Deregulation of Retroelements as an Emerging Therapeutic Opportunity in Cancer

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Review

Deregulation of Retroelements as an Emerging Therapeutic Opportunity in Cancer Charles A. Ishak,1 Marie Classon,2 and Daniel D. De Carvalho1,3,* Nearly half of the human genome is comprised of repetitive elements that are tightly regulated to protect the host genome from deleterious consequences associated with their inappropriate activation. Cancer cells often misexpress these elements, in part, due to decreases in DNA methylation. Recent discoveries suggest that tumor suppressor proteins contribute to repression of repetitive elements, and their functional inactivation promotes repeat element misexpression during carcinogenesis. Recent findings also suggest that increased expression of repetitive elements beyond a threshold of tolerance can augment cancer therapy responses. Such advances, reviewed here, paint a picture in which deregulated expression of repetitive genome elements not only contributes to the development of cancer but may also provide a tumor-specific Achilles heel for cancer treatment. Host Defense Mechanisms Control Expression and Propagation of Viral Repeat Elements Nearly half of the human genome is comprised of repetitive sequences derived in part from exogenous retroviruses that successfully incorporated themselves into the host germline. Fulllength and fragmented copies of these viral genomes have propagated throughout host genomes to produce repeating instances of their sequences [1]. Major types of repeat elements are depicted in Figure 1 and include autonomous and nonautonomous retrotransposons as well as DNA transposons. The regulatory elements of these repetitive sequences can control host gene expression and have, in some instances, undergone positive selection within the germline to serve as de novo open reading frames or regulatory elements for transcriptional programs in development, innate immunity, and other settings [2,3]. Co-option or exaptation of repeat-derived regulatory elements underlies the intersection of long-terminal repeats (LTRs) with
20% of all functional binding sites for numerous transcription factors in mammalian cells [4,5]. Uncontrolled expression and propagation of full-length and fragmented repetitive sequences can also confer deleterious mutagenic effects on the host genome and thereby decrease cellular fitness. Therefore, colonization strategies of mobile repetitive elements coevolved with a myriad of host defense mechanisms that counteract mobile element expression and propagation. In this context, it is interesting to note that recent studies implicate de-regulate expression of repetitive elements in the origin and evolution of a number of diseases that include autoimmune diseases and cancer [6]. Deregulation of repetitive elements also occurs in aging humans [7], which may explain the often-late onset of many such diseases. Host defense strategies against mobile repetitive sequences can generally be classified into two categories: transcriptional and post-transcriptional. Transcriptional defenses prevent misexpression of repetitive sequences, and involve transcription factors that coevolve with the integration and mutation of repeat elements. For example, the large diversity of zinc-finger

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Highlights Low response rates to immunotherapy in multiple tumor types highlight the need for improved treatment. Functional inactivation of tumor suppressors and loss of DNA methylation cause misregulation of repetitive genomic elements early in oncogenesis. Tumors employ multiple tolerance strategies to evade negative selection due to decreased fitness and increased immunogenicity. DNA demethylating agents induce viral mimicry beyond a threshold of tolerance and increase cancer immunotherapy response. Tumor-specific compensatory silencing of repeat elements may present a therapeutic opportunity to maximize cancer treatment responses. Tumor-specific compensatory antiviral signaling mechanisms may also present therapeutic opportunities.

1 Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada 2 AccIntus, Mill Valley, CA 94941, USA 3 Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 2M9, Canada

*Correspondence: [email protected] (D.D. De Carvalho).

https://doi.org/10.1016/j.trecan.2018.05.008 © 2018 Elsevier Inc. All rights reserved.

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Arrangement

Type

Subtype

Class (% genome) SINE (15%)

Non-LTR LINE (21%)

Type I LTR

Interspersed Type II

RepeƟƟve elements

ERV (9%) DNA transposon (3%) Satellite (3%)

Tandem

VNTR (0.2%)

Family

Alu ID L1 CR1 ERV1 Gypsy Mariner PiggyBac

Canonical structure (length)

pA

Pol III

(∼300 bp)

Pol II 5′ UTR

ORF1

Pol II LTR

gag

Pol II TIR

Transposase

α-satellite Telomeric

I II III IV

Simple repeat microsatellite

I

ORF2

pol pA TIR

env

pA 3′ UTR

LTR

(∼6 kb)

pA (6-11 kb)

(1-3 kb) (10–300 bp)

(2–6 bp)

Figure 1. Classification and Organization of Repetitive Elements in the Human Genome. Major types of repetitive elements shown are classified according to chromosomal distribution pattern, mechanism of propagation, and the presence or absence of certain functional domains. Two examples of families are shown per class, and families are further stratified into subfamilies. Listed abundances were determined from human genome 19 [114]. Differences in major categories between multiple repetitive element classification systems underscore recent calls for a renewed consensus towards a single classification system [115]. For example, SVA composite elements that contain SINE, VNTR and Alu sequences are not shown. Full-length canonical structures are shown with corresponding polymerases and polyadenylation signals indicated. However, the majority of repetitive elements, such as LINE-1 or ERV families, primarily exist as partial fragments throughout the human genome. Abbreviations: ERV, endogenous retrovirus; LINE-1, long interspersed nuclear element 1; LTR, long terminal repeat; ORF, open reading frame; SINE, short interspersed nuclear element; UTR, untranslated region; VNTR, variable number of tandem repeats.

proteins in the human genome [8,9] is seemingly the result of an evolutionary arms race initiated by integration of newly colonizing repetitive elements coupled with escape mutations that diminish transcription factor recognition. These transcription factors recruit chromatin regulatory complexes that modify DNA and histone tails to establish transcriptionally repressive heterochromatin over repeat-rich genomic regions [8,10]. These defense mechanisms have largely been characterized in the context of early development and evolution, and are elaborated upon in subsequent sections of this article. Post-transcriptional responsive defense strategies are employed upon compromised transcriptional silencing of repetitive elements [11]. Such defenses include host restriction factors, exemplified by the APOBEC3 cytidine deaminase, which directly targets repetitive transcripts for degradation [12], as well as factors that prevent proper RNA processing [13]. Host defenses that engage exogenous viral transcripts are also activated upon deregulation of endogenous repeat elements (Figure 2) [14]. These innate immune responses have predominantly been characterized within the context of exogenous viral infection, where nucleic acids of foreign origin are detected by endosomal or cytosolic pattern recognition receptors that initiate an interferon (IFN)-driven immune response to cull affected cell populations [15–17]. The discovery that the cellular host engages a similar signaling pathway upon misexpression of endogenous retroviruses forms the basis for a therapeutic approach coined viral mimicry that uses clinical agents to induce expression of endogenous viral repeats that stimulate an antiviral

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5

MAVS M MA AVS VS MDA MA MAVS

MAVS

MAVS

IRF7

IRF3

P IRF7

P IRF3

AnƟgen processing AnƟgen presentaƟon CytolyƟc T-cell acƟvity Immunogenic cell death

5 MDA dsRNA

DNMTi HDACi

Cytoplasm Nucleus

Repeat expression MutaƟonal burden

P

ERV

FuncƟonal inacƟvaƟon of tumor suppressor proteins

Loss of epigeneƟc repression

P IRF7

ERV

ISGs ISGs

Interferon sƟmulated genes

Figure 2. Signal Transduction of Viral Mimicry and dsRNA Antiviral Response. Derepression of repetitive sequences, such as endogenous retroviruses, permits expression of repetitive transcripts that form dsRNA structures. Cytosolic dsRNA sensors, such as MDA-5, bind dsRNAs and localize to the mitochondria to promote MAVS aggregation and the initiation of a signaling cascade that promotes phosphorylation, dimerization, and nuclear localization of IRFs. Endosomal dsRNA sensors that activate immune responses are not shown. IRF transcription factors activate genes that coordinate an antiviral immune response that synergize with enhanced production of repeatderived neoantigens to enhance visibility of affected cells towards the immune system. These responses may occur from intrinsically derived events that perturb silencing of repetitive sequences. Therapeutic viral mimicry utilizes clinical agents that perturb epigenetic silencing of repetitive sequences, such as DNA demethylating agents (DNMTi) or other epigenetic therapies (exemplified by HDACi), to promote the dsRNA response as a means of enhancing tumor immunogenicity. Abbreviations: DNMTi, DNA methyltransferase inhibitor; dsRNA, double-stranded RNA; ERV, endogenous retrovirus; HDACi, histone deacetylase inhibitor; IRF, interferon regulatory factor; ISG, interferon-stimulated gene; MAVS, mitochondrial antiviral-signaling protein; MDA5, melanoma differentiation-associated protein 5.

response [14]. Thus far, therapeutic viral mimicry has been best characterized in cancer cells treated with DNA demethylating agents or histone deacetylase (HDAC) inhibitors (Figure 2). DNA demethylating agents derepress endogenous retroviruses that form double-stranded (ds) RNAs, such as LTR12C family members expressed from previously nonannotated cryptic transcriptional start sites [14,18,19]. Upon detection of dsRNA, cytosolic RIG-I-like receptors (RLRs) RIG-I and MDA5 activate mitochondrial antiviral-signaling protein (MAVS) that induces nuclear localization of IFN regulatory factors (IRFs) to initiate a type I or type III IFN response; a mechanism similar to that observed in virus-infected cells [14,18]. Analogous to Toll-likereceptor-induced IFN responses, RLR-induced IFN responses increase immunogenicity or visibility of cancer cells to cytolytic T cells, and cause immunogenic cell death [20]. This response to DNA demethylating agents has been validated in numerous human cancers that include colorectal cancer, ovarian cancer, promyelocytic leukemia, and hepatocellular carcinoma [21–24]. The robust activation of viral mimicry across multiple human cancers forms the basis of ongoing clinical trials that explore DNA demethylating agents and HDAC inhibitors as inducers of therapeutic viral mimicry towards augmentation of immunotherapy responses. A number of recent reports explore the underlying basic biology of this antiviral response and highlight a plethora of new questions and considerations (see Outstanding Questions). For

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example, does the viral mimicry response provide a checkpoint used to cull populations with compromised epigenetic regulation of repetitive elements? Does expression of these repeat elements also result in the expression of neoantigens? Furthermore, are the epigenetic regulatory mechanisms that silence repetitive elements upon DNA hypomethylation in developmental settings re-engaged in tumors? If so, can the differences in the regulation of these repeat elements in normal somatic tissue and DNA hypomethylated cancers be exploited in cancer therapy? This review attempts to put some of these questions into context.

Compensatory Heterochromatinization Maintains Silenced Repeat Elements during Early Development Dormant tandem and interspersed repetitive elements are maintained in host tissues through repressive constitutive heterochromatin most concentrated over centromeric and telomeric regions. Constitutive heterochromatin is characterized by DNA methylation as well as histone H3 lysine 9 trimethylation (H3K9me3) and histone H4 lysine 20 trimethylation (H4K20me3) on N-terminal tails of hypoacetylated H3 and H4 histones [25]. DNA methyltransferases (DNMTs) deposit methyl moieties predominantly on carbon 5 of cytosine residues adjacent to a guanosine that forms the repetitive unit of CpG dinucleotides [26]. Germline knockout models distinguish DNMTs involved in de novo DNA methylation (DNMT3s) from those involved in the maintenance of DNA methylation (DNMT1) during DNA replication when passive demethylation can occur. Initial characterization of cultured embryonic stem cells (ESCs) from mice deficient in these DNMTs revealed selective reductions in CpG methylation, predominantly at minor satellite repeats and a number of retrotransposons, with limited disruption to transcriptional repression (Table 1). The minor perturbances of repetitive element expression upon DNMT deletion in ESCs hints at repressive compensatory mechanisms that could involve histone modifications associated with constitutive heterochromatin upon disruption of DNA methylation. Such compensation occurs upon genome-wide DNA hypomethylation in early development and in germ cells where the H3K9 histone methyl transferase (HMT) SET domain bifurcated (SETDB1) is required for transcriptional repression of repeat elements and cell survival [27]. Additional compensatory mechanisms maintain transcriptionally silenced repeats upon ablation of H3K9 HMTs. Deletion of SETDB1 in ESCs reduces H3K9me3 at pericentric satellite repeats, long interspersed nuclear elements (LINEs), and endogenous retrovirus (ERV) classes I and II in early embryos and during gametogenesis [27–31]. Deletion of suppressor of variegation 3-9 homologs -1 and -2 (SUV39h1/2) also diminishes H3K9me3 at pericentric major satellites and telomeric repeats, but not at LTR retrotransposons [32–34]. In both cases, impacted elements exhibit minimal expression changes. Deletion of the H3K9me1/2 HMT G9a/EHMT2 in ESCs is even less consequential as repeats exhibit ostensibly normal silencing [35,36]. H3K9me3 spreads to new repeat integrations, in part through engagement of the human silencing hub (HUSH) complex that contains the H3K9 methyl reader MPP8, and promotes H3K9me3 deposition at young LINE-1 elements enriched within introns of transcriptionally active genes [37,38]. H3K9 methylation also recruits transcriptional repressors, such as the histone H3.3 chaperon complex ATRX–DAXX, to silence repetitive sequences [39,40]. Disruption of ATRX–DAXX activity derepresses a limited number of repetitive sequences [41]. Deletion of H4K20 HMTs reduces H4K20me3 predominantly at pericentric major satellites, with minimal transcriptional deregulation of these repeats [42]. In all of these instances, minimal effects on repeat element expression suggest the existence of compensatory silencing mechanisms. In addition to constitutive heterochromatin, factors that establish a more readily reversible form of chromatin compaction referred to as facultative heterochromatin also repress repeat 4

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Table 1. Effect of Germline Murine Epigenetic Writer Ablation on Regulation of Repetitive Elements Mutant

Diminished mark

Cell type

Repetitive elements modified

Overt phenotypes

Refs

Dnmt1 (hypomorph)

CpGme

MEFs, tumors

Minor satellites

Lymphomas with MMTV integrations

[94,95]

Dnmt1/

CpGme

ESCs

Minor satellites, certain IAP LTRs

E9.5 lethal

[96,97]

Dnmt3a/

CpGme

ESCs

N/A

Lethal 4 weeks post birth

[98]

Dnmt3b/

CpGme

ESCs

Minor satellites

E9.5 lethal

[98]

Dnmt3l

CpGme

ESCs

Certain IAP LTRs and L1 elements

Viable

[99,100]

Dnmt3cIAP/IAP

CpGme

P20 testis

Certain ERV-K and L1 elements

Viable

[101]

Setdb1/ (conditional)

H3K9me3

ESCs

Major satellites, select LINEs and ERV-I/II elements

Phenotypes vary depending upon tissue targeted

[27,28,30]

Suv39h1/2/

H3K9me3

ESCs, MEFS

Major satellites, telomeres

Viable (subMendelian); B cell lymphoma

[32–34,102,103]

Suv4-20h1/

H4K20me2

MEFS

Major satellites

Viable

[42,104]

Suv4-20h2/

H4K20me3

MEFS

Major satellites

Viable

[42,104]

G9a

CpGme

ESCs

Major satellites, MLV, IAP, L1Md-A2

E9.5–E12.5 lethal

[35,36,105]

Glp/

CpGme

ESCs

Major satellites, MLV, IAP, L1Md-A2

E9.5 lethal

[35,106]

chip/

/

/

elements in some contexts. H3 methylation at lysine 27 (H3K27me) by the enhancer-of-zeste homolog (EZH) HMTs EZH1 or EZH2, that participate in the multimeric polycomb repressive complex 2 (PRC2), can compensate for disrupted constitutive heterochromatin in ESCs [43]. For example, loss of the H3K9 methyl transferases SUV39h1/2 in ESCs results in elevated PRC2 and H3K27me3 enrichment at repetitive sequences [44]. Studies also demonstrate that DNA methylation and H3K27 methylation are interdependent in some contexts, although precise mechanistic links require further characterization. For example, Dnmt1, Dnmt3a, and Dnmt3b triple-knockout ESCs exhibit pronounced reductions in CpG methylation in tandem with compensatory PRC2-mediated H3K27me3 deposition at pericentric repeats [27,45]. Facultative heterochromatization of repetitive sequences upon acute induction of DNA demethylation in ESCs and tumor cells makes it unlikely that compensation is a phenomenon confounded in germline knockouts due to long-term adaptation [46,47]. Collectively, these loss-of-function approaches detail an intricate network of compensatory repressive mechanisms that can be employed to prevent expansion of populations with promiscuous expression of repetitive sequences.

Repetitive Elements in Cancer Causes and Consequences of Repetitive Element Reactivation in Cancer The development of a tumor requires, amongst other criteria, the capacity for self-sustained cellular growth as well as an ability to evade programmed cell death, cellular senescence, and immune surveillance. Despite the compensatory potential of epigenetic silencing, misexpression of repeat elements can compromise genome integrity and thereby contribute Trends in Cancer, Month Year, Vol. xx, No. yy

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to the initiation of tumorigenesis [15,48]. Disruption of epigenetic homeostasis in cancer cells often makes it difficult to determine whether repeat misexpression may have been a cause or consequence of tumorigenesis for a given tumor. However, misexpression of repeat elements can also activate an immune response against the developing tumor. Therefore, mechanisms to control misexpression of repeat elements upon widespread perturbances to the DNA methylation landscape in normal cells likely undergo positive selection in tumors [49]. Since the early discovery of increased DNA hypomethylation in tumors, stable expression of repetitive sequences throughout tumorigenesis has come to be perceived as a determinant of disease progression rather than a silent byproduct [50–52]. The contribution of repetitive sequences to tumorigenesis reflects potential vulnerabilities associated with host domestication strategies. Just as the host exapts LTRs to serve as de novo open reading frames or regulatory elements that fine tune transcription, cancers can engage in oncoexaptation to create oncogenic fusion transcripts or promote aberrant activation of proto-oncogenes. Examples of oncoexaptation include expression of the oncogenic LTR–IRF5 chimeric transcript, or cis-activation of the CSF1R proto-oncogene by the mammalian apparent LTR retrotransposon (MaLR) LTR in Hodgkin’s lymphoma [53,54]. Compromised transcriptional repression also permits re-integration of tandem or interspersed repeats that cause mutagenesis. Such loss-of-function or gain-of-function mutations have been observed in numerous cancers that involve autonomous LINE-1 retrotransposons or centromeric satellite repeats that propagate through RNA intermediates [51,55–58]. More recently, the piggyBac DNA transposase PGBD5 has also been implicated in tumorigenesis through deletion of the SMARCB1 tumor suppressor in rhabdoid tumors [52]. In addition to direct mutagenesis, meiotic and mitotic defects that promote chromosomal instability often accompany repetitive element derepression for reasons that remain unclear, but may be due to perturbed heterochromatinization [59]. Repetitive element reactivation in premalignant lesions suggests that misregulation of repeats may occur in tandem with early oncogenic events that drive the development of a tumor [51,60]. Context for this phenomenon may be derived from recent discoveries that establish a role for tumor suppressor proteins as negative regulators of repetitive genomic regions [61]. For example, functional disruption of two well-established tumor suppressor proteins, the retinoblastoma tumor suppressor (pRB) and p53, can result in increased expression of repeat elements [62,63]. Although early studies attribute the tumor suppressive functions of pRB and p53 to regulation of proliferative control and the induction of DNA repair or cell cycle and cell death checkpoints upon DNA damage, repeat-silencing may comprise an overlooked facet of their tumor suppressor function. In the case of pRB, best characterized as a negative regulator of cell cycle genes [64], new observations suggest a role for pRB in the regulation of repeats mediated by a cyclin-dependent kinase (CDK)-resistant interaction with the E2F1 transcription factor that recruits EZH2 and Condensin II to repetitive sequences [62,65]. During mouse development, germline disruption of this pRB–E2F1 interaction is associated with mitotic defects and gH2AX deposition at major satellite repeats normally occupied by pRB–Condensin II, as well as dispersion of H3K27me3 from LINEs, ERVs, and major satellite repeats regulated by pRB–EZH2 [62,66]. Furthermore, the onset of spontaneous splenic and mesenteric lymphomas following disruption of this pRB–E2F1 interaction suggests that pRB-mediated regulation of repetitive sequences may contribute to the tumor suppressive function of pRB. Analogous to pRB, p53 participates in multiple epigenetic repressive complexes required to establish both DNA and histone modifications, and multiple p53 cistrome datasets reveal 6

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extensive p53 occupancy at intergenic retrotransposons [67–69]. This occupancy is likely functional since p53-loss or hotspot TP53 mutations are associated with deregulated expression of repeat elements in numerous organisms [63,70]. However, it remains to be determined whether p53 silencing of repeats directly contributes to its tumor suppressive properties. In addition to pRB and p53, an expanding list of tumor suppressors are implicated in the regulation of repetitive sequences. For example, inactivation of the von Hippel–Lindau tumor suppressor (VHL) disrupts CpG methylation of the HERV-E 50 LTR resulting in HERV-E misexpression in renal cell carcinoma [71,72]. ATRX and H3.3 mark retrotransposons and telomeres in ESCs, although consensus regarding roles for ATRX or H3.3 in retrotransposon silencing remains to be established [40,41,73,74]. For example, it remains unclear whether tumor-specific mutations in ATRX and H3.3 result in deregulation of repeat elements in tumors. Collectively, these examples suggest interplay between repeat element expression, and the activation of oncogenes or the inactivation of tumor suppressor proteins. Cancer Cells Tolerate Higher Levels of Repetitive Element Expression The relaxed repression of repeat elements in tumor cells highlights a curious paradox: repetitive element misexpression may promote tumorigenesis, and yet, can also reduce cellular fitness and induce an antitumor immune response. This raises the possibility that cancers actively manipulate the threshold or efficacy of endogenous viral mimicry induction (Figure 3). Although a growing body of literature correlates expression of repeat elements, mutational burden, and IFN response gene expression in tumor cells, the tolerance to viral mimicry in cancer cells remains poorly understood [18,23]. It is interesting to note that splenocytes with germline disruption of the CDK-resistant pRB–E2F1 interaction exhibit transcriptional responses associated with IFN activation that suggest an attempt to mount a viral mimicry response that precedes lymphomagenesis [62]. Likewise, misexpression of retrotransposons upon Trp53 deletion induces IRF7 and activates a type I IFN response in mouse embryonic fibroblasts (MEFs) [70]. Despite activation of an innate immune response in p53-deficient MEFs, pronounced misexpression of repetitive sequences in p53-deficient murine thymic lymphomas, human Wilms’ tumors, and colorectal tumors corroborates the notion that cancers may tolerate endogenous viral mimicry responses [23,63,70]. Such viral mimicry evasion by tumor cells may involve a multipronged strategy wherein different cancer cell subpopulations employ distinct defensive mechanisms. Some of these approaches may involve mechanisms borrowed from ESCs or somatic cells that must tolerate increased repetitive element expression during specific developmental stages. For example, LINE-1 retrotransposition throughout neuronal development evades negative selective pressure from the immune system to establish somatic mosaicism within the brain [75,76]. While tumors exhibit a perturbed epigenetic landscape with reduced DNA methylation in repetitive regions, maintenance of some repeat element repression in tumors may still be important since innate immune responses against repeat-derived transcripts diminish cancer cell fitness [14,18]. In this context it is important to note that it is still somewhat unclear whether compensatory mechanisms, similar to those observed during development, play a role in repeat element repression in tumors. Epigenetic regulators often exhibit altered expression levels in tumor cells [77], and certain cancers selectively deploy some of these regulatory factors to maintain heterochromatin formation over repeat elements. For example, the H3K9 HMT SETDB1 is highly expressed in a number of cancer cell types, as compared to normal tissue, and SETDB1 deletion in acute myeloid leukemia (AML) cell lines diminishes H3K9me3 at LINEs and ERVs. Reduced H3K9me3 upon SETDB1 loss in AML permits repeat misexpression that Trends in Cancer, Month Year, Vol. xx, No. yy

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(A)

(B)

Development

Tumor cell

CpG CpGme Histone trimethylaƟon Compensatory repression

ERV ERV

Cancer therapeuƟcs

Enhanced dsRNA tolerance

EpigeneƟc therapy

Drugtolerant persisters

ERV

ERV

EpigeneƟc therapy

ERV

H3K9me3 H3K27me3 Maintenance of cell fitness

dsRNA paƩern recogniƟon receptors? Maintenance of cell fitness

H3K9me3 / H3K27me3 Maintenance of cell fitness

ERV

AnƟtumor signaling cascades Immunogenicity / immunogenic cell death Cancer cell fitness

(C) Tolerance of dsRNA expression

ESCs

SomaƟc cells

Drug-tolerant persisters

Untreated cancer cells

Figure 3. Cancer Cells Tolerate Endogenous Viral Mimicry. (A) DNA hypomethylation early in development is accompanied by compensatory histone modifications that heterochromatinize and silence mobile repetitive sequences. Bursts of transposon activation upon initial DNA hypomethylation do not suffice to negatively select ESC populations that exhibit enhanced dsRNA tolerance. (B) Early events in oncogenesis perturb epigenetic homeostasis and permit derepression of repetitive sequences. Cancers likely engage multiple strategies to circumvent the negative selection pressures of viral mimicry. Certain cancer therapeutics, such as erlotinib, select for drug-tolerant persister populations that exhibit selective heterochromatinization of retrotransposons and serve as founders for therapeutic relapse. Other evasion mechanisms may elevate tolerance thresholds required to induce dsRNA antiviral signaling. Epigenetic therapies that further increase dsRNAs beyond elevated tolerance thresholds induce immunogenic cell death for a number of cancer cell types. (C) ESCs and drug tolerant persisters occupy opposite ends of the spectrum for tolerance of dsRNA responses. Further characterization will define where certain somatic cells or untreated cancer cells are positioned along this spectrum, and identify events that direct this positioning, such as mutations in the JAK–STAT pathway or epigenetic machinery. Abbreviations; dsRNA, doublestranded RNA; ERV, endogenous retrovirus; ESCs, embryonic stem cells; H3K9me3, H3 lysine 9 trimethylation; H3K27me3, H3 lysine 27 trimethylation.

activates an MDA-5/RIG-I-dependent innate immune response and cell death [78]. It remains unclear whether SETDB1 plays a similar role in other tumor types, and whether other chromatin regulators impose selective and compensatory repression of repeat elements or downstream response pathways in blood or solid tumors. In many settings, stress-activated repeat element expression promotes cellular or organismal adaptation to changing conditions. Repetitive element misexpression and retrotransposition have the potential to contribute to regulatory variation in gene expression in response to stress. In this context, it is interesting to note that numerous targeted cancer therapeutics as well as chemotherapeutic agents activate repeat element expression in cancer cells [79,80]. The mechanism behind this activation is not well understood; although, studies using CDK4/6 inhibitors in breast cancer attribute repeat activation to pRB-mediated repression of DNMT1 [80]. In some cancer cells, such activation may contribute to cell death. However, some cells within a heterogeneous cancer cell population survive during otherwise lethal drug exposures through maintenance of epigenetic repression of repeat elements (Figure 3) [79,81]. For example, SETDB1-mediated repression of LINE-1 elements is required for the survival of drug-tolerant persisters [79]. Such studies suggest that modulation of epigenetic repression of repeat elements may promote more durable drug responses not only in the context of

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immunotherapy, but also in the context of other treatments [82]. Indeed, small clinical studies suggest that DNA demethylating agents or HDAC inhibitors may enhance response to other therapies. Interestingly, combined carboplatin and anti-programmed death-1 demonstrated enhanced efficacy in lung cancer clinical trials, and it can be speculated as to whether such combinatorial effects are due to the ability of carboplatin to induce repeat elements expression and viral mimicry [83].

Emerging Therapeutic Opportunities and Considerations Correlation between repeat expression and IFN responses in tumors, together with the identification of DNA demethylating agents and HDAC inhibitors as inducers of viral mimicry, underlies the rationale for a number of clinical trials that combine epigenetic modulators with immune checkpoint inhibitors [23]. Pending the conclusion of these trials, it is important to consider a number of points that may influence efficacy. For example, these drugs may increase expression of repeat elements in normal tissues and promote autoimmunity, which may impact the therapeutic index of DNA demethylating drugs. Furthermore, tumors may engage mechanisms other than DNA methylation to repress repeats, or downregulate MHC expression to compensate for excessive repeat deregulation. It is also important to consider tumor-specific alterations downstream of viral mimicry signaling pathways. Such considerations may be relevant in patients that relapse on immune checkpoint therapy and harbor mutations in JAK–STAT signaling components that would impede transduction of cytokineinduced signaling [84]. Future studies of compensatory mechanisms that control repeat element expression or responses downstream of viral mimicry in tumors, before or after treatment, may reveal new therapeutic opportunities specific to certain tumor types or genotypes. Tumor Cell-Intrinsic Factors That May Affect Viral Mimicry Efficacy A better understanding of the mechanisms that determine repetitive element silencing or viral mimicry responses in tumors will likely reveal biomarkers that can instruct and identify tools to more effectively induce therapeutic viral mimicry. For example, p53-deficient MEFs elicit immune responses to retrotransposon activation at lower doses of DNA demethylating agents, likely due to already elevated retroviral transcripts present upon p53 deficiency [70]. This observation has been recapitulated in p53-deficient human colorectal cancer cell lines and AML [22,23]. This phenomenon suggests a lower therapeutic index for DNA demethylating agents in therapeutically induced viral mimicry in p53-deficient tumors. These observations exemplify why the utilization of biomarkers in combination with targeting nonredundant repeat element silencing mechanisms may augment induction of viral mimicry in some cancers. Similar to p53, functional status of the pRB tumor suppressor protein or EZH2-dependent H3K27me3 distribution may also serve as indicators to select an appropriate therapeutic viral mimicry approach. For example, viral mimicry induction through CDK4/6 inhibition in breast cancer requires the presence of functional pRB [80]. Furthermore, discovery of pRB–EZH2mediated silencing of repetitive sequences in somatic cells suggests that EZH2 inhibitors may be the next class of agents adopted to elicit dsRNA responses [62]. Indeed, increased transcription of 1q12 pericentromeric satellite II DNA in A375 cells treated with the EZH2 inhibitor GSK126 indicates that this may be the case [85]. Taken together, these studies suggest that the functional status of pRB or EZH2 may also inform decisions as to when to target EZH2 and H3K27me3 versus an alternative nonredundant silencing mechanism in order to maximize viral mimicry induction in a therapeutic setting.

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Alterations in chromatin modifiers are common in human tumors but, akin to loss-of-function ESC studies, it is unclear whether these alterations serve to control the expression of repetitive elements. However, compensatory repetitive element silencing mechanisms best characterized in early development may offer additional therapeutic targets for viral mimicry induction. These may include histone methyltransferases, histone demethylases, or epigenetic reader proteins that bind methylated DNA or histones. Future studies will determine whether these potential drug targets offer more precise ways to induce viral mimicry specifically in tumor cells as compared to DNA demethylating agents or HDAC inhibitors. Leveraging Repetitive Elements as Sources of Tumor-Associated Antigens It has long been known that cancer patients can develop antibodies to proteins expressed from endogenous retroviral families such as HERV-K [86]. Targeting these repeat-derived tumor-associated antigens can promote tumor regression. For example, studies in metastatic renal cell carcinoma patients reveal that inactivation of the VHL tumor suppressor corresponds with CpG hypomethylation of the HERV-E 50 LTR that permits hypoxiainducible-factor-2a-induced expression of a HERV-E-derived peptide [72]. This peptide forms a tumor-associated antigen, designated CT-RCC-1, with HLA-A11 as the corresponding MHC class I restricting allele. Strikingly, this antigen stimulates a cytotoxic T cell response that underlies sustained tumor regression observed in patients with advanced disease following hematopoietic stem cell transplantation [71]. Such studies suggest that the effects achieved from autologous T cell transfer may be augmented by agents that derepress repetitive elements. Indeed, DNA demethylating agents and HDAC inhibitors can enhance expression of the CT-RCC-1 antigen [72] and future studies will determine how widespread such phenomena may be. Retrotransposon-derived tumor associated antigens have been identified in numerous human tumors, with confirmed induction of cytotoxic T cell responses (Table 2). In response to these discoveries, CAR+ T cells have been engineered to target HERV-K-derived antigens prevalent in melanomas and breast cancer, and encouragingly, vaccines against these antigens have been deemed largely safe [87–90]. Future studies will determine how effective such strategies will be clinically. Maximizing opportunities to target repeat-derived, tumor-associated antigens demands higher-throughput approaches to identify viable targets. Putative repeat-derived

Table 2. Immune Responses to Tumor-Associated Antigens Derived from HERVs Repeat type

Repeat family

Peptide source

Presenting human cancer type

Immune responses

Refs

LTR

HERV-K

env

Melanoma, breast cancer, ovarian, lymphoma, teratocarcinoma

Engineered CAR-T cells kill HERV-K+ melanoma cells and inhibit metastasis of breast cancer cells. Vaccination inhibits murine tumor growth.

[87–89,107]

LTR

HERV-K

gag

Prostate cancer

Vaccination against HERV-K Gag delays subcutaneous tumor growth of HERV-K+ murine renal cell carcinoma cells

[108–111]

LTR

HERV-E

env

Clear cell renal cell carcinoma

Promotes cytolytic T cell activation and tumor regression in patients

[71,112]

LTR

HERV-H

env

Gastrointestinal and colorectal cancers

Promotes CD8+ T cell proliferation and IFN-g secretion

[113]

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tumor antigens have been identified from TCGA RNA-seq datasets by assessing transcriptional signatures in ERV-positive tumors that correspond with cytotoxic immune responses [91]. More direct approaches encompass direct capture of presented antigens derived from noncoding regions [92]. Future studies that combine these strategies with novel approaches will likely yield the most promising repeat-derived antigens to assess for induction of cytotoxic T cell responses. It remains to be determined whether clinically induced derepression of repetitive elements generates antigens in a tumor-specific manner, and whether this strategy will be effective in tumors that downregulate MHC to reduce neoantigen presentation.

Concluding Remarks Clinically induced derepression of genomic repeat elements harbors the potential to enhance the immunogenicity of cancer cells and augment the response to a number of immunotherapy approaches [14,18,23,93]. Enhanced repeat element expression may also increase responses to other drugs [79]. However, identification of the most effective means to specifically deregulate repeat element expression in tumor cells requires an improved understanding of the underlying regulatory mechanisms in cancer tissues as compared to normal somatic tissues. Discovery of tumor-specific DNA hypomethylation as well as tumor-suppressor-based repeat silencing suggests that derepression of repetitive sequences can occur synonymously with the earliest events of oncogenesis [62,63]. Derepression of repeat elements can impart mutagenic consequences such as oncoexaptation and retrotransposition that promote genomic instability during tumorigenesis. In response, cancers may manipulate dosage, engage compensatory silencing mechanisms, or establish tolerance towards viral mimicry responses induced by repetitive transcripts in order to evade antitumor immune responses. These compensatory mechanisms may differ according to tumor origin and genotype, underscoring the importance of biomarker identification in future efforts to identify vulnerabilities in cancer cell populations. As basic cancer biology reveals novel therapeutic opportunities in tumor cells, the potential role for viral mimicry in tumor development and therapy highlights the need to re-examine mechanisms of tolerance to expressed repetitive elements. Active repeat elements can be detected in differentiated tissues from multiple organisms, including adult human neurons [62,63,75], suggesting that there are biological contexts in which viral mimicry does not promote an immune response. Such observations raise several questions. For example, is it possible that different cell types and tissues harbor different thresholds for the induction of the dsRNA response? A spectrum of repeat silencing stringencies or thresholds of innate immune response induction across cell types and developmental stage may reflect different risks associated with permitting genomically unstable populations to populate the host organism (Figure 3). For instance, the consequences of repeat derepression and retrotransposition in ESCs would carry over to every cell in the organism. In contrast, the requirements for a somatic cell may differ, since derepression of repeat elements and retrotransposition would not alter the genetic composition of every cell in the organism. When selecting therapeutic strategies to derepress repeat elements, it is also important to consider that repetitive genomic regions exhibit DNA hypomethylation in aging somatic tissues [7].

Outstanding Questions How are cell-type specific thresholds for induction of the dsRNA immune responses established in development and cancer? How do cancer cells evade endogenous viral mimicry responses upon misregulation of repetitive elements? Can the identification of biomarkers and targeting of signaling components, beyond epigenetic regulators, enhance therapeutic viral mimicry responses? Which classes of repetitive elements are reactivated by each type of epigenetic therapy (DNA demethylating agents, HDAC inhibitors, polycomb inhibitors, H3K9 methyltransferase inhibitors, etc.)? What classes of repetitive elements directly activate specific pattern recognition receptors (MDA5, RIG-I, TLR3, etc.)? Does the viral mimicry response provide a physiological epigenetic checkpoint to cull populations with compromised heterochromatin/DNA methylation? Does expression of these repeat elements also result in the expression of neoantigens? Are other epigenetic mechanisms engaged in silencing repeat elements upon treatment with DNA demethylating drugs in a process similar to early development? Do combination epigenetic therapies provide a better mechanism for viral mimicry induction in light of tumorspecific compensatory silencing?

In summary, recent developments in tumor biology and studies of tumor therapy responses reveal important roles for repetitive elements often overlooked in expression and epigenetic studies of tumors. Altogether, the advances reviewed here describe the framework for a novel epigenetic checkpoint, in which the viral mimicry response serves as a physiological mechanism to recognize and eliminate cells with deleterious loss of heterochromatin that otherwise Trends in Cancer, Month Year, Vol. xx, No. yy

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Proposed epigeneƟc checkpoint

ERV ERV Loss of repressive heterochromaƟn

Loss of repressive heterochromaƟn

ERV ERV ERV expression / dsRNA formaƟon

• • • •

CpG CpGme

RetrotransposiƟon Mutagenesis Genomic instability Cancer iniƟaƟon?

Viral mimicry Immunogenic cell death/ clearance by immune system

Figure 4. The Biology of Viral Mimicry Indicates a Possible Epigenetic Checkpoint. Early events in oncogenesis, such as tumor suppressor inactivation and loss of DNA methylation, perturb epigenetic homeostasis and cause genome-wide loss of heterochromatin. Loss of heterochromatin promotes mutagenesis, DNA damage, and ultimately compromises genome integrity to establish conditions appropriate for the initiation of tumorigenesis. Intrinsic mechanisms utilized to sense and mitigate consequences associated with loss of heterochromatin remain poorly described. Discovery of the underlying biology of viral mimicry responses suggests that repetitive elements interspersed throughout the genome serve the host as signals of heterochromatin loss. Upon perturbed heterochromatinization, transcripts expressed from certain repetitive elements form dsRNAs that initiate viral mimicry responses and immunogenic cell death to prevent the expansion of populations susceptible to development of genomic instability. Thereby, viral mimicry signaling can serve as a checkpoint for heterochromatin loss. Abbreviations: dsRNA, doublestranded RNA; ERV, endogenous retrovirus.

would permit genomic instability, mutagenesis, and possibly facilitate cancer initiation (Figure 4). Mechanisms that counteract this response in tumor cells may also present novel therapeutic opportunities. In light of these advances, we suggest that the repetitive regions of the genome may play a more central role in cancer initiation and responses to cancer therapies that include immunotherapy. Acknowledgments This work was supported by Canadian Institutes of Health Research (CIHR) New Investigator Salary Award (201512MSH360794-228629), Helen M Cooke professorship from Princess Margaret Cancer Foundation, Canada Research Chair, CIHR Foundation Grant (FDN 148430), NSERC (489073), and Ontario Institute for Cancer Research (OICR) with funds from the province of Ontario to DDC. A Princess Margaret Excellence Fellowship supports CAI.

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