Epigenetics during EMT in lung cancer: EZH2 as a potential therapeutic target

Author’s Accepted Manuscript Epigenetics during EMT in lung cancer: EZH2 as a potential therapeutic target Anastasios Dimou, Toros Dincman, Emilie Evanno, Robert M. Gemmill, Joëlle Roche, Harry A. Drabkin www.elsevier.com

PII: DOI: Reference:

S2468-2942(16)30137-X http://dx.doi.org/10.1016/j.ctarc.2017.06.003 CTARC58

To appear in: Cancer Treatment and Research Communications Received date: 3 November 2016 Revised date: 5 June 2017 Accepted date: 15 June 2017 Cite this article as: Anastasios Dimou, Toros Dincman, Emilie Evanno, Robert M. Gemmill, Joëlle Roche and Harry A. Drabkin, Epigenetics during EMT in lung cancer: EZH2 as a potential therapeutic target, Cancer Treatment and Research Communications, http://dx.doi.org/10.1016/j.ctarc.2017.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Epigenetics during EMT in lung cancer: EZH2 as a potential therapeutic target

Anastasios Dimou1,2, Toros Dincman1,2, Emilie Evanno3, Robert M Gemmill1, Joëlle Roche4, Harry A. Drabkin1*

1

Division of Hematology-Oncology, Medical University of South Carolina

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co-equal first authors

3

LNEC, Université de Poitiers, F-86073 Poitiers, France

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Laboratoire Ecologie et Biologie des Interactions, Equipe SEVE, Université de Poitiers, UMR

CNRS 7267, F-86073 Poitiers, France

[email protected] [email protected]

*

Corresponding author. Harry A Drabkin (or Robert M Gemmill), Medical University of South

Carolina, 39 Sabin St., MSC 635, Charleston, SC USA 29425, Tel.: (843) 792-4643; fax 843792-0644. 1

ABSTRACT Cancer is a disease of dysregulated growth and differentiation, along with the ability of the cancer cell to invade, metastasize and escape immune surveillance. Altered transcription factor activity from mutations, gene fusions, copy number alterations and growth factor signaling contributes substantially to the malignant phenotype. In addition, transcriptional deregulation has increasingly been shown to result from alterations in epigenetic modifying genes. In part, epigenetic regulation includes DNA methylation, as well as the covalent modification of histones by druggable targets that control chromatin accessibility. One epigenetic modifier, EZH2, is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2) that methylates lysine 27 of histone H3 (H3K27) associated with gene silencing. Of note, the activities of PRC2 and the nucleosome remodeling complex, SWI/SNF, are antagonistic and mutations in the SWI/SNF subunits, BRG1 and ARID1a, result in EZH2 dependency and sensitivity to EZH2 inhibitors. Mutations of BAP1, seen frequently in mesothelioma, have also been associated with EZH2 inhibitor sensitivity. In lung cancer and other malignant diseases, the upregulation of EZH2 is often a poor prognostic factor. However, in some cellular contexts, loss of EZH2/PRC2 function has also been linked to disease progression. Thus, understanding the biology and regulation of EZH2 is critical for the rational use of EZH2 inhibitors.

Keywords: EMT, Epigenetics, Polycomb Repressive Complex 2, SWI/SNF, NSCLC, SCLC, SETD2, chromatin modifications

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1.

Introduction Lung cancer is the leading cause of cancer deaths world-wide1. Large-scale genomic

studies have identified recurrent alterations in lung cancer and malignant mesothelioma. In many cases, these are in epigenetic regulators affecting chromatin structure. Epigenetic regulation is normally dynamic, reversible and includes the incorporation of histone variants, covalent histone modifications, nucleosome re-positioning, DNA methylation and changes in the expression of non-coding RNAs. The impact of epigenetic changes includes gene expression, reactivation of endogenous retroelements and genomic instability. The nucleosome is the basic chromatin unit consisting of a protein core formed by two copies of histones H2A, H2B, H3 and H4, encircled by 180-200 bp of DNA (Fig. 1). In addition, histone H1 binds DNA at its entry and exit points stabilizing the nucleosome2. The amino termini of histones undergo covalent modifications, the nature of which forms a “histone code” that affects transcription of nearby genes3. A conceptual classification of epigenetic modifiers includes “writers” that add modifications, “erasers” that remove them and “readers”, which recognize modifications and recruit various adapters. From a therapeutic standpoint, these processes are druggable4. Several reviews on epigenetic abnormalities in lung cancer have been published5-9. For example, smoking has been shown to cause overexpression of DNA methyltransferases (DNMTs) during lung carcinogenesis. Subsequently, hypermethylation of cytosine in CpG islands, as well as alterations that affect nucelosome remodeling, lead to silencing of tumor suppressor genes, such as p16INK4A. In addition, changes in histone methylation and acetylation correlate with prognosis in patients. Lastly, histone modifier proteins and miRNAs are often altered and critically affect lung carcinogenesis and response to therapy 6,7,10-13. In this review,

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we focus on EZH2 (Enhancer of Zeste 2), along with its role in lung cancer and the Epithelial to Mesenchymal Transition (EMT). EZH2is the catalytic subunit of the Polycomb Repressor Complex 2 (PRC2), which methylates histone H3 at lysine 27 (K27) to repress gene expression. Overexpression of EZH2 is frequently encountered in many tumor types including lung cancer and is often associated with an adverse prognosis.

2.1 EZH2 and the Polycomb Repressor Complexes Polycomb Group (PcG) proteins are predominantly transcriptional repressors acting to compact chromatin. Two multi-subunit PcG core complexes have been identified, PRC2 and PRC1, the latter of which is comprised of at least six distinct entities incorporating alternative subunits. EZH2 is a member of the PRC2 complex, which has three additional core components, SUZ12, EED and RBBP4. A high-resolution crystal structure for the Ezh2-Suz12-Eed complex has been determined14, shown schematically in Fig. 2A. This has provided molecular understanding of how the components interact, how H3K27 tri-methylation causes a positive feedback to enhance Ezh2 activity, and how lysine 27 to methionine mutations in histone H3, present in pediatric brain tumors, inhibit the enzymatic activity of EZH2. In the PRC2 complexes of some tissues, EZH1 is utilized instead of EZH2. EZH1 is ubiquitously expressed and shares 65% overall identity with EZH2. Of interest, current EZH2 inhibitors are generally less active against EZH1, and EZH1 may affect chromatin compaction independently of the methyltransferase cofactor, SAM15. In the mouse, Prc2 deficiency causes limited and specific developmental defects16. For example, this results in Cdkn2a (p16) upregulation and cell-cycle arrest. Of note, most genes upregulated by Prc2 deficiency are involved in the determination of

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tissue specificity and their promoters are typically bivalent, containing both active (H3K4me2/3) and inactive (H3K27me3) marks16. A genome wide analysis using antibodies to precipitate chromatin bound by PRC1 and PRC2 complexes in mouse embryonal stem cells identified Hox genes, as expected, as well as other homeodomain containing transcription factors (Gata, Fox, Sox, Tbx) and developmental genes 17. Likewise, in human ES cells, the PRC2 complex was found to occupy promoters and silence developmental genes like FOX, SOX, and TBX. In both human and mouse, activation of PcG target genes is linked to specific lineage differentiation. PRC2 binding sites were shown to overlap with the targets of SOX2, OCT4 and NANOG, known stem cell transcription factors. Of interest, in a mouse lung cancer model, deletion of the homeobox transcription factor, Ttf1/Nkx2-1, was recently shown to result in loss of pulmonary differentiation with a shift towards a mucin-producing gastric phenotype18. This seems reminiscent of the role of Prc2 in regulating tissue-specific genes, suggesting there may be layers of gene regulation involved in moving a cell from an embryonal to differentiated tissue-specific state. Thus, we speculate that less differentiated lung tumors could result from either increased Prc2 activity or Ttf-1/Nkx2-1 loss. The function of PRC2 sets the stage for the activity of PRC1, an E3 ubiquitin ligase with varying subunit composition that mono-ubiquitylates lysine 119 of histone H2A. A schematic of PRC1 containing either RING1A or RING1B and PCGF2 (PRC1.2) or PCGF4/BMI1 (PRC1.4)19 is shown in Fig. 2B. Together with one of several chromobox (CBX) proteins, these alternative PRC1 complexes are recruited to sites containing tri-methylated H3K27. The monoubiquitylation of histone H2A at K119 leads to silencing of developmental genes. The canonical process begins with H3K27 methylation by PRC2, which is subsequently recognized by a PRC1 CBX subunit and followed by Ring1A/1B-mediated ubiquitylation (Fig 3). Other PRC1

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complexes exist that are able to compact chromatin and mono-ubiquitylate H2A-K119 without a CBX subunit and without binding to tri-methylated H3K27. The subunit diversity of PRC120 contributes to multiple alternative regulatory pathways converging upon the H2A-K119-Ub mark, emphasizing its significance. Mono-ubiquitylation of H2AK119 inhibits lysine methyltransferases targeting H3K36, including SETD2, ASH1L, NSD1 and NSD2

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. PRC1 also recruits the KDM2 family of H3K36 demethylases (FBXL11,

FBXL10) providing a second mechanism for reducing H3K36 methylation. Conversely, H3K36me3 inhibits PRC2 22-24. Thus, methylation of H3K27 and H3K36 is largely mutually exclusive (Fig. 3). Of note, there is phenotypic similarity involving overgrowth between the developmental loss of EZH2 activity (Weaver Syndrome) and Sotos Syndrome, which results from loss of function mutations in H3K36 methyltansferases (e.g., SETD2 localized in 3p21, a region of frequent LOH in lung cancer, and NSD1 on chr 5). Although rare, SCLC was reported in a young non-smoking individual with Sotos syndrome25, suggesting that Polycomb dysregulation and genes affected by H3K36 methylation may be involved. Recently, the tumor suppressor protein, ZMYND11, was shown to localize to sites of H3K36 methylation established by SETD226. Furthermore, knockdown of ZMYND11 caused upregulation of genes specifically enriched in SCLC, thus providing a potential mechanistic link to Sotos syndrome. Not surprisingly, the effects of EZH2 overexpression on tumor progression are multifaceted, including downregulation of lineage-specific and tumor suppressor genes, as well as DNA repair pathways and senescence. For instance, EZH2 downregulates the cell-cycle inhibitor, p16INK4A, the tumor suppressor E-cadherin, as well as the dsDNA break repair protein, RAD51, which in turn leads to genomic instability27-29. EZH2 inhibition resulted in loss of non-

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canonical NF-kB signaling, which induced cellular senescence through de-repression of p16 and p2130. In prostate and breast cancer cells, EZH2 overexpression was associated with increased invasion31,32, which may involve reducing the expression of RAF1 kinase inhibitor protein (RKIP), a metastasis suppressor33. In contrast to gene suppression, EZH2 upregulates two kinesins, KIF2C and KIF22, which have been linked to invasion34. Thus, the tumor-promoting effects of EZH2 are multi-faceted.

2.2 SWI/SNF Complex The SWI/SNF complex is an important epigenetic regulator of gene expression and often has competing roles with PRC2. This competitive interaction can lead to unopposed PRC2 function when there is loss of SWI/SNF activity. Among the best studied functions of SWI/SNF is nucleosome sliding. During this process, proteins of the SWI/SNF complex bind the nucleosome and disrupt DNA-histone contacts utilizing energy from ATP hydrolysis. This alters the accessibility of DNA to transcription factors. Although the SWI/SNF complex is often an activator, it can also be involved in gene silencing by nucleosome movements that mask binding sites35. The composition of human SWI/SNF varies with the cellular context36 and contains a single ATPase, either BRM (SMARCA2) or BRG1 (SMARCA4). In addition, SWI/SNF complexes are distinguished by their DNA binding component; SWI/SNF-A binds to DNA with ARID1A or ARID1B, whereas SWI/SNF-B binds to DNA via ARID2. BRG1 is a frequent mutation target in lung cancer. In its wild-type state, BRG1 can antagonize c-MYC and is required for retinoic acid-induced differentiation37-40. Mutations in various SWI/SNF subunits are found in multiple tumors types, including in approximately 20% of lung cancers (cBioportal database; http://www.cbioportal.org). Of note, loss of function mutations are particularly

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common in poorly differentiated lung cancers. For example, adenocarcinomas with BRG1 loss have a solid growth pattern and are poorly-differentiated with EMT features41. Furthermore, up to one-third of large cell and pleomorphic lung carcinomas have alterations of BRG1 or other SWI/SNF subunits42. In contrast, EGFR mutant tumors, and tumors with high TTF-1 (NKX2-1) or E-cadherin expression, are rarely associated with BRG1 mutations.

2.3 Genetic antagonism between SWI/SNF and PRC2 Antagonism between SWI/SNF and Polycomb complexes was first described in Drosophila, with mutant PcG phenotypes suppressed by mutations in Swi/Snf43,44. In human malignant rhabdoid tumors, the SWI/SNF protein SNF5 (SMARCB1), is commonly mutated. In these tumors, SNF5 was shown to counteract the effect of PcG proteins at the p15-p16 locus (INK4b-ARF-INK4a)45. Changing the balance between SWI/SNF and PcG complexes also affected binding of the DNA methyltransferase, DNMT3B, and altered the DNA methylation state. In addition, in the mouse, Snf5 was shown to directly suppress Ezh246, providing an explanation for increased EZH2 levels in human cancer cells with SNF5 loss. In rhabdoid tumors, SNF5 loss results in disassembly of SWI/SNF at gene enhancers that promote cell differentiation, while sparing the complex’s activity at super enhancers, which are associated with cell identity 47. Two recent studies shed further light on the genetic antagonism between SWI/SNF and the PRC complexes 48,49. With the aid of Chemical Inducer of Proximity (CIP) technology, the investigators showed that SWI/SNF complex evicts PRC2 from occupying and silencing the promoter of Oct4 in MEFs. In this system, the eviction of Polycomb complexes by SWI/SNF depends on the ATPase activity of BRG1 and requires SNF5. This mechanism explains how

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SNF5 mutations lead to the loss of p16INK4A expression. On the other hand, in synovial sarcomas, a fusion protein, SS18/SSX, enhances the occupancy of SWI/SNF and the eviction of Polycomb proteins from the oncogene, SOX2. Thus, different mutations affecting SWI/SNF can alternatively de-activate a tumor suppressor gene (INK4) or induce an oncogene (SOX2).

2.4 Mutations of epigenetic modifying genes in lung cancer Mutations of epigenetic modifying genes have frequently been identified in lung cancer. Among 178 squamous cancers, mutations of the H3K4 methyltransferase, MLL2, were present in 20%50. As discussed below, both ARID1a and BRG mutations sensitize tumors to EZH2 inhibitors. Interestingly, ARID1a mutations were recently identified as drivers of colon cancer independent of APC mutations 51. Mutations of the H3K36 methyltransferase, SETD2, have been associated with DNA hypermethylation and diminished expression of p16INK4a/CDKN2A52, suggesting that DNA methylation inhibitors (and EZH2 inhibitors) might be therapeutically beneficial. To illustrate the fact that SETD2 has pleiotropic effects, it was recently shown to methylate tubulin and its deletion was shown to result in genomic instability53. Among >400 adenocarcinomas, mutations in the SWI/SNF components, ARID1a or BRG1, as well as SETD2, occurred in ~ 20% of cases52. In addition, mutations of DOT1L, a H3K79 methyltransferase, were identified in 3% of cases54. The high frequency of BRG1 alterations in large-cell neuroendocrine and pleomorphic carcinomas was discussed earlier. Small-cell lung cancer represents about 15% of total lung cancer cases and the most frequently mutated genes are p53 and RB. In addition to cell-cycle control and inhibition of the stem-cell factors, OCT4 and SOX255,56, RB loss is associated with EZH2 upregulation57. In mesothelioma, mutations of the SWI/SNF component, SMARCB1 (SNF5), and the histone 2A

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deubiquitinase, BAP1, are frequent58. Like ARID1a and BRG1, tumors with BAP1 mutations are sensitive to EZH2 inhibitors59 and EZH2 inactivation has been shown to block the growth of tumors with SNF5 mutations46.

2.5 EZH2 upregulation in lung cancer Elevated EZH2 in lung cancer is common and associated with poor prognosis. When assessed at the protein level by immunohistochemistry, EZH2 was upregulated in 45-60% of lung tumors60-63. The initial focus on EZH2 upregulation came from gene expression profiles in prostate cancer, where EZH2 was found to be the top upregulated gene in metastatic versus localized disease64. EZH2 overexpression by IHC was subsequently reported in other malignant diseases, including lung cancer, where it was associated with aggressive tumor characteristics, advanced stage and a poor prognosis60-63. In cell lines and patient-derived xenografts, EZH2 inhibition attenuated cell-cycle progression, growth and invasion62,63,65,66. Interestingly, tobacco smoke causes upregulation of Wnt signaling by recruiting EZH2 to suppress Dickkopf-1, a Wnt antagonist67. At the transcriptional level, EZH2 is suppressed by RB protein. At least in part, this explains the upregulation of EZH2 in SCLC, a disease with near-universal mutation of RB55,57. KRAS mutations in NSCLC are also associated with EZH2 upregulation, but act in a mutationspecific manner68. Codon 12 alterations that replace glycine with cysteine had significantly higher EZH2 expression than other substitutions. Mechanistically, cysteine mutants were preferentially associated with ERK activation, which increased binding of phospho-ELK1 to the EZH2 promoter69. Taken together, mis-expression of EZH2 and unopposed EZH2 activity in

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tumors with loss of function SWI/SNF mutations are distinct but overlapping mechanisms of EZH2 pro-tumorigenic function.

2.6 Non-Polycomb roles of EZH2 involving AR, STAT3 and ROS1 degradation The relevance of EZH2 to cancer progression encompasses PRC2 dependent and PRC2independent functions. Thus, not all EZH2 activity is associated with PRC2 and H3K27 methylation. EZH2 interacts with both transcription factors and cell surface receptors to affect their function. In castrate-resistant prostate cancer, a set of EZH2-upregulated genes was identified that was independent of H3K27 methylation70. Most of these genes were jointly bound by EZH2 and the androgen receptor (AR). EZH2 and AR were observed to co-sediment in a high molecular weight complex distinct from other PRC2 subunits. In this complex, EZH2 was phosphorylated on the AKT site, serine 21, and mutation of Ser21 to alanine inhibited growth of castrate-resistant prostate cancer cells. These results suggest AKT inhibition may restrict the ability of EZH2 to associate with AR and re-sensitize cells to androgen deprivation. EZH2 was also shown to bind and activate STAT3 in glioblastoma-derived cell lines71. This involved STAT3 methylation and led to upregulation of the stem cell factors, NANOG and SOX2. EZH2 Ser21 phosphorylation was also necessary for its interaction with STAT3 and cells containing an EZH2 S21A mutation were deficient in tumorsphere formation. In many NSCLC cell line models, STAT3 is activated by pathways such as EGFR, IL-6 and MET. In addition, STAT3 inhibition induces apoptosis72. Whether EZH2 plays a role in STAT3 activation in these pathways is an open question. In various species, the conserved H3K27 site methylated by EZH2 occurs in the context of Arg-Lys-Ser. Using a bioinformatics approach, related sequences were identified in other

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proteins, including the retinoic acid orphan receptor alpha (RORα), a transcription factor and known tumor suppressor in breast cancer 73. EZH2-dependent mono-methylation of RORα was shown to result in its subsequent degradation by the proteasome. Although the enzymatic activity of EZH2 on RORα is fundamentally different from its role in H3K27 tri-methylation, a smallmolecule inhibitor of EZH2 might nevertheless upregulate RORα, while simultaneously impairing gene silencing by the PRC2 complex. In breast cancer specimens, RORα and EZH2 levels are inversely correlated, and EZH2 inhibition, as well as RORα overexpression, suppress growth of tumors in vitro.

2.7 Mutations affecting EZH2 and the H3K27 demethylase, UTX Activating mutations of EZH2 have been described in non-Hodgkin lymphomas (NHL) and melanoma. In NHL, heterozygous mutations involving tyrosine 641 and alanine 677 were reported in 10-24% of cases74,75. Similar activating mutations occur in melanoma, although less frequently76. Amplification of EZH2 occurs in 5% of melanomas and both mutations and amplification were associated with sensitivity to EZH2 inhibitors. Like other targeted therapies, however, secondary resistance mutations have already been described77,78. EZH2 and the related EZH1, as the major H3K27 methyl-transferases, have their activity opposed by the two demethylases, Jumonji D3 (JMJD3/KDM6B) and UTX (KDM6A) (Fig. 3). Despite their common target, JMJD3 function has been linked to oncogene expression, while UTX activity upregulates tumor suppressor genes79. In T-cell ALL containing a UTX mutation, this dichotomy provided a therapeutic opportunity for use of a dual UTX/JMJD3 inhibitor79. UTX mutations were also reported in lung cancer cell lines80. Moreover, from the cBio-Portal database (http://www.cbioportal.org), deletions and mutations (predominantly truncating) were

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identified in 10% of 178 squamous carcinomas of the lung. In breast cancer stem cells, UTX loss was found to induce EMT by upregulation of SNAIL, ZEB1 and ZEB281. To our knowledge, the role of UTX in lung cancer stem cells has not been investigated.

2.8 Post-translational Regulation of EZH2 The regulation of EZH2 function occurs at multiple levels, including expression modification by micro-RNAs, long non-coding RNAs, phosphorylation and acetylation. For example, H3K27me3 levels are lower in cell lines with activated AKT. In a seminal work in breast cancer cell lines, AKT was shown to phosphorylate EZH2 at Ser2182, which downregulated EZH2 methylation of histone H3K27. In addition to the previously discussed association with AR, EZH2 Ser21 phosphorylation has also been reported in drug resistant multiple myeloma cells83. Here, the interaction of tumor cells with the extra-cellular matrix resulted in AKT-mediated EZH2 phosphorylation, which led to de-repression of BCL-2, IGF1 and HIF1α. Other phosphorylation sites have been identified in EZH2. In prostate cancer, the Cyclin Dependent Kinases 1 and 2 (CDK1, CDK2) phosphorylate EZH2 at Thr350, which preserved H3K27me3 at target genes, such as HOXA9 and DAB2IP84. At a cellular level, Thr350 phosphorylation enhanced migration. Recently, EZH2 was shown to be phosphorylated on Tyr244 by JAK3 in natural killer/T-cell lymphoma cells85. Like Ser21 phosphorylation by AKT, Tyr244 phosphorylation resulted in the activation of non-canonical EZH2 targets, while PRC2-mediated H3K27 methylation was reduced. EZH2 can also be acetylated, for example at Lys348 by p300/CBP Associated Factor (PCAF) 86. In lung cancer cells, this was shown to enhance EZH2 protein stability and target gene suppression. Conversely, SIRT1 was shown to

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de-acetylate EZH2. Collectively, these modifications act to regulate lung cancer cell invasion and migration.

2.9 EZH2 regulation by non-coding RNAs NextGen DNA sequencing studies indicate that only 2% of the genome codes for proteins, whereas 70% is transcribed into non-coding RNAs (ncRNAs). The RNAs are classified by size, with 200 nucleotides representing the dividing line between small and large. These molecules have emerged as a novel class of regulators affecting most physiological processes. MicroRNAs (miRNAs) are 21-23 bp in length and play important roles controlling the translation and degradation of messenger RNAs87-89. More than 2,000 miRNAs have been identified in humans which are estimated to regulate about 30% of coding genes. Four miRNAs, miR-101, miR-138, miR-26a and let-7c, have been reported to affect EZH2 (Fig. 4A). MiR-101 and miR-138 levels are inversely correlated with EZH2 in NSCLC; their overexpression was shown to inhibit proliferation, invasion and sensitize cells to chemotherapy by directly repressing EZH211,12. Alterations in miR-26a have been reported in Burkitt’s lymphoma90 and rhabdomyosarcoma91. In A549 lung cancer cells, miR-26a inhibited EZH2, which led to apoptosis, reduced proliferation and metastasis92. Let-7c participates in a feedback loop with EZH2 (Fig. 4A). EZH2 directly binds the Let-7c promoter and inhibits its expression93. In turn, reduced Let-7c results in up-regulation of the histone demethylase, JMJD1, which positively regulates EZH294. The suppression of Let-7c by EZH2 facilitates NSCLC tumorigenesis by upregulating HOXA1, ITGB3 and MAP4K313,95. Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts. An estimated 16,000 genes encode > 28,000 distinct lncRNAs (ENCODE Project Consortium). In

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addition, alternative splicing may affect their local architecture and target binding96. lncRNAs are involved in many processes, including recruitment of chromatin-modifying proteins to target sites97. As shown in Fig. 4B, lncRNAs implicated in Polycomb regulation include HOTAIR (HOX transcript antisense RNA), ANRIL (antisense non-coding RNA in the INK4 locus), PVT1 (plasmacytoma variant translocation 1) and MALAT1/NEAT2 (metastasis associated lung adenocarcinoma transcript 1)98. HOTAIR associates with PRC2 and LSD1 to silence specific loci that inhibit metastasis99,100. Its expression is upregulated in different cancers, including NSCLC and SCLC, and high expression is associated with advanced disease, metastases and a poor prognosis101,102. ANRIL, which directly interacts with the PRC2 subunit, SUZ12, is involved in p15 silencing103. Together with the chromodomain protein CBX7, a PRC1 subunit, ANRIL mediates p16 silencing104. ANRIL is also up-regulated in NSCLC and correlates with advanced stage and poor outcome105. PVT1 is located at 8q24.21 and is often co-amplified with c-MYC106. PVT1 is upregulated in NSCLC and is associated with advanced disease and poor survival107. Of note, PVT1 recruits EZH2 to inhibit expression of LATS2 (large tumor suppressor kinase 2), an upstream component of the growth-inhibiting Hippo pathway. Lastly, MALAT1 is induced by TGFβ and interacts with SUZ12 and EZH2 to suppress target loci108,109. MALAT1 was one of the first lncRNAs identified as a negative prognostic marker in lung cancer and is a critical regulator of metastasis110,111.

2.10 Epigenetics and EMT Loss of H3K27 acetylation, which otherwise antagonizes H3K27 methylation, was the most prominent observed histone effect following forced ZEB1 expression in lung cancer cells112. EZH2 and components of the Polycomb complexes have been linked to EMT and a

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stem-cell phenotype113-116. In immortalized NMuMG breast cells, Tgfβ induced Sox4, which in turn upregulated Ezh2 and led to EMT117. Forced Ezh2 expression was able to rescue EMT when Sox4 was knocked down. In patients with node-negative breast cancer, high SOX4 and EZH2 levels were correlated with a poor outcome117. In vitro, stem-like cells grow as tumorspheres. In epidermal squamous carcinomas, both EZH2 knockdown and small-molecule EZH2 inhibitors reduced spheroid formation and the ability of the cells to migrate and invade118. In glioblastoma, TGFβ led to the induction of the stem-cell factor SOX2 via upregulation of SOX4. Similar to EZH2, EZH1 has also been implicated in H3K27 tri-methylation, especially involving developmental genes and cell fate commitment119. EZH1 also binds the Prc2 components, EED and SUZ12 and in tissues such as pituitary, EZH1 is the sole H3K27 methylase. Thus, while EZH2 is usually the major H3K27 methylase and this activity is required for stem-cell function, specific EZH2 inhibitors may not be effective in tumors that also express EZH1.

2.11 DAB2IP: an important mediator of EZH2-induced EMT Ras-GAPs facilitate the hydrolysis of Ras-bound GTP and thereby reduce Ras signaling. There are 14 human Ras-GAPs120, which include the gene for DAB2 interacting protein (DAB2IP). Importantly, DAB2IP is directly suppressed by EZH2 and replacing DAB2IP in EZH2-expressing cells blocked in vivo tumorigenesis121. In patient samples, DAB2IP DNA methylation was significantly correlated with increased tumor size and lymph node metastasis. In a mouse model, mutant Ras induced immortalized prostate epithelial cells to form non-invasive tumors120. DAB2IP inhibition facilitated the formation of invasive, metastatic adenocarcinomas, driven in part by EMT.

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2.12 PRC2 as a barrier to inflammation and EMT in KRAS mutant lung cancer Elevated SOX4 or EZH2 levels are poor prognostic features in breast cancer117,122,123. However, despite the multifaceted role of EZH2 in tumorigenesis, loss of EZH2 or other PRC2 components can be pro-tumorigenic in certain contexts124. Wassef et al, reported that while high EZH2 expression was an adverse prognostic factor in breast cancer, these levels appeared to be the consequence of proliferation 124. In support of this, a subset of breast tumors with low EZH2 levels were shown to have a worse prognosis. In a mouse model, Ezh2 loss led to changes in gene expression that on a single-cell basis appeared stochastic and non-responsive to subsequent Ezh2 replacement. In a mouse lung cancer model, elevated Ezh2 expression was present in some tumors with KrasG12D mutations125 and forced Ezh2 overexpression increased tumor formation and shortened survival. However, in a combined KrasG12D / p53 deletion model, loss of the Prc2 component, Eed, was associated with more aggressive disease. This loss was associated with the expression of inflammatory cytokines, such as IL-6 and G-CSF, macrophage infiltration, EMT changes, local invasion and lymph node metastasis. In both the mouse model and human lung cancer cells with mutant KRAS, either EED loss or EZH2 inhibition by a small molecule induced an EMT phenotype. Thus, in a context-dependent manner, loss of PRC2 function leads to more aggressive disease with an inflammatory phenotype with EMT changes. In patients with lung adenocarcinoma, high levels of IL-6 and G-CSF have also been associated with a poor prognosis126.

2.13 Therapeutic inhibition of EZH2 With increasing understanding of the role of EZH2 in cancer progression, its inhibition is an increasingly attractive therapeutic strategy. However, a key recurrent theme is that it requires

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the right molecular context to be successful. A number of EZH2 inhibitors (e.g., GSK126, EPZ6438) are in clinical testing. Recently, a comprehensive review of EZH2 inhibitors has been reported127. A brief presentation of EZH2 inhibitors in phase I studies is presented in Table I. Here, we review the use of EZH2 inhibitors in three scenarios involving mutations in BRG1, ARID1a or BAP1, which occur in lung cancer and mesothelioma, as well as the combined use of EZH2 and PD1 inhibitors. Additionally, we discuss EZH2 inhibition in SCLC as a means to overcome chemotherapy resistance. As noted, high EZH2 expression is a poor-prognostic feature in lung cancer. In patient tumors, topoisomerase II was identified as a high-ranking EZH2 correlated gene128. Importantly, EZH2 inhibition sensitized certain lung cancer cell lines to the topoisomerase II inhibitor, etoposide128. However, in other cell lines, EZH2 inhibition was protective. Further analysis demonstrated that of 14 cell lines with mutations in the SWI/SNF component, BRG1 (SMARCA4), 12 were sensitized to etoposide by EZH2 inhibition. In contrast, cell lines with wild-type BRG1 were either not sensitized by an EZH2 inhibitor or, conversely, demonstrated protection. As discussed above, BRG1 mutations are uncommon in TTF-1/NKX2-1 positive tumors, whereas they are particularly frequent in large-cell and pleomorphic carcinomas42. The SWI/SNF component, ARID1a, is mutated in 7% of lung adenocarcinomas, a frequency similar to overall BRG1 mutations52. EZH2 inhibitors were identified in a screen of compounds that selectively inhibited ARID1a mutant tumors129. A combination of gene expression studies and sites that underwent H3K27 tri-methylation led to the identification of PI3-kinase inhibitory protein 1 (PI3KIP1), which normally suppresses AKT phosphorylation. PI3KIP1 expression is controlled by the opposing effects of SWI/SNF and PRC2 complexes, such that ARID1a mutations downregulate PI3KIP1 levels leading to increased AKT activity.

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Conversely, EZH2 inhibition results in the upregulation of PI3KIP1 thereby suppressing AKT. Of note, PI3KIP1 knockdown cells were resistant to EZH2 inhibition. In contrast, cells with myristoylated PI3K and upregulated AKT activity were more sensitive to an EZH2 inhibitor, presumably because they were addicted to AKT signaling. In this regard, PTEN mutations, which preferentially occur in SCLC130-132, would be expected to have increased sensitivity to EZH2 inhibitors. A third mutation scenario involves BAP1, which is frequently altered in mesothelioma, uveal melanoma and renal carcinoma59. BAP1 was identified as a BRCA1-associated protein and subsequently shown to be a histone 2A deubiquitinating enzyme, acting on H2AK119-Ub (Fig. 3). In a mouse hematopoietic tumor model, Bap1 deletion causes upregulation of Ezh2 and other Prc2 components59. In this model, Ezh2 inhibition counteracted the effect of deleting Bap1 on tumor development. Mechanistically, Bap1 loss was associated with decreased H4K20 methylation at the Ezh2 locus. Conversely, Bap1 replacement increased H4K20 methylation and inhibited Ezh2 expression. Moreover, in Bap1 mutant cells, overexpression of the H4K20 methyltransferase SETD8 downregulated Ezh2 expression, inhibited proliferation and induced apoptosis. A recent study has evaluated the effect of EZH2 epigenetic silencing on immune evasion by ovarian cancer cells133. H3K27 methylation, in addition to DNA methylation, was shown to repress tumor cell production of the T-helper (TH1) chemokines, CXCL9 and CXCL10, which bind the CXCR3 receptor and stimulate T-cell tumor infiltration. Levels of EZH2 and the DNA methyltransferase, DNMT1, were found to be negatively correlated with tumor-infiltrating CD8+ T-cells. Moreover, the combination of EZH2 and DNMT1 inhibitors resulted in a CXCR3dependent anti-tumor effect. In various treatment models, combined EZH2 and DNMT1

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inhibition, plus a therapeutic T-cell component (i.e., anti-PD-L1 or adoptive transfer of T-cells), resulted in substantially greater anti-tumor activity than a single or dual agent approach 133. Moreover, in human colon cancer cells, EZH2-mediated CXCL9 and 10 silencing, along with impaired effector T-cell infiltration, has also been reported134. Thus, the combination of EZH2 and DNA methylation inhibitors together with immune checkpoint blockade may be beneficial. Lastly, Gardner et al 135 modeled resistance in SCLC in a series of patient derived xenografts (PDXs). Interestingly, the mutational profile, copy number alteration and gene expression signatures were similar between the chemo-resistant and chemo-sensitive tumors, suggesting changes in a limited number of genes that mediated the resistant phenotype. Of note, Schlafen family member 11 (SLFN11), a previously recognized marker of chemotherapy sensitivity, was epigenetically suppressed in the resistant tumors by EZH2. Importantly, EZH2 inhibition raised SLFN11 levels and reversed chemo-resistance.

3.0 Concluding remarks Successful development of epigenetic treatment strategies in patients with lung cancer requires an understanding of the complexity of epigenetic regulation in tumor biology. Figure 5 summarizes the characteristics of EZH2 discussed in this review and illustrates the relationships between oncogenic drivers of lung cancer, EZH2 regulation and the consequences for cancer hallmarks. In the proper context, EZH2 inhibitors represent a promising novel approach for patients with lung cancer. The molecular cues that regulate EZH2 function set the stage for testing EZH2 inhibition in clinical trials. As discussed, loss of function mutations in BAP1 or the SWI/SNF components BRG1 and ARID1a sensitize tumors to EZH2 inhibitors and therefore define molecularly a patient population for a clinical trial with these drugs. The universal loss of

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RB and the link of EZH2 with drug resistance in SCLC, raises the potential of testing the combination of EZH2 inhibition with chemotherapy in patients with this type of lung cancer. Conversely, EZH2 inhibitors may have detrimental effects in tumors where EZH2 loss is associated with an inflammatory tumor phenotype as is the case of NSCLC with KRAS and p53 mutations. The complexity of possible phenotypes related to EZH2 upregulation in various molecular contexts, underscores the importance of exploratory analysis of specimens from patients participating in clinical trials with EZH2 inhibitors.

Acknowledgements Support to HD and RG provided by DOD LC150622

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4.1 Fig 1. A Schematic Overview of the Nucleosome. The nucleosome is composed of eight histone subunits (an octamer consisting of two copies each of H2A, H2B, H3 and H4) wrapped with 180 to 200 bps of DNA (green). Histone H1 stabilizes the DNA coils on the histone octamer. Lysine (K) residues within the amino-terminal portions of each histone are subject to post-translational modifications that influence chromatin compaction and gene expression. 4.2 Fig 2. Composition of the Polycomb Repressor Complexes. (A) The PRC2 core complex includes four subunits; EZH2, SUZ12, EED and Rbbp4 (RbAp64/48 ). The approximate positions of each subunit are based upon the crystal structure of an activated complex14. Auxiliary subunits (AEBP2, Jarid2, PCL1, PCL2 and PCL3) also exist that enhance the activity or influence binding to target genes. As indicated, histone H3 tri-methylated at K27 binds to EED and acts as an allosteric activator for the SET domain methyltransferase. Also, a K27M mutant of histone H3 binds and inhibits the SET domain. AKT-mediated phosphorylation of EZH2 on Ser21 has been associated with interactions involving other proteins outside of the PRC2 complex and with methylation of non-histone substrates. (B) The PRC1 complex is more variable than initially thought. It has three core subunits consisting of one of six PCGFs, a RING1A or RING1B subunit, and RYBP or YAF2. The PRC1 complexes, PRC1.2 and 1.4, containing PCGF2 or PCGF4, respectively, also associate with one of many chromobox (CBX) subunits along with PHC (1 to 3) and SCHM1 or SCHML1 or L2.

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4.3 Fig 3. Sequential functions of PRC2 and PRC1.2/1.4. A histone octamer is shown with the relevant N-terminal lysine residues of histones H3 and H2A. (1) PRC2 is recruited to target genes where the SET domain tri-methylates lysine residue 27. This activity is opposed by two specific demethylases, JMJD3 and UTX. (2) Tri-methylation of H3K27 creates an interaction site for the CBX subunits of the PRC1.2 & PRC1.4 complexes. (3) PRC1 then modifies H2AK119 by attaching a mono-ubiquitin moiety via the RING1A or RING1B subunits. Ubiquitylated H2A-K119 in turn can inhibit methyltransferases targeting H3K36, such as SETD2. Conversely, H2A-K119-Ub is a substrate for the deubiquitinase, BAP1. PRC1.2/PRC1.4 also recruits H3K36 demethylases including KDM2B while H3K36me3 inhibits the methyltransferase activity of PRC2 and serves as a recruitment site for ZMYND11, a tumor suppressor. 4.4 Fig 4. Non-coding RNAs regulate PRC2. (A) Within the PRC2 complex, EZH2 is regulated by several microRNAs, three of which inhibit its expression. The relationship between EZH2 and let-7c is more complex with mutual inhibition creating a feedback loop. High let-7c inhibits the positive EZH2 regulator, JMJD1. It also inhibits a number of pro-tumorigenic molecules such as HOXA1, ITGB3 and MAP4K3. However, because EZH2 binds and inhibits the let-7c promoter, genetic alterations that increase EZH2 levels, such as RB loss or selected KRAS mutations, lead to inactivation of this negative regulator. As a result, EZH2 levels are constitutively high along with enhancement of other tumor promoting factors. Positive interactions are indicated by green arrows, while inhibitory effects are indicated by red bars. (B) The indicated long non-coding RNAs interact with PRC2, helping to target its methyltransferase activity to groups of genes involved in the indicated pathways. 4.5 Fig 5. Multifaceted role of EZH2 in cancer. Genetic alterations which promote EZH2 function are frequently encountered in various lung cancer histologies. The role of EZH2

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upregulation has pleiotropic effects in cancer biology including induction of EMT, stemness, genomic instability and drug resistance while inhibiting senescence. RB: Retinoblastoma, g.o.f. gain of function, l.o.f. loss of function, EMT: Epithelial to Mesnchymal Transition, SLFN11: Schlafen family member 11, RKIP: RAF1 kinase inhibitor protein, DAB2IP: DAB2 Interacting Protein, Ras-GAP: Ras GTPase Activating Protein.

Table I. Inhibitors of EZH2 in Clincal Trials

Compound

EPZ-6438

EPZ-6438

EPZ-6438

Clinical Trial A Phase II, Multicenter Study of the EZH2 Inhibitor Tazemetostat in Adult Subjects With INI1Negative Tumors or Relapsed/Refractory Synovial Sarcoma A Phase 1 Study of the EZH2 Inhibitor Tazemetostat in Pediatric Subjects With Relapsed or Refractory INI1-Negative Tumors or Synovial Sarcoma An Open-Label, Multicenter, Phase 1/2 Study of E7438 (EZH2 Histone Methyl Transferase [HMT] Inhibitor) as a Single Agent in Subjects With Advanced Solid Tumors or With Bcell Lymphomas

Sponsor

ClinicalTrials.gov Phase Status Identifier

Epizyme, Inc.

NCT02601950

II

20152018

Epizyme, Inc.

NCT02601937

I

20152018

Epizyme, Inc.

NCT01897571

I/II

20132017

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GSK 126

CPI-1205

A Study to Investigate the Safety, Pharmacokinetics, Pharmacodynamics and Clinical Activity of GSK2816126 in Subjects With Relapsed/Refractory Diffuse Large B Cell Lymphoma, Transformed Follicular Lymphoma, Other Non-Hodgkin's Lymphomas, Solid Tumors and Multiple Myeloma A Phase 1 Study of CPI1205, a Small Molecule Inhibitor of EZH2, in Patients With B-Cell Lymphomas

GSK

NCT02082977

I/II

20142017

Constellation Pharmaceuticals

NCT02395601

I

20152016

Table II: Potential Therapeutic targets in the PRC2/PRC1 pathway

Complex

Protein(s)

function

Phenotype promoted by inhibition

PRC2

EZH2 SUZ12 EED RBBP4

H3K27 trimethylase PRC2 subunit PRC2 subunit PRC2 subunit

differentiated differentiated differentiated differentiated

PRC1

RING1A/B PCGF2/4 RYBP/YAF2 CBX2-8 PHC1-3 SCMH1/L12

H2AK119 E3 Ubiquitin ligase PRC1 subunit PRC1 subunit binds H3K27me3 PRC1 subunit

differentiated differentiated differentiated differentiated differentiated

PRC1 subunit

differentiated

KDM2B KDM2A BAP1 JMJD3 UTX

H3K36 demethylase H3K36 demethylase H2AK119 deubiquitylase H3K27 demethylase H3K27 demethylase

differentiated differentiated stem-like stem-like stem-like

others

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SETD2 NSD1 NSD2 ASHL1 ZMYND11

H3K36 methyltransferase H3K36 methyltransferase H3K36 methyltransferase H3K36 methyltransferase tumor suppressor binds H3K36me3

stem-like stem-like stem-like stem-like differentiated

Highlights 

Alterations and dysregulation of epigenetic genes are frequent in NSCLC and SCLC.



In the Polycomb Repressor Complex, EZH2 methylates lysine 27 of histone H3 to suppress transcription.



SWI/SNF and PRC2 complexes are antagonistic; SWI/SNF mutations predispose to EZH2 inhibitor sensitivity.



When phosphorylated at key residues, EZH2 has oncogenic activity independent of PRC2.



Depending on cell context, either gain or loss of PRC2 activity is associated with disease progression.

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33

34

35

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