Targeting Histone Demethylases: A New Avenue for the Fight against Cancer

663MMonographs

Abstract

In addition to genetic disorders, epigenetic alterations have been shown to be involved in cancer, through misregulation of histone modifications. Miswriting, misreading, and mis-erasing of histone acetylation as well as methylation marks can be actually associated with oncogenesis and tumor proliferation. Historically, methylation of Arg and Lys residues has been considered a stable, irreversible process due to the slow turnover of methyl groups in chromatin. The discovery in recent years of a large number of histone Lys demethylases (KDMs, belonging to either the amino oxidase or the JmjC family) totally changed this point of view and suggested a new role for dynamic histone methylation in biological processes. Since overexpression, alteration, or mutation of a number of KDMs has been found in many types of cancers, such enzymes could represent diagnostic tools as well as epigenetic targets to modulate for obtaining novel therapeutic weapons against cancer. The first little steps in this direction are described here.

Keywords: LSD1, Jumonji-containing enzymes, FAD, 2-oxoglutarate, cancer

In addition to genetic alterations, it is now well established that epigen-etic dysfunctions can be involved in

the pathogenesis and development of cancer. Epigenetic regulation of gene expression involves two major mecha-nisms, DNA methylation at the CpG islands of gene promoters and chromatin remodeling. Chromatin folding and its switch from heterochromatin, transcrip-tionally silent, to euchromatin, tran-scriptionally active, and vice versa, is finely tuned by three categories of pro-teins: (1) enzymes that add chemical marks to histone substrates, the so-called writers such as histone acetyltransfer-ases (HATs) and histone methyltransfer-ases (further distinct into protein arginine methyltransferases [PRMTs] and histone lysine methyltransferases [HKMTs]); (2) enzymes that remove such chemical covalent modification from histone substrates—namely, “eras-ers” such as histone deacetylases (HDACs) and histone lysine demethyl-ases (KDMs, the lysine-specific demeth-ylases [LSDs], and the Jumonji C [JmjC] families; Fig. 1); and (3) proteins that recognize and react to specific modified histone residues at epigenetic code level, the “readers.”1-4

Aberrant expression of writer or eraser enzymes has been implicated in the course of tumor initiation and progres-sion, and indeed 2 HDAC inhibitors, vorinostat (suberoylanilide hydroxamic acid, SAHA) and romidepsin (depsipep-tide, FK-228), have been approved by Food and Drug Administration (FDA) for the treatment of refractory cutaneous T cell lymphoma and actually are in clinical trials, alone or in combination, for a vari-ety of both hematological and solid tumors.5

KDMs are the most recently discov-ered families of erasers. Methylation of lysine residues at histone substrates was long retained an irreversible reaction. In 2004, the first protein showing mamma-lian KDM activity was reported to be a flavin-containing amino oxidase (AO) that specifically demethylated mono- or dimethylated lysine 4 at histone H3 (H3K4me1 and H3K4me2) and has been named LSD1 (lysine-specific demethylase 1)/KDM1A (Fig. 2A).6,7 More recently, a second flavin-dependent H3K4me1/2 demethylase called LSD2/AOF1/KDM1B has been identified in mammals.8 In 2006, a larger and more versatile family of KDMs structurally different from LSD1 was identified, all

containing as a signature motif the JmjC domain and all working through a Fe2+/2-oxoglurate (2-OG) mechanism for cataly-sis (Fig. 2B).9,10 The reaction goes on generating a superoxide radical by the complex Fe2+/2-OG, which hits the C2 atom of 2-OG, leading to its decarboxyl-ation to succinate and formation of a Fe4+-oxo species. Afterwards, the Fe4+-oxo species abstracts a hydrogen from the N-methyl group of the amine substrate while it is reduced to Fe3+. The last two steps involve the generation of a carbi-nolamine intermediate on the substrate, which leaves formaldehyde and the amine with one methyl group less (Fig. 2B).

Recently, overexpression or mutation of KDMs has been linked to mis-erased histone methyl modifications and, from a clinical point of view, to many types of

Pasteur Institute—Cenci-Bolognetti Foundation, Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy

Corresponding Authors:Dante Rotili and Antonello Mai, Pasteur Institute—Cenci-Bolognetti Foundation, Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro, 5, 00185, Rome, Italy Email: [email protected]; [email protected]

Targeting Histone Demethylases: A New Avenue for the Fight against Cancer

Dante Rotili and Antonello Mai

Genes & Cancer2(6) 663 –679© The Author(s) 2011Reprints and permission: sagepub.com/journalsPermissions.navDOI: 10.1177/1947601911417976http://ganc.sagepub.com

664 Genes & Cancer / vol 2 no 6 (2011)M Monographs

cancer (Table 1). Thus, such enzymes could represent new potential diagnostic tools as well as therapeutic targets in oncology, and compounds able to inhibit KDMs could be of considerable interest as novel anticancer agents.

The Lys-Specific Demethylase LSD1LSD1/BHC110/AOF2/KDM1A was identified for the first time as a stable component of the BRAF-HDAC tran-scriptional co-repressor complex con-taining also the REST co-repressor, CoREST, already known for a repres-sive role for neuronal genes in nonneu-ronal cells.11-14 LSD1 consists of an N-terminal SWIRM domain and an AO domain containing 2 sites to bind the methylated substrate and the flavin ade-nine dinucleotide (FAD) cofactor, respectively. Between these 2 sites in the AO domain, there is the active site of the enzyme, and from them 2 antiparallel α-helices project away as a unique fea-ture forming the Tower domain, crucial for the interaction of LSD1 with CoR-EST and for the ability to demethylate nucleosomal substrates.15 Indeed, LSD1

alone only demethylates H3K4 in his-tone substrates lacking associated DNA and requires CoREST to catalyze nucleosomal demethylation.14,16,17 The reaction starts with generation of an iminium cation intermediate from the methylated amino group of Lys, which is in turn hydrolyzed to yield a carbinol-amine that spontaneously degrades to give formaldehyde and the demethyl-ated amine (Fig. 2A). Since such mecha-nism of demethylation requires a protonated nitrogen to initiate the reac-tion, the demethylation is limited to dimethyl and monomethyl Lys. The FAD cofactor, reduced during the reac-tion to FADH

2, is reoxidized by O

2 in a

following step that leads to production of H

2O

2 (Fig. 2A).7,18,19 The X-ray struc-

ture of the complex between LSD1 and H3K4me2 has been solved, and the observed tight constraints on the H3 N terminus account for no more than 3 amino acids to be present on the N- terminal side of the methyllysine in the active site of the enzyme, near to the FAD cofactor. This, combined with sequence-specific interactions between the active site and Arg2, Thr3, and Gln5, provides an explanation for the observed

specificity of LSD1 toward the H3K4me1 and H3K4me2 substrates.20-22 When bound to androgen receptor (AR), LSD1 seems to change its substrate specificity: under AR signaling, protein kinase C β1 (PKCβ

1) is recruited at the receptor and

phosphorylates threonine 6 at histone H3 (H3T6).23 Such histone H3 modification blocks the H3K4 demethylating activity of LSD1, which in this case catalyzes the H3K9me1 and H3K9me2 (2 repressive markers) demethylation, playing a role of co-activator.24,25 Such a role seems to be corroborated by the cooperation of LSD1 and JMJD2C, a H3K9me3 demethylase, into a specific demethylase complex on AR target genes, which was found to co-localize with AR in normal prostate as well as in prostate carcinomas.26 Thus, depending on the its binding partners and its substrates, LSD1 seems to be able to have a role as a co-repressor (H3K4 demethylation) or a co-activator (H3K9 demethylation). In addition, LSD1 has been shown to act on nonhistone sub-strates. LSD1-mediated demethylation of K370me2 in the tumor suppressor p53 prevents its interaction with the co- activator 53BP1 (p53-binding protein 1) and represses its transcriptional activa-tion and apoptosis induction.27 Demeth-ylation of DNMT1 by LSD1 stabilizes the enzyme from protein degrading and is essential for maintaining global DNA methylation in embryonic stem (ES) cells.28,29 Interestingly, many types of cancer show upregulation of both DNMT1 and LSD1, but a real link between the 2 epigenetic enzymes in such pathologies has to be established. The retinoblastoma protein 1 (RB1) regulator myosin phosphatase target subunit 1 (MYPT1) is also demethylated by LSD1 at Lys442, losing in such way its stability and leading to enhancement of RB1 phosphorylation.30

In 2009, the second FAD-dependent demethylase, AOF1/LSD2/KDM1B, was identified in mammals.8 Similar to LSD1, LSD2 contains a conserved SWIRM domain required for its catalytic activity and specifically demethylates H3K4me1 and H3K4me2; nevertheless, its repress-ing activity seems to be unrelated to the

Figure 1. Schematic representation of H3 and H4 histone tails, with the most important Lys residues involved in mono-, di-, and trimethylation (circles). The most frequent KDM families involved in demethylation of specific methyl markers are shown.

665Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

H3CNCH3

+

H3K4 N

N

NH

N

O

OR

H3C

H3C NH

N

NH

HN

O

OR

H3C

H3C

FAD FADH2

O2 H2O2

A

LSD1

H3CNCH2

H3K4

H3CNCH2

H3K4

H3CNH

H3K4

H2O

OH

HCHO

H2CNH

H3K4

N N

HNNO

O

R

CH3H3C

HN N

HNNHO

O

R

CH3H3C

FAD

FADH2

O2

H2O2

LSD1

+H2O

HNCH2

H3K4

HNH

H3K4

OH

HCHO

B

H3CN

R

CH3

CH3+

O-

-OO

OO

FeIIHis

Glu/AspHis

+

O-

-OO

OO

FeIIIHis

Glu/AspHis

O2

OO

FeIV

His

Glu/AspHis

+ CO2

O-O

OO-

O

H2CN

R

CH3

CH3+

+

FeIIIHis

Glu/AspHis

O-O

OO-

OH

H2O

H2CN

R

CH3

CH3+

+

FeIIHis

Glu/AspHis

O-O

OO-

HON

R

CH3

CH3+H

+ HCHO

.

Figure 2. Catalytic mechanism of LSD1 (A) and JmjC (B) enzymes.

demethylase function.31 Different from LSD1, LSD2 lacks the Tower domain and thus it is not able to bind CoREST; it forms active complexes with euchro-matic histone methyltransferases such as G9a and NSD3 as well as cellular factors involved in transcription elongation rather than with HDACs, and it is local-ized at the gene body level rather than at the promoters.32 With respect to LSD1, LSD2 has a more restricted expression pattern, it being abundant essentially in growing oocytes and being required for de novo DNA methylation of some imprinted genes.33 Thus, considering the involvement of both the 2 flavin-contain-ing amino oxidases LSD1 and LSD2 with

DNMTs and de novo DNA methylation (see also above and below), it seems fea-sible that a real functional link between DNA methylation and histone demethyl-ation would exist. Very recently, LSD2 has been reported to promote H3K9me2 in addition to H3K4me2 demethylation, leading to control of stimulus-induced recruitment of NF-κB to the MDC and IL12B promoters and activation of these inflammatory genes.34

LSD1 in CancerAberrant expression of LSD1 has been shown in many types of cancers. In par-ticular, LSD1 expression is upregulated

in bladder, small cell lung, and colorectal clinical cancer tissues when compared with the corresponding nonneoplastic tis-sues.35 In bladder carcinogenesis, LSD1 is highly overexpressed even in tumors at an early grade, thus suggesting LSD1 to be one of the initiators of the whole pro-cess. Although estrogen receptor (ER)–positive breast tumors are efficiently treated with antihormonal therapy, ER-negative breast tumors are usually treated with nonselective cytotoxic drugs, and they generally have a less favorable prognosis. LSD1 expression has been found strongly upregulated in ER-negative breast cancers, so LSD1 could be suggested as a predictive bio-marker for aggressive tumor biology and tumor recurrence during therapy.36 Pharmacological (by tranylcypromine, clorgyline) downregulation of LSD1 in ER-negative breast cancer led to growth inhibition, and LSD1 silencing by siRNA resulted in increased expression of the tumor suppressor p21WAF1/CIP1 and in downregulation of the pro-prolifera-tive genes CCNA2 and ERBB2.36 LSD1 expression was also highly upregulated in poorly differentiated neuroblastoma, it being strongly associated with adverse clinical outcome and inversely corre-lated with differentiation.37 Conversely, LSD1 was not expressed in benign gan-glioneuroblastomas/ganglioneuromas or in nonmalignant cells such as stromal tissue or infiltrating leukocytes. Down-regulation of LSD1 in vitro with both siRNA and monoamine oxidase (MAO) inhibitors (pargyline, clorgyline, tranyl-cypromine) in SH-SY5Y cells led to growth inhibition and differentiation with an increase of H3K4 methylation, and tranylcypromine treatment in vivo reduced the growth of neuroblastoma xenograft in nude mice,37 indicating that LSD1 may provide not only a predictive marker for aggressive biology but also a novel therapeutic target for the treatment of aggressive cancer.

On the other side of the coin, LSD1 was shown to be recruited by the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex that in such way possesses at the same time ATPase,

666 Genes & Cancer / vol 2 no 6 (2011)M Monographs

Table 1. Histone Lysine Demethylases (KDMs): Specificity, Transcriptional Effects, and Potential Links to Cancer

Cluster

Names

Demethylase Activity

Transcriptional Effects

Cancer Alterations and Possible Mechanisms

Putative Cancer Roles

References

KDM1 KDM1A; LSD1; BHC110; AOF2

H3K4me2/1 Repression Overexpression in breast, small cell lung, colorectal, prostate, neuro-blastoma, and bladder cancer

Putative oncogene 35-37

H3K9me2/1 Activation KDM1B; LSD2;

AOF1H3K4me2/1 Repression NR NR

KDM2 KDM2A; JHDM1A; FBXL11

H3K-36me2/1

Repression Reduced expression in prostate cancer; suppression of NF-κB-dependent growth of colon can-cer cells; effects on chromosomal segregation

Context-dependent pro- and anti-onco-genic functions

76-84

KDM2B; JHDM1B; FBXL10

H3K-36me2/1

Repression Overexpression in lymphomas and adenocarcinomas; downregula-tion in glioblastoma multiform; mutation in hematological tumors; cell protection against ultraviolet (UV)–induced apoptosis, oxidative stress, and spontaneous muta-genesis

Context-dependent pro- and anti-onco-genic functions

76-84

H3K4me3 (?)

Repression 74, 76

KDM3 KDM3A; JMJD1A; JH-DM2A; TSGA

H3K9me2/1 Activation Co-activator of androgen receptor (AR)–mediated transcription; un-der hypoxia increases a subset of hypoxia-inducible genes enhanc-ing tumor growth

NR 85-90, 93

KDM3B; JMJD1B; JHDM2B; 5qNCA

H3K9me2/1 Activation 5q31 (JMJD1B) frequent deletion in cancer

Candidate tumor suppressor

91, 92

KDM3C; JMJD1C; JHDM2C; TRIP8

NR Reduced expression in breast cancer

Candidate tumor suppressor

91, 92

KDM4 KDM4A; JMJD2A; JHDM3A

H3K9me3/2 Activation Overexpression in prostate cancer Putative oncogene 26, 94-102

H3K-36me3/2

Repression

KDM4B; JMJD2B; JHDM3B

H3K9me3/2 Activation Overexpression in prostate cancer Putative oncogene 26, 94-102

H3K-36me3/2

Repression

KDM4C; JMJD2C; JHDM3C; GASC1

H3K9me3/2 Activation Overexpression/amplification in prostate cancer, esophageal squamous cell carcinoma, desmoplastic medulloblastoma, metastatic lung sarcomatoid carcinoma, mucosa-associated lymphoid tissue (MALT) lympho-ma, and breast cancer; increasing genomic instability; transcriptional activation of oncogenes (e.g., NOTCH1, MDM2) and of AR target genes

Putative oncogene 26, 94-102

H3K-36me3/2

Repression

(continued)

667Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

Cluster

Names

Demethylase Activity

Transcriptional Effects

Cancer Alterations and Possible Mechanisms

Putative Cancer Roles

References

KDM5 KDM5A; JA-RID1A; RBP2

H3K4me3/2 Repression Translocation: NUP98-JARID1A fusion protein; overexpression in gastric cancer; key effec-tor of pRB-mediated cell cycle withdrawal and differentiation; el-evated expression in drug-tolerant subpopulation of cancer cells

Conflicting reports and likely context-dependent pro- and anti-oncogenic functions

103-111

KDM5B; JA-RID1B; PLU-1

H3K4me3/2 Repression Overexpression in breast, testis, prostate, ovary, and esopha-geal cancer; silencing of tumor suppressor genes (e.g., BRCA1, CAV1, HOXA5)

Putative oncogene 112-114

KDM5C; JA-RID1C; SMCX

H3K4me3/2 Repression Regulation of human papilloma virus (HPV) oncogene expression

NR 67, 116

KDM5D; JA-RID1D; SMCY

H3K4me3/2 Repression NR 67

KDM6 KDM6A; UTX H3K-27me3/2

Activation Downregulation; inactivating somatic mutations in multiple myeloma, esophageal squamous cell carci-nomas, and renal cell carcinomas; transcriptional activation of pRB binding proteins contributing to the pRB-mediated cell cycle arrest

Candidate tumor suppressor

117-120, 123, 126, 127

KDM6B; JMJD3 H3K-27me3/2

Activation Downregulation in subsets of hu-man cancers; 17p13.1 (JMJD3) frequent deletion in cancer; transcriptional activation of INK4A/ARF region promoting senescence

Candidate tumor suppressor

117-120, 124, 125, 128

NR, not reported.

Table 1. (continued)

deacetylase, and demethylase activities, influencing various pathways compris-ing transforming growth factor β (TGFβ) signaling, cell growth, and migration and invasion.38 Through a negative correlation with the TGFβ1 level, highly relevant in epithelial-mesenchymal tran-sitions and invasion, LSD1 suppressed breast cancer metastatic potential in vitro and in vivo. Thus, depending on a variety of factors such as the biological context, the type and the grade of the tumor, and the presence of different sub-strates, LSD1 seems to have either onco-genic or tumor-suppressive functions, and further studies are needed to better manage this point.

Putative Mechanisms for LSD1-Mediated OncogenesisMechanisms by which overexpression of LSD1 leads or contributes to tumor

formation could involve its capacity to silence tumor suppressor genes as a tran-scriptional co-repressor, mainly through H3K4 demethylation. The physical association of LSD1 with HDACs at the co-repressor complex contributes to repress transcription of common sets of genes. This interplaying activity has been confirmed by experiments made on LNCaP prostate cancer cells treated with pan- (vorinostat) and class I selec-tive (HDAC42, entinostat) HDAC inhibitors.39 Such compounds led to increased H3K4 methylation levels and suppression of expression of the JmjC demethylase RBP2 and LSD1 through downregulation of Sp1 expres-sion. Moreover, shRNA-mediated silencing of the class I HDAC isozymes 1, 2, 3, and 8, but not that of the class IIb isozyme HDAC6, mimicked the drug effects on H3K4 methylation and H3K4 demethylases, which could be reversed

by ectopic Sp1 expression.39 Further evidence of the role of LSD1 as a tran-scriptional co-repressor involved in can-cer development came from the recruitment, from the CoREST/HDACs/LSD1 complex, of the oncoprotein ZNF217, able to negatively regulate the tumor suppressor gene p15INK4B.40 Simi-larly, the DNA binding factor SNAIL, which correlates with high tumor grade and nodal metastasis and is also predic-tive of a poor outcome in patients with breast cancer, interacts through its SNAG domain with the AO domain of LSD1/CoREST to repress E-cadherin in a variety of cancer cell lines and forms the ternary complex SNAIL/LSD1/CoREST in 116 primary breast cancer samples.41,42 Functionally, LSD1 is linked to DNA methylation and subse-quent gene silencing. DNMT3L, a non-catalytic paralog of de novo DNMT3A and -3B, specifically interacts with the

668 Genes & Cancer / vol 2 no 6 (2011)M Monographs

histone H3 tail only when H3K4 is unmethylated, and thus the formation of a complex LSD1/DNMT3L/DNMT3A is highly reliable.43 Alternatively, LSD1 has been reported to demethylate the Lys res-idue of DNMT1, thus preserving it from degradation.28 Interestingly, LSD1 and DNMT1 are both upregulated in many types of cancers, but whether the first is responsible for the increased expression of the latter is still unknown. However, further evidence of this DNMT1-LSD1 interplay came from the effect of combi-nation treatment of LSD1 inhibitors and DNMT inhibitors against colon cancer xenograft mice, resulting in a great inhi-bition of the growth of the tumor.44 Another possibility for explaining the oncogenic potential of LSD1 is its effect on the tumor suppressor p53. p53 is a nonhistone substrate that is demethylated by LSD1 at Lys370. Methylated Lys370 in p53 is important for its binding to the transcriptional co-activator p53-binding protein–1 (PBP-1), and its demethylation led to inhibition of the p53 apoptotic activity, whereas the knockdown of LSD1 increased p21WAF1/CIP1 and MDM2, well-known p53 target genes.27 A further putative oncogenic mechanism of LSD1 resides into its capability to trigger Myc-induced transcription through transient H3K4 demethylation.45 The local DNA oxidation given by the LSD1 catalytic activity and the following production of hydrogen peroxide gave the recruitment of 8-oxoguanine-DNA glycosylase (OGG1) and of the nuclease Ape1 on the E-box chromatin sequence, leading to the assembly of the transcription initiation complex of the oncogene Myc.45

LSD1 InhibitorsSubstrate Analogues

From the knowledge that LSD1 is a FAD-dependent AO that catalyzes the oxidative demethylation of mono-methyl- and dimethyl-K4 residue at his-tone H3, as well as H3-tail peptides requiring at least 15 amino acid residues for efficient reaction, the propargyl- Lys-derivatized peptide 1 was designed as a mechanism-based LSD1 inhibitor

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

NR

COOH

1, R = H2, R = CH3

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

N

COOH

3

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

HN

COOH

4

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

NR2

COOH

5, R = H6, R = CH3

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

HN

COOH

R

R'

7, R = Cl, R' = H8, R = H, R' = Cl

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

HN

COOH

9

NH2

Figure 3. Substrate analogues as LSD1 inhibitors.

(Fig. 3). Actually, 1 exhibited Ki = 16.6

and then 0.77 µM against LSD1, show-ing a clear time- and concentration-dependent inhibition of the enzyme.46,47

Mechanistic studies made on 1 revealed that its LSD1 inactivation involves amine oxidation followed by Michael addition of the FADH

2 N5 to the

propargylic imine (Fig. 4).48 The struc-ture of the similar covalent adduct involv-ing FADH

2 and 2 (Fig. 3), the N-methyl

analogue of 1, was then confirmed by X-ray crystal structure of the LSD1/2 complex.20 In addition to the propargyl residue, other warheads taken from more than 50 years of anti-MAO research story have been applied to the H3- peptide scaffold to explore their ability to inhibit LSD1.47

Although aziridine and exo- and endo-cyclopropylamine units (compounds 3-6) failed to inactivate LSD1, the cis- and trans-3-chloropropyl groups (compounds 7 and 8) gave to the H3-peptide skeleton similar ability as 1 to inhibit LSD1 (K

i =

0.95 and 0.76 µM, respectively), and the hydrazine-Lys-4 H3-21 peptide 9 showed the highest enzyme inhibition, reaching K

i = 0.00435 µM (Fig. 3).

Also for the chloroallyl- and hydrazine-H3-peptides 7-9, a mechanism of sui-cide inactivation has been proposed (Fig. 4).47

Polyamine Analogues

The AO domain of LSD1 shares 20% sequence similarity with those of the FAD-dependent MAO and PAO (poly-amine oxidases such as spermine oxi-dase [SMO] and N1-acetylpolyamine oxidase), with the catalytic domains of LSD1 and SMO showing 60% similarity in amino acid sequences. As compounds bearing guanidine groups were shown to inhibit both SMO and other polyamine oxidases, a pioneer series of (bis)guani-dines and (bis)biguanides, previously synthesized as potential antitrypano-somal agents,49 were tested as inhibitors of LSD1 (Fig. 5).50 Among them,

669Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

compounds 10 and 11 showed high, noncompetitive LSD1 inhibition at <2.5 µM and were able to induce the reex-pression of aberrantly silenced tumor suppressor genes in colon cancer cells in vitro, together with an increase of H3K4me2 and acetyl-H3K9 marks and a decrease of the repressive H3K9me1 and H3K9me2 marks. The same effects on recombinant LSD1 as well as human colon cancer HCT116 and RKO cell growth were observed by a series of octamines and decamines (see com-pounds 12 and 13 as the most potent derivatives), reported by the same research group (Fig. 5).44 In addition, 12 showed striking synergy in combination with the DNMT inhibitor 5-azacytidine against HCT116 tumors in vivo, without significant overall toxicity as indicated by animal weight. The series of poly-amine analogues was further expanded with some (bis)urea and (bis)thiourea derivatives, such as compounds 14-18 (Fig. 5).51 Two of the most potent LSD1 inhibitors among them (i.e., 15 and 16) were also able to inhibit the growth of Calu-6 non–small cell lung carcinoma cells in vitro with GI

50 = 10.3 and 9.4 µM,

respectively.51

Anti-MAO-Based Small Molecules

Due to the high sequence similarity between LSD1 and MAO, some known anti-MAO agents were tested against LSD1. Pargyline (Fig. 6), a propargyl-amine-based anti-MAO initially sug-gested as a valuable LSD1 inhibitor prototype, failed in further studies to inhibit the Lys demethylase.21,52 Phenel-zine (Fig. 6), an anti-MAO agent bearing a hydrazine moiety, was initially reported as a weak LSD1 inhibitor, and when rein-vestigated on its inhibiting activity, it resulted in having highly improved potency against LSD1 (K

i = 17.6 µM),

similar to that displayed versus MAOs.47

Tranylcypromine (trans-2-phenylcy-clopropyl-1-amine, trans-2-PCPA) (Fig. 6) was reported to inhibit LSD1 with a K

i value ranging from 477 to 22 µM,

depending on the type of assay, the time of addition, the substrate, and the

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

NR

COOH

1, R = H2, R = CH3

FAD FADH2

LSD1

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

NR

COOH

+

N

N

NH

HN O

O

H3C

H3C

R

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

HN

COOH

R

R'

7, R = Cl, R' = H8, R = H, R' = Cl

FAD FADH2

LSD1

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

N

COOH

R

R'

H+

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

HN

COOH

9

NH2

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

N

COOH

NH2FAD FADH2

LSD1

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

N

COOH

N

+

FAD

FADH2

NH

QTARKSTGGKAPRKQLA

O

ARTH2N1 21

N

COOH

N

NH

HN

O

OH3C

H3C

R

- N2

Figure 4. Proposed mechanism of inactivation of LSD1 by substrate analogue inhibitors.

enzyme source used for testing.52-58 Nevertheless, trans-2-PCPA became a useful lead to develop highly potent small-molecule inhibitors of LSD1. Some trans-2-PCPA analogues carrying 4-bromo, 4-methoxy, and 4-trifluorome-thoxy substitutions at the benzene ring led to compounds (19-21) with similar or slightly improved activity against LSD1.59 Interestingly, the introduction of a bulky, branched substituent at the 4 position of the benzene ring, able to occupy the wide space present in the

LSD1 cavity surrounding the FAD cofactor and the AO domain, furnished tranylcypromine-based compounds such as 22-27 endowed with high potency against LSD1 and scarce or no activity against MAOs.55,56 Compounds 22 and 23 were also reported to be active against a panel of cancer cell lines with growth inhibition 50% (GI

50) values ranging

from 6.0 to 67 µM (Table 2),55 whereas 26 was able to inhibit cell growth and induce differentiation in murine acute promyelocytic leukemia (APL) blasts as

670 Genes & Cancer / vol 2 no 6 (2011)M Monographs

a single agent and in APL-derived NB4 cells in synergy with retinoic acid (RA).56 The perfluorinated trans-2-PCPA ana-logue 28 (trans-2-pentafluorophenylcy-clopropylamine, 2-PFPA), 21-fold more potent than the parent compound against LSD1, is allowed to identify through structure-based drug design new fluorine-containing trans-2-PCPA analogues (29-32) with single-digit- to sub-micromolar IC

50 values against LSD1.57 Trans-

2-PCPA and analogues inhibit LSD1, forming a covalent linkage with the FAD cofactor. Different adducts between trans-2-PCPA and FAD have been postu-lated (Fig. 7).53,54,60

As trans-2-PCPA is the racemic mix-ture of 2 enantiomers, (+)-trans-2-PCPA and (–)-trans-2-PCPA, we prepared large amounts of trans- and cis-2-PCPA, separated the 2 trans- and 2 cis-enantio-mers, and tested them against LSD1 and MAOs, determining their binding to

H3C NH

NH

NH

NH

NH

NHCH3

NH3C N

CH3

NH

NH

NH

NH

NH

NH

NH NH

( )5 NH

NH

NH

NH

10

11

NH

HN

12H3C

NHNH

CH3( )4( )4

NH

HN

H3C ( )4 ( )4HN

NH

CH313

HN

HN

HN

HN

HN( )n

HN

14, n = 115, n = 216, n = 5

S S

HN

HN

HN

HN

HN( )5

HN

17S S

HN

HN

HN

HN

HN N( )2

HN

18O OO

N

O

Figure 5. Polyamine analogues as LSD1 inhibitors.

FAD through X-ray crystallography.56 Thus, we found that the 2 trans-enantio-mers of 2-PCPA were slightly more potent than the 2 cis congeners against LSD1, with (–)-trans-2-PCPA being 1.7-fold more efficient than (+)-trans-2-PCPA (Fig. 7). Conversely, against MAO-B, (+)-trans-2-PCPA was 20-fold more potent than the corresponding (–) enantiomer, highlighting the crucial role of stereochemistry in MAO-B inhibition by trans-2-PCPA. From X-ray crystal structures, it was clear that (–)-trans-2-PCPA connects its carbonyl carbon in a covalent bond with the flavin N5 atom, positioning its phenyl ring in the core of the substrate-binding pocket, above the flavin ring. Differently, (+)-trans-2-PCPA links the flavin N5 atom through the phe-nyl-substituted carbon of the inhibitor, pointing the phenyl ring away from the flavin ring (Fig. 8). Due to the higher potency shown by the (–) enantiomer,

when the racemic trans-2-PCPA is crys-tallized with LSD1, the enzyme selects this enantiomer for the binding with FAD, and the N5-carbonyl adduct is obtained, according to the literature.56,60

LSD1 Activators?

Two immunomodulatory drugs, pomalid-omide and lenalidomide (Fig. 9), have been reported to induce p21WAF1 expres-sion in Burkitt lymphoma and multiple myeloma cell lines, leading to cell cycle arrest. This p21WAF1 induction is gener-ated by modification of the chromatin structure at the p21 promoter involving a switch from H3K9 methylation (a repres-sive mark) to H3K9 acetylation (an acti-vation mark of transcription). As LSD1 silencing reduced the observed pomalid-omide and lenalidomide-induced p21 upregulation, LSD1 has been suggested to be involved in this effect of p21 induc-tion through demethylation of H3K9, and pomalidomide and lenalidomide could act as direct LSD1 activators or indirectly through activation of a third partner required for LSD1 activation.61

The Jumonji C Family of Lysine DemethylasesThe second and largest class of histone lysine demethylases (KDMs) to be dis-covered is the Fe+2/2-OG-dependent fam-ily of KDMs.9,10,62 These enzymes belong to a much larger enzyme superfamily, the 2-OG oxygenases, whose members (about 70-80 genes in the human genome) catalyse a diverse range of oxidation reactions, using 2-OG, molecular oxy-gen, and Fe2+ as co-substrates/co-factors (Fig. 2B).63 In humans, the two main reactions catalyzed by 2-OG oxygenases identified so far are protein/nucleotide hydroxylation and N-methyl demethyl-ation.64 In particular, the 2-OG-dependent histone lysine demethylases are members of the Jumonji family of 2-OG oxygen-ases, which have common structural fea-tures (e.g., with respect to 2-OG binding residues) and comprise nonhistone modi-fying enzymes such as factor inhibiting HIF (FIH).65 The Jumonji protein family

671Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

is named this way because it contains the conserved Jumonji C (JmjC) catalytic domain that was first identified in the so-called Jumonji protein (JARID2).66 In fact, jumonji means cruciform in Japa-nese, and the gene was so named because mice with a genetrap inserted in the Jumonji locus develop an abnormal cross-like neural tube.66 Among the 30 JmjC domain-containing proteins identi-fied so far within the human genome, about 20 have been published to demeth-ylate specific lysines in the histone proteins.

The JmjC histone lysine demethylases catalyze the hydroxylation at the carbon of the Nε-methyl group to give an unsta-ble hemiaminal intermediate that rapidly breaks down, leading to the release of formaldehyde and the demethylated lysine residue.67 Unlike LSD1, the hydroxylation-based mechanism of the JmjC KDMs does not require a protonat-able lysine ε-amine group, enabling these enzymes to demethylate all 3 lysine methylation states (tri-, di- and mono-methylation) at H3K4, H3K9, H3K27, and H3K36, as well as H1K26.67 Six dif-ferent subfamilies (JMJD1s, JMJD2s, JARID1s, UTX/Y-JMJD3, PHFs, and FBXLs) of JmjC histone demethylases have been identified, which have different histone sequence and methyla-tion state selectivities.67,68 For instance, KDMs of the JmjC domain-containing 2 (JMJD2) subfamily are selective for the demethylation of the tri- and di-Nε-methylation states of specific lysines on histone H3, whereas other subfamilies (e.g., the PHF and FBXL subfamilies) are selective for the di- and mono-Nε-methylated states and do not accept the trimethylated state. Recent crystallo-graphic analyses have shown that varia-tions in the size of the active site region binding the Nε-methyl group are in part responsible for the methylation state selectivity.69 Selectivity for a particular histone sequence seems to be conferred by binding interactions directly with the catalytic domain and, at least for some enzymes, by the presence of other non-catalytic domains that target the catalytic

NCH3

pargyline

NHNH2

phenelzine

NH2

tranylcypromine,trans-2-PCPAK i = 477 µM (ref. 59)IC50 = 32 µM (ref. 55)K i = 271 mM (ref. 56)IC50 = 184 µM (ref. 57)

NH2

NH2 NH2

Br

O F3CH3C

19K i = 566 µM (ref. 59)

20K i = 188 µM (ref. 59)

21K i = 352 µM (ref. 59) NH2

O

HN ONH

O

NH2O

HN O

O

HN

NH2

22IC50 = 1.9 µM (ref . 55)

23IC50 = 2.5 µM (ref . 55)

HNO

O

NH2

HN

O

24K i = 1.1 µM (ref. 56)

25K i = 1.9 µM (ref. 56)

NH2

HN

O

NH

O

NH2

HN

O

NH

O

O

NH26

K i = 1.3 µM (ref. 56)

27K i = 40 µM (ref. 56)

O

NH2

FF

F

FF

28, 2-PFPAIC50 = 8.9 µM (ref . 57)

NH2FO

29IC50 = 8.1 µM (ref . 57)

NH2FO

F

R

30, R = H, IC50 = 0.99 µM (ref. 57)31, R = CH2NH2, IC50 = 4.1 µM (ref. 57)32, R = C2H5, IC50 = 1.9 µM (ref. 57)

Figure 6. Anti-MAO-based LSD1 inhibitors.

Table 2. Antiproliferative Activity of trans-2-PCPA, 22, and 23 on a Panel of Cancer Cell Lines

GI50

, µM

Cancer cell line trans-2-PCPA 22 23

HeLa cervix cancer >500 30 13HCT116 colon cancer >1000 67 43PC3 prostate cancer 600 17 6KYSE150 esophagus cancer 260 18 11SH-SY5Y neuroblastoma 500 43 27

672 Genes & Cancer / vol 2 no 6 (2011)M Monographs

one to particular residues, as recently demonstrated for PHF8.69-71 In fact, this histone demethylase has a plant homeo-box domain (PHD), in which binding the Nε-trimethylated form of the K4 of the histone H3 increases the activity of the catalytic domain with regard to demethylation of the Nε-dimethylated H3K9. Mounting evidence (especially works with peptides) suggests that some JmjC demethylases have also nonhistone substrates, a conceivable finding given the rising data on nonhistone proteins that are Nε-methylated.72

Despite the functional characteriza-tion of many JmjC histone demethyl-ases being still at a relatively early stage, recent evidence suggests for these enzymes many important biological roles ranging from the regulation of cellular differentiation and development to the control of neuronal function, from senes-cence process modulation to the control of genome stability and cancer cell prolif-eration.64,67,68 Jumonji histone demethyl-ases are being targeted for inhibition by small molecules, both with a view to developing “chemical probes” potentially

useful in functional assignments and for the eventual development of pharmaceu-ticals, with the most likely application being cancer.18,73 In fact, the misregula-tion of JmjC KDMs has significantly being implicated in cancer initiation and progression, and this will be reviewed here by analyzing the involvement of dif-ferent subfamilies of the JmjC histone lysine demethylases.

The KDM2 Cluster (FBXL Subfamily)

KDM2A/JHDM1A (JmjC domain–con-taining histone demethylation protein 1A)/FBXL11 (F-box and leucine-rich repeat protein 11) was the first JmjC domain–containing protein shown to be a histone lysine demethylase. Human KDM2A and KDM2B/JHDM1B/FBXL10 have been shown to catalyze H3K36me2/me1 demethylation.74,75 In addition, mamma-lian FBXL10 has been suggested to act as an H3K4me3 demethylase,76,77 but reports on its activity toward H3K4me3 are conflicting.74,76 Both proteins contain an F-box domain in addition to 2 leucine-rich repeat (LRR) domains.76 Retroviral inser-tional mutagenesis within the FBXL10/JHDM1B gene has been shown to cause lymphoma in BLM (Bloom syndrome RecQ protein-like-3 DNA helicase)– deficient mice, indicating KDM2B as a candidate tumor suppressor.78 Other stud-ies have proposed FBXL10, along with FBXL11, as a putative proto-oncogene.79 Expression data from human cancers show overexpression of KDM2B in lymphomas and adenocarcinomas79 but reduced expression of KDM2A and KDM2B in prostate cancer and in the most aggressive of the primary brain tumors, the glioblas-toma multiform, respectively.76,80 Cell cul-ture studies support the view that KDM2 proteins might have context-dependent pro- and anti-oncogenic functions.

NH2

NH2

(1S,2R)-(+)-t rans-2-PCPAK i (LSD1) = 284 µMK i (MAO-B) = 4.4 µM

LSD1N

N

NH

HN

O

OR

H3C

H3CC4aN5

CHO

(1R,2S)-(-)-trans-2-PCPAK i (LSD1) = 168 µMK i (MAO-B) = 89 µM

LSD1N

N

NH

HN

O

OR

H3C

H3CC4aN5

O

NH2

(+/-)-trans-2-PCPAK i (LSD1) = 271 µMK i (MAO-B) = 16 µM

LSD1N

N

NH

HN

O

OR

H3C

H3CC4aN5

O

Figure 8. Structures of flavin adenine dinucleotide (FAD)–tranylcypromine adducts obtained by the trans-2-PCPA enantiomers.

NH

N

NH

N

O

OR

H3C

H3C

OHC

NH

N

NH

N

O

OR

H3C

H3C

OHC

C4aC4a

see ref. 53see ref. 53

N

N

NH

N

O

OR

H3C

H3CC4a

HO

see ref. 54

N5N

N

NH

HN

O

OR

H3C

H3CC4aN5

O

see ref. 60

Figure 7. Proposed structures for the flavin adenine dinucleotide (FAD)–tranylcypromine adducts in the LSD1 complex.

NNH

O

O

O

NH2lenalidomide

NNH

O

O

O

NH2pomalidomide

O

Figure 9. Structures of pomalidomide and lenalidomide.

673Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

KDM2A can suppress NF-κB-dependent growth of colon cancer cells,81 whereas KDM2B has been shown to inhibit proliferation of tumor cells corre-lating with repression of rRNA genes.76 By contrast, KDM2B prevents premature senescence in MEFs (mouse embryonic fibroblasts) through a mechanism that likely involves repression of the Ink4b gene.74,77,82 MEFs ectopically expressing KDM2A or KDM2B bypass stress-induced senescence, depending on an intact JmjC domain, and KDM2B coop-erates with Ras in oncogenic transforma-tion.74,79 In addition to the proliferative control, KDM2 proteins are important for genomic stability. KDM2B has been demonstrated to protect cells against ultraviolet (UV)–induced apoptosis, oxidative stress, and spontaneous

mutagenesis,78,83,84 whereas depletion of FBXL11 was reported to influence chro-mosomal segregation through effects exerted in the heterochromatic areas of the genome.80 Taken together, mamma-lian KDM2 demethylases have been sug-gested to have context-dependent tumor-promoting and tumor-inhibiting functions.

The KDM3 Cluster (JMJD1 Subfamily)

KDM3A/JHDM2A/JMJD1A (Jumonji domain–containing 1a)/TSGA (testis- specific gene A) is a histone demethyl-ase specific for H3K9me2/me1.85 Two JMJD1A homologues exist in human cells: KDM3B/JHDM2B/JMJD1B and JMJD1C/JHDM2C/TRIP8 (thyroid receptor interacting protein 8). JMJD1A

features an LXXLL motif that is a signa-ture involved in nuclear receptor inter-actions.86 The expression of JMJD1A is very significant in testes and has been implicated in demethylation of H3K9me2 of AR target genes.85 Thus, JMJD1A was found to interact with the AR in a ligand-dependent manner. Inhi-bition of JMJD1A expression in the prostate cancer cell line LnCaP led to an increase in H3K9me2 in a subset of AR target genes, including PSA, NKX3.1, and TMPRSS22, and a decrease in their expression.85 These results show that JMJD1A acts as a co-activator of AR-mediated transcription.

Studies of genetrap and knockout mice have demonstrated important roles for KDM3A in germ cell development and metabolism.87-90 JMJD1A is highly and dynamically expressed during sper-matogenesis, and male Kdm3a genetrap mice are infertile, with small testes and a severe reduction in sperm count. This defect has been linked to KDM3A posi-tively regulating expression of genes involved in sperm chromatin condensa-tion and maturation through demethyl-ation of H3K9me2/me1.88,89 Kdm3a knockout mice also exhibit an adult-onset obesity phenotype, and in this con-text, KDM3A has been implicated in the transcriptional control of metabolic genes in muscle and adipose tissues.87,90 Little is known about the implication of JMJD1s in tumorigenesis. Frequent deletion of 5q31 locating the JMJD1B gene or reduced expression of JMJD1C in various malignancies suggests possi-ble roles of KDM3 proteins in tumor suppression.91,92 Recent studies have shown that JMJD1A is the target of hypoxia-inducible transcription factors, and under hypoxia, JMJD1A increases a subset of hypoxia-inducible genes enhancing tumor growth.93

The KDM4 Cluster (JMJD2 Subfamily)

Although the potential role of JMJD1s in cancer remains controversial, the JMJD2 subfamily members are mainly regarded as oncogenes. Their

HOOC NH

COOH

O

N

COOH

COOH

NOG 2,4-PDCA

HOOC NH

COOH

O

OS

O O

Br

N-oxalyl-D-tyrosine derivative

NOH

COOH

8-hydroxyquinoline derivative

NN

COOH

COOMe

2,2'-bipyridil derivative

O

O

HO

HOOH

quercetin

N SS

S

S

N

disulfiram

SeN

O

ebselen

HN

ONHOH

O

O N OH

O

OH

NMe

Me8

N -(3-carboxypropyl)hydroxamicacid derivative

SAHA

Figure 10. Structures of JmjC KDM inhibitors.

674 Genes & Cancer / vol 2 no 6 (2011)M Monographs

oncogenic potential could result from demethylating heterochromatic H3K9me3/2, an important mark for for-mation and maintenance of heterochroma-tin and somehow of genomic stability. In fact, KDM4 (JMJD2) proteins were the first published demethylases that showed activity toward trimethylated lysines. There are 4 members of the KDM4 cluster in mammalian cells: KDM4A/JHDM3A/JMJD2A, KDM4B/JHDM3B/JMJD2B, KDM4C/JHDM3C/JMJD2C/GASC1 (gene amplified in squamous cell carci-noma 1), and KDM4D/JHDM3D/JMJD2D. The JMJD2 proteins catalyze the demethylation of H3K9me3/me2 and/or H3K36me3/me2, with the substrate specificity differing between subfamily members.94-98 Moreover, the KDM4 pro-teins have recently been demonstrated to demethylate H1.4K26me3/me2.99

Mounting evidence correlates human KDM4 family members—especially KDM4C—to malignant transformation. Significantly, amplification of the KDM4C locus has been reported for esophageal squamous carcinomas, medulloblastomas, and breast cancers, and amplification of KDM4B is detected in medulloblasto-mas.100-102 Gene expression analysis further shows that JMJD2A-C is overex-pressed in prostate cancer.94 Depletion of JMJD2C from cancer cells, expressing high levels of it, resulted in inhibition of cell growth of several tumor cell lines,26,94,101 whereas overexpression of GASC1 in human nontransformed mam-mary epithelial cells resulted in pheno-typic alterations that are hallmarks of neoplastic transformation, including growth factor–independent proliferation and anchorage-independent growth in soft agar.101 Introduction of the JMJD2C gene in normal breast MCF10A cells could increase higher capacity to gener-ate mammospheres, a phenotype of can-cer stem cells, suggesting that JMJD2C acts as a transforming gene.101 However, the genomic targets and cellular functions of the mammalian KDM4 subfamily members are still for the most part uncharacterized. To date, different studies have shown that KDM4C can

function as a transcriptional activator by removing the heterochromatic H3K9me3 at promoter regions, which is generally associated with transcriptional repres-sion.26 Analysis of genes altered by over-expression of GASC1 in MCF10A- GASC1 cells revealed NOTCH1 as a tar-get gene of GASC1.101 Induction of NOTCH signaling promotes self-renewal of normal human mammary stem cells. This may support the finding that the phenotype of cancer stem cells was increased in MCF10A-GASC1 cells. The involvement of GASC1 in tumorigenesis has been supported further by a recent report demonstrating the functional inter-action between GASC1 and AR in pros-tate carcinoma.26 It was known that LSD1 and JMJD1A are also required for tran-scriptional activation of AR responsive genes and for proliferation of prostate cancer cells. Although LSD1 and JMJD1A only demethylate mono- and dimethylated H3K9, GASC1 is espe-cially capable of efficiently demethylat-ing trimethylated H3K9, inducing a robust cooperative stimulation of AR transcriptional activity. Thus, specific modulation of GASC1 activity alone, or in combination with LSD1, may be a promising therapeutic strategy to control AR activity in tissues where AR has a pivotal physiological role.26 Clearly, these observations suggest also critical functions not only in cancer but also dur-ing development. In summary, growing evidence suggests important roles for human JMJD2B and JMJD2C in tumori-genesis, indicating these proteins as potential targets for the development of new-generation anticancer agents.

The KDM5 Cluster (JARID1 Subfamily)

The JARID1 subfamily, specific for the demethylation of H3K4me3/2, encom-passes 4 enzymes: KDM5A/JARID1A (Jumonji, AT-rich interactive domain 1A)/RBP2 (retinoblastoma-binding pro-tein 2), KDM5B/JARID1B/PLU-1, KDM5C/JARID1C/SMCX (selected mouse cDNA on the X), and KDM5D/JARID1D/SMCY.67 Despite the fact that

all members of KDM5 cluster catalyze the demethylation of the same histone mark, they appear to have exclusive func-tional properties probably because of their different expression profiles and presence in distinct protein complexes. RBP2 is broadly expressed in diverse tis-sues and has been linked by target gene mapping and functional studies to regula-tion of differentiation, cell cycle progres-sion, and mitochondrial function.103-107 The KDM5A interaction partners identi-fied so far include RBP-J (recombination signal-binding protein–Jk), Sin3, c-Myc, the tumor suppressor pRB (retinoblas-toma protein), and PcG proteins of the PRC2 complex, which harbor H3K27 methyltransferase activity.105,106,108-110 Lit-tle is known at present about the role of JARID1A/RBP2 in human cancers, and the few available reports are contradic-tory. RBP2 has been shown to be a key effector of pRB-mediated cell cycle with-drawal and differentiation by interacting with the tumor suppressor pRB.103 Recently, RBP2 protein was shown to be an integral part of the core Notch-RBP-J repressor complex, contributing to switch off the Notch signaling.109 In contrast to its tumor suppressor function, a recent report showed that it is overexpressed in gastric cancer and that its inhibition leads to cellular senescence of gastric and cer-vical cancer cells by de-repressing cyclin-dependent kinase inhibitors such as p27, p21, and p16.107 Recently, enhanced Jarid1A/Rbp2 expression was found to contribute to drug tolerance with respect to a receptor tyrosine kinase inhibitor in a non–small cell lung carcinoma (NSCLC) cell system, whereas RBP2 knockdown significantly reduced the number of the drug-tolerant population.111 These drug-tolerant NSCLC cells displayed reduced levels of H3K4me2 and H3K4me3, which is reminiscent of the prognostic value of reduced H3K4me3 in NSCLC. This also indicates that acquired drug resistances are accompanied by epigene-tic changes, and combining a chromatin-modifying agent with a single, rationally targeted agent would prevent the devel-opment of drug resistance.

675Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

Although significant expression of PLU-1 (JARID1B) is limited to testis and only low levels of PLU-1 are seen in other normal adult tissues, its expression increases markedly in pathological con-ditions such as malignant breast cancer, metastatic prostate cancer, and testis cancer.112,113 Based on its overexpres-sion in cancer, PLU-1 was suggested as a testis cancer antigen. Supporting an oncogenic potential at a cellular level, PLU-1 knockdown slowed down the proliferation of MCF-7 breast cancer cells and tumor growth of mammary carcinoma cells in a syngenic mouse mammary tumor model.114 Like LSD1, PLU-1 was shown to be involved in silencing a series of tumor suppressor genes previously linked to breast cancer, including SFN (14-3-3-σ), BRCA1, CAV1, and HOXA5. Although this is consistent with a transcription repres-sive role for this protein, JARID1B can also act as a transcriptional co-activator for the androgen receptor.112,113 Involve-ment in hormone signaling is consistent with high expression levels in testis, ovary, and mammary glands of pregnant females.112

JARID1C/SMCX (KDM5C) has been implicated in X-linked mental retardation and epilepsy.115 However, its role in the pathogenesis of human papil-lomavirus (HPV)–associated cancers was recently identified by a genome-wide siRNA screen that found, among other genes, JARID1C/SMCX as one of the E2-dependent regulators of HPV oncogene expression.116

The KDM6 Cluster (UTX/Y-JMJD3 Subfamily)

The KDM6 cluster in human cells con-sists of KDM6A/UTX (ubiquitously transcribed X chromosome tetraticopep-tide repeat protein), UTY, and KDM6B/JMJD3. KDM6A and KDM6B are his-tone demethylases specific for H3K27me3/me2, whereas no activity has been reported for UTY so far.117-120

The tri- and dimethylation of H3K27, which are catalyzed by the polycomb group (PcG) proteins, are important

repressive histone marks. The PcG genes have been characterized as oncogenes and are frequently overexpressed or amplified in cancer. Their oncogenic potential is mainly mediated through PcG-mediated H3K27 methylation and epigenetic inacti-vation of the INK4A-ARF locus.121,122 The INK4A-ARF locus encodes tumor suppressor genes p16INK4A and p14ARF, which are key regulators of cellular senescence.

Both the H3K27 demethylases KDM6A and KDM6B are suggested as tumor suppressor genes by functioning antagonistically to the oncogenic PcG proteins. Although JMJD3 and UTX have the same catalytic activity, they most likely have, as the JARID1 proteins, different biological functions. Overex-pression of either leads to cell cycle arrest in fibroblasts, dependent on intact cata-lytic domains.123-125 Although knock-down of KDM6A confers an immediate growth advantage to fibroblasts,123 deple-tion of KDM6B enables MEFs to over-come the senescence barrier.124,125 The growth arrest induced by KDM6A has been linked to transcriptional activation of components of the pRB pathway. In fact, a genome-wide chromatin occu-pancy analysis has shown that many pRB-binding proteins, including HMG-box protein 1 (HBP1), are UTX target genes.123 UTX, by means of preventing H3K27 methylation and silencing HBP1, is able to enforce cell cycle arrest by pRB. Consistently, UTX depletion results in elevated levels of proliferation. UTX, the first reported mutated histone demeth-ylase gene associated with cancer, is a frequent target of somatic mutations in human cancers with high prevalence in multiple myeloma, esophageal squa-mous cell carcinomas, and renal cell carcinomas.126,127

It has been shown that JMJD3, but not UTX, contributes to the activation of INK4A-ARF by removing H3K27me3 during induction of oncogene- or stress-induced senescence.124,125 KDM6B expression in fibroblasts is induced by oncogenes of the RAS-RAF pathway, suggesting that it could provide an

important barrier to tumorigenesis. Accordingly, KDM6B levels are high in melanocytic nevi, which constitute pre-neoplastic lesions, whereas data mining indicates that KDM6B expression is decreased in subsets of human can-cers.124,125 Moreover, it appears that a large part of the genetic lesions leading to p53 loss likewise causes the deletion of JMJD3. The JMJD3 gene is located on chromosome 17, in close proximity to the p53 tumor suppressor gene. The allelic loss at 17p13.1, including both p53 and JMJD3, was also significantly correlated with more aggressive tumor behaviour.128 Taken together, genetic lesions and/or decreased expression of JMJD3 might contribute to the develop-ment of some human cancers, most likely by epigenetic silencing of the INK4A-ARF tumor suppressor locus.

JmjC Histone Demethylase InhibitorsPotent and specific inhibitors of the Jumonji C domain–containing histone lysine demethylases have not been iden-tified yet, and the available structure-activity relationship (SAR) data are limited.129-131 However, the efforts to target the cofactors essential for the activity of this class of enzymes have provided first promising results.129-131 The 2-oxoglutarate analogue, N-oxalyl-glycine (NOG), can weakly inhibit JMJD2C and the catalytic core of JMJD2A (Fig. 10).132 JMJD2A was also found to be specifically inhibited by dis-ruption of its Zn2+ binding site by Zn-ejecting compounds such as disulfiram and ebselen (Fig. 10).133 Interestingly, Zn-ion ejection seemed to be selective for the JMJD2 subfamily as other KDMs do not contain a Zn-binding site near their catalytic domain.133 In a recent study employing binding analyses by non-denaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography, a set of N-oxalyl-D-ty-rosine derivatives was investigated for the inhibition of the JMJD2 (KDM4)

676 Genes & Cancer / vol 2 no 6 (2011)M Monographs

subfamily of histone demethylases. Some of the inhibitors were shown to be selec-tive for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2.130 Other 2-OG mimetics endowed with a promis-ing JMJD2 inhibitory potency are the 2,4-pyridindicarboxylic acid (PDCA), a fragment-size inhibitor with (sub)micro-molar activity against JMJD2s and some of its derivatives,129,131 and a 2,2′-bipyri-dyl derivative containing a 4-carboxylate group (Fig. 10).129 A recent screen of a small library of compounds provided as JmjC KDM inhibitors some flavonoid- and catechol-type molecules, which have the limitation of being promiscuous inhibitors affecting a wide range of enzy-matic targets (Fig. 10).134 Quantitative high-throughput screening of a ~236,000-member collection of diverse molecules led to the identification of 8-hydroxy-5-carboxyquinoline as a potent inhibitor of JMJD2 enzymes via chelation of the active-site iron and endowed with activity against JMJD2A in cell-based studies.135 The well- known HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was shown to be also a micromolar inhibitor of the recombinant enzyme JMJD2E (annotated as a pseudogene) that, for its close anal-ogy in terms of sequence identity to the catalytic domain of the other JMJD2 enzymes, is easily amenable to large-scale inhibition studies (Fig. 10).129 Recently, some hydroxamic acids bear-ing as a unique feature an N-propionic acid unit have been reported by Hamada and co-workers136 as selective inhibitors of JMJD2A and JMJD2C over PHD1 and PHD2 enzymes (Fig. 10). Two cell- permeable pro-drugs of the best inhibitor of this series displayed a synergistic cell growth inhibition in combination with a tranylcypromine-based LSD1 inhibitor in different cancer cell lines (LNCaP, PC3, and HCT116), suggesting clinical poten-tial for this type of combination in anti-cancer chemotherapy.136

Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of inter-est with respect to the research, authorship, and/or publication of this article.

FundingThe author(s) received no financial support for the research, authorship, and/or publication of this article.

References 1. Strahl BD, Allis CD. The language of covalent his-

tone modifications. Nature. 2000;403(6765):41-5.

2. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293(5532):1074-80.

3. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128(4):693-705.

4. Mai A, Altucci L. Epi-drugs to fight cancer: from chemistry to cancer treatment, the road ahead. Int J Biochem Cell Biol. 2009;41(1):199-213.

5. Mai A. Small-molecule chromatin-modifying agents: therapeutic applications. Epigenomics. 2010;2(2):307-24.

6. Shi Y, Lan F, Matson C, et al. Histone demeth-ylation mediated by the nuclear amine oxidase homolog LSD1. Cell. 2004;119(7):941-53.

7. Forneris F, Binda C, Vanoni MA, Mattevi A, Battaglioli E. Histone demethylation catalysed by LSD1 is a flavin-dependent oxidative process. FEBS Lett. 2005;579(10):2203-7.

8. Karytinos A, Forneris F, Profumo A, et al. A novel mammalian flavin-dependent histone demethyl-ase. J Biol Chem. 2009;284(26):17775-82.

9. Tsukada Y, Fang J, Erdjument-Bromage H, et al. Histone demethylation by a family of JmjC domain-containing proteins. Nature. 2006; 439(7078):811-6.

10. Klose RJ, Kallin EM, Zhang Y. JmjC-domain-containing proteins and histone demethylation. Nat Rev Genet. 2006;7(9):715-27.

11. Hakimi MA, Bochar DA, Chenoweth J, Lane WS, Mandel G, Shiekhattar R. A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes. Proc Natl Acad Sci U S A. 2002;99(11):7420-5.

12. You A, Tong JK, Grozinger CM, Schreiber SL. CoREST is an integral component of the CoR-EST–human histone deacetylase complex. Proc Natl Acad Sci U S A. 2001;98(4):1454-8.

13. Battaglioli E, Andres ME, Rose DW, et al. REST repression of neuronal genes requires compo-nents of the hSWI.SNF complex. J Biol Chem. 2002;277(43):41038-45.

14. Lee MG, Wynder C, Cooch N, Shiekhattar R. An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature. 2005; 437(7057):432-5.

15. Forneris F, Binda C, Battaglioli E, Mattevi A. LSD1: oxidative chemistry for multifaceted functions in chromatin regulation. Trends Bio-chem Sci. 2008;33(4):181-9.

16. Yang M, Gocke CB, Luo X, et al. Structural basis for CoREST-dependent demethylation of nucleo-somes by the human LSD1 histone demethylase. Mol Cell. 2006;23(3):377-87.

17. Shi YJ, Matson C, Lan F, Iwase S, Baba T, Shi Y. Regulation of LSD1 histone demethyl-ase activity by its associated factors. Mol Cell. 2005;19(6):857-64.

18. Spannhoff A, Hauser AT, Heinke R, Sippl W, Jung M. The emerging therapeutic potential of

histone methyltransferase and demethylase inhibi-tors. ChemMedChem. 2009;4(10):1568-82.

19. Culhane JC, Cole PA. LSD1 and the chemistry of histone demethylation. Curr Opin Chem Biol. 2007;11(5):561-8.

20. Yang M, Culhane JC, Szewczuk LM, et al. Struc-tural basis of histone demethylation by LSD1 revealed by suicide inactivation. Nat Struct Mol Biol. 2007;14(6):535-9.

21. Forneris F, Binda C, Vanoni MA, Battagli-oli E, Mattevi A. Human histone demethylase LSD1 reads the histone code. J Biol Chem. 2005;280(50):41360-5.

22. Forneris F, Binda C, Dall’Aglio A, Fraaije MW, Battaglioli E, Mattevi A. A highly spe-cific mechanism of histone H3-K4 recognition by histone demethylase LSD1. J Biol Chem. 2006;281(46):35289-95.

23. Metzger E, Imhof A, Patel D, et al. Phosphory-lation of histone H3T6 by PKCbeta(I) con-trols demethylation at histone H3K4. Nature. 2011;464(7289):792-6.

24. Metzger E, Wissmann M, Yin N, et al. LSD1 demethylates repressive histone marks to pro-mote androgen-receptor-dependent transcription. Nature. 2005;437(7057):436-9.

25. Garcia-Bassets I, Kwon YS, Telese F, et al. His-tone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell. 2007;128(3):505-18.

26. Wissmann M, Yin N, Muller JM, et al. Coop-erative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression. Nat Cell Biol. 2007;9(3):347-53.

27. Huang J, Sengupta R, Espejo AB, et al. p53 is regulated by the lysine demethylase LSD1. Nature. 2007;449(7158):105-8.

28. Wang J, Hevi S, Kurash JK, et al. The lysine demethylase LSD1 (KDM1) is required for main-tenance of global DNA methylation. Nat Genet. 2009;41(1):125-9.

29. Yuan B, Zhang J, Wang H, et al. 6-Thioguanine reactivates epigenetically silenced genes in acute lymphoblastic leukemia cells by facilitating proteasome-mediated degradation of DNMT1. Cancer Res. 2011;71(5):1904-11.

30. Cho HS, Suzuki T, Dohmae N, et al. Demeth-ylation of RB regulator MYPT1 by histone demethylase LSD1 promotes cell cycle progres-sion in cancer cells. Cancer Res. 2011;71(3): 655-60.

31. Yang Z, Jiang J, Stewart DM, et al. AOF1 is a histone H3K4 demethylase possessing demeth-ylase activity-independent repression function. Cell Res. 2010;20(3):276-87.

32. Fang R, Barbera AJ, Xu Y, et al. Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation. Mol Cell. 2010;39(2):222-33.

33. Ciccone DN, Su H, Hevi S, et al. KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature. 2009;461(7262):415-8.

34. van Essen D, Zhu Y, Saccani S. A feed-forward circuit controlling inducible NF-kappaB target gene activation by promoter histone demethyl-ation. Mol Cell. 2010;39(5):750-60.

677Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

35. Hayami S, Kelly JD, Cho HS, et al. Overexpres-sion of LSD1 contributes to human carcinogen-esis through chromatin regulation in various cancers. Int J Cancer. 2011;128(3):574-86.

36. Lim S, Janzer A, Becker A, et al. Lysine-specific demethylase 1 (LSD1) is highly expressed in ER-negative breast cancers and a biomarker predicting aggressive biology. Carcinogenesis. 2011;31(3):512-20.

37. Schulte JH, Lim S, Schramm A, et al. Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy. Cancer Res. 2009;69(5):2065-71.

38. Wang Y, Zhang H, Chen Y, et al. LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast cancer. Cell. 2009;138(4):660-72.

39. Huang PH, Chen CH, Chou CC, et al. Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases. Mol Pharmacol. 2011;79(1):197-206.

40. Thillainadesan G, Isovic M, Loney E, Andrews J, Tini M, Torchia J. Genome analysis identifies the p15ink4b tumor suppressor as a direct target of the ZNF217/CoREST complex. Mol Cell Biol. 2008;28(19):6066-77.

41. Lin Y, Wu Y, Li J, et al. The SNAG domain of Snail1 functions as a molecular hook for recruit-ing lysine-specific demethylase 1. EMBO J. 2010;29(11):1803-16.

42. Lin T, Ponn A, Hu X, Law BK, Lu J. Require-ment of the histone demethylase LSD1 in Snai1-mediated transcriptional repression during epithelial-mesenchymal transition. Oncogene. 2010;29(35):4896-904.

43. Ooi SK, Qiu C, Bernstein E, et al. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature. 2007;448(7154):714-7.

44. Huang Y, Stewart TM, Wu Y, et al. Novel oli-goamine analogues inhibit lysine-specific demethylase 1 and induce reexpression of epi-genetically silenced genes. Clin Cancer Res. 2009;15(23):7217-28.

45. Amente S, Bertoni A, Morano A, Lania L, Avvedimento EV, Majello B. LSD1-mediated demethylation of histone H3 lysine 4 trig-gers Myc-induced transcription. Oncogene. 2010;29(25):3691-702.

46. Culhane JC, Szewczuk LM, Liu X, Da G, Mar-morstein R, Cole PA. A mechanism-based inacti-vator for histone demethylase LSD1. J Am Chem Soc. 2006;128(14):4536-7.

47. Culhane JC, Wang D, Yen PM, Cole PA. Com-parative analysis of small molecules and histone substrate analogues as LSD1 lysine demethylase inhibitors. J Am Chem Soc. 2010;132(9):3164-76.

48. Szewczuk LM, Culhane JC, Yang M, Majumdar A, Yu H, Cole PA. Mechanistic analysis of a sui-cide inactivator of histone demethylase LSD1. Biochemistry. 2007;46(23):6892-902.

49. Bi X, Lopez C, Bacchi CJ, Rattendi D, Woster PM. Novel alkylpolyaminoguanidines and alkylpolyaminobiguanides with potent antitry-panosomal activity. Bioorg Med Chem Lett. 2006;16(12):3229-32.

50. Huang Y, Greene E, Murray Stewart T, et al. Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes. Proc Natl Acad Sci U S A. 2007;104(19):8023-8.

51. Sharma SK, Wu Y, Steinbergs N, et al. (Bis)urea and (bis)thiourea inhibitors of lysine-specific demethylase 1 as epigenetic modulators. J Med Chem. 2010;53(14):5197-212.

52. Lee MG, Wynder C, Schmidt DM, McCafferty DG, Shiekhattar R. Histone H3 lysine 4 demeth-ylation is a target of nonselective antidepressive medications. Chem Biol. 2006;13(6):563-7.

53. Schmidt DM, McCafferty DG. Trans-2-phe-nylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1. Biochemistry. 2007;46(14):4408-16.

54. Yang M, Culhane JC, Szewczuk LM, et al. Structural basis for the inhibition of the LSD1 histone demethylase by the antidepressant trans-2-phenylcyclopropylamine. Biochemistry. 2007;46(27):8058-65.

55. Ueda R, Suzuki T, Mino K, et al. Identifica-tion of cell-active lysine specific demethyl-ase 1–selective inhibitors. J Am Chem Soc. 2009;131(48):17536-7.

56. Binda C, Valente S, Romanenghi M, et al. Bio-chemical, structural, and biological evaluation of tranylcypromine derivatives as inhibitors of his-tone demethylases LSD1 and LSD2. J Am Chem Soc. 2010;132(19):6827-33.

57. Mimasu S, Umezawa N, Sato S, Higuchi T, Ume-hara T, Yokoyama S. Structurally designed trans-2-phenylcyclopropylamine derivatives potently inhibit histone demethylase LSD1/KDM1. Bio-chemistry. 2010;49(30):6494-503.

58. Benelkebir H, Hodgkinson C, Duriez PJ, et al. Enantioselective synthesis of tranylcypromine analogues as lysine demethylase (LSD1) inhibi-tors. Bioorg Med Chem. 2011;19(12):3709-16.

59. Gooden DM, Schmidt DM, Pollock JA, Kabadi AM, McCafferty DG. Facile synthesis of substi-tuted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and mono-amine oxidases A and B. Bioorg Med Chem Lett. 2008;18(10):3047-51.

60. Mimasu S, Sengoku T, Fukuzawa S, Umehara T, Yokoyama S. Crystal structure of histone demeth-ylase LSD1 and tranylcypromine at 2.25 A. Bio-chem Biophys Res Commun. 2008;366(1):15-22.

61. Escoubet-Lozach L, Lin IL, Jensen-Pergakes K, et al. Pomalidomide and lenalidomide induce p21 WAF-1 expression in both lymphoma and multiple myeloma through a LSD1-mediated epigenetic mechanism. Cancer Res. 2009;69(18):7347-56.

62. Klose RJ, Zhang Y. Regulation of histone meth-ylation by demethylimination and demethylation. Nat Rev Mol Cell Biol. 2007;8(4):307-18.

63. Loenarz C, Schofield CJ. Expanding chemical biology of 2-oxoglutarate oxygenases. Nat Chem Biol. 2008;4(3):152-6.

64. Loenarz C, Schofield CJ. Physiological and bio-chemical aspects of hydroxylations and demeth-ylations catalyzed by human 2-oxoglutarate oxygenases. Trends Biochem Sci. 2011;36(1):7-18.

65. Hewitson KS, McNeill LA, Riordan MV, et al. Hypoxia-inducible factor (HIF) asparagine

hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem. 2002;277(29):26351-5.

66. Takeuchi T, Yamazaki Y, Katoh-Fukui Y, et al. Gene trap capture of a novel mouse gene, jumonji, required for neural tube formation. Genes Dev. 1995;9(10):1211-22.

67. Cloos PA, Christensen J, Agger K, Helin K. Eras-ing the methyl mark: histone demethylases at the center of cellular differentiation and disease. Genes Dev. 2008;22(9):1115-40.

68. Pedersen MT, Helin K. Histone demethylases in development and disease. Trends Cell Biol. 2011;20(11):662-71.

69. McDonough MA, Loenarz C, Chowdhury R, Clifton IJ, Schofield CJ. Structural studies on human 2-oxoglutarate dependent oxygenases. Curr Opin Struct Biol. 2010;20(6):659-72.

70. Yue WW, Hozjan V, Ge W, et al. Crystal struc-ture of the PHF8 Jumonji domain, an Nepsilon- methyl lysine demethylase. FEBS Lett. 2010; 584(4):825-30.

71. Horton JR, Upadhyay AK, Qi HH, Zhang X, Shi Y, Cheng X. Enzymatic and structural insights for substrate specificity of a family of Jumonji histone lysine demethylases. Nat Struct Mol Biol. 2010;17(1):38-43.

72. Ponnaluri VK, Vavilala DT, Putty S, Gutheil WG, Mukherji M. Identification of non-histone sub-strates for JMJD2A-C histone demethylases. Bio-chem Biophys Res Commun. 2009;390(2):280-4.

73. Varier RA, Timmers HT. Histone lysine methyla-tion and demethylation pathways in cancer. Bio-chim Biophys Acta. 2011;1815(1):75-89.

74. He J, Kallin EM, Tsukada Y, Zhang Y. The H3K36 demethylase Jhdm1b/Kdm2b regulates cell pro-liferation and senescence through p15(Ink4b). Nat Struct Mol Biol. 2008;15(11):1169-75.

75. Lagarou A, Mohd-Sarip A, Moshkin YM, et al. dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 2008;22(20):2799-810.

76. Frescas D, Guardavaccaro D, Bassermann F, Koyama-Nasu R, Pagano M. JHDM1B/FBXL10 is a nucleolar protein that represses transcription of ribosomal RNA genes. Nature. 2007;450(7167):309-13.

77. Tzatsos A, Pfau R, Kampranis SC, Tsichlis PN. Ndy1/KDM2B immortalizes mouse embryonic fibroblasts by repressing the Ink4a/Arf locus. Proc Natl Acad Sci U S A. 2009;106(8):2641-6.

78. Suzuki T, Minehata K, Akagi K, Jenkins NA, Cope-land NG. Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice. EMBO J. 2006;25(14):3422-31.

79. Pfau R, Tzatsos A, Kampranis SC, Serebren-nikova OB, Bear SE, Tsichlis PN. Members of a family of JmjC domain-containing oncoproteins immortalize embryonic fibroblasts via a JmjC domain-dependent process. Proc Natl Acad Sci U S A. 2008;105(6):1907-12.

80. Frescas D, Guardavaccaro D, Kuchay SM, et al. KDM2A represses transcription of centromeric satellite repeats and maintains the heterochro-matic state. Cell Cycle. 2008;7(22):3539-47.

81. Lu T, Jackson MW, Wang B, et al. Regulation of NF-kappaB by NSD1/FBXL11-dependent

678 Genes & Cancer / vol 2 no 6 (2011)M Monographs

reversible lysine methylation of p65. Proc Natl Acad Sci U S A. 2010;107(1):46-51.

82. Gearhart MD, Corcoran CM, Wamstad JA, Bardwell VJ. Polycomb group and SCF ubiqui-tin ligases are found in a novel BCOR complex that is recruited to BCL6 targets. Mol Cell Biol. 2006;26(18):6880-9.

83. Koyama-Nasu R, David G, Tanese N. The F-box protein Fbl10 is a novel transcriptional repressor of c-Jun. Nat Cell Biol. 2007;9(9):1074-80.

84. Polytarchou C, Pfau R, Hatziapostolou M, Tsi-chlis PN. The JmjC domain histone demethylase Ndy1 regulates redox homeostasis and pro-tects cells from oxidative stress. Mol Cell Biol. 2008;28(24):7451-64.

85. Yamane K, Toumazou C, Tsukada Y, et al. JHDM2A, a JmjC-containing H3K9 demethyl-ase, facilitates transcription activation by andro-gen receptor. Cell. 2006;125(3):483-95.

86. Heery DM, Kalkhoven E, Hoare S, Parker MG. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature. 1997;387(6634):733-6.

87. Inagaki T, Tachibana M, Magoori K, et al. Obe-sity and metabolic syndrome in histone demeth-ylase JHDM2a-deficient mice. Genes Cells. 2009;14(8):991-1001.

88. Liu Z, Zhou S, Liao L, Chen X, Meistrich M, Xu J. Jmjd1a demethylase-regulated histone modi-fication is essential for cAMP-response element modulator-regulated gene expression and sper-matogenesis. J Biol Chem. 2010;285(4):2758-70.

89. Okada Y, Scott G, Ray MK, Mishina Y, Zhang Y. Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogen-esis. Nature. 2007;450(7166):119-23.

90. Tateishi K, Okada Y, Kallin EM, Zhang Y. Role of Jhdm2a in regulating metabolic gene expression and obesity resistance. Nature. 2009;458(7239):757-61.

91. Wolf SS, Patchev VK, Obendorf M. A novel vari-ant of the putative demethylase gene, s-JMJD1C, is a coactivator of the AR. Arch Biochem Bio-phys. 2007;460(1):56-66.

92. Hu Z, Gomes I, Horrigan SK, et al. A novel nuclear protein, 5qNCA (LOC51780) is a can-didate for the myeloid leukemia tumor suppres-sor gene on chromosome 5 band q31. Oncogene. 2001;20(47):6946-54.

93. Krieg AJ, Rankin EB, Chan D, Razorenova O, Fernandez S, Giaccia AJ. Regulation of the histone demethylase JMJD1A by hypoxia-inducible factor 1 alpha enhances hypoxic gene expression and tumor growth. Mol Cell Biol. 2010;30(1):344-53.

94. Cloos PA, Christensen J, Agger K, et al. The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature. 2006;442(7100):307-11.

95. Fodor BD, Kubicek S, Yonezawa M, et al. Jmjd2b antagonizes H3K9 trimethylation at pericentric heterochromatin in mammalian cells. Genes Dev. 2006;20(12):1557-62.

96. Klose RJ, Yamane K, Bae Y, et al. The tran-scriptional repressor JHDM3A demethylates tri-methyl histone H3 lysine 9 and lysine 36. Nature. 2006;442(7100):312-6.

97. Lin CH, Li B, Swanson S, et al. Heterochro-matin protein 1a stimulates histone H3 lysine 36 demethylation by the Drosophila KDM4A demethylase. Mol Cell. 2008;32(5):696-706.

98. Whetstine JR, Nottke A, Lan F, et al. Rever-sal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell. 2006;125(3):467-81.

99. Trojer P, Zhang J, Yonezawa M, et al. Dynamic histone H1 isotype 4 methylation and demeth-ylation by histone lysine methyltransferase G9a/KMT1C and the Jumonji domain–con-taining JMJD2/KDM4 proteins. J Biol Chem. 2009;284(13):8395-405.

100. Ehrbrecht A, Muller U, Wolter M, et al. Com-prehensive genomic analysis of desmoplas-tic medulloblastomas: identification of novel amplified genes and separate evaluation of the different histological components. J Pathol. 2006;208(4):554-63.

101. Liu G, Bollig-Fischer A, Kreike B, et al. Genomic amplification and oncogenic proper-ties of the GASC1 histone demethylase gene in breast cancer. Oncogene. 2009;28(50):4491-500.

102. Northcott PA, Nakahara Y, Wu X, et al. Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma. Nat Genet. 2009;41(4):465-72.

103. Benevolenskaya EV, Murray HL, Branton P, Young RA, Kaelin WG Jr. Binding of pRB to the PHD protein RBP2 promotes cellular differ-entiation. Mol Cell. 2005;18(6):623-35.

104. Lopez-Bigas N, Kisiel TA, Dewaal DC, et al. Genome-wide analysis of the H3K4 histone demethylase RBP2 reveals a transcriptional program controlling differentiation. Mol Cell. 2008;31(4):520-30.

105. Pasini D, Hansen KH, Christensen J, Agger K, Cloos PA, Helin K. Coordinated regulation of transcriptional repression by the RBP2 H3K4 demethylase and Polycomb-Repressive Com-plex 2. Genes Dev. 2008;22(10):1345-55.

106. van Oevelen C, Wang J, Asp P, et al. A role for mammalian Sin3 in permanent gene silencing. Mol Cell. 2008;32(3):359-70.

107. Zeng J, Ge Z, Wang L, et al. The histone demeth-ylase RBP2 is overexpressed in gastric cancer and its inhibition triggers senescence of cancer cells. Gastroenterology. 2010;138(3):981-92.

108. Secombe J, Li L, Carlos L, Eisenman RN. The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth. Genes Dev. 2007;21(5):537-51.

109. Liefke R, Oswald F, Alvarado C, et al. Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex. Genes Dev. 2010;24(6):590-601.

110. Defeo-Jones D, Huang PS, Jones RE, et al. Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product. Nature. 1991;352(6332):251-4.

111. Sharma SV, Lee DY, Li B, et al. A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell. 2010;141(1):69-80.

112. Barrett A, Madsen B, Copier J, et al. PLU-1 nuclear protein, which is upregulated in breast cancer, shows restricted expression in normal

human adult tissues: a new cancer/testis anti-gen? Int J Cancer. 2002;101(6):581-8.

113. Xiang Y, Zhu Z, Han G, et al. JARID1B is a histone H3 lysine 4 demethylase up-regulated in prostate cancer. Proc Natl Acad Sci U S A. 2007;104(49):19226-31.

114. Yamane K, Tateishi K, Klose RJ, et al. PLU-1 is an H3K4 demethylase involved in transcrip-tional repression and breast cancer cell prolif-eration. Mol Cell. 2007;25(6):801-12.

115. Tahiliani M, Mei P, Fang R, et al. The histone H3K4 demethylase SMCX links REST target genes to X-linked mental retardation. Nature. 2007;447(7144):601-5.

116. Smith JA, White EA, Sowa ME, et al. Genome-wide siRNA screen identifies SMCX, EP400, and Brd4 as E2-dependent regulators of human papillomavirus oncogene expression. Proc Natl Acad Sci U S A. 2010;107(8):3752-7.

117. Agger K, Cloos PA, Christensen J, et al. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and develop-ment. Nature. 2007;449(7163):731-4.

118. De Santa F, Totaro MG, Prosperini E, Notar-bartolo S, Testa G, Natoli G. The histone H3 lysine-27 demethylase Jmjd3 links inflamma-tion to inhibition of polycomb-mediated gene silencing. Cell. 2007;130(6):1083-94.

119. Lan F, Bayliss PE, Rinn JL, et al. A histone H3 lysine 27 demethylase regulates animal pos-terior development. Nature. 2007;449(7163): 689-94.

120. Lee MG, Villa R, Trojer P, et al. Demethyl-ation of H3K27 regulates polycomb recruit-ment and H2A ubiquitination. Science. 2007;318(5849):447-50.

121. Kotake Y, Cao R, Viatour P, Sage J, Zhang Y, Xiong Y. pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK-4alpha tumor suppressor gene. Genes Dev. 2007;21(1):49-54.

122. Bracken AP, Kleine-Kohlbrecher D, Dietrich N, et al. The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev. 2007;21(5):525-30.

123. Wang JK, Tsai MC, Poulin G, et al. The histone demethylase UTX enables RB-dependent cell fate control. Genes Dev. 2010;24(4):327-32.

124. Agger K, Cloos PA, Rudkjaer L, et al. The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence. Genes Dev. 2009;23(10):1171-6.

125. Barradas M, Anderton E, Acosta JC, et al. His-tone demethylase JMJD3 contributes to epigen-etic control of INK4a/ARF by oncogenic RAS. Genes Dev. 2009;23(10):1177-82.

126. Dalgliesh GL, Furge K, Greenman C, et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature. 2010;463(7279):360-3.

127. van Haaften G, Dalgliesh GL, Davies H, et al. Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer. Nat Genet. 2009;41(5):521-3.

679Histone demethylase inhibitors and cancer / Rotili and Mai MMonographs

128. Nigro ND, Vaitkevicius VK, Considine B Jr. Dynamic management of squamous cell cancer of the anal canal. Invest New Drugs. 1989;7(1):83-9.

129. Rose NR, Ng SS, Mecinovic J, et al. Inhibi-tor scaffolds for 2-oxoglutarate-dependent histone lysine demethylases. J Med Chem. 2008;51(22):7053-6.

130. Rose NR, Woon EC, Kingham GL, et al. Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches. J Med Chem. 2010;53(4):1810-8.

131. Thalhammer A, Mecinovic J, Loenarz C, et al. Inhibition of the histone demethylase JMJD2E by 3-substituted pyridine 2,4-dicarboxylates. Org Biomol Chem. 2011;9(1):127-35.

132. Chen Z, Zang J, Kappler J, et al. Structural basis of the recognition of a methylated histone tail by JMJD2A. Proc Natl Acad Sci U S A. 2007;104(26):10818-23.

133. Sekirnik R, Rose NR, Thalhammer A, Seden PT, Mecinovic J, Schofield CJ. Inhibition of the histone lysine demethylase JMJD2A by ejection of structural Zn(II). Chem Commun (Camb). 2009;(42):6376-8.

134. Sakurai M, Rose NR, Schultz L, et al. A minia-turized screen for inhibitors of Jumonji histone demethylases. Mol Biosyst. 2010;6(2):357-364.

135. King ON, Li XS, Sakurai M, et al. Quantitative high-throughput screening identifies 8-hydroxy-quinolines as cell-active histone demethylase inhibitors. PLoS One. 2010;5(11):e15535.

136. Hamada S, Suzuki T, Mino K, et al. Design, syn-thesis, enzyme-inhibitory activity, and effect on human cancer cells of a novel series of Jumonji domain–containing protein 2 histone demeth-ylase inhibitors. J Med Chem. 2010;53(15): 5629-38.