The NSD family of protein methyltransferases in human cancer

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The NSD family of protein methyltransferases in human cancer

The NSD family of protein lysine methyltransferases consists of NSD1, NSD2/WHSC1/MMSET and NSD3/WHSC1L1. NSD2 haploinsufficiency causes WolfHirschhorn syndrome, while NSD1 mutations lead to the Sotos syndrome. Recently, a number of studies showed that the NSD methyltransferases were overexpressed, amplified or somatically mutated in multiple types of cancer, suggesting their critical role in cancer. These enzymes methylate specific lysine residues on histone tails and their dysfunction results in epigenomic aberrations which play a fundamental role in oncogenesis. Furthermore, NSD1 was also reported to methylate a nonhistone protein substrate, RELA/p65 subunit of NF-κB, implying its regulatory function through nonhistone methylation pathways. In this review, we summarize the current research regarding the role of the NSD family proteins in cancer and underline their potential as targets for novel cancer therapeutics.

Theodore Vougiouklakis1, Ryuji Hamamoto1, Yusuke Nakamura1 & Vassiliki Saloura*,1 1 Section of Hematology/Oncology, Department of Medicine, The University of Chicago, 5841 S. Maryland Ave, MC2115 Chicago, IL 60637, USA *Author for correspondence: vsaloura@ bsd.uchicago.edu

Keywords: H3K36 • lysine methyltransferase • methylation • NSD1 • NSD2/MMSET/WHSC1 • NSD3/WHSC1L1 • SET domain

The NSD protein lysine methyltransferase (KMT) family is composed of three members, NSD1 (KMT3B), NSD2 (WHSC1/MMSET) and NSD3 (WHSC1L1), which are primarily known to regulate gene expression through methylation of lysine 36 on histone H3 (H3K36) [1] . The protein structure of the NSD KMTs is similar and consists of multiple common domains, that is a catalytic Suppressor of variegation 3–9, Enhancer of zeste and Trithorax (SET) domain, a high-mobility-group box, two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomain zinc fingers. The SET domain is responsible for the methyltransferase activity of the NSDs, while the PWWP and plant homeodomain-type zinc finger domains are involved in chromatin and protein–protein interactions. Biochemically, methylation of lysine residues increases their hydrophobicity and basicity. Methylated lysines have been postulated to serve as docking sites for ‘reader’ proteins, such as the plant homeodo-

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main-, Tudor- and chromodomain-containing proteins, which subsequently mediate downstream transcriptional events [2,3] . In vitro and in vivo data support that NSD1, NSD2 and NSD3 mono- and dimethylate H3K36, generating H3K36me1 and H3K36me2. H3K36me2 also regulates H3K36me3 levels by providing a substrate for tri-methylating enzymes, such as SETD2 [1,4] . The biological function of H3K36 methylation in humans has not been fully elucidated yet, however it has been highly correlated with actively transcribed genomic regions [5–7] . NSD1-knockout mice demonstrate embryonic lethality [8] , while NSD2-knockout mice exhibit a phenotype similar to Wolf–Hirschhorn syndrome and die shortly after birth [9] , providing evidence for the nonredundant functions of these enzymes, despite the fact that they both di-methylate H3K36. Interestingly, a recent study using peptide arrays to screen substrates of NSD1 identified H1.5K168 as a substrate with a stronger methylation signal

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Review Vougiouklakis, Hamamoto, Nakamura & Saloura compared with H3K36 in vitro [10] . This methylation was also confirmed at the cellular level. In addition, it is important to keep in mind that nonhistone substrates may also be methylated by this group of proteins. For example, NSD1 exhibits nonhistone methyltransferase activity for the RELA/p65 subunit of NF-κB [11] . Based on the above, it is possible that the NSD KMTs have substrates other than H3K36 which may render functional nonredundancy. Regarding additional potential histone substrates of the NSD KMTs, it is important to note that there has been considerable disagreement among previous reports for H3K4, H3K27 and H4K20. These discrepancies may be explained by differences in assay conditions, such as enzyme sources (e.g., full length vs partial enzyme), substrates (e.g., recombinant histone peptides, histones, octamers or nucleosomes) and the platforms implemented to assess methylation (e.g., mass spectrometry vs antibodies) [12] . Another important point to make is that a number of other methyltransferases are also known to mono- and di-methylate H3K36, such as SETD2, SETD3, ASH1L, SETMAR and SMYD2. However, the mechanism by which specificity of the transcriptional events downstream of each of these enzymes and the NSD KMTs is achieved remains unclear. It is possible that specificity may be rendered by unique histone modification patterns and the formation of protein complexes specific to each of these enzymes. Over the past decade, accumulating evidence has unveiled the potential roles of chromatin modifying enzymes in cancer development and progression. Among these enzymes, the NSDs have been reported to carry frequent genetic alterations or to have aberrant expression in a variety of cancer types. In this review, we focus on the most recent advances elucidating the functions of the NSD methyltransferases in human tumorigenesis. NSD1 Structure & biological function

NSD1, nuclear receptor binding Su(var)3–9, Enhancer of zeste and Trithorax domain protein 1, is the largest of the NSD family members. It consists of 2696 aa (Figure 1) and the gene is located on chromosome 5q35. The mouse Nsd1 gene was first discovered by Huang et al. [13] using a two-hybrid screen system in an attempt to find proteins that interact with the ligandbinding domain of retinoic acid receptor α (RAR-α). They found that Nsd1 interacted with the LBDs of RAR-α, thyroid hormone and vitamin D3 receptors in the absence of the respective ligands, and with the estrogen receptor in the presence of estrogen. Its human homologue NSD1 was first reported as a fusion partner of the nucleoporin gene NUP98 in de novo childhood

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acute myeloid leukemia [14] . This protein is normally expressed in adult brain, kidney, spleen, thymus and skeletal muscle tissues. It possesses two nuclear receptor interaction domains and binds to nuclear receptors, such as the androgen receptor, the estrogen receptor and the retinoic acid receptor [15] . Heterozygous, loss-of-function or truncating mutations in NSD1 are associated with two autosomal dominant overgrowth genetic disorders, the Sotos and Beckwith–Wiedemann syndromes (Table 1) . The Sotos syndrome is characterized by craniofacial abnormalities, advanced bone age, variable learning disabilities and a propensity to malignancies, including squamous cell carcinoma [16] , while the Beckwith–Wiedemann syndrome is characterized by exomphalos, macroglossia and a higher risk of embryonal tumors. The mechanisms through which NSD1 mutations induce these syndromes are unknown. In this context, a recent study sought to elucidate the NSD1 downstream pathways that could explain the phenotypic features of Sotos syndrome patients, and suggested that downregulation of the MAPK/ERK pathway activity could explain the statural overgrowth and accelerated skeletal maturation in these patients [17] . Further research is needed to delineate the molecular events and resultant phenotypes driven by NSD1 mutations in Sotos syndrome. Cancer relevance

Numerous studies have reported the role of NSD1 in acute myeloid leukemia (AML). The cryptic NUP98NSD1 fusion gene t(5;11)(q35;p15.5) encodes for a chimeric protein consisting of the FG-repeat domain of NUP98 which binds to the histone acetyltransferase CBP/p300 and the carboxyterminal end of NSD1 which includes the SET domain [18] . This unique combination allows for dual acetyltransferase and methyltransferase activity. NUP98-NSD1 was detected in 16.1% of pediatric cytogenetically normal AML and 2.3% of cytogenetically normal adult AML, and portends poor prognosis [19–21] . Wang et al. showed that NUP98-NSD1 hindered cellular differentiation and induced immortalization of myeloid progenitor cells and AML in vivo through upregulation of stem-cell renewal oncogenes HOXA7, HOXA9, HOXA10 and MEIS1 (Figure 2) [18] . This was mediated through the NUP98-FG-repeat and the SET domains of NSD1, which allows for maintenance and colocalization of H3K36 methylation and H3/H4 acetylation in regulatory elements adjacent to HOXA7 and HOXA9. NUP98-NSD1 also antagonizes EZH2 binding and H3K27-mediated transcriptional repression of the HOXA locus. NSD1 has also been described as an important regulatory factor of NF-κΒ activity through direct methylation of RELA, a component of NF-κΒ [11] .

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PWWP PHD NSD1

Review

SET 2696 aa

NSD2 type II

1365 aa

NSD3L

1437 aa

Figure 1. Structure of NSD protein methyltransferases. Colored shapes represent conserved domains in the proteins. PHD: Plant homeodomain; PWWP: Proline-tryptophan-tryptophan-proline domain; SET: Su(var)3–9, Enhancer of zeste, Trithorax domain.

Lu et al. found that NSD1 activates RELA by methylation of lysines K218 (K218me1) and K221 (K221me2) in HT29 colon cancer cells, while the protein lysine demethylase FBXL11 inactivates RELA through demethylation of these lysines (Figure 2) . Exposure of cells to IL-1β and TNF-α induced methylation of K218 and K221 by NSD1, which was enhanced by overexpression of NSD1 or by decreased expression of FBXL11. Knockdown of FBXL11 led to increased proliferation of cancer cells in a K218 and K221 methylation-dependent manner. These data suggest a possible role of NSD1 as an oncogene and a mediator of inflammatory responses. Interestingly, a few reports imply the function of NSD1 as a tumor suppressor rather than an oncogene. NSD1 silencing through CpG island-promoter hypermethylation was frequently observed in neuroblastomas and gliomas, and predicted poor outcome in patients with high-risk neuroblastoma [22] . Restoration of NSD1 expression in neuroblastoma and glioma cells with abrogated NSD1 function led to decreased colony formation and cellular proliferation, suggesting a tumor-suppressive effect mediated by NSD1. In addition, NSD1 was found to directly repress the promoter of MEIS1, a critical oncogene for neuroblastoma. In support of the role of NSD1 as a tumor suppressor gene, recent comprehensive genomic analysis of 279 squamous cell carcinomas of the head and neck (SCCHN) (The Cancer Genome Atlas Network) revealed recurrent loss-of-function mutations (10.9%) and focal homozygous deletions in NSD1, which suggests a tumor suppressor function, particularly in HPVnegative tumors [23,24] . Widely distributed NSD1 mutations were also found in endometrial (9.5%) and gastric (9.1%) adenocarcinomas [25] . On the other hand, renal


clear cell carcinoma showed a high frequency of recurrent NSD1 amplifications (17.6%), which would be more consistent with an oncogenic function of NSD1. Based on the above, it is possible that NSD1 may function either as an oncogene or a tumor suppressor depending on the cellular context and coexisting alterations in other chromatin modifiers. NSD2 Structure & biological function

NSD2 is also known as multiple myeloma SET domain (MMSET) or Wolf–Hirschhorn syndrome candidate 1 (WHSC1) and it has the lowest molecular weight in the NSD family, consisting of 1365 aa (Figure 1) . Due to alternative splicing, it yields three protein isoforms: NSD2 type I (short NSD2, 647 aa), NSD2 type II (long NSD2, 1365 aa) and RE-IIBP (interleukin-5 response element II binding protein, 584 aa). NSD2 type II and RE-IIBP possess a SET domain and share an identical 584 aa region in the carboxy-terminal end, while NSD2 type I and NSD2 type II have a common amino-terminal end. The absence of the SET domain in NSD2 type I implies no methyltransferase activity in this isoform. Stec et al. [26] first mapped and discovered the NSD2 gene in the smallest region of overlap between Wolf– Hirschhorn syndrome patients on the distal short arm of chromosome 4 (4p16.3). Stec proposed NSD2 as the gene responsible for the Wolf–Hirschhorn syndrome and he also described it as the gene fused with the promoter of the heavy chain immunoglobulin IgH in the t(4;14)(p16.3;q32.3) translocation observed in multiple myeloma patients. NSD2 is expressed in adult testis and thymus, as well as in highly proliferating embryonic tissues, though its biological and physiologic functions are not precisely known. Haploinsufficiency of

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Table 1. NSD protein lysine methyltransferases. Enzyme

Histone substrate

Nonhistone substrate

Developmental syndromes

Frequencies of genetic alterations in cancer (%)

Pathways

NSD1

H3K36

p65 subunit of NF-κB

Sotos syndrome, Beckwith– Wiedemann syndrome

NSD1 amplification: Renal clear cell carcinoma (17.6), Adrenocortical carcinoma (4.5), Uterine carcinosarcoma (3.6), Hepatocellular carcinoma (3.1) NSD1 deletions: Lung squamous cell carcinoma (2.8), Bladder urothelial carcinoma (2.4), Head and neck squamous cell carcinoma (1.4), Lung adenocarcinoma (1.2) NSD1 mutations: Head and neck squamous cell carcinoma (10.9), Uterine corpus endometrial carcinoma (9.5), Skin cutaneous melanoma (7.4), Colorectal adenocarcinoma (6.9)

NF-κB signaling pathway, stemcell renewal by HOXA genes

NSD2

H3K36

Wolf–Hirschhorn NSD2 amplification: syndrome Uterine carcinosarcoma (17.9), Ovarian serous cystadenocarcinoma (7.4), Bladder urothelial carcinoma (5.5), Pancreatic adenocarcinoma (3.3) NSD2 deletions: Stomach adenocarcinoma (2.3), Lung squamous cell carcinoma (2.2), Diffuse large B-cell lymphoma (2.1), Head and neck squamous cell carcinoma (2) NSD2 mutations: Colorectal adenocarcinoma (9.7), Skin cutaneous melanoma (8.3), Stomach adenocarcinoma (5.6), Bladder urothelial carcinoma (5.5)

Cell cycle progression by NEK7, epithelial mesenchymal transition, EZH2 micro-RNA-NSD2 axis, γ-H2AXMDC1 pathway, c-MYC signaling pathway, NFκB signaling pathway, WNTsignaling pathway

NSD3

H3K36

Cell cycle progression by NEK7 and CCNG1, WNT-signaling pathway

NSD3 amplification: Lung squamous cell carcinoma (16.9), Breast invasive carcinoma (13), Bladder urothelial carcinoma (11), Head and neck squamous cell carcinoma (9.3) NSD3 deletions: Prostate adenocarcinoma (5.8), Hepatocellular carcinoma (3.6), Uterine carcinosarcoma (3.6), Bladder urothelial carcinoma (2.4) NSD3 mutations: Colorectal adenocarcinoma (5.6), Stomach adenocarcinoma (5.6), Uterine corpus endometrial carcinoma (5.4), Pancreatic adenocarcinoma (4.4)

NSD2 results in the Wolf–Hirschhorn syndrome, a rare developmental disorder characterized by growth retardation, learning disabilities, heart defects and craniofacial deformities (Table 1) [27] . The mechanisms underlying this phenotype are elusive. In this regard, Nimura et al. recently showed that Nsd2 interacts with the stem-cell related factors Sall1, Sall4 and Nanog in

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embryonic stem cells, and the cardiac transcription factor Nkx2–5 in embryonic hearts, and regulates the expression of their downstream genes [9] . They also showed that Nsd2 and Nkx2–5 double-heterozygous mutant mice had more frequent heart defects compared with their single-heterozygous mutant counterparts, and inferred that Nsd2 mediates normal embryonic



NSD1 Cancer growth and proliferation

NUP98-NSD1 fusion gene

TNFα

RELB/p50

NF-κΒ K218

Me Me

Me

Me Me

TWIST1 IQGAP1 TIAM1

Me Me Me

K20

γ-H2AX-MDC1 pathway

CCND1

FBXL11

miR-126* Silencing

H3K36me2

AC

c-MYC

H3

H4

H2A

H2B

c-MYC

3’

TCF4

WNT pathway

β- catenin NEK7

NF-κΒ

NSD2

NSD2 mRNA H3K36me2

miR-203 miR-26a miR-31 NEK7

H3K36me2 p300

H3K36me2

Cell cycle progrssion

NUP98

K36

KAP1 HDAC1

53BP1

EMT

Me Me Me K36

NSD1

K221

H4

Prostate cancer H3

CBP/p300

NF-κΒ

RELA/p65

DNA damage

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HOXA7 HOXA9 HOXA10 MEIS1 mRNA

Immortalization of myeloid progenitor cells

IL-6 IL-8, VEGFA, BCL-2 c-MYC, CCND1

EZH2 EZH2-micro-RNA-NSD2 axis

CCNG1

IRX3 + TBL1X

NSD3

SRFP1

WNT pathway

Figure 2. Schema of NSD family functions. (A) NUP98-NSD1 activates expression of HOXA7, HOXA9, HOXA10 and MEIS1 through methylation of histone H3K36. NSD1 also methylates lysines K218 and K221 of RELA/p65 of the NF-κB component and promotes NF-κB activity. (B) NSD2 regulates the TWIST1, WNT and NEK7 pathways through methylation of H3K36, and the γ-H2AX-MDC1 pathway through methylation of H4K20. NSD2 expression is suppressed by miR-203, miR-26a and miR-31 which are transcriptionally repressed by EZH2 through methylation of histone H3K27. NSD2 also interacts with NF-κB and the acetyltransferase p300, and regulates NF-κB target genes, such as IL-6 and IL-8, through methylation of H3K36. Additionally, NSD2 cooperates with KAP1 and HDAC1 and suppresses the expression of miR-126*, which leads to an increase in c-MYC expression. (C) NSD3 regulates the expression of NEK7 and CCNG1 through methylation of H3K36, which activates cell cycle progression. NSD3 also promotes the WNT pathway.

heart development through transcriptional repression of Nkx2–5. These data provide initial evidence for the function of NSD2 during embryonic development, and underline the need for further studies to elucidate the NSD2 functions under physiologic conditions. Cancer relevance

Emerging evidence has accumulated with regard to the involvement of NSD2 in cancer pathogenesis. According to our expression profile analysis, overexpression of NSD2 mRNA was observed in 13 cancer types compared with their normal counterparts [28] . Hudlebusch et al. studied the expression of NSD2 protein by immunohistochemistry in 3774 tumor samples of various cancer types and found that colon, small cell lung, skin and bladder cancers had significantly higher levels of NSD2 compared with controls [29] . In bladder cancer, high levels of NSD2 expression were cor-


related with higher histological grade and T stage, but not with survival. Overexpression of NSD2 was also associated with higher grade in oligodendroglioma, breast, prostate and head and neck cancers [30] . NSD2 overexpression has also been shown to correlate with adverse clinicopathological features in endometrial and hepatocellular cancer. Xiao et al. showed that high levels of NSD2 protein were correlated with higher stage, grade, myometrial invasion, lymph node metastasis and poor overall and disease-free survival in endometrial cancer [31] . High levels of NSD2 were also associated with shorter overall survival in HCC [32] . The oncogenic role of NSD2 was first reported in multiple myeloma (MM). The t(4;14)(p16;q32) translocation is observed in approximately 15–20% of MM, it is the second most frequent translocation in this disease and is associated with poor prognosis [33] . This translocation results in overexpression

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Review Vougiouklakis, Hamamoto, Nakamura & Saloura of both NSD2 and fibroblast growth factor receptor 3 (FGFR3), however approximately 30% of MM patients overexpress only the NSD2 gene, suggesting its pivotal role in the disease [33–35] . Seminal work by the Licht group has shown that NSD2 overexpression induces a genome-wide increase in H3K36me2 and is associated with actively transcribed genes, such as genes related to cell death, the p53 pathway, cell cycle, DNA repair and integrin-mediated signaling [36] . NSD2 knockdown leads to apoptosis, marked attenuation of colony formation and diminished tumor growth in vivo in MM cells bearing the t(4;14) translocation [36] . Kuo et al. similarly showed that t(4;14)driven overexpression of NSD2 is sufficient to induce oncogenic transformation, mediated through global genome reprogramming of H3K36me2 and subsequent transcriptional activation of cancer-associated and cell migration signaling pathways [37] . Overexpression of NSD2 in t(4;14)-negative myeloma cell lines also led to increased proliferation rates, anchorage-independent growth and global increase in H3K36me2 levels. These data support the oncogenic function of NSD2 in MM and provide rationale for the therapeutic targeting of this enzyme in MM patients with the t(4;14) translocation. In the context of identifying activating NSD2 mutations, recent studies reported a recurrent glutamic acid to lysine mutation (E1099K) in the cleft between the SET and post-SET domains of NSD2, which leads to enhanced methyltransferase activity of NSD2. Particularly, Oyer et al. examined the Cancer Cell Line Encyclopedia (CCLE) database and found the E1099K mutation in eight lymphoid cell lines (1 MM and six acute lymphoblastic leukemia [ALL]) [38] . In a study by Jaffe et al. [39] , sequencing analysis of more than 1000 pediatric cancer genomes revealed that E1099K is predominantly encountered in t(12;21) ETV6-RUNX1 pediatric B-cell ALL (14%). On the contrary, no NSD2 E1099K mutations were identified in a series of 30 adult patients with ALL, as well as in the TCGA database of solid tumors, suggesting that this mutation is clinically relevant mostly in pediatric ALL [39] . The augmented methyltransferase activity of E1099K NSD2 is supported by the fact that Jaffe et al. showed increased H3K36me2 levels in CCLE cell lines with the E1099K NSD2 mutation using mass spectrometry analysis [39] . Consistently, comparison of the catalytic domains of E1099K and wild-type NSD2 confirmed enhanced enzymatic activity of the E1099K compared with wild-type NSD2 by means of quantification of S-adenosyl-lhomocysteine production by the respective catalytic domains. In the same context, Oyer et al. [38] showed that MM and ALL cell lines with the NSD2 E1099K

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mutation had significantly higher H3K36me2 as well as decreased H3K27me3 levels compared with wildtype cells by Western blotting analysis [38] . To identify genes regulated by E1099K NSD2, Oyer et al. utilized and compared gene expression data from the CCLE database between E1099K NSD2 and wild-type NSD2 cell lines. Genes involved in epithelial to mesenchymal transition (EMT), embryonic development, cell cycle regulation and microtubule disassembly were found to be significantly upregulated in the E1099K NSD2 cell lines. Interestingly, TWIST1, a previously reported downstream target of NSD2 involved in EMT, was found to be the most potently upregulated gene (21-fold change) [40] . E1099K NSD2 was also shown to increase colony formation more than wild-type NSD2, when transfected in NSD2-knockout cells [39] . The above data support the oncogenic activity of the E1099K NSD2 mutant and underline its potential as a therapeutic target in pediatric ALL. A number of studies have attempted to elucidate the specific molecular pathways mediated by NSD2 and the interplay between NSD2 and other histone modifiers. The data presented below underline the diversity of the NSD2 functions in cancer. The EZH2-micro-RNA-NSD2 histone methyltransferase axis

Asangani et al. demonstrated a tight relationship between EZH2 and NSD2, which is regulated through microRNAs [41] . The microRNAs miR-203, miR-26a and miR-31 normally function as tumor suppressors by decreasing the stability of NSD2 mRNA. The study suggests that EZH2-mediated H3K27 trimethylation suppresses the expression of miR-203, miR-26a and miR-31 and this leads to upregulation of NSD2 mRNA (Figure 2) [41] . This finding is further supported by the observation that EZH2 mRNA and protein levels correlated with NSD2 levels in various cancer types, including colon, prostate, pancreatic cancer and lymphoma, while siRNA-mediated knockdown of EZH2 was associated with decreased levels of NSD2. In addition, EZH2 was found to induce invasive properties in normal breast and prostate cells, which was mediated through the increased expression of NSD2 [41] . These findings are intriguing, given that EZH2 has been known to mediate its oncogenic activity by suppressing the expression of tumor suppressors. The study proposes a unique microRNA-mediated mechanism through which EZH2 upregulates the expression of another histone modifier, NSD2, which functions as an oncogene. In a relevant study, Popovic et al. examined the relationship between overexpression of NSD2 and levels



of H3K27me3, a product of EZH2 [42] . They reported that despite a global decrease in H3K27me3 attributed to overexpression of NSD2, specific genomic loci, including Polycomb targets, showed increased H3K27me3 levels mediated by EZH2 recruitment. These loci included c-MYC-related genes and were perceived to contribute to myelomagenesis given the sensitivity of NSD2 overexpressing MM cells to EZH2 inhibition. Furthermore, the authors suggest that NSD2 activates gene expression by antagonizing EZH2 through induction of H3K36me2, which is mutually exclusive with H3K27me3. The above data raise questions regarding the potential role of the EZH2 pathway as an escape mechanism in the context of NSD2 inhibition and whether dual EZH2 and NSD2 targeting would provide the best antineoplastic effect in NSD2-overexpressing cancers.

cells [43] . Specifically, a micro-RNA screening in t(4;14) MM cells revealed miR-126* as one of the important targets regulated by NSD2. miR-126* was also shown to recognize the 3′-UTR untranslated region of c-MYC, leading to inhibition of its translation and thus a decrease in c-MYC protein levels (Figure 2) . Expression of miR-126* in t(4;14) MM cells suppressed cell proliferation. NSD2, together with KAP1 co-repressor and histone deacetylases HDAC1/2, binds to the promoter of miR126* and induces H3K9 tri-methylation and subsequent silencing of miR-126*, resulting in upregulation of c-MYC. NSD2 activates the WNT-signaling pathway through direct interaction with β-catenin

A recent study by Ezponda et al. supported the role of NSD2 in the invasive potential of prostate cancer cells. TWIST1 (twist family bHLH transcription factor 1), a basic helix-loop-helix transcription factor, upregulates N-cadherin which characterizes EMT. NSD2 dimethylated H3K36 in the gene body of the metastasis related gene TWIST1 and upregulated its transcription, thus promoting EMT and migratory properties in prostate cancer cells [40] . NSD2-mediated TWIST1 transcriptional upregulation was also observed in t(4;14)-positive MM cells.

In this study, we proposed that NSD2 may activate the WNT-signaling pathway through its direct interaction with β-catenin [28] . Co-immunoprecipitation and mass spectrometry revealed that NSD2 interacts with β-catenin and its known partners IQGAP1 (IQ motif containing GTPase activating protein 1) and TIAM1 (T-cell lymphoma invasion and metastasis 1) which control cell motility, adhesion and migration properties. NSD2 knockdown was associated with decreased expression of cyclin D1 (CCND1), a downstream target of the β-catenin/Tcf-4 complex. We concluded that the interaction between NSD2 and β-catenin transcriptionally regulates CCND1 through H3K36 tri-methylation of the CCND1 promoter, thereby showing a possible synergistic cooperation of β-catenin and NSD2.

NSD2 promotes cell cycle progression through direct transcriptional upregulation of NEK7

NSD2 interacts with NF-κB & activates its downstream genes

Our group recently showed that NSD2 was overexpressed in 73% of 149 SCCHN tissues and overexpression was associated with malignant transformation of SCCHN tumors [30] . NSD2 knockdown resulted in growth suppression and apoptosis of both HPV-positive and HPV-negative cells, and a concomitant attenuation of both H3K36me2 and H3K36me3 levels. NIMA-related-kinase-7 (NEK7), a serine/ threonine protein kinase required for microtubule nucleation activity of the centrosome, mitotic spindle formation and cytokinesis, was identified as a direct downstream target gene of NSD2 (Figure 2) . This was further supported by cell cycle arrest and a decrease in phosphorylated histone H3 serine 10 levels, induced by NSD2 knockdown and its downstream NEK7.

Yang et al. found that NSD2 interacts with NF-κB and the acetyltransferase p300, and induces di- and tri-methylation of H3K36 at the promoter regions of genes regulated by NF-κB in castration-resistant prostate cancer cells [44] . The methylation activity of NSD2 was necessary for the transactivation of these genes. Chromatin immunoprecipitation (ChIP) assays supported that NSD2 co-occupied the promoter regions of NF-κB-regulated genes, such as IL-6, IL-8, VEGFA, BCL-2, BIRC5, c-MYC and CCND1. In support of this, TNF-α stimulation of prostate cancer cells induced recruitment of NSD2 in NF-κB-binding promoter regions. On the contrary, NSD2 knockdown led to decreased recruitment of RELA to NF-κB-binding promoter regions. Interestingly, NSD2 was induced by cytokines, such as TNF-α and IL-6, through NF-κB. NSD2 in turn activates NF-κB which constitutes a positive feedback mechanism and results in constitutive activation of the NF-κB pathway. In addition, NSD2 levels correlated strongly with RELA levels in

NSD2 induces epithelial–mesenchymal transition through upregulation of TWIST1

NSD2 induces cell growth through miR-126* mediated overexpression of c-MYC in MM cells

Min et al. reported a novel mechanism by which NSD2 upregulates c-MYC through microRNAs in MM


Review

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Review Vougiouklakis, Hamamoto, Nakamura & Saloura prostate adenocarcinoma tissues. Overall, these data support that NSD2 is activated by proinflammatory cytokines and functions as a transcriptional coactivator for NF-κB, mediating the NF-κB signaling (Figure 2) . NSD2 is required for the recruitment of TP53BP1 to sites of DNA damage response

Recent reports have supported the role of NSD2 in the DNA damage response system. Pei et al. reported that NSD2 is recruited at sites of DNA damage through the γ-H2AX-MDC1 (mediator of DNA damage checkpoint 1) pathway, and in turn recruits TP53BP1 [45] . TP53BP1 recognizes and is recruited at sites with H4K20me2, which was found to be induced by NSD2 around DNA regions of double strand breaks. Given that the majority of studies support H3K36 and not H4K20 as a substrate of NSD2, the described role of NSD2 in DNA damage response is considered to be controversial. Hajdu et al. showed that shRNA-mediated depletion of NSD2 in HCT116 colon cancer cells led to increased sensitivity of the cells to DNA damaging agents, such as hydroxyurea, camptothecin and mitomycin C, as well as to ionizing radiation [46] . In addition, NSD2 co-localized with γ-H2AX at sites of DNA damage, indicating a possible role of NSD2 in sensing or repairing DNA damage. Furthermore, the DNA damage response system remained intact in the absence of NSD2, but the DNA damage-repair signaling was prolonged, as indicated by persistent phosphorylated CHK1, RPA32 and γ-H2AX in cells with depletion of NSD2. These results suggest that in the absence of NSD2, cells exposed to DNA damaging factors may generate more DNA damage, repair the DNA damage less efficiently, or both. NSD3 Structure & biological function

The third member of the family, NSD3, also known as Wolf–Hirschhorn syndrome candidate 1-like 1 (WHSC1L1), is located on chromosome 8p12 (Figure 1) . NSD3 has two main isoforms, NSD3L (long NSD3,1437 aa) and NSD3S (short NSD3, 645 aa). NSD3L and NSD3S share a common 620 aa N-terminal part, while NSD3S lacks a SET domain. NSD3 was first isolated, cloned and described by Angrand et al. as the third NSD family member which was amplified in several breast cancer cell lines and primary breast carcinomas [47] . In contrast to the NSD1 and NSD2 genes, no NSD3-knockout mouse phenotype has been described yet and no relevant overgrowth syndromes attributed to defects in this gene have been described in humans (Table 1) .

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Cancer relevance

NSD3 is the least characterized member among the NSD family. NSD3 has been identified as a gene frequently amplified in breast cancer cell lines [47] . In a more recent study, Liu et al. performed genomic and transcriptomic analysis of various protein methyltransferases in 958 breast cancer samples using the TCGA database, and found that NSD3 was the fourth most frequently amplified methyltransferase [48] . NSD3 was significantly overexpressed in the nonbasal breast cancer subtypes and showed the highest frequency of amplification in the luminal B subtype (19%). High NSD3 mRNA levels were also associated with worse survival (p = 0.0231). Similarly, a number of studies have associated the 8p11-12 amplification with poor grade, high Ki-67 proliferation index and poor prognosis in breast cancer [49–51] . Chen et al. also utilized the TCGA database and identified NSD3 as a putative oncogenic driver, as it was found to display a high gene copy number and mRNA expression in a total of 14 cancer types, including squamous lung (21%) and breast carcinoma (15%) [52] . Higher NSD3 copy numbers were strongly correlated with NSD3 mRNA levels. The TCGA database has also revealed frequent NSD3 amplifications in bladder cancer (11%) and SCCHN (recurrent, 9%) [25] . NSD3 has also been described in the chromosomal translocation t(8;11)(p11.2;p15) which results in the NSD3-NUP98 fusion protein in AML and in chemotherapy- or radiation-related myelodysplastic syndrome [53,54] . Yang et al. reported that transduction of NSD3 in immortalized nontransformed mammary epithelial cells resulted in clonogenicity and induced morphological features of disorganized acini [55] . In addition, shRNA-mediated knockdown of NSD3 attenuated cellular proliferation and led to decreased cell survival in 8p11–12 amplified breast cancer cells, supporting the oncogenic potential of this enzyme in breast cancer. Furthermore, NSD3 augmented the expression of transcription factors IRX3 (Iroquois Homeobox 3) and TBL1X (Transducin (Beta)-Like 1X-Linked), which are known to positively regulate the WNT-signaling pathway, and downregulated SRFP1 (Secreted Frizzled-Related Protein 1), a negative regulator of the WNT-signaling pathway, in embryonic stem cells. As mentioned above, NSD2 has also been reported to regulate the WNT-signaling pathway through its interaction with β-catenin, indicating that NSD2 and NSD3 may have some functional redundancy. Another study supporting the function of NSD3 as an oncogene was conducted by our group, which showed that knockdown of NSD3 resulted in growth



suppression and arrested cell growth at the G2/M phase of the cell cycle in lung adenocarcinoma and bladder cancer cells [56] . Expression analysis after NSD3 knockdown identified CCNG1 (cyclin G1) and NEK7 as potential downstream genes in both lung adenocarcinoma and bladder cancer cells. Notably, both CCNG1 and NEK7 enhance cell cycle progression and concordantly NSD3 knockdown caused G2/M arrest. It is interesting that NEK7 was also found to be a direct downstream target of NSD2, also indicating potential functional redundancy between NSD2 and NSD3. Rahman et al. reported that NSD3 interacts with bromodomain (BRD) proteins to regulate transcription of its downstream genes via its histone methyltransferase activity. NSD3 binds to the extraterminal (ET) domain of BRD4, regulating transcription of BRD4 through a pTEFb-independent manner [57] . Depletion of either NSD3 or BRD4 attenuated H3K36me3 levels in genes regulated by BRD4, such as CCND1. In addition, the recruitment of NSD3 to target genes was mediated through BRD4. Overall, this study supported that the transcriptional activity of BRD4 is not only modulated through its C-terminal domain by pTEFb, but also through the extraterminal domain by other transcription modulators, including NSD3. Bromodomain proteins, BRD3 and BRD4, and NSD3 have also been identified as fusion oncoproteins with the nuclear protein in testis (NUT) gene in the rare NUT midline carcinoma (NMC) malignancy, an aggressive subtype of squamous cell carcinoma. In a study utilizing a new patient derived NSD3-NUT cell line, NSD3-NUT was found to bind to BRD4 and utilize its chromatin reading function [58] . BRD bromodomain inhibitors, such as JQ1, were shown to induce differentiation and growth arrest of this cell line. These data provide rationale to use BET inhibitors for the treatment of NSD3-NUT midline carcinoma cancers. As NSD3

Review

has also been found to be an important component of the BRD4 complex, BET inhibition could warrant further exploration for the treatment of NSD3 overexpressing cancers. Conclusion & future perspective The advent of large scale genomic and transcriptomic analyses of cancer has revealed that the NSD methyltransferases harbor frequent genetic alterations, which has attracted attention regarding their importance in cancer pathogenesis. Over the past 5 years, a number of functional analyses have underlined the critical role of these enzymes in tumorigenesis. This review has highlighted the potential of the NSD methyltransferases for drug development. Another important point to consider is that the function of protein methyltransferases is not only mediated through histone methylation, but also through nonhistone substrate methylation [3] , which brings into concept the presence of intricate methylation cascades in accordance to the presence of phosphorylation cascades [3,59–63] . While current evidence has only revealed RELA/p65 as a nonhistone substrate for NSD1, further investigation to identify nonhistone substrates of the NSD members is necessary to elucidate the full spectrum of the functions of these enzymes. The specific functions of the NSDs in cancer pathogenesis are versatile and have been perceived as mostly nonredundant to one another given their nonoverlapping pattern of genetic alterations. Still, there are examples whereby the NSDs seem to have some functional redundancy, as highlighted in this review; for example, NEK7 and the WNT-signaling pathway as downstream targets for both NSD2 and NSD3, and NF-κB as an interacting partner for both NSD1 and NSD2. Further studies are needed to clarify the degree of redundancy, as this will be an important factor to consider for the clinical application of NSD-specific inhibitors.

Executive summary Key conclusions • Genomic alterations or aberrant expression of the NSD protein lysine methyltransferases (KMTs) is evident in various types of cancer. • NSD1 may function either as an oncogene or a tumor suppressor depending on the cellular context and coexisting alterations in other chromatin modifiers. • NSD2 regulates cell cycle progression, DNA damage repair and epithelial to mesenchymal transition. • NSD3 seems to function as an oncogene, though its specific biological functions in cancer remain to be elucidated.

Future perspective • The NSD methyltransferases show promise as targets for anticancer drug development. • Further investigation into the exact mechanisms of action and to identify nonhistone substrates of the NSD KMTs is necessary to elucidate the full spectrum of the functions of these enzymes. • Further studies are needed to clarify the degree of redundancy among the NSD KMTs, as this will be an important factor to consider for the clinical application of NSD-specific inhibitors.


10.2217/EPI.15.32

Review Vougiouklakis, Hamamoto, Nakamura & Saloura Financial & competing interests disclosure Y Nakamura is a stock holder and a scientific advisor of Oncotherapy Science and also has research grants from Oncotherapy Science. The authors have no other relevant affiliations or financial involvement with any organization or entity

with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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