Histone regulation

Histone modifications play a major role not only in transcription but in all DNA-templated processes¹. The activity of many proteins that modify or bind certain histone modifications is misregulated in cancer. We will discuss below essential histone modifications related to cancer.

Key histone proteins and their modifications involved in cancer.

Histone acetylation

​​The N-terminal acetylation of lysine residues is a major histone modification involved in transcription, chromatin structure, and DNA repair. Acetylation adds a negative charge to lysine residues; and these negative charges repel negatively charged DNA, which results in a relaxed chromatin structure. The open chromatin conformation allows transcription factor binding and significantly increases gene expression².

Histone acetylation is a highly dynamic process regulated by two competing enzymatic families: the histone lysine acetyltransferases (KATs) and the histone deacetylases (HDACs).

KATs family has two major classes: type-A and type-B. Type-A KATs are primarily nuclear and are classified into the GNAT, MYST, and CBP/p300 families. Type-B KATs are predominantly cytoplasmic and modify free histones³. Many KATs are shown to be implicated in neoplastic transformation. Also, KATs function as transcription co-factors for several tumor suppressors, eg CBP/p300 acetylate p53⁴.

Histone deacetylases (HDACs) are the family of 18 enzymes that reverse lysine acetylation and restore the positive charge on the histone side chain. The HDACs are overexpressed in numerous cancers, which results in a global loss of histone acetylation and silenced tumor suppressor gene expression.

The HDACs are divided into four major classes, depending on their sequence homology and structure. Class I HDACs (1, 2, 3, 8) and class II HDACs (4–7, 9, 10) have been reported to play roles in oncogenesis. HDAC4 is found to be a prognostic marker for pancreatic and ovarian cancers⁵, whereas HDAC5 may be a marker for liver, renal and cervical cancers⁶. Furthermore, numerous HDACs inhibitors are currently being developed as potential anti-cancer therapies⁷. HDAC8 is a promising novel target in cancer, and many potent HDAC8-selective inhibitors have been reported to exhibit cellular activity and anticancer effects⁸.

The histone acetylation marks on lysine residues are read by small protein modules, called “readers”. These protein modules are called bromodomains and include BRD2, BRD3, and BRD4, which are members of the BET family. BET proteins are essential for transcriptional elongation and cell-cycle regression. BET bromodomains represent a promising therapeutic target in cancer. Several studies demonstrated the possibility of the development of highly specific and chemically distinct small molecules against the BET family^9,10,11.

Histone methylation

Histones are methylated on the side chains of arginine, lysine, and histidine residues. Methylation, unlike acetylation and phosphorylation, does not alter the overall charge of the molecule.

The best-studied histones, implicated in lysine methylation, include H3K4, H3K9, H3K27, H3K36, and H3K79. Methylation is associated with activation or deactivation of gene expression¹¹. Importantly, different methylation states (eg monomethyl me1, dimethyl me2, trimethyl me3) of the same lysine residue may have distinct functional consequences. For example, H3K4me2/3 usually spans the transcriptional start site of active genes¹², whereas H3K4me1 modification is associated with active enhancers¹³. Similarly, H3K9me1 is found at active genes, while H3K9me3 is associated with gene repression¹⁴.

Histone methylation of lysine residues is carried out by highly specific enzymes called the histone lysine methyltransferases (KMT). Cytogenetic and NGS studies of various cancer genomes have demonstrated recurrent translocations and/or coding mutations in many KMTs, including MLL family members. Also, recurrent coding mutations have been reported in several histone demethylases such as KDM5A (JARID1A), KDM5C (JARID1C), and KDM6A (UTX). These recurrent mutations may significantly alter the catalytic activity of the methyltransferases or demethylases. Additionally, as many of these enzymes contain chromatin-reader motifs, the mutations may affect the ability of these proteins to survey and bind epigenetic modifications³.

DNA modifications

Changes in both global and individual gene methylation patterns are often found in cancer, and methylation patterns can distinguish tumor types. Global DNA hypomethylation is commonly observed in cancer genomes. However, hypermethylation is frequently detected in specific CpG-rich regions leading to the silencing expression of tumor suppressors. 5%–10% of normally unmethylated CpG promoter islands become abnormally methylated in various cancer genomes. CpG island methylation plays an important role in transcriptional regulation and is commonly altered during malignant transformation^14,15

Methylation of the 5-carbon of cytosine nucleotides is carried out by a family of DNA methyltransferases (DNMTs), which includes 3 active DNMTs in higher eukaryotes. DNMT1 is a maintenance methyltransferase that recognizes hemimethylated DNA generated during DNA replication and then methylates newly synthesized CpG dinucleotides, whose partners on the parental strand are already methylated¹⁶. DNMT3a and DNMT3b function primarily as de novo methyltransferases to establish DNA methylation during embryogenesis¹⁷. Recurrent loss-of-function mutations in DNMT3A have been reported in up to 25% of patients with acute myeloid leukemia (AML)¹⁸.

Key DNA modifications critical in cancer.

​DNA methylation provides a platform for several methyl-binding proteins, including MBD1, MBD2, and MeCP2. These proteins in turn function to recruit histone-modifying enzymes to coordinate the chromatin-templated processes¹⁹. MBD1, MBD2, and MeCP2 all act as transcriptional repressors and are implicated in various cancers. Thus, MeCP2 has been identified as a frequently amplified oncogene in several cancer types, including breast, lung, cervical, and uterine cancers²⁰. MBD1 is involved in tumor development and progression of multiple cancers, including lung, prostate, and colorectal cancers²¹. Finally, MBD2 is essential for the maintenance and spread of DNA methylation at CpG islands and shores in cancer²².

DNA hydroxymethylation is another type of DNA modification, in which the hydrogen atom at the C5-position in cytosine is replaced by a hydroxymethyl group. The ten-eleven translocation (TET 1–3) family of proteins carries out DNA hydroxymethylation in mammals by catalyzing the successive oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC)^23,24. TET genes, and especially TET2, are frequently mutated and show reduced expression in various cancers. Thus, TET2 was found to mutate recurrently in numerous hematopoietic malignancies, including AML, MPD, MDS, CMML^25,26,27. However, it is unknown how the TET proteins contribute to preventing the onset and maintenance of these malignancies²⁸.

RNA modifications

Non-coding RNAs are essential for normal development and may play a fundamental role in epigenetic regulation²⁹. Small non-coding RNAs have a high degree of sequence conservation across species and are involved in transcriptional and post-transcriptional gene silencing through specific base-pairing with their targets. Long non-coding RNAs show poor sequence conservation across species and may act as molecular chaperones for various chromatin regulators³⁰.

Similar to DNA, RNA is subject to a covalent modification such as methylation. Among all RNA modifications, N6-methyladenosine (m6A) is the most common, abundant, and conserved internal transcriptional modification, especially within eukaryotic messenger RNAs (mRNAs). m6A RNA modifications play an important role in RNA transcription, processing, translation, and metabolism and are associated with multiple diseases such as obesity, infertility, and cancer. In cancer, these modifications have been linked to the oncogenesis of multiple malignancies, including AML, glioblastoma, lung cancer, and others³¹.

Key RNA modifications involved in cancer.

m6A methylation is a dynamic and reversible process coordinated by methyltransferases, such as METTL3, demethylases, and identifiers. METTL3 is involved in all stages of the life cycle of RNA. It plays a key role in pre-mRNA splicing, 3′-end processing, nuclear export, translation regulation, mRNA decay, and microRNA processing³². METTL3 affects tumor formation by regulating the m6A modification in the mRNAs of critical oncogenes or tumor suppressors. For example, METTL3 acts as an oncogenic signal in lung adenocarcinoma and as a tumor suppressor in glioblastoma^33,34. METTL3 was also shown to be upregulated in various solid tumors, including hepatocellular carcinoma, associated with poor prognosis³⁵.

RNA epigenetics (or epitranscriptomics) can present new opportunities for early-stage diagnostics and treatment of cancer. Thus, since METTL3 has been detected in multiple cancer types, targeting m6A-associated proteins might constitute a new form of cancer treatment³⁶. For instance, novel small molecule inhibitors of METTL3 have been demonstrated as an effective therapeutic strategy in AML³⁷.

Polycomb and chromatin remodeling

Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes. PcG proteins have been linked to cancer development and progression; several cancer types demonstrate the deregulation of the PcG protein expression and function.

PcG proteins selectively repress gene expression during embryonic development by the formation of multi-subunit complexes called polycomb repressive complexes (PRCs). These complexes regulate chromatin organization and keep it in a transcriptionally inactive state⁴¹. PRC targets consist of diverse genes encoding transcription factors, signaling proteins, receptors, and regulators involved in all major developmental pathways. The PRCs comprise of PRC1 and PRC2 (with additional variants also being reported), which induce covalent post-translational histone modifications. While the PRC1 subunits catalyze the monoubiquitination of histone H2A at lysine 119 (H2AK119Ub1), the PRC2 subunits catalyze di- and trimethylation of histone H3 at lysine 27 (H3K27me2/3). Both histone modifications are associated with transcriptional silencing⁴².

Key polycomb and chromatin remodeling proteins involved in cancer.

​PRC2 activity is often misregulated in various human cancers and thereby became a focus for therapeutic drug development. PRC2 complex contains several proteins, including EZH2, EED, and SUZ12. EZH2 is a PRC2 component that catalyzes di- and trimethylation of H3K27. Extensive evidence has linked EZH2 to cancer development and progression. EZH2 overexpression and gain-of-function and loss-of-function mutations were identified in various cancer types. Furthermore, EZH2 overexpression is correlated with a worse progression of several cancers, including prostate cancer40. The overexpression of EZH2 and its mutations in various cancers motivated the development of small-molecule inhibitors of EZH2, which are currently a hot topic in drug development⁶. Thus, several EZH2 inhibitors have been developed and are being investigated in clinical trials.

Several other important epigenetic targets are involved in the interactions with the PRC2 complex, including histone methyltransferases DOT1L and KMT2A and histone demethylase KDM1A. KDM1A is highly expressed in several cancers and is specifically required for the terminal differentiation of hematopoietic cells⁴¹.

DOT1L, H3K79 methyltransferase, is a promising epigenetic target that plays an important role in mixed lineage leukemia. Furthermore, deletion and somatic mutations of the DOT1L gene are found in several types of solid cancers, including melanoma, colorectal cancer, and ovarian cancer^42,43.

Lysine methyltransferase 2 (KMT2, also known as MLL) methylates H3K4 to promote genome accessibility and transcription. KMT2 genes are frequently mutated in many human cancers and are associated with some of the most common and deadly solid tumors, such as lung and colon carcinomas^44,45,46.

Summary

Key polycomb and chromatin remodeling proteins involved in cancer.

​PRC2 activity is often misregulated in various human cancers and thereby became a focus for therapeutic drug development. PRC2 complex contains several proteins, including EZH2, EED, and SUZ12. EZH2 is a PRC2 component that catalyzes di- and trimethylation of H3K27. Extensive evidence has linked EZH2 to cancer development and progression. EZH2 overexpression and gain-of-function and loss-of-function mutations were identified in various cancer types. Furthermore, EZH2 overexpression is correlated with a worse progression of several cancers, including prostate cancer40. The overexpression of EZH2 and its mutations in various cancers motivated the development of small-molecule inhibitors of EZH2, which are currently a hot topic in drug development⁶. Thus, several EZH2 inhibitors have been developed and are being investigated in clinical trials.

Several other important epigenetic targets are involved in the interactions with the PRC2 complex, including histone methyltransferases DOT1L and KMT2A and histone demethylase KDM1A. KDM1A is highly expressed in several cancers and is specifically required for the terminal differentiation of hematopoietic cells⁴¹.

DOT1L, H3K79 methyltransferase, is a promising epigenetic target that plays an important role in mixed lineage leukemia. Furthermore, deletion and somatic mutations of the DOT1L gene are found in several types of solid cancers, including melanoma, colorectal cancer, and ovarian cancer^42,43.

Lysine methyltransferase 2 (KMT2, also known as MLL) methylates H3K4 to promote genome accessibility and transcription. KMT2 genes are frequently mutated in many human cancers and are associated with some of the most common and deadly solid tumors, such as lung and colon carcinomas^44,45,46.

References

1. Kouzarides, T. Chromatin modifications and their function Cell 128 ,693-705 (2007)
2. Roth, S. Y., Denu, J. M., Allis, C. D. Histone acetyltransferases Annu. Rev. Biochem. 70 ,81-120 (2001)
3. Dawson, M. A., Kouzarides, T. Cancer epigenetics: from mechanism to therapy Cell 150 ,12-27 (2012)
4. Shi, D., et al. CBP and p300 are cytoplasmic E4 polyubiquitin ligases for p53 Proc Natl Acad Sci USA 106 ,16275-16280 (2009)
5. Bennett, R. L., Licht, J. D. Targeting epigenetics in cancer Annu Rev Pharmacol Toxicol 58 ,187-207 (2018)
6. Chakrabarti, A., et al. Targeting histone deacetylase 8 as a therapeutic approach to cancer and neurodegenerative diseases Future Med Chem 8 ,1609-1634 (2016)
7. Dawson, M. A., et al. . Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia Nature 478 ,529-533 (2011)
8. Filippakopoulos, P., et al. Selective inhibition of BET bromodomains Nature 468 ,1067-1073 (2010)
9. Nicodeme, E., et al. Suppression of inflammation by a synthetic histone mimic Nature 468 ,1119-1123 (2010)
10. Barski, A., et al. High-resolution profiling of histone methylations in the human genome Cell 129 ,823-837 (2007)
11. Heintzman, N. D., et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression Nature 459 ,108-112 (2009)
12. Baylin, S. B., Jones, P. A. A decade of exploring the cancer epigenome - biological and translational implications Nat. Rev. Cancer. 11 ,726-734 (2011)
13. Robertson, K. D. DNA methylation and human disease Nat. Rev. Genet. 6 ,597-610 (2005)
14. Li, E., Bestor, T. H., Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality Cell 69 ,915-926 (1992)
15. Okano, M., Bell, D. W., Haber, D. A., Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development Cell 99 ,247-257 (1999)
16. Ley, T. J., et al. DNMT3A mutations in acute myeloid leukemia N. Engl. J. Med. 363 ,2424-2433 (2010)
17. Klose, R. J., Bird, A. P. Genomic DNA methylation: the mark and its mediators Trends Biochem. Sci. 31 ,89-97 (2006)
18. Neupane, M., et al. MECP2 is a frequently amplified oncogene with a novel epigenetic mechanism that mimics the role of activated RAS in malignancy Cancer Discov. 6 ,45-58 (2016)
19. Parry, L., Clarke, A. R. The roles of the methyl-CpG binding proteins in cancer Genes Cancer 2 ,618-630 (2011)
20. Stirzaker, C., et al. Methyl-CpG-binding protein MBD2 plays a key role in maintenance and spread of DNA methylation at CpG islands and shores in cancer Oncogene 36 ,1328-1338 (2017)
21. He, Y-F., et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA Science 333 ,1303-1307 (2011)
22. Ito, S., et al. . Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine Science 333 ,1300-1303 (2011)
23. Cimmino, L., Abdel-Wahab, O., Levine, R. L., Aifantis, I. TET family proteins and their role in stem cell differentiation and transformation Cell Stem Cell 9 ,193-204 (2011)
24. Delhommeau, F., et al. Mutation in TET2 in myeloid cancers N Engl J Med 360 ,2289-2301 (2009)
25. Langemeijer, S. M. C., et al. Acquired mutations in TET2 are common in myelodysplastic syndromes Nat Genet 41 ,838-842 (2009)
26. Rasmussen, K. D., Helin, K. Role of TET enzymes in DNA methylation, development, and cancer Genes Dev 30 ,733-750 (2016)
27. Amaral, P. P., Dinger, M. E., Mercer, T. R., Mattick, J. S. The eukaryotic genome as an RNA machine Science 319 ,1787-1789 (2008)
28. Wang, K. C., Chang, H. Y. Molecular mechanisms of long noncoding RNAs Mol. Cell. 43 ,904-914 (2011)
29. Chen, X. Y., Zhang, J., Zhu, J. S. The role of m6A RNA methylation in human cancer Mol cancer 18 ,103 (2019)
30. Han, J., et al. METTL3 promote tumor proliferation of bladder cancer by accelerating pri-miR221/222 maturation in m6A-dependent manner Mol Cancer 18 ,110 (2019)
31. Visvanathan, A., Somasundaram, K. mRNA traffic control reviewed: N6-methyladenosine (m(6)A) takes the driver’s seat Bioessays 40 , (2018)
32. Wang, S., et al. . Roles of RNA methylation by means of N(6)-methyladenosine (m(6)A) in human cancers Cancer Lett 408 ,112-120 (2017)
33. Chen, M., et al. RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2 Hepatology 67 ,2254-2270 (2018)
34. He, L., et al. The dual role of N6-methyladenosine modification of RNAs is involved in human cancers Cell Mol. Med 22 ,4630-4639 (2018)
35. Tzelepis, K., et al. . Pharmacological inhibition of the RNA m6a writer METTL3 as a novel therapeutic strategy for acute myeloid leukemia Blood 134 , (2019)
36. Levine, S. S., King, I. F., Kingston, R. E. Division of labor in polycomb group repression Trends Biochem. Sci 29 ,478-485 (2004)
37. Wang, W., et al. . Polycomb group (PcG) proteins and human cancers: multifaceted functions and therapeutic implications Med Res Rev 35 ,1220-1267 (2015)
38. Kim, K. H., Roberts, C. W. . Targeting EZH2 in cancer Nat Med 22 ,128-134 (2016)
39. Sprussel, A., et al. Lysine-specific demethylase 1 restricts hematopoietic progenitor proliferation and is essential for terminal differentiation Leukemia 26 ,2039-2051 (2012)
40. Cerami, E., et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data Cancer Discov. 2 ,401-404 (2012)
41. Gao, J., et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal Sci Signal 6 , (2013)
42. Kandoth, C., et al. Mutational landscape and significance across 12 major cancer types Nature 502 ,333-339 (2013)
43. Ding, L., et al. Somatic mutations affect key pathways in lung adenocarcinoma Nature 455 ,1069-1075 (2008)
44. Cancer Genome Atlas Network . Comprehensive molecular characterization of human colon and rectal cancer Nature 487 ,330-337 (2012)