underlines the phenotypic variability of cells ty"). Histone modifications are involved in the neutraliza- belonging to the same genome. Therefore, identification tion/deneutralization of the positive charges present on of epigenetic marks and their bio-implications in tumors histones and alter chromatin structure by determining an represents a crucial step toward defining new therapeutic on–off state of transcription. In particular, histone acetylation is conducted by histone acetyltransferase enzymes (HAT), an example of writers. HATs acetylate conserved Authors' Affiliations: 1Dipartimento di Patologia Generale, Seconda amino acids on histones through the transfer of an acetyl Universita di Napoli, Vico L. De Crecchio; and 2Istituto di Genetica e group from Acetil-CoA to form e-N-acetyl-lysine, thus neu- Biofisica, Adriano Buzzati Traverso, CNR-IGB, Via P. Castellino, Napoli, Italy tralizing positive cells. Histone acetylation is a posttransla- tional modification linked to transcriptional activation. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Conversely, histone deacetylases (HDAC), an example of erasers, define the removal of acetyl groups from an e-N- Corresponding Author: Lucia Altucci, Dipartimento di Patologia Generale, Seconda Universita di Napoli, Vico L. De Crecchio 7, 80138, Napoli, Italy. acetyl-lysine amino acid on a histone, thus preventing Phone: 39-0815667569; Fax 39-081450169; E-mail: transcription by increasing positive charges of histone tails [email protected] and thus determining high-affinity binding with DNA. The doi: 10.1158/1078-0432.CCR-12-2037 fact that modulation by writers and erasers can also involve 2012 American Association for Cancer Research. nonhistone substrates adds a further level of complexity (4).

www.aacrjournals.org OF1

Conte and Altucci

Therefore, a great deal of attention has been focused on leukemia (CLL; 17, 19, 21, 22) have been reported to chromatin-modifying enzymes to define their deregulation display HDAC3 overexpression. The class IIa histone dea- in cancer and their potential as therapeutic targets. cetylase HDAC4 is expressed in a tissue-specific manner and promotes the growth of colon cancer cells through repres- HATs and cancer sion of the cell-cycle regulator p21 (23). HDAC5 down- HATs belong to one of two main categories. A-type HATs regulation has been reported in lung cancer (24), although acetylate nucleosomal histones within chromatin, whereas its overexpression has been found in colon cancer (25). B-type HATs play a much broader role in the cell. A-type Downregulated expression levels of HDAC6 have been HATs are classified into 4 families sharing sequence homol- observed in lymphoma (26), whereas higher expression ogy within the HAT domain and include Gcn5/P300/CBP- levels are associated with oral squamous cell cancer (27). associated factor (PCAF; ref. 5), MYST (6), p300/CBP (7), The role of HDAC8 has been investigated in CLL (chronic and Rtt109, the latter reported to be mycotic specific (8). lymphoid leukemia) in children (28). Of the class III In prostate cancer both p300 and CREB-binding protein HDACs, SIRT1 is involved in carcinogenesis and above all (CBP) are overexpressed, thus altering androgen receptor– in age-related neoplasms. In particular, sirtuins are corre- responsive gene modulation. CBP is also involved in trans- lated to aging, cancer, and stress response. SIRT1 overex- location t(8;16) in which monocytic leukemia zinc finger pression has been found in prostate, colon, and skin cancers protein (MOZ), an acetyltransferase localized on 8p11, is as well as in acute myeloid leukemia (AML; 29). In addition fused to CBP (9). Mutations in p300 (more frequently to SIRT1, other sirtuins (such as SIRT4 and 7) are linked to C-terminal truncations) have been found in many tumors cancer development (30). Conversely, low levels of SIRT2 such as breast, pancreatic, colorectal, gastric, cervical, and have been observed in gastric carcinoma and in gliomas. For ovarian cancers (10–12). Recently, downregulation of some sirtuins, such as SIRT3, the scenario is complicated by PCAF has been correlated to gastric carcinoma progression the fact that both upregulation and downregulation have linked to poor clinical outcome. Mutation in Tip60 is been reported in different forms of breast cancer. SIRT4 loss associated with the development of prostate cancer through may contribute to diabetes, a major risk factor for cancer. deregulation of DNA repair and resistance to apoptosis. SIRT6, which also displays ADP-ribosyltransferase activity, PCAF and Gcn5 are overexpressed in diseases of the central is widely overexpressed in brain and skeletal muscle. Final- nervous system in pediatric patients and Wilms tumors ly, SIRT7 promotes active transcription of rRNA genes and (13). Rtt109, correlated to modifications of histones H3 in lower levels of this enzyme have been found in nonproli- K9, K27, and K56, plays a critical role in nucleosome ferating tissues such as heart, brain, and skeletal muscle assembly (Supplementary Table S1 and Fig. S1). (Table 1).

Global (de)acetylation, HDACs, and cancer PRMTs, KMTs, KDMs, and cancer The balance between HDAC and HAT activities plays a Histones are methylated by enzymes including arginine crucial role in regulating gene expression. Future studies will methyltransferases (PRMT) and lysine methyltransferases be needed to verify whether the modulation of chromatin (KMT). The presence or absence of methyl marks on specific acetylation is causally linked to cancer development or if it is histones is crucial for gene expression regulation and has caused by complex epi-deregulations. Whether cancers implications in carcinogenesis. PRMT1 is an important characterized by global alterations of chromatin acetylation component of mixed-lineage leukemia (MLL) oncogenic state may benefit (and how) from an epi-based therapeutic fusion proteins (31). PRMT6 is responsible for H3R2 (his- approach is an issue that still needs to be addressed further tone H3 Arginine 2) methylation, counteracting H3K4me3 (Supplementary Table S2). HDACs play a key role in gene (histone H3 lysine 4 trimethylation) deposition. High levels regulation and therefore in human cancers, such as leuke- of PRMT1 have been observed in breast and colon cancers mia (14). HDACs are divided into 4 classes: class I HDACs (32). PRMT2 can interact with estrogen receptor and acts as (HDAC1, 2, 3, and 8), which are homologous to Saccharo- a strong coactivator of androgen receptor. PRMT3 is myces cerevisae Rpd3; class II (further divided into class IIa involved in the regulation of protein synthesis, whereas and IIb) HDACs (HDAC4, 5, 6, 7, 9, and 10), which are PRMT4 (CARM1, coactivator-associated arginine methyl- homologous to yeast Hda1; class III HDACs (sirtuins) transferase1) is known to control the arginine-regulated including enzymes homologous to yeast Sir2 involved in mechanism of transcription. CARM1 methylates histone modulation of longevity, metabolism, and physiology; H3, and the mutation in the presumed binding domain class IV comprising only HDAC11, which shares homology decreases methyltransferase and p160 coactivator regula- with class I and II. HDACs are overexpressed in several tion (33). CARM1 is overexpressed in breast tumors and is cancers. In particular, HDAC1 is highly expressed in many essential for estrogen-induced cell-cycle progression (34). malignancies including gastric, colorectal, hepatic, breast, PRMT5 is highly expressed in a wide variety of lymphoma and pancreatic cancer (15–17). HDAC2 has been found and leukemia cells as well as in gastric carcinoma and mutated in colon cancer and is overexpressed in esophageal, immortalized fibroblast cells (35). prostate, and gastrointestinal carcinomas (18–20). Many Many KMTs are strongly associated with cancer. Lysine other cancers associated with poor prognosis, such as pros- methylation occurs in a very large number of histones, tate, gastric, colorectal cancer, and chronic lymphocytic deposited by KMTs and removed by lysine demethylases

OF2 Clin Cancer Res; 18(20) October 15, 2012 Clinical Cancer Research

Cancer Epigenome and Therapeutic Strategies

(KDM). EZH2, an H3K27 KMT and polycomb repressive HDAC inhibitors are therefore considered a potential strat- complex 2 component, is highly expressed in several solid egy to reverse epigenetic aberrations associated with cancer, tumors such as primary prostate cancer (36) and in pro-B and many compounds have been tested in clinical trials cells (37). Overexpression of SUV39H1/2 (KMT1A/B) has (46). For an in-depth description of the role of HDAC been reported in dietary-induced tumors (38) and in colon inhibitors in cancer we refer the reader to refs. 47–49 and cancer cells. The KMT G9a contributes to histone H3 lysine 9 references therein. Whether isotype-selective HDAC inhi- (H3K9) dimethylation, involved in suppressor-gene silenc- bitors offer greater therapeutic benefits (or lower adverse ing. G9a is overexpressed in various cancers such as leuke- effects) than broadly acting HDAC inhibitors (pan HDAC mia, prostate, and lung cancer as well as hepatocellular inhibitors) remains one of the issues to be addressed in the carcinoma (39). KMT1D (Eu-HMTase1) is overexpressed in clinic (Supplementary Table S3 and Fig. S1). gland tumors (40), whereas SETDB1/ESET cooperates with DNA methyltransferase in the silencing of promoter regions HAT inhibitors in tumors via trimethylation of H3K9. Numerous muta- Few and poorly validated small molecules modulating tions and rearrangements of MLL1 (KMT2A) have been HATs are currently available. In the past decade, 2 substrates observed in leukemogenesis. MLL4 (KMT2D) is involved analogous to peptide-CoA conjugates, Lys-CoA and H3- in liver oncogenesis in hepatitis B patients. In addition, CoA-20, have been identified as powerful p300 and PCAF misregulation of histone demethylases also causes or con- inhibitors. However, their metabolic instability precludes tributes to cancer. In breast carcinoma, the downregulation their use as anticancer drugs. Other PCAF inhibitors include of KDM1 (LSD1) is correlated to the onset of metastasis. isothiazolones and analogs that act as specific acetylation The aberrant regulation of jumonji domain demethylases inhibitors in a dose- and time-dependent manner, and their has been found in various cancer cell lines. KDM2B anticancer properties have been studied in liver cancer cells. (JHDM1B/FBXL11) abolishes the dimethylation state of Polyisoprenylated benzophene derivates (PBD) have been histone H3 lysine 36 dimethylation (H3K36me2) or his- proposed as candidates for HAT modulation. Garcinol is a tone H3 lysine 4 trimethylation (H3K4me3) by causing the B-type PBD active against viruses, bacteria, gastric ulcers, downregulation of several proteins involved in the cell cycle and cancer, such as colon adenocarcinoma (50). Garcinol such as p14, p15, and p16 in T-cell lymphomas. The KDM5 derivates based on iso-garcinol have been synthesized. family, including RBP2 (KDM5A), PLU1 (KDM5B), and These include LTK-13, -14, and the disulphonyl-substituted SMCX (KDM5C), is overexpressed in a wide variety of derivate LTK-19. Another promising HAT inhibitor is ana- cancers such as gastric, cervical, lung, breast, prostate, and cardic acid, the main component of cashew nut shell liquid. kidney cancer as well as leukemias. Another family of lysine This compound is able to reduce breast cancer cell prolif- demethylases, KDM8 or JMJD5, targets H3K36me2. This eration by inhibiting ERa-DNA binding (51). Gcn5-specific enzyme is overexpressed in breast, thyroid, adrenal, blad- inhibitors, Y-butyrolactones, have also been synthesized. der, and liver cancers (41) and plays a key role in modu- Some studies suggest that Gcn5 inhibition mediated by lating cell proliferation (Fig. 1 and Table 1). these compounds occurs in vitro in several cancers such as leukemia, melanoma, ovarian, renal, prostate, and breast cancers as well as in colon cancer cells (52). Curcumin, an DNA methylation and cancer inhibitor of p300, regulates tumor suppressor pathways and DNA methylation has frequently been described as a triggers mitochondrial-mediated death in tumors. Curcu- static silencing event. DNA methylation is not, however, min has been reported to be active in the prevention and static, as recently shown by the variable 5mC-oxidation treatment of kidney, lung, ovarian, cervical, and liver can- kinetics at distinct genomic/functional loci and by the fact cers. The development of HAT inhibitors is less advanced that 2HG (product of IDH-1 and -2) mutation has been than that of HDAC inhibitors, probably because HATs found in leukemia (42). are less overexpressed in cancer and more often mutated In recent years genome-scale mapping of methylation has compared with HDACs. Nevertheless, the identification revealed that DNA methylation is involved in different of selective HAT inhibitors for mutated forms of HAT in (epi)-genetic settings. Methylation is catalyzed by DNA cancer may offer a valid therapeutic strategy with expected methyltransferases (DNMT). Several cancers are associated features of tumor specificity. with quantitative or positional alterations of DNA methylation. Furthermore, DNMT3a mutations have been found PRMT, KMT, and KDM inhibitors in AML (43). Thus, DNA methylation is the subject of PRMT alterations correlate with many cancers. Hence, intense studies aimed at better understanding alterations molecules able to modulate methyltransferase activity are involved in the transformation of normal cells into pro- desirable for cancer treatment. Chaetocin, a fungal myco- cancerous cells (44, 45). toxin, is an SUV39H1 methyltransferase inhibitor reported to exert antimyeloma activity in interleukin 6-dependent Clinical-Translational Advances and -independent myeloma cell lines, and in vivo (53). HDAC inhibitors BIX01294 was the first small-molecule inhibitor of G9a Much attention has been focused on HDAC inhibitors and GLP. BIX01294 has been reported to inhibit the histone mainly because HDACs are often overexpressed in cancer. methyltransferase EHMT2, which acts as a corepressor for

www.aacrjournals.org Clin Cancer Res; 18(20) October 15, 2012 OF3

Conte and Altucci

PRMT, HAT KMT inhibitors inhibitors

Histone methylation Histone acetylation

SUV39H1/2, MLL1/2, Ash1 PRMT6 PRMT1 Gcn5, CBP, p300, PCAF, MOZ/MYST3, G9a, SETDB1 Set1, Smyd3 MORF/MYST4, Tip60, Rtt109

R2 R3 K5 K36 G9a, SUZ12 K4 K8 K27 EED-EZH2 K15 K9 K12 K9 K12 K5 K14 K27 Set2 K16 K18 K23 H1 K12 K36 H3 H4 H2A H2B

Writers H2B H2A K36 Erasers K9 Blockers K4 K36 H4 H3 K14 K5 K12 K56 K9 K20 H1 K8 K16

KDM2B/JHD KDM1/LSD1 KDM8/JMJD5 SET8/SUV4 HDAC1, HDAC2, HDAC3. SIRT1 M1B

Histone demethylation Histone deacetylation

KDM HDAC inhibitors inhibitors

Figure 1. Schematic representation of histone acetylation and methylation: chromatin marks and epi-enzyme deregulation in cancer. Chromatin enzymes able to deposit or erase an epigenetic mark are indicated as writers (teal blue) and erasers (purple), respectively. Epi-drugs able to inhibit the activity of chromatin enzymes are indicated as blockers (brown). For histone methylation: Ash1, absent, small, or homeotic discs 1G9a; EED-EZH2, enhancer of zeste homolog 2; EHMT2, euchromatic histone-lysine N-methyltransferase 2; MLL1/2, lysine N-methyltransferase 2; PRTMT1 and PRMT6, protein arginine N-methyltransferase 1 and 6; Set1 and Set 2, spin echo T1 and T2 sequences; SETDB1, SET domain bifurcated 1; Smyd3, SET domain-containing protein with histone methyltransferase activity on histone H3 K4; SUV39H1/2, suppressor of variegation 3 to 9 (Drosophila) homolog 1/2; SUZ12, suppressor of zeste 12 protein homolog. For histone acetylation: CBP, CREB binding protein; Gcn5, general control of amino-acid synthesis, yeast, homolog-like 2; p300, protein 300; MOZ, monocytic leukemia zinc finger protein; MYST3 and MYST4, MYST histone acetyltransferase (monocytic leukemia) 3 and 4; PCAF, p300/CBP-associated factor; Rtt109, regulator of Ty1 transposition protein 109; Tip60, HIV1 Tat interacting protein. For histone demethylation: JKDM8, jumonji domain of human lysine-specific demethylase 8; JMJD5, jumonji domain-containing protein 5; KDM1 and KDM2B, lysine-specific demethylase 1 (LSD1 and LSD2B); M1B, methylation-inhibited binding protein 1; SET8, SET domain-containing protein 8; SUV4, suppressor of variegation 4 homolog 2. For histone deacetylation: HDAC1, HDAC2, and HDAC3; SIRT1, silent information regulator 1.

OF4 Clin Cancer Res; 18(20) October 15, 2012 Clinical Cancer Research

Cancer Epigenome and Therapeutic Strategies

Table 1. HDACs, SIRTs, HMTs, and KDMs in cancer

Functions and alterations in cancer HDAC class I HDAC1 Downregulated in colorectal primary tumors; upregulated in breast, prostate, gastric, and hepatic cancers HDAC2 Truncating mutation in coion, gastic, and endometrial cancers; overexpressed in prostate and gastrointestinal cancers HDAC3 Overexpressed in chronic lymphocytic leukemia HDAC8 Target for neuroblastoma differentiation

HDAC class IIa HDAC4 Downregulated in lung carcinoma and overexpressed in colon, prostate, and breast cancers HDAC5 Downregulated in lung cancer and upregulated in colon diseases HDAC7 High expression in childhood acute lymphoblastic leukemia (ALL) and colorectal cancer HDAC9 Overexpressed in medulloblastoma

HDAC class IIb HDAC6 Downregulated in lymphoma; high expression in oral squamous cancer HDAC10 Reduced expression affects the prognosis of lung cancers

HDAC class III SIRT1 Upregulated in AML, prostate, breast, and colon cancers; downregulated in colorectal cancer SIRT2 Reduced expression in human brain tumoral cells SIRT3 Is a promoter of cell proliferation and survival in oral cancer carcinogenesis SIRT4 Role in downregulating insulin secretion SIRT5 Overexpressed in pancreatic cancer SIRT6 Its loss contributes to the accumulation of mutations SIRT7 Increased expression in breast cancer; low levels in heart, brain, and skeletal muscle

HDAC class IV HDAC11 Regulates OX40 ligand expression in Hodgkin lymphoma

Main HMTs and KDMs PRMT1 Overexpressed in breast and bladder cancers PRMT2 Is associated with ERa and is upregulated in breast cancer PRMT4 (CARM1) Overexpressed in breast and prostate cancers PRMT5 Involved in the mechanisms of apoptosis; overexpressed in lymphoma PRMT6 Overexpressed in prostate and cervical cancers KMT1 A/B (SUV39 H1/2) Overexpressed in colorectal cancer G9a (KTM1C) Overexpressed in leukemia and lung cancer KMT1D (Eu-HMTase1) Overexpressed in gland tumors SETDB1/ESET (KMT1E) Cooperation with DNA methyltransferase silencing of promoter regions in tumor cells KMT2A (MLL1) Involved in leukemogenesis mutations KMT2D (MLL4) Related to liver carcinogenesis SMYD2 Involved in maintaining an undifferentiated status of ALL-AF9–induced AML KDM1 (LSD1) Correlates with the production of metastasis JHDM1A (KDM2A) Upregulated in colorectal cancer cells JMJD2A (KDM4A) Involved in prostate cancer progression specific transcription factors and is strongly overexpressed Tranylcypromine (trans-2-phenylcyclopropylamine) and in bladder carcinomas (54). A large virtual screening effort its analogues are among the best known LSD1 (or KDM1) has been carried out to identify PRMT inhibitors using the inhibitors. Tranylcypromine has been proposed for the ChemBridge compound collection containing more than treatment of sarcomas as well as fibrous and peripheral 300,000 compounds. Among these, acyl derivatives of p- nerve sheath tumors (56). Furthermore, the therapeutic aminosulfonamides and dapsone have been selected. In potential of these 2 inhibitors has been reported for the particular, dapsone has been suggested for the treatment of treatment of promyelocytic leukemia (57). The KMT inhib- glioblastoma (55). Intensive efforts are currently ongoing itor 3-Deazaneplanocin A (DZNep) interferes with the for the identification of novel selective PRMT inhibitors. polycomb-repressive complex 2 and induces apoptosis in

www.aacrjournals.org Clin Cancer Res; 18(20) October 15, 2012 OF5

Conte and Altucci

cancer cells, for example in AML. Moreover, inhibition of of well-studied marks such as histone methylation on H3K4 EZH2 by DZNep reduces proliferation in breast cancer cell or DNA methylation. Only in a few cases has a "true" lines (58). In AML, cotreatment with DNZep and panobi- hierarchy been successfully established, such as the require- nostat, an HDAC inhibitor, exerts an apoptotic effect on ment of H2B ubiquitylation for the deposition of the active primary leukemia cells but not on normal cells (59). histone mark H3K4me3 (65). Mutual exclusiveness has been reported for repressive DNA methylation and histone DNMT inhibitors H3 lysine 27 trimethylation (H3K27me3) on CpG-islands The Food and Drug Administration has approved the (66). A well-known example of hierarchy is the dependence DNMT inhibitors 5-azacytidine (azacytidine) and 5-aza-2- of 5-hydroxymethylation of cytosines on 5-methylcytosine deoxycytidine (decitabine) for myelodysplastic syndromes as its substrate. (60). Decitabine has also been used to force reexpression of Translating our current knowledge from bench to bed- silenced estrogen receptor in triple-negative breast cancer. side will involve identifying and developing new forms of Another important DNMT inhibitor is zebularine, which is "targeted" treatment, where large areas of chromatin within selectively incorporated into malignant cells but not into cancer cells are likely to be influenced by the forced mod- normal cells. Cancers such as ovarian and cervical carcino- ification of their epigenetic status. This consideration should mas can be treated with the DNMT inhibitor hydralazine lead to a reevaluation of the therapeutic scheme for existing (61). Besides nucleoside DNMT inhibitors, nonnucleoside epi-based cancer treatments as well as to a cautious patient targeted molecules directly inhibiting DNMTs have been stratification currently complicated by the presence of very developed. Among these, the phosphorothioate antisense few, if any, epi-biomarkers of response to treatment and oligonucleotide MG98, currently being investigated in a prognosis. For example, only the predictive power of HR23B phase I study, has been tested in patients with high-risk for clinical response to HDAC inhibitors in cutaneous T- myelodysplasia and AML (62). RG108 displays demethy- cell lymphoma seems to be confirmed (67). Whether within lating activity comparable with that of zebularine in lym- this complex framework the anticancer action of epi-based phoid, myeloid, and colorectal cancers. The quinoline- modulators can be linked to "pure" epi-effects alone and/or based compound, SGI-1027, inhibits DNMT1, DNMT3a, to what extent nonepigenetic action needs to be evaluated and DNMT3b and has been proposed for hepatocellular, remains unclear. Furthermore, the fact that apparently cervical, prostate, and breast cancer in vitro (63). Finally, the opposite deregulations of the same epi-target have been antibiotics mithramycin A and nanaomycin have recently reported complicates the interpretation of the cancer epi- been reported to inhibit DNMT3B reactivation (64). genome. These contradictory findings might be explained by the hypothesis that opposite quantitative epi-alterations Targeting the complexity of epigenetic modifications may lead, in some cases, to similar readouts in cancer, Misregulation of the physiologic pattern of chromatin causing or contributing to a complex disorder of chromatin modifications (mainly DNA methylation and histone mod- (and related gene expression) and its potential combinato- ifications) can induce the promotion and progression of rial patterns. As things stand, further knowledge is needed to cancer. Chromatin modifications may be regulated by wri- mine the real value of some of the epi-target deregulations in ters and erasers and interpreted by readers. These events cancer as well as to identify differences and specificities both give rise to a complex epi-modulated scenario, where both for cancer disease and epi-modifiers. An additional and single modifications/single enzymes and the interplay with largely unexplored level of complexity is presented by the other histone marks/enzymatic complexes (possibly mutu- modulation of nonhistone targets by small epi-molecules ally exclusive or additive) need to be taken into account to and by the fact that very few epi-based treatments (mainly achieve a better understanding of epigenetic deregulation in HDAC inhibitors and DNA demethylating agents) have malignancies. In recent years a large number of high- actually entered the clinic. Lessons learned from the use of throughput screening-based studies in cancer models have HDAC inhibitors in cancer treatment suggest, for instance, highlighted both the importance of specific histone marks that modulation of histone acetylation should be consid- and epi-enzymes in cancer and their interconnection with ered as a readout of the effects of treatment and not, as other marks in wide areas of chromatin, suggesting the initially proposed, as a parameter of clinical response to importance of the positioning (potentially mutually exclu- treatment. Thus, one parameter of choice might be repre- sive) of marks and chromatin modifiers in cancer patho- sented by the quantitatively unbalanced presence of writers/ genesis and progression. Such scenarios point to the exis- readers/erasers (overexpression or silencing) or by a specific tence of a very complex "code" of mechanisms and their mutation in one of these enzymes. However, although the deregulation in cancer, underlining the difficulties involved selection of patients on the basis of HDAC expression levels in evaluating these mechanisms as therapeutic targets. might prove useful, at least in in vitro settings, this parameter Additional complexity arises from the difficulty in deter- does not always seem to be predictive of a better response. mining a hierarchy in epigenetic mark deposition. The fact The scenario might, however, be different when epi- that new modifications are still being uncovered makes the enzymes are mutated in cancer. This type of epigenetic combinatorial repertoire of epigenetic marks appear end- modification is currently under investigation to identify less. Currently, detailed knowledge of a hierarchy—mutual and validate biomarkers and may allow patient stratifica- exclusiveness or interdependence—is restricted to a handful tion. It also offers the exciting possibility of synthesizing

OF6 Clin Cancer Res; 18(20) October 15, 2012 Clinical Cancer Research

Cancer Epigenome and Therapeutic Strategies

selective small molecules able to modulate only the mutated promising approach (68), as could the creation of hybrid enzyme, thus acquiring features of tumor-selective action. molecules able to act broadly on a class of epi-modifiers and Interestingly, different categories of enzymes appear to act to simultaneously mark a specific non–epi-target within differently: HDACs, for example, are more often quantita- cancer cells. This last approach, which still needs to be fully tively modulated in cancer (with the exception of HDAC2 validated, might "remodulate" chromatin in a more tar- mutation), whereas HATs seem more frequently mutated. geted manner. However, the implications of this difference still need to be investigated. Moreover, a further level of complexity is Disclosure of Potential Conflicts of Interest added by new discoveries continuously being made in this No potential conflicts of interest were disclosed. field: Novel chromatin marks are identified and the efforts Authors' Contributions for mining these targets (alone and within the context of Conception and design: L. Altucci others) may rapidly change our view. For example, hydro- Writing, review, and/or revision of the manuscript: M. Conte, L. Altucci xymethyl cytosine, along with its modulation, is at present Study supervision: L. Altucci the focus of discussions aimed at understanding its mode of Acknowledgments action and its potential role in cancer. The authors apologize to authors whose work could not be cited due to Finally, it is debated whether more selective or broader- restrictions in the number of references. The authors thank C. Fisher for acting chromatin modulators should be chosen as an linguistic editing of the manuscript. approach to epi-treatment in cancer. Although mutated targets might benefit from a selective epi-drug approach Grant Support This work was supported by EU: Blueprint (282510), ATLAS (221952); (better if active exclusively on the mutant), a broad mod- Epigenomics Flagship Project EPIGEN (MIUR-CNR); the Italian Association ulator might present greater advantages in the case of for Cancer Research (AIRC 11812); Italian Ministry of University and concomitant alterations of different chromatin modifiers Research (PRIN_2009PX2T2E_004); PON002782; PON0101227. and marks. Multiple epi-modulators (targeting more than Received June 20, 2012; revised August 6, 2012; accepted August 8, 2012; one class of enzymes, for instance) may also represent a published OnlineFirst August 17, 2012.

References 1. Janzen WP, Wigle TJ, Jin J, Frye SV. Epigenetics: tools and technol- 14. Timmermann S, Lehrmann H, Polesskaya A, Harel-Bellan A. Histone ogies. Drug Discov Today Technol 2010;7:e59–65. acetylation and disease. Cell Mol Life Sci 2001;58:728–36. 2. Wilting RH, Dannenberg JH. Epigenetic mechanisms in tumorigenesis, 15. Choi JH, Kwon HJ, Yoon BI, Kim JH, Han SU, Joo HJ, et al. Expression tumor cell heterogeneity and drug resistance. Drug Resist Updat profile of histone deacetylase 1 in gastric cancer tissues. Jpn J Cancer 2012;15:21–38. Res 2001;92:1300–4. 3. Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to 16. Rikimaru T, Taketomi A, Yamashita Y, Shirabe K, Hamatsu T, therapy. Cell 2012;150:12–27. Shimada M, et al. Clinical significance of histone deacetylase 1 4. Kouzarides T. Chromatin modifications and their function. Cell 2007; expression in patients with hepatocellular carcinoma. Oncology 128:693–705. 2007;72:69–74. 5. Liu X, Wang L, Zhao K, Thompson PR, Hwang Y, Marmorstein R, et al. 17. Weichert W, Roske A, Gekeler V, Beckers T, Stephan C, Jung K, et al. The structural basis of protein acetylation by the p300/CBP transcrip- Histone deacetylases 1, 2 and 3 are highly expressed in prostate tional coactivator. Nature 2008;451:846–50. cancer and HDAC2 expression is associated with shorter PSA relapse 6. Sapountzi V, Cote J. MYST-family histone acetyltransferases: beyond time after radical prostatectomy. Br J Cancer 2008;98:604–10. chromatin. Cell Mol Life Sci 2011;68:1147–56. 18. Ropero S, Fraga MF, Ballestar E, Hamelin R, Yamamoto H, Boix- 7. Bowers EM, Yan G, Mukherjee C, Orry A, Wang L, Holbert MA, et al. Chornet M, et al. A truncating mutation of HDAC2 in human cancers Virtual ligand screening of the p300/CBP histone acetyltransferase: confers resistance to histone deacetylase inhibition. Nat Genet 2006; identification of a selective small molecule inhibitor. Chem Biol 2010; 38:566–9. 17:471–82. 19. Fritzsche FR, Weichert W, Roske A, Gekeler V, Beckers T, Stephan C, 8. Stavropoulos P, Nagy V, Blobel G, Hoelz A. Molecular basis for the et al. Class I histone deacetylases 1, 2 and 3 are highly expressed in autoregulation of the protein acetyl transferase Rtt109. Proc Natl Acad renal cell cancer. BMC Cancer 2008;8:381. Sci U S A 2008;105:12236–41. 20. Chang HH, Chiang CP, Hung HC, Lin CY, Deng YT, Kuo MY. Histone 9. Sobulo OM, Borrow J, Tomek R, Reshmi S, Harden A, Schlegelberger deacetylase 2 expression predicts poorer prognosis in oral cancer B, et al. MLL is fused to CBP, a histone acetyltransferase, in therapy- patients. Oral Oncol 2009;45:610–4. related acute myeloid leukemia with a t(11;16)(q23;p13.3). Proc Natl 21. Krusche CA, Wulfing P, Kersting C, Vloet A, Bocker W, Kiesel L, et al. Acad Sci U S A 1997;94:8732–7. Histone deacetylase-1 and -3 protein expression in human breast 10. Iyer NG, Ozdag H, Caldas C. p300/CBP and cancer. Oncogene 2004; cancer: a tissue microarray analysis. Breast Cancer Res Treat 2005; 23:4225–31. 90:15–23. 11. Muraoka M, Konishi M, Kikuchi-Yanoshita R, Tanaka K, Shitara N, 22. Moreno DA, Scrideli CA, Cortez MA, de Paula Queiroz R, Valera ET, Chong JM, et al. p300 gene alterations in colorectal and gastric daSilvaSilveiraV,etal.Differential expression of HDAC3, HDAC7 carcinomas. Oncogene 1996;12:1565–9. and HDAC9 is associated with prognosis and survival in child- 12. Bryan EJ, Jokubaitis VJ, Chamberlain NL, Baxter SW, Dawson E, hood acute lymphoblastic leukaemia. Br J Haematol 2010;150: Choong DY, et al. Mutation analysis of EP300 in colon, breast and 665–73. ovarian carcinomas. Int J Cancer 2002;102:137–41. 23. Wilson AJ, Byun DS, Nasser S, Murray LB, Ayyanar K, Arango D, et al. 13. Armas-Pineda C, Arenas-Huertero F, Perezpenia-Diazconti M, Chico- HDAC4 promotes growth of colon cancer cells via repression of p21. Ponce de Leon F, Sosa-Sainz G, Lezama P, et al. Expression of PCAF, Mol Biol Cell 2008;19:4062–75. p300 and Gcn5 and more highly acetylated histone H4 in pediatric 24. Osada H, Tatematsu Y, Saito H, Yatabe Y, Mitsudomi T, Takahashi T. tumors. J Exp Clin Cancer Res 2007;26:269–76. Reduced expression of class II histone deacetylase genes is

www.aacrjournals.org Clin Cancer Res; 18(20) October 15, 2012 OF7

Conte and Altucci

associated with poor prognosis in lung cancer patients. Int J Cancer 46. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone 2004;112:26–32. deacetylase inhibitors. Nat Rev Drug Discov 2006;5:769–84. 25. Ozdag H, Teschendorff AE, Ahmed AA, Hyland SJ, Blenkiron C, 47. Dell'aversana C, Lepore I, Altucci L. HDAC modulation and cell death in Bobrow L, et al. Differential expression of selected histone modifier the clinic. Exp Cell Res 2012;318:1229–44. genes in human solid cancers. BMC Genomics 2006;7:90. 48. Venugopal B, Evans TR. Developing histone deacetylase inhibitors as 26. Gloghini A, Buglio D, Khaskhely NM, Georgakis G, Orlowski RZ, anti-cancer therapeutics. Curr Med Chem 2011;18:1658–71. Neelapu SS, et al. Expression of histone deacetylases in lymphoma: 49. Dickinson M, Johnstone RW, Prince HM. Histone deacetylase inhibi- implication for the development of selective inhibitors. Br J Haematol tors: potential targets responsible for their anti-cancer effect. Invest 2009;147:515–25. New Drugs 2010;28 Suppl 1:S3–20. 27. Sakuma T, Uzawa K, Onda T, Shiiba M, Yokoe H, Shibahara T, et al. 50. Hong J, Kwon SJ, Sang S, Ju J, Zhou JN, Ho CT, et al. Effects of Aberrant expression of histone deacetylase 6 in oral squamous cell garcinol and its derivatives on intestinal cell growth: inhibitory effects carcinoma. Int J Oncol 2006;29:117–24. and autoxidation-dependent growth-stimulatory effects. Free Radic 28. Amann JM, Nip J, Strom DK, Lutterbach B, Harada H, Lenny N, et al. Biol Med 2007;42:1211–21. ETO, a target of t(8;21) in acute leukemia, makes distinct contacts with 51. Schultz DJ, Wickramasinghe NS, Ivanova MM, Isaacs SM, Dough- multiple histone deacetylases and binds mSin3A through its oligo- erty SM, Imbert-Fernandez Y, et al. Anacardic acid inhibits estrogen merization domain. Mol Cell Biol 2001;21:6470–83. receptor alpha-DNA binding and reduces target gene transcription 29. Carafa V, Nebbioso A, Altucci L. Sirtuins and disease: the road ahead. and breast cancer cell proliferation. Mol Cancer Ther 2010;9: Front Pharmacol 2012;3:4. 594–605. 30. Ashraf N, Zino S, Macintyre A, Kingsmore D, Payne AP, George WD, 52. Selvi BR, Mohankrishna DV, Ostwal YB, Kundu TK. Small molecule et al. Altered sirtuin expression is associated with node-positive breast modulators of histone acetylation and methylation: a disease perspec- cancer. Br J Cancer 2006;95:1056–61. tive. Biochim Biophys Acta 2010;1799:810–28. 31. Cheung N, Chan LC, Thompson A, Cleary ML, So CW. Protein 53. Isham CR, Tibodeau JD, Jin W, Xu R, Timm MM, Bible KC. Chae- arginine-methyltransferase-dependent oncogenesis. Nat Cell Biol tocin: a promising new antimyeloma agent with in vitro and in vivo 2007;9:1208–15. activity mediated via imposition of oxidative stress. Blood 2007; 32. Papadokostopoulou A, Mathioudaki K, Scorilas A, Xynopoulos D, 109:2579–88. Ardavanis A, Kouroumalis E, et al. Colon cancer and protein argin- 54. Cho HS, Kelly JD, Hayami S, Toyokawa G, Takawa M, Yoshimatsu M, ine methyltransferase 1 gene expression. Anticancer Res 2009;29: et al. Enhanced expression of EHMT2 is involved in the proliferation of 1361–6. cancer cells through negative regulation of SIAH1. Neoplasia 2011;13: 33. Chen D, Ma H, Hong H, Koh SS, Huang SM, Schurter BT, et al. 676–84. Regulation of transcription by a protein methyltransferase. Science 55. Kast RE, Scheuerle A, Wirtz CR, Karpel-Massler G, Halatsch ME. The 1999;284:2174–7. rationale of targeting neutrophils with dapsone during glioblastoma 34. Frietze S, Lupien M, Silver PA, Brown M. CARM1 regulates estrogen- treatment. Anticancer Agents Med Chem 2011;11:756–61. stimulated breast cancer growth through up-regulation of E2F1. Can- 56. Schildhaus HU, Riegel R, Hartmann W, Steiner S, Wardelmann E, cer Res 2008;68:301–6. Merkelbach-Bruse S, et al. Lysine-specific demethylase 1 is highly 35. Pal S, Vishwanath SN, Erdjument-Bromage H, Tempst P, Sif S. Human expressed in solitary fibrous tumors, synovial sarcomas, rhabdomyo- SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and sarcomas, desmoplastic small round cell tumors, and malignant negatively regulates expression of ST7 and NM23 tumor suppressor peripheral nerve sheath tumors. Hum Pathol 2011;42:1667–75. genes. Mol Cell Biol 2004;24:9630–45. 57. Binda C, Valente S, Romanenghi M, Pilotto S, Cirilli R, Karytinos A, et al. 36. Yu J, Yu J, Rhodes DR, Tomlins SA, Cao X, Chen G, et al. A polycomb Biochemical, structural, and biological evaluation of tranylcypromine repression signature in metastatic prostate cancer predicts cancer derivatives as inhibitors of histone demethylases LSD1 and LSD2. J outcome. Cancer Res 2007;67:10657–63. Am Chem Soc 2010;132:6827–33. 37. Su IH, Basavaraj A, Krutchinsky AN, Hobert O, Ullrich A, Chait BT, et al. 58. Tan J, Yang X, Zhuang L, Jiang X, Chen W, Lee PL, et al. Pharmacologic Ezh2 controls B cell development through histone H3 methylation and disruption of Polycomb-repressive complex 2-mediated gene repres- Igh rearrangement. Nat Immunol 2003;4:124–31. sion selectively induces apoptosis in cancer cells. Genes Dev 2007; 38. Pogribny IP, Tryndyak VP, Muskhelishvili L, Rusyn I, Ross SA. Methyl 21:1050–63. deficiency, alterations in global histone modifications, and carcino- 59. Fiskus W, Wang Y, Sreekumar A, Buckley KM, Shi H, Jillella A, et al. genesis. J Nutr 2007;137:216S–22S. Combined epigenetic therapy with the histone methyltransferase 39. Watanabe H, Soejima K, Yasuda H, Kawada I, Nakachi I, Yoda S, et al. EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase Deregulation of histone lysine methyltransferases contributes to onco- inhibitor panobinostat against human AML cells. Blood 2009;114: genic transformation of human bronchoepithelial cells. Cancer Cell Int 2733–43. 2008;8:15. 60. Kuendgen A, Lubbert M. Current status of epigenetic treatment in 40. Aniello F, Colella G, Muscariello G, Lanza A, Ferrara D, Branno M, et al. myelodysplastic syndromes. Ann Hematol 2008;87:601–11. Expression of four histone lysine-methyltransferases in parotid gland 61. Yildirim E, Zhang Z, Uz T, Chen CQ, Manev R, Manev H. Valproate tumors. Anticancer Res 2006;26:2063–7. administration to mice increases histone acetylation and 5-lipoxygen- 41. Hsia DA, Tepper CG, Pochampalli MR, Hsia EY, Izumiya C, Huerta SB, ase content in the hippocampus. Neurosci Lett 2003;345:141–3. et al. KDM8, a H3K36me2 histone demethylase that acts in the cyclin 62. Klisovic RB, Stock W, Cataland S, Klisovic MI, Liu S, Blum W, et al. A A1 coding region to regulate cancer cell proliferation. Proc Natl Acad phase I biological study of MG98, an oligodeoxynucleotide antisense Sci U S A 2010;107:9671–6. to DNA methyltransferase 1, in patients with high-risk myelodysplasia 42. Matarese F, Carrillo-de Santa Pau E, Stunnenberg HG. 5-Hydroxy- and acute myeloid leukemia. Clin Cancer Res 2008;14:2444–9. methylcytosine: a new kid on the epigenetic block? Mol Syst Biol 63. Datta J, Ghoshal K, Denny WA, Gamage SA, Brooke DG, Phiasivongsa 2011;7:562. P, et al. A new class of quinoline-based DNA hypomethylating agents 43. Ribeiro AF, Pratcorona M, Erpelinck-Verschueren C, Rockova V, reactivates tumor suppressor genes by blocking DNA methyltransfer- Sanders M, Abbas S, et al. Mutant DNMT3A: a marker of poor ase 1 activity and inducing its degradation. Cancer Res 2009;69: prognosis in acute myeloid leukemia. Blood 2012;119:5824–31. 4277–85. 44. Schausberger E, Hufnagl K, Parzefall W, Gerner C, Kandioler-Eck- 64. Jinawath A, Miyake S, Yanagisawa Y, Akiyama Y, Yuasa Y. Tran- ersberger D, Wrba F, et al. Inherent growth advantage of (pre)malignant scriptional regulation of the human DNA methyltransferase 3A and 3B hepatocytes associated with nuclear translocation of pro-transforming genes by Sp3 and Sp1 zinc finger proteins. Biochem J 2005;385: growth factor alpha. Br J Cancer 2004;91:1955–63. 557–64. 45. Toyota M, Yamamoto E. DNA methylation changes in cancer. Prog Mol 65. Berger SL. The complex language of chromatin regulation during Biol Translational Sci 2011;101:447–57. transcription. Nature 2007;447:407–12.

OF8 Clin Cancer Res; 18(20) October 15, 2012 Clinical Cancer Research

Cancer Epigenome and Therapeutic Strategies

66. Brinkman AB, Gu H, Bartels SJ, Zhang Y, Matarese F, Simmer F, et al. inhibitor-based therapy. Proc Natl Acad Sci U S A 2010;107: Sequential ChIP-bisulﬁte sequencing enables direct genome-scale 6532–7. investigation of chromatin and DNA methylation cross-talk. Genome 68. Nebbioso A, Pereira R, Khanwalkar H, Matarese F, Garcia-Rodriguez J, Res 2012;22:1128–38. Miceli M, et al. Death receptor pathway activation and increase of ROS 67. Khan O, Fotheringham S, Wood V, Stimson L, Zhang C, Pezzella production by the triple epigenetic inhibitor UVI5008. Mol Cancer Ther F, et al. HR23B is a biomarker for tumor sensitivity to HDAC 2011;10:2394–404.

www.aacrjournals.org Clin Cancer Res; 18(20) October 15, 2012 OF9

Molecular Pathways: The Complexity of the Epigenome in Cancer and Recent Clinical Advances

Mariarosaria Conte and Lucia Altucci

Clin Cancer Res Published OnlineFirst August 17, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-2037

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2012/08/17/1078-0432.CCR-12-2037.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2012/10/06/1078-0432.CCR-12-2037. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.