Mutated Chromatin Regulatory Factors As Tumor Drivers in Cancer Carl Koschmann1,2, Felipe J

Published OnlineFirst January 6, 2017; DOI: 10.1158/0008-5472.CAN-16-2301 Cancer Review Research

Mutated Chromatin Regulatory Factors as Tumor Drivers in Cancer Carl Koschmann1,2, Felipe J. Nunez2,3, Flor Mendez3, Jacqueline A. Brosnan-Cashman4, Alan K. Meeker4,5, Pedro R. Lowenstein2,3, and Maria G. Castro2,3

Abstract

Genes encoding proteins that regulate chromatin structure and DNA alterations in CRFs and how these influence tumor DNA modifications [i.e., chromatin regulatory factors (CRF)] and chromatinstructureandfunction,whichinturnleadsto genes encoding histone proteins harbor recurrent mutations in tumorigenesis. We also discuss the clinical implications and most human cancers. These mutations lead to modifications in review concepts of targeted treatments for these mutations. tumor chromatin and DNA structure and an altered epigenetic Continued research on CRF mutations will be critical for our state that contribute to tumorigenesis. Mutated CRFs have now future understanding of cancer biology and the development been identified in most types of cancer and are increasingly and implementation of novel cancer therapies. Cancer Res; 77(2); regarded as novel therapeutic targets. In this review, we discuss 1–7. 2017 AACR.

Introduction chromatin, the affinity of the DNA for the histones, and the chemical modification of histone tails and DNA (4). As an In recent years, there has been an increased interest in the example, methyl and acetyl groups can be added to and removed impact of epigenetics on tumor biology. Epigenetic modifications from specific residues of the histone 3 amino-terminal tail (e.g., can alter tumor gene expression independently of alterations in H3K27me3 denotes three methyl groups added to the 27th lysine the tumor DNA sequence. Changes in DNA methylation, histone residue). The DNA can be modified as well; methylation of a modifications, and nucleosome composition or placement play a cytosine within a CpG dinucleotide causes transcriptional silenc- critical role in tumor biology and progression. These epigenetic ing or activation, depending on the proximity to the gene (4, 6–9). changes can be driven by environmental changes and factors in The regulation of chromatin structure is tightly controlled by the tumor cells' microenvironment (1). As we have continued to CRFs, which ultimately maintain genome integrity and patterns build our understanding of epigenetic pathways in cancer, we of gene expression. There are three broad categories of chro- have circled back to the tumor DNA itself. Genes that encode matin-regulating proteins, which we will discuss herein: (i) proteins that regulate chromatin structure and DNA modifica- ATP-dependent chromatin remodeling complexes, which tions [i.e., chromatin regulatory factors (CRF)] and genes encod- insert, remove, and move nucleosomes along the DNA; (ii) ing histone proteins themselves harbor recurrent mutations in histone tail modifiers, which posttranslationally modify his- human cancers. These mutations lead to modifications in tumor tone tails by inserting or removing methyl, acetyl, and other chromatin and DNA structure, leading, in turn, to an altered groups; and (iii) DNA methyltransferase/demethylases, which epigenetic state and expression program that contribute to tumor- can alter DNA methylation (Fig. 1; refs. 3, 4). igenesis (2–5). These mutated CRFs have now been identified in Not surprisingly, proteins that are so centrally involved in most types of cancer and are increasingly regarded as novel targets patterns of gene expression can dramatically disrupt cellular for cancer treatment (2–5). behavior when mutated. Protein-altering changes in genes encod- Gene expression in eukaryotes is regulated by several mechan- ing CRFs (including point mutations, amplifications, deletions, isms, which include the placement of the nucleosome on the and fusions) are capable of locking cancer cells in an abnormal epigenetic state that promotes perpetual self-renewal without differentiation (4, 6, 8, 10). Certain CRF-altering mutations 1Department of Pediatrics, Division of Pediatric Hematology-Oncology, Univer- 2 behave as driver mutations in many human malignancies, some- sity of Michigan Medical School, Ann Arbor, Michigan. Department of Neuro- – surgery, University of Michigan Medical School, Ann Arbor, Michigan. 3Depart- times as the only driving tumor mutation (3 5). In addition, ment of Cell and Developmental Biology, University of Michigan Medical School, enzymes that generate metabolites used by CRFs can be mutated, Ann Arbor, Michigan. 4Department of Pathology, Johns Hopkins University, which can contribute to cancer development. As an example, Baltimore, Maryland. 5Department of Urology, Johns Hopkins University, Balti- multiple human cancers harbor a gain-of-function point muta- more, Michigan. tion in the active site of isocitrate dehydrogenase 1 (IDH1), Corresponding Author: Maria G. Castro, University of Michigan School of resulting in production of the metabolite 2-hydroxyglutarate Medicine, 1150 W. Medical Center Drive, MSRB II, Room 4570, Ann Arbor, MI rather than a-ketoglutarate (Fig. 1). This blocks a-ketogluta- 48109-5689. Phone: 734-764-7052; Fax: 734-764-7051; E-mail: rate–dependent demethylases, increases H3K9 and H3K27 meth- [email protected] ylation and DNA hypermethylation, and blocks tumor cell dif- doi: 10.1158/0008-5472.CAN-16-2301 ferentiation (11). In addition, recurrent gain-of-function point 2017 American Association for Cancer Research. mutations in histone variants HIST1H3A (H3.1) and H3F3A

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Enzyme that generates Histone tail modiﬁer metabolite inhibiting CRF (e.g. MLL1, EZH2, NSD2) (e.g. mutant IDH1 -> 2HG)

DNA methyl- DNA transferases demethylase (e.g. DNMT3A) (e.g. TET2) K27 K36 K79

Histone tail methylation

Mutated CRF DNA methylation ATP-dependent chromatin Transcription remodeling complex Histone methytransferase/ Demethylase DNA methytransferase/ Demethylase ATP-dependent chromatin remodeling Enzyme that generates complexes metabolite inhibiting CRF (e.g. SWI/SNF)

Protein-altering change in Alteration of: tumor DNA (point • DNA methylation Epigenetic state with altered mutation, ampliﬁcation, • Histone tail methylation expression program deletion, fusion) of a or acetylation (e.g. perpetual tumor cell self- chromatin remodeling • Nucleosome composition renewal without differentiation) factor (CRF) or placement

Figure 1. Schematic demonstrates how alterations in the coding DNA of CRFs result in tumor cell proliferation. Protein-altering changes in tumor DNA (e.g., point mutation, ampliﬁcation, deletion, fusion) of a CRF can lead to downstream epigenetic changes elsewhere on the tumor DNA. The main classes of chromatin remodeling factors are (i) ATP-dependent chromatin remodeling complexes (red triangles), which can insert, remove, and move nucleosomes along DNA; (ii) histone tail modiﬁers (green and red boxes), which can modify histone tails to insert or remove methyl and acetyl groups; and (iii) DNA methyltransferase/ demethylases (green and red circles), which can alter cytosine methylation on DNA. In addition, enzymes can generate metabolites (orange diamonds) that block the activity of other CRFs, such as 2-hydroxyglutarate production blocking TET2 activity in IDH1-mutated tumors. Mutations in CRFs lead to alterations in DNA methylation or histone tail methylation/acetylation in tumor cells or tumor precursor cells. This can result in an epigenetic state with an altered expression program (e.g., perpetual tumor cell self-renewal without differentiation).

(H3.3) result in critical downstream epigenetic alterations, con- alterations in nine genes have been found to impact H3K27me3 tributing to tumor growth in pediatric glioblastoma, chondro- levels, all of which are mutually exclusive (10). Recent research blastoma, and undifferentiated sarcoma (12, 13). A murine has gained signiﬁcant insight into the role of CRFs' mutations in model of one of these mutations, H3 lysine 36 to methionine tumorigenesis. (H3K36M) mutation, resulted in the inhibition of H3K36 methyltransferases, a mesenchymal differentiation block, and the gen- ATP-Dependent Chromatin Remodeling eration of murine undifferentiated sarcomas (13). A recent analysis of data from sequencing studies noted fre- Complexes quent mutations of chromatin remodeling components in 36 ATP-dependent chromatin remodeling complexes are highly cancer types (7). In a survey of multiple human cancers, the conserved from yeast to humans (14). They utilize an ATPase proportion found to have mutations in CRFs varied by tumor subunit to mobilize nucleosomes along DNA, removing histones type, with some subtypes harboring a CRF mutation in nearly all from nucleosomes, and replacing histones with other histone cases examined (3). Although mutations in CRFs as a group are variants, all of which can result in dramatic effects on transcrip- seen fairly frequently, mutations at any individual gene may be tional activity (5, 14). There are four classes of chromatin remo- found only rarely. As an example, at least 12 individual DNA deling ATPase complexes, all of which have similar ATPase

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domains but have distinct chromatin-interacting domains and expression of EZH2 are seen in acute myeloid leukemia (AML) additional individual components. Genetic alterations in com- and are associated with a higher risk of disease progression or ponents of ATP-dependent chromatin remodeling complexes are recurrence (23). Mutated EZH2 is felt to be oncogenic in this quite common in human malignancies (7). setting through static repression of differentiation programs in Members of the switch/sucrose nonfermentable (SWI/SNF) leukemic stem cells (24). Egr1 encodes a zinc-finger transcription complex are mutated in more than 30% of multiple human factor that plays a central role in the development of myeloid cells. cancers (Fig. 1; refs. 5, 14). For example, loss-of-function muta- Egr1 is repressed by EZH2, and overexpression of Egr1 allows tions in the SMARCB1 (SNF5) subunit of the SWI/SNF complex myeloid cells to differentiate (24). Interestingly, EZH2 has also are seen in almost all rhabdoid tumors, frequently as the only been reported as a tumor suppressor. Missense, nonsense, and somatic DNA alteration (5). Loss of SNF5 can be found as a frameshift mutations in EZH2 that affect SET domain activity are germline mutation, which increases susceptibility to multiple present in myelodysplastic syndrome and AML and correlate with early childhood cancers. Tumors that develop in this familial poor prognosis in myelodysplastic syndrome (25). Therefore, form lose the remaining wild-type allele, consistent with Knud- current evidence indicates a dual role for EZH2 as either a sontwo-hitmodelofoncogenesisforatumorsuppressor(5, proto-oncogene or tumor suppressor, depending on the tumor 14). Loss of SNF5 leads to elevated expression of the polycomb cell context. Adding complexity, EZH2 mutations may result in gene EZH2, leading to widespread increase in H3K27me3 and redistribution of H3K27me3, rather than uniform increase or tumor cell-cycle progression (15). EZH2 will be discussed in decrease (Fig. 1; ref. 26). A gain-of-function point mutation in greater detail in the "histone tail modifiers" section below. EZH2 (EZH2Y646F) is found in melanoma and some B-cell lym- Mutations in the SWI/SNF–like protein ATRX contribute to phomas, and expression of this mutation in mouse B cells and cancer development through a distinct mechanism. ATRX is melanocytes induces tumor growth through widespread redistri- responsible for deposition of the histone variant H3.3 to pericen- bution of H3K27me3 (both increased and decreased at different tromeric and telomeric heterochromatin (16). Recurrent loss-of- loci; ref. 26). Nevertheless, EZH2 alterations are tumorigenic and function mutations in ATRX have been identified in pancreatic may prove to be an important therapeutic target. neuroendocrine tumors, pediatric glioblastoma, adult low-grade Mixed lineage leukemia (MLL) is another histone methyltrans- glioma, and at least 15 other human cancer types (17, 18). All ferase that is frequently altered in human tumors. MLL1 catalyzes tumors require maintenance of telomere length to sustain unlim- the methylation of H3K4, resulting in transcriptional activation of ited cell division, most frequently through upregulation of telo- genes involved in embryogenesis and hematopoiesis (27). Altera- merase (18). A minority of cancers use non-telomerase–based tions in MLL1 are tumorigenic in leukemia and are found in 70% mechanisms, collectively termed alternative lengthening of telo- of infants with acute lymphoblastic leukemia (ALL) and 35% of meres (ALT). ALT uses a homologous recombination–based infants with AML (27). MLL1 is frequently translocated, and the method to lengthen telomeres (19). Mutations of ATRX are highly prognosis of MLL-rearranged AML is highly dependent on the correlated with ALT in human cancers; in glioma, they are mutu- translocation partner (27). MLL-rearranged leukemias display ally exclusive with activating telomerase gene promoter muta- overexpression of MLL-controlled homeobox (HOX) genes, tions (17, 20). Loss of ATRX leads to changes in the telomeric which may play a central role in oncogenesis (27). Recent work chromatin state due to loss of H3.3 deposition and damage and has shown that various MLL fusion protein complexes recruit the recombination at the telomeres (21). In addition, cells with loss of DOT1-Like (DOT1L) complex to genes known to play a role in functional ATRX display increased genomic instability, which leukemogenesis. DOT1L is a histone methyltransferase that may contribute to an increased mutation rate and the acceleration methylates H3K79, resulting in deregulated transcription and of tumorigenesis (17). leukemia cell growth (28). Other MLL genes are also mutated in childhood cancers. The histone methyltransferases MLL2 and fi MLL3 are collectively mutated in 18% of pediatric medulloblas- Histone Tail Modi ers tomas, again demonstrating the role of epigenetic dysregulation The modification of histone tail residues constitutes another of developmental pathways in the pathogenesis of pediatric important mechanism of epigenetic regulation of gene expres- tumors (29). sion. Methylation and acetylation are the most well-described The nuclear receptor–binding SET domain (NSD) is a family of examples, but other alterations may play just as important roles. three histone methyltransferases that contribute to cancer devel- The addition of crotonyl groups to lysine (crotonylation) has been opment via gene translocation events. Translocations between the reported to be a more potent transcriptional activator than acet- nucleocytoplasmic transport gene NUP98 and the carboxyl ter- ylation (22). However, the dynamic mechanisms of histone minus of the histone methyltransferase domain NSD1 are recur- methylation have been the most prominently studied histone rently found in AML (30). The FG-repeat domain of NUP98 binds alteration in human cancers, including the regulation of histone to the histone acetyltransferase domain of NSD1 to create the methylation by lysine methyltransferases (KMT) and demethyl- NUP98-NSD1 fusion protein with acetyltransferase and histone ation by lysine demethylases (KDM). The SET domain of KMTs methyltransferase activity (30). NUP98-NSD1 blocks cellular catalyzes the methyl transfer from S-adenosyl methionine to differentiation and increases the expression of HoxA genes asso- histone tail lysine residues and is a CRF that is of particular ciated with promotion of stem cell self-renewal in hematopoietic importance in many human leukemias. progenitors (30). This fusion protein is present in 16.1% of Enhancer of zeste 2 subunit (EZH2) is a subunit of the SET pediatric cytologically normal (CN)-AML and 2.3% of adult domain proteins that catalyzes the methylation of the histone tail CN-AML and is associated with a poor prognosis (31). residue H3K27. In its normal state, EZH2 maintains transcrip- Another genetic alteration affecting the NSD family is the tional repression and the long-term self-renewal of hematopoietic t(4;14) translocation, which results in overexpression of NSD2 stem cells (HSC; ref. 23). Gain-of-function mutation and over- (32). NSD2 preferentially dimethylates H3K36, and in multiple

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myeloma cell lines, NSD2 overexpression results in increased rupting hematopoietic differentiation and generating a suscepti- H3K36 dimethylation and decreased H3K27 trimethylation bility to myeloid transformation (38). However, similar to (Fig. 1; ref. 32). These global changes increase chromatin acces- DNM3TA, studies in TET2-deficient mice demonstrate that the sibility and alter the expression of genes involved in cell growth, TET2 mutation alone is not enough to produce AML (41). adhesion, DNA repair, and apoptosis (32). A gain-of-function Finally, according to recent studies, CRF mutations can alter mutation in NSD2 has also been reported in pediatric ALL (33). gene expression and promote tumor development through aber- The mutation is located in the SET domain and enhances methyl- rant hypermethylation on enhancer regions outside of gene transferase activity, resulting in increased global levels of H3K36 promoters or CpG islands (42). DNA methylation levels on and decreased levels of H3K27me3 (33). Leukemia cells harbor- enhancers can alter chromatin environment and cause down- ing the NSD2 mutation in vitro display increased H3K36 dimethy- regulation of nearby genes, resulting in oncogenic transformation lation, and NSD2 knockdown inhibits leukemic cell growth (33). (42). Future research may uncover further associations between In addition, in a xenograft model, NSD2 knockdown decreases somatic lesions and methylation of enhancer regions. H3K36 methylation and inhibits tumor growth (33). Clinical Significance of Mutated CRFs DNA Methyltransferases/Demethylases With a greater understanding of the frequency and pathophys- In the third main category of altered CRFs, we highlight factors iology of mutated CRFs in cancer, there is a growing interest in involved in the regulation of methyl group placement and remov- their clinical applications. Mutated CRFs impart important prog- al, on the DNA. Methylation of CpG islands in promoter regions is nostic and treatment considerations. Often, the same mutation often associated with transcriptional silencing (34). However, can harbor unique prognostic and treatment implications in methylation can occur outside of promoter CpG islands and may different tumor types. Mutated CRFs have been found in prema- not necessarily be correlated with gene silencing (34). DNA lignant uterine, skin, and cervical lesions (3, 43, 44). With further methyltransferase 3 a (DNMT3A) catalyzes the methylation of validation in clinical studies, these mutated genes and proteins cytosine in CpG dinucleotides to 5-methylcytosine (5-mC). can be used as biomarkers for cancer risk. In addition, the presence DNMT3A mutations are found in approximately 20% of patients of a mutated CRF may increasingly define prognostic or clinical with AML, the majority being heterozygous missense mutations subgroups. A recent study proposed that alteration in a gene that altering the amino acid R882 (34). DNMT3A mutations are more regulates chromatin or transcription (including RUNX1, ASXL1, common in CN-AML and are associated with older age and higher and MLL), the so-called "chromatin–spliceosome group," defines white blood cell count at diagnosis (34). The DNMT3A mutation a distinct prognostic and clinical subgroup in AML (45). reduces methyltransferase activity, leading to focal hypomethyla- Most excitingly, targeted therapies and clinical trials aimed to tion by disrupting the ability of wild-type DNMT3A to form exploit cancer-associated CRF mutations, and epigenetic changes functional tetramers (Fig. 1; ref. 35). Loss of DNMT3A in murine are being pursued. In applying epigenetic-based therapies, the HSCs results in a considerable expansion of the stem cell pool and presence of a mutated CRF is an important consideration. If gradual loss of HSC differentiation capacity upon serial trans- epigenetic changes not driven by mutations in a CRF (e.g., those plantation; however, as mice transplanted with DNMT3A null caused by environmental stressors or factors in the tumor micro- HSCs do not develop leukemia, additional genetic alterations environment) are more reversible than those driven by mutated appear to be necessary for tumorigenesis (36). DNMT3A R882 CRFs, then tumors without mutated CRFs will be more responsive mutations are associated with a poor prognosis for relapse-free to epigenetic-based therapies (1, 6). On the other hand, assuming survival and poor overall survival (34). tumors with mutated CRFs, especially those without additional Somatic alterations in tumors are also found in DNA demethy- driving mutations, are the most addicted to (or most unilaterally lases, as is the case of Tet methylcytosine dioxygenase 2 (TET2). driven by) their epigenetic changes for tumor survival, such TET2 belongs to a family of TET proteins, which are dioxygenases tumors (e.g., a tumor with an isolated SWI/SNF mutation) will able to catalyze the conversion of 5-mC to 5-hydroxymethylcy- be the most susceptible to treatments aimed at downstream tosine (5-hmC), leading to DNA demethylation in tightly regu- epigenetic changes (4). lated areas of chromatin (37). TET2 loss-of-function mutations The mainstay of epigenetic therapies thus far has been based on are present in about a quarter of AML patients and have been targeting the epigenetic modification that has led to the cancer associated with poorer prognosis in patients with intermediate- cells' altered epigenetic state (6, 8, 46). Histone deacetylase risk cytogenetic lesions (38). The binding and ubiquitylation of (HDAC) inhibitors have been used to reinsert acetyl groups to TET2 by the ubiquitin ligase VprBP are critical for TET2 function, tumors harboring low histone tail acetylation (46). In preclinical promoting the binding of TET2 to DNA. TET2-inactivating muta- tumor models, histone tail acetylation can result in relaxed tumor tions target ubiquitylation sites or residues necessary for VprBP chromatin structure, which leads to increased expression of dif- binding, disrupting VprBP regulation of TET2 (37). TET2 muta- ferentiation or apoptotic programs in cancer cells (8, 47). HDAC tions are associated with reduced global 5-hmC levels and inhibitors can reverse these changes (46). increased levels of 5-mC in AML patients (Fig. 1; ref. 39). Inter- Unfortunately, targeting epigenetic changes clinically has prov- estingly, TET2 mutations are mutually exclusive with IDH1 muta- en to be less than straightforward, as the impact of a mutated CRF tions in AML patients, consistent with a common hypermethyla- may be unique to the cancer type. As an example, breast and tion phenotype (40). Confirming the relevance of these lesions, bladder cancers frequently harbor amplification and gain-of- AML subtype and clinical outcome can be predicted by the DNA function mutations of EZH2, resulting in increased H3K27me3 methylation pattern of leukemic cells (39). (10). Meanwhile, loss-of-function mutations of EZH2 have been The pathogenesis of TET2 mutations has been explored in found in myeloid malignancies, myelodysplastic syndrome, and murine models, where TET2 loss induces HSC self-renewal, dis- head and neck squamous cell carcinomas, which result in

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decreased H3K27me3 (10). EZH2 inhibitors that are able to ref. 56). The small-molecule BET inhibitor JQ1 has shown efficacy effectively reduce H3K27me3 should be applied only to those in preclinical models of NMC and is currently being tested in with confirmed increased EZH2 or H3K27me3 expression. multiple human cancers (some harboring BRD3/4 overexpression In addition, epigenetic agents, such as HDAC inhibitors, can rather than fusion) (56). As referenced above, DOT1L plays a have off-target effects on other nuclear proteins and are highly critical role in MLL-driven leukemia, and DOT1L inhibitors are dose dependent (47). At high doses, HDAC inhibitors cause currently in trial in patients with MLL-rearranged leukemia (56). double-stranded DNA breaks and are likely more cytotoxic. This Finally, EZH2 inhibitors are being tested in rhabdoid tumors and may explain why many trials using HDAC inhibitors at high doses lymphomas with loss of SNF5 (57), where EZH2 is upregulated as have resulted in toxicities, with no significant clinical efficacy discussed above. Importantly, a genome-scale shRNA screen of when used as monotherapy for human cancers (6). SWI/SNF–deficient tumor cells showed variable dependence on Clinical efficacy and FDA approval for HDAC inhibitors and EZH2 expression, with less dependence in those with cooccurring demethylating agents has thus far been limited to a few cancer RAS mutations (58). Further work may demonstrate that these indications (multiple myeloma, myelodysplastic syndrome, and cooccurring mutations may dictate response to EZH2 inhibition certain T-cell lymphomas; ref. 6). Recent work has demonstrated clinically. there may be some utility to these agents in gliomas harboring certain mutations. IDH1-mutant glioma cells have been shown to Conclusions demonstrate inhibition of growth in vivo in response to the DNMT In conclusion, epigenetic modifications play a central role in inhibitor decitabine (48) and the demethylating drug 5-azacyti- cancer biology and in the development of new cancer therapies. dine (49). Pharmacologic inhibition of the histone K27 demethy- Tumors harboring DNA alterations in CRFs represent perhaps the lase JMJD3 results in restored H3K27me3 and inhibition of most pure form of this concept. It is also possible that most brainstem gliomas in vivo, alone (50) and in combination with epigenetic changes in cancer are, in fact, the downstream result of the HDAC inhibitor panobinostat (51). mutations elsewhere in the tumor DNA with an association yet to The potential utility of HDAC inhibitors may be fully realized be uncovered (59). As a possible testament to this, in a survey of in current studies exploring their use in combination with other somatic copy number alterations in more than 4,000 tumor anticancer therapeutics. A recent phase I trial of CUDC-907, a samples, genes involved in epigenetic regulation (but not previ- small molecule designed to inhibit both HDAC and PI3K ously recognized as tumor drivers) were the most frequently enzymes, showed safety and some responses among patients with identified group to have focal amplifications (60). Undoubtedly, refractory lymphomas and multiple myeloma, two tumors that continued research on CRF mutations will be critical for our future frequently harbor genetic alterations amenable to targeting both understanding of cancer biology and the development of novel of these pathways (52). There is preclinical evidence that epige- cancer therapeutics. netic agents can reverse chemoresistance in cancer stem-like cells, opening the door to efficacy in combination with radiation or Disclosure of Potential Conflicts of Interest traditional chemotherapeutics (53). HDAC inhibitors can induce No potential conflicts of interest were disclosed. expression of genes in immune pathways, including those involved with IFN response, antigen presentation, cytokine pro- Grant Support duction, and inflammation (54). Assuming expression of these This work was supported by NIH/National Institute of Neurological genes makes cancer cells more targetable to immune cells, Disorders & Stroke (NIH/NINDS) grants R37-NS094804, R01-NS074387, immune-based therapies will be more effective after HDAC R01-NS057711, R21-NS091555, and BioInterfaces Institute, University of inhibitor priming. This principle is currently being tested in adult MichiganU042841 (M.G. Castro), NIH/NINDS grants R01-NS054193, R01- lung cancer trials (6, 54, 55). NS061107, R01-NS082311, and R21-NS084275 (P.R. Lowenstein), 1-R01-EB- Newer small-molecule epigenetic agents are increasingly being 022563-01 (M.G. Castro and P.R. Lowenstein), the Department of Neuro- developed and tested, some of which directly target mutated surgery, University of Michigan Medical School, the Michigan Institute for Clinical and Health Research, NIHUL1-TR000433, University of Michigan CRFs. Small molecules targeting the 2-hydroxyglutarate produc- Cancer Biology Training Grant, NIH/NCIT32-CA009676, University of Michi- tion of mutant IDH1 are in clinical trials, with promising early gan Training in Clinical and Basic Neuroscience, NIH/NINDST32-NS007222, results in AML (2, 6). Molecules designed to target SET domain the University of Michigan Medical Scientist Training Program, NIH/NIGMS proteins have demonstrated the ability to upregulate H3K27me3, (National Institute of General Medicine Sciences) T32-GM007863, and very leading to apoptosis in EZH2-mutated tumor cells (56). These are generous support from Phil F. Jenkins. C.K. was supported by NIH/NINDS grant now in early-phase trials for recurrent lymphomas and solid K08-NS099427-01, NIH/NICHD grant 4K12HD028820-24, and the University of Michigan Janette Ferrantino Investigator Award. A.K. Meeker was supported by tumors. Other CRFs being targeted are the bromodomain and NIH/NCI grant R01-CA172380-01. J.A. Brosnan-Cashman was supported by grants extra terminal (BET) family genes, BRD3 and BRD4. BET bromo- from the Rally Foundation for Childhood Cancer Research and The Truth 365. domains recognize and spread acetylation of histones, and their fusion to NUT (nuclear protein in testis) has been shown to Received August 19, 2016; revised October 4, 2016; accepted October 8, 2016; promote tumor growth in 80% of NUT midline carcinoma (NMC; published OnlineFirst January 6, 2017.

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www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF7

Mutated Chromatin Regulatory Factors as Tumor Drivers in Cancer

Carl Koschmann, Felipe J. Nunez, Flor Mendez, et al.

Cancer Res Published OnlineFirst January 6, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-2301

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