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New lysine methyltransferase drug targets in cancer Tobias Wagner & Manfred Jung
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Recent preclinical studies suggest that inhibitors of histone methyltransferases represent promising drug candidates for cancer therapy. A spate of recent studies on histone methyltransferases has revealed interesting links to human disease. Since 2010, when histone methyltransferases were last reviewed in Nature Biotechnology1, several more family members have been associated with cancers, and a number of small-molecule inhibitors have been reported. Here we highlight these new developments, in particular, the identification of seven new inhibitors for five promising drug targets—G9a, Dot1L, SMYD2, EZH2 and Set7/9. Histone methyltransferases modify key lysines and arginines on histones and thereby influence patterns of gene expression. Eleven human arginine methyltransferases have been identified, and over 50 human genes are known to encode lysine methyltransferases. We will focus on lysine methylation because arginine methylation has been recently reviewed2 and because new drug candidates with relatively high selectivity and potency are available for lysine methyltransferases. Histone lysine methyltransferases modify specific lysine residues on histone tails, but unbiased approaches have revealed that they are more promiscuous and act on a wider range of substrates than expected. Modified lysine residues exist in a mono-, di- or trimethylated state (Fig. 1), each of which is generated by different lysine methyl transferases. As proteins that bind methylated lysines can distinguish between different levels of methylation, the degree of methylation conveys biological information. The five histone methyltransferases discussed have been the focus of significant research efforts in the past few years. Notably, histone modifications that they catalyze have been identified, as have many non-histone targets. From a therapeutic perspective, overexpression of these enzymes has been linked to various forms of cancer and other diseases, underscoring their potential functions in regulating cellular processes, signaling pathways and cancer development. Tobias Wagner and Manfred Jung are in the Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany. M.J. is also at the Freiburg Institute of Advanced Studies (FRIAS), University of Freiburg, Germany. e-mail:
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Small-molecule inhibitors are important tools for testing the roles of methyltransferases in disease and for investigating their ‘druggability’ (Table 1). Whereas the first histone lysine methyltransferases were characterized in 2000, the first inhibitors were not developed until 2005. These inhibitors had various drawbacks, such as limited potency and selectivity or poor pharmacokinetic properties. Since then, substantial progress by the pharmaceutical industry and many academic groups has led to improved compounds, especially with regard to potency and selectivity. The latter is important because different histone methyltransferases may have opposite effects on gene expression or may affect the expression of different target genes. In recent years, detailed molecular biology studies have been used to validate targets of histone methyltransferases, and combinations of screening and medicinal chemistry have led to optimized inhibitors, which in turn helped to define more precisely the sets of target genes. The G9a methyltransferase catalyzes the methylation of the histone protein H3 on lysine 9 (H3K9), which has an important role in early embryogenesis, the propagation of imprinting and the control of DNA methylation. In addition, G9a regulates centrosome duplication, presumably through chromatin structure. In cancer, G9a is upregulated in lung, prostate and hepatocellular carcinoma. An early G9a inhibitor, BIX-01294, which has micromolar potency, was last year optimized to yield UNC0638 (ref. 3), which has nanomolar potency and selectivity only for G9a and its closest homolog, GLP. UNC0638 reduced clonogenicity in MCF7 breast cancer cells3. The high potency of UNC0638 in vitro and in vivo, as well as its remarkable selectivity toward other protein methyltransferases, make this compound a helpful tool for further investigations of G9a, and UNC0638 analogs may be candidates for therapeutic evaluation as a cancer therapy. Very recently, a third G9a inhibitor, BRD4770, was shown to induce senescence in pancreatic adenocarcinoma4. Another promising anticancer target is Dot1L, which methylates lysine 79 on the tail of histone H3 (H3K79). A recent report showed that Dot1L is involved in the pathogenesis of
NH2
NH 2
NH2 NH2
Lysine methyltransferases G9a Dot1L SMYD2 EZH2 Set7/9
Small-molecule inhibitors
CH3 H2N+ H + N C 3 H2
CH3 CH3 N+ CH3
H N+ CH3 CH3
Normal human physiology
Other disease processes
Glucose response
HIV transcription
Cancer Hormone-dependent breast cancer Pancreatic adenocarcinoma Mixed lineage leukemia B-cell lymphoma Kaposi’s sarcoma
Figure 1 Protein lysine methylation. Lysine residues on histones and other proteins (yellow circles) are modified by methyltransferases, which may add one, two or three methyl groups (blue ovals). Recent studies have revealed intriguing links between histone lysine methyltransferases and human disease. Since 2010, several promising small-molecule inhibitors of these enzymes have been reported, and pharmaceutical companies and academic groups are actively pursuing inhibitors for use as therapeutics (Table 1).
leukemia driven by translocations of the mixed lineage leukemia (MLL) gene5. In an MLLAF9 animal model, the disease was dependent on Dot1L activity, and genetic abrogation of Dot1L led to lower expression of the target fusion gene MLL-AF9 (AF9 is also known as MLLT3) and to differentiation of leukemic blasts in vivo5. Moreover, a highly potent smallmolecule inhibitor of Dot1L, EPZ004777 (IC50 = 0.4 nM), recapitulated in vitro and in vivo the positive effects of genetically inactivating this gene6. Although EPZ004777 resembles the natural cosubstrate for methylation, S-adenosyl methionine, it was selective for Dot1L, which is somewhat surprising because the activity of other methyltransferases also depends on S-adenosyl methionine. In an MLL xenograft
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ne w s an d vie w s Table 1 Recent developments in histone methyltransferase inhibitors Enzyme
Modification
Inhibitor
Selected institutions/companies with documented efforts
Disease indications
G9a/GLP
H3K9me1/2 p53K373
3,4
H3K79
AZ505 H2B H3 H4 p53K370 RbK860 Hsp90K616me1 H3K27me1/2/3 Not clearly specified, GlaxoSmithKline; Epizyme; Constellation see GSK patent14 Pharmaceuticals
Various cancers, mixed lineage leukemia Various cancers
5,6
SMYD2
Boehringer Ingelheim, Research Institute of Molecular Pathology (IMP) Vienna; Structural Genomics Consortium; Broad Institute GlaxoSmithKline Epizyme AstraZeneca
Various cancers
Dot1L
BIX-01294 UNC0638 BRD4770 EPZ004777
EZH2
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© 2012 Nature America, Inc. All rights reserved.
Set7/9
H3K4me1 Tat ER
PDB 4e47
Structural Genomics Consortium
model, treatment with EPZ004777 led to histone hypermethylation and an increase in overall survival, and the compound was well tolerated in healthy mice6. It remains to be seen whether molecules with better pharmacokinetic properties and similar potency and selectivity can be created and shown to be efficacious in human patients. SMYD2 methylates histones and other proteins, including the tumor suppressor p53. The oncogenic potential of SMYD2 was revealed in 2009 with the finding that higher SMYD2 levels in patients with esophageal squamous cell carcinoma correlated with lower rates of survival. More recently, the first SMYD2 inhibitor—AZ505—which has submicromolar activity in vitro and is selective toward SMYD3, Dot1L, G9a and Set7/9, was described (ref. 7). Structures of SMYD2 alone and bound to AZ505 have been determined7, but functional data on the consequences of chemical inhibition of SMYD2 have not been reported. EZH2, which trimethylates lysine 27 on the tail of histone H3 (H2K27), is a major component of the polycomb repressor complex 2 and has been linked to the formation and progression of many cancers through overexpression and mutation8. Recent work has revealed that mutation of tyrosine 641 (e.g., in B-cell lymphoma) increases the cellular level of trimethylated H3K27, whereas the wild-type enzyme has a higher capacity to monomethylate the unmethylated lysine9,10. Moreover, inactivating mutations are linked to myeloid neoplasms11,12, and upregulation in endothelial cells infected with Kaposi’s sarcoma–associated herpes virus promotes angiogenesis in Kaposi’s sarcoma13. There are many ongoing efforts to develop direct inhibitors of EZH2 (in addition to the indirect inhibitor 3-deazaneoplanocin (DZnep)). Initial reports (ref. 14 and AACR abstracts (2012) 4700) have described inhibitors that compete with S-adenosyl methionine,
reducing global H3K27 trimethylation. In B-cell lymphoma cells harboring activating mutated EZH2, treatment with an EZH2 inhibitor decreased cell proliferation. Finally, Set7/9, which monomethylates lysine 4 on the tail of histone H3 (H3K4), has been implicated in the response following transient hyperglycemia and may be involved in the formation of a diabetic memory (that is, persistent damage to vasculature and nerves that is induced by prolonged periods of hyperglycemia). Set7/9 also methylates the HIV protein Tat and contributes to HIV transcription, and it methylates the estrogen receptor, facilitating estrogen-driven gene transcription. Therefore, it may qualify as a drug target for diseases such as type II diabetes, AIDS and hormone-dependent breast cancer. So far, there have been no publications on Set7/9 inhibitors, but an X-ray crystal structure of Set7/9 bound to an inhibitor was recently deposited by the Structural Genomics Consortium (Protein database entry 4e47). Many studies are currently in progress to profile histone modifications on a genomewide level and correlate these patterns with health and disease1. A central controversy is whether these marks are causative or merely symptomatic of chromatin accessibility and hence transcriptional activation15. Another major question concerns the redundancy of histone methyltransferases. Key lysine residues in histone tails can be methylated by several methyltransferase isotypes, and it is unclear whether broad or highly selective inhibitors will be needed for therapy. The promising results with histone methyltransferases suggest another class of potential drug target, the histone demethyltransferases. Active histone demethylation also contributes to epigenetic regulation, and many of the histone demethylases have been implicated in important diseases, especially cancer. The anticancer effects of histone demethylase inhibitors have already been proven in
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Various cancers, especially B-cell lymphoma; angiogenesis in Kaposi’s sarcoma
References
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8–14
Breast cancer, diabetes (type II), HIV
animals16,17. Notably, these results raise concerns about the potential side effects of therapeutically boosting histone methylation levels through methyltransferase inhibitors. For example, the G9a inhibitor UNC0638 reactivates silent retroviral inserts3. This may be useful in the context of cellular reprogramming strategies, but it raises safety issues about the use of histone methyltransferase inhibitors in patients. Much remains to be learned about histone methyltransferases and their potential as therapeutics and diagnostics. We look forward with great interest to further developments, especially the first clinical studies of histone methyltransferase inhibitors, which may begin soon. ACKNOWLEDGMENTS We thank the Deutsche Krebshilfe (Nr. 107898) for funding of our work on histone methyltransferases. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Kelly, T.K., De Carvalho, D.D. & Jones, P.A. Nat. Biotechnol. 28, 1069–1078 (2010). 2. Bissinger, E.M. et al. Med. Chem. Commun. 1, 114–124 (2010). 3. Vedadi, M. et al. Nat. Chem. Biol. 7, 566–574 (2011). 4. Yuan, Y. et al. ACS Chem. Biol. advance online publication, doi:10.1021/cb300139y (26 April 2012). 5. Bernt, K.M. et al. Cancer Cell 20, 66–78 (2011). 6. Daigle, S.R. et al. Cancer Cell 20, 53–65 (2011). 7. Ferguson, A.D. et al. Structure 19, 1262–1273 (2011). 8. Chang, C.J. & Hung, M.C. Br. J. Cancer 106, 243–247 (2012). 9. Sneeringer, C.J. et al. Proc. Natl. Acad. Sci. USA 107, 20980–20985 (2010). 10. Morin, R.D. et al. Nat. Genet. 42, 181–185 (2010). 11. Chase, A. & Cross, N.C.P. Clin. Cancer Res. 17, 2613–2618 (2011). 12. Zhang, J. et al. Nature 481, 157–163 (2012). 13. He, M. et al. Cancer Res. advance online publication, doi:10.1158/0008-5472.CAN-11-2876 (16 May 2012). 14. Burgess, J. et al. Azaindazoles. WO2012/005805 A1 (2012). 15. Henikoff, S. & Shilatifard, A. Trends Genet. 27, 389–396 (2011). 16. Willmann, D. et al. Int. J. Cancer Preprint at (2012). 17. Schenk, T. et al. Nat. Med. 18, 605–611 (2012).
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