Meeting Report: the Role of the Mobilome in Cancer Daniel Ardeljan1,2, Martin S

Published OnlineFirst July 18, 2016; DOI: 10.1158/0008-5472.CAN-15-3421 Cancer Meeting Report Research

Meeting Report: The Role of the Mobilome in Cancer Daniel Ardeljan1,2, Martin S. Taylor3, Kathleen H. Burns1,4, Jef D. Boeke5, Michael Graham Espey6, Elisa C. Woodhouse6, and Thomas Kevin Howcroft6

Abstract

Approximately half of the human genome consists of repetitive in developmental and pathologic contexts, including many sequence attributed to the activities of mobile DNAs, including types of cancers. However, we have limited knowledge of the DNA transposons, RNA transposons, and endogenous retro- extent and consequences of L1 expression in premalignancies viruses. Of these, only long interspersed elements (LINE-1 or and cancer. Participants in this NIH strategic workshop consid- L1) and sequences copied by LINE-1 remain mobile in our species ered key questions to enhance our understanding of mechan- today. Although cells restrict L1 activity by both transcriptional isms and roles the mobilome may play in cancer biology. and posttranscriptional mechanisms, L1 derepression occurs Cancer Res; 76(15); 1–4. Ó2016 AACR.

Introduction demonstration of retrotransposition in cultured HeLa cells (2). A somatically acquired L1 insertion was shown to disrupt the Mobile DNAs, including DNA transposons, RNA transposons, adenomatous polyposis coli (APC) tumor suppressor gene in a and endogenous retroviruses, are highly abundant sequences that case of colorectal cancer (3). Nearly a decade of advances in DNA make up a major portion of eukaryotic genomes. Although sequencing have both underscored the importance of L1 activity human genomes no longer possess active DNA transposons, in causing heritable variation through retrotransposition in the which integrate via a "cut-and-paste" mechanism, they are instead germline and also demonstrated that widespread somatic retro- enriched in active retrotransposons that integrate via RNA inter- transposition occurs in many cancers. On September 25, 2015, the mediates. Human retrotransposons include long and short inter- NCI (Rockville, MD) sponsored a strategic workshop to assess the spersed elements (LINE and SINE), composite SINE-R, VNTR, and potential impact of somatic retrotransposition on cancer initia- long terminal repeat (LTR) elements. LINEs and LTRs are auton- tion and progression. A panel of experts in mammalian mobile omous or protein-coding elements, although only LINE-1 (L1) is DNAs, chaired by K.H. Burns and J.D. Boeke, considered recent considered competent to mobilize in humans. L1 sequences advances and challenges in the field. The goals were to define key represent approximately 17% of the human genome; however, research priorities and discuss ways to accelerate our understand- the vast majority of the estimated 500,000 copies is incapable of ing of mobile DNAs in cancer. The following is a summary of the mediating retrotransposition due to substantial truncations in the key topics discussed. 50 region. In contrast, recently inserted, full-length L1s encode proteins that can "copy-and paste" L1 sequences to new locations Retroelement Biology and Its Regulation in the genome. In addition to mobilizing L1 RNA (in cis), L1 0 proteins can also transpose other repetitive elements and endog- L1 is an approximately 6-kb DNA sequence comprised of a 5 enous RNAs in trans. untranslated region (UTR); two sense stranded open reading Interest in L1 relationships to cancer biology dates back to the frames (ORF), which encode two proteins, ORF1p and ORF2p; 0 0 1980s. Early L1 work included sequence characterization of a3 UTR; and a poly(A) homopolymer. The 5 UTR is rich in CpG expressed full-length L1 in teratocarcinoma cells (1) and the dinucleotides and contains an antisense promoter activity and a recently discovered antisense ORF protein dubbed ORF0 (4, 5). Transcribed L1 mRNA is exported to the cytoplasm as a bicistronic transcript that encodes both ORF1p, an RNA-binding protein, and 1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins Uni- versity School of Medicine, Baltimore, Maryland. 2Medical Scientist ORF2p, which possesses endonuclease and reverse transcriptase Training Program, Johns Hopkins University School of Medicine, Bal- domains required for L1-mediated retrotransposition. ORF1p timore, Maryland. 3Department of Pathology, Massachusetts General and ORF2p form a ribonucleoprotein (RNP) complex with target 4 Hospital, Boston, Massachusetts. Department of Pathology, Johns RNA (i.e., L1, Alu, SVA) or endogenous mRNAs in the cytoplasm Hopkins University School of Medicine, Baltimore, Maryland. 5Institute for Systems Genetics, New York University Langone Medical Center, and gains access to the nucleus through a poorly understood New York, New York. 6Division of Cancer Biology, NCI, NIH, Rockville, process. Transposition of L1 sequences to new positions in the Maryland. genome takes place through target-primed reverse transcription Corresponding Author: Elisa C. Woodhouse, NCI, 9609 Medical Center Dr., (TPRT). In this reaction, the ORF2p endonuclease first nicks the Tumor Biology and Metastasis Branch, Room 6W416, Bethesda, MD 20892- DNA double helix at a TTTT/A consensus motif to allow for an 9748. Phone: 240-276-6220; Fax: 240-276-7861; E-mail: interaction between the DNA and the poly(A) tail of the mRNA [email protected] and then uses this DNA to prime a reverse transcription reaction. doi: 10.1158/0008-5472.CAN-15-3421 Canonical L1-mediated integrations can be recognized by target Ó2016 American Association for Cancer Research. site duplications flanking the insertion site, the presence of poly-A

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tracts at the 30 end of the insertion, and in the case of processed cellular and organismal aging and, by extension, how this relates pseudogenes their lack of intronic sequences. to cancer. It is important to consider that although derepression of J.D. Boeke discussed the identification of host proteins that retrotransposons in advanced age may have negligible conse- interact with L1 ORF1 and ORF2 proteins with the hypothesis that quences for the fecundity of a species, its occurrence may create this will elucidate mechanisms of TPRT and promote a better susceptibility to age-related diseases. Retroelements, including L1, understanding of how cells contend with LINE-1 activity (6). He have been shown to increase expression as a consequence of aging, also described a transgenic mouse model harboring an inducible a change that can be attenuated by caloric restriction in mice (14). L1 gene-trap cassette useful for forward genetic screens in cancer. Vera Gorbunova (University of Rochester, Rochester, NY) dis- These mice surprisingly demonstrated L1 activity–dependent, cussed the role of SIRT6, a chromatin regulator that mono-ADP- nonheritable coat color phenotypes, suggesting cell populations ribosylates KAP1 and PARP1 and deacetylates histones, in regu- highly susceptible to somatic L1 activity (7). Tim Bestor (Colum- lating L1 expression. SIRT6 binds L1 50UTR sequences in the bia University, New York, NY) provided evidence for an L1- genome and directs heterochromatin formation to suppress tran- derived gene involved in control of L1 activity, designated scription. Exposure to ionizing radiation or oxidative stress results ECAT11 or L1TD1. Evidence suggests that ECAT11/L1TD1 is a in a displacement of SIRT6 away from L1 50UTRs to sites of DNA partially rearranged L1 ORF1, under strong selection in species strand breaks, thereby allowing reexpression of L1 genes. These with active L1, and neutral selection in species without active L1. findings suggest a relationship between accumulated DNA dam- John Moran (University of Michigan, Ann Arbor, MI) emphasized age and increased L1 expression with age (15). the need to elucidate L1 genomic insertion patterns, highlighting a L1 activity also seems to be influenced by environmental key technical challenge in the field. Somatically acquired L1 factors, such as inversion of day–night cycles that disrupt circadian insertions, he explained, may be targeted to specific genomic pathways. Victoria Belancio (Tulane University, New Orleans, LA) intervals owing to biases of either the retroelement or the chro- highlighted the role of the circadian-responsive melatonin recep- matin state of a particular cell, that is, certain intervals may be tor 1 (MT1) in regulating L1 activity. MT1 is capable of reducing L1 more accessible than others. Alternatively, de novo insertions may transcript and protein levels and also reduces L1-mediated retro- occur randomly and subsequently be subject to natural selection transposition when overexpressed in cell culture systems. Fur- pressures during the expansion of a cell lineage. A need for more thermore, perfusion of human prostate cancer xenografted in in vivo experimental model systems to query the L1 life cycle, its nude rats with serum collected from human subjects at night, interactions with host factors, and characteristics of insertion site which contains high levels of melatonin, suppressed L1 expres- preferences was stressed. sion in tumor cells (16). In contrast, L1 RNA is expressed when tumors are perfused with serum collected from the same subjects Roles of Retroelements in Neurobiology during the daytime or at night after bright light exposure. Over the past several years, a number of groups have focused on analyses of somatic retrotransposition insertions in neurons, both Retroelement Activity in Cancer in Drosophila (8, 9) and in single-cell analyses of human neurons A direct link between somatic L1 activity and genome instability (10–12). At this point in time, the field is largely in agreement that associated with oncogenesis has been difficult to assess. One case somatic retrotransposition can cause some genetic variation in study in 1992 reported a somatic L1 insertion in the last exon of neuronal populations. However, differences in methodologies, APC as a causal factor in the subject's development of colon cancer both during the genome amplification required for single-cell (3). Despite this observation, the significance of recent reports sequencing and in the bioinformatic analyses, have resulted in demonstrating that L1 insertions are found in many human substantial discrepancies in estimations of how much L1 retro- clinical tumor samples by next-generation sequencing is not yet transposition contributes to somatic variation and, by extension, clear (17–27). Major points for discussion that have emerged in its significance to the biology of mature neurons. Alice Eunjung the L1 field include the prevalence of L1 expression and activity in Lee (Harvard Medical School, Boston, MA) reported that even many cancers and whether L1 expression or retrotransposition infrequent somatic insertions recovered by sampling different affects cancer progression. areas of the brain can inform models of neural development K.H. Burns began the discussion by highlighting an interro- using shared insertion sites as a marker of common cell ancestries gation of ORF1p protein expression by IHC. This revealed tumor- (11). Others are considering roles for retrotransposons in the specific localization to be a common feature of many types of central nervous system (CNS) that are not necessarily tied high-grade malignant cancers (21). She described follow-up stud- to their insertion sites. Specifically, Alysson Renato Muotri ies showing extensive somatic L1 retrotransposition in pancreatic (University of California, San Diego, La Jolla, CA) discussed adenocarcinoma. Somatic L1 insertions occur with varying rates how unchecked L1 activity may be seen in the pathogenesis of in the course of the clonal evolution and metastasis of these certain human neurologic conditions. He described how loss of 0 0 malignancies (22). She also proposed that inherited polymorph- TREX1, a cytosolic 3 -5 DNA exonuclease involved in innate isms in repetitive element insertions may account for a portion of immunity, led to increased L1 activity and decreased survival of heritable cancer risks. Susan Logan (New York University, New human neurons, at least in tissue culture. See the article by York, NY) presented work from her laboratory on germline Richardson and colleagues (2014) for a review of L1 activity in mechanisms suppressing L1 activity. A yeast two-hybrid screen neuronal tissue (13). using androgen receptor (AR) as bait led to the discovery of AR- trapped clone 27 (ART-27; ref. 28), which in subsequent studies Effects of Aging and the Environment was found to interact with unconventional prefoldin RPB5 inter- An additional emerging area of interest is in understanding the actor (URI) to regulate AR target genes (29). She described a extent of somatic retroelement expression and transposition in transcriptional repressor complex comprised of ART-27, URI, and

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KRAB-associated protein 1 (KAP1), the latter of which has been somatic retrotransposition in many human cancers. Models of implicated in L1 regulation. ART-27 conditional knockout cancer initiation and progression presume the involvement of resulted in a loss of germ cells in mouse testes. Whether the loss genomic stresses, and the extent to which the mobilome acts as of germ cells was due to toxicity associated with excessive L1 a contributor to these processes remains an area open for activity will be explored in future studies. Prescott Deininger further study. Arguably, the most imperative question is wheth- (Tulane University, New Orleans, LA) emphasized technical er this activity causes or promotes malignancy. Furthermore, challenges in understanding L1 RNA expression. He stressed the why such a vast heterogeneity of retrotransposition activity is importance of identifying characteristics that distinguish L1 loci as evident both between tumor types and even among tumors of potentially retrotransposition competent ("hot") versus quies- thesametypeisunknown.Whetherthesedifferencescanbe cent. Additional technical challenges discussed include limits to attributed to the inherited complement of active L1 loci or our ability to uniquely map mobile element reads from recently differences in how tumors control L1 expression and activity is inserted L1 sequences and the issue that many active L1 are not not clear. Beyond L1, analysis of other mobile elements, Alu represented in the reference genome. Solving these problems will and SVA, as well as more ancient sequences, has yet to be allow for a deeper understanding of the role of specific L1 loci in thoroughly undertaken in cancer. disease. The session concluded with Peter Park (Harvard Medical There are several needs in the field at the moment. In particular, School, Boston, MA) presenting his group's analysis of sequencing curated databases with robust quality control mechanisms to data generated by The Cancer Genome Atlas (TCGA) consortium catalog these structural variants will be essential for continued suggesting some recurrent somatic L1 integration sites in cancers progress. Standardization of accurate and accessible methods for and the potential impact of L1 insertions on the expression of calling insertions in genomic sequencing data would enable more surrounding gene loci. He underscored a need for standardization samples to be compared and promote future functional explo- among analytic pipelines and new and innovative ways for ration of these sequences. More development in the challenging storing, analyzing, and sharing data, considering that TCGA alone area of single cell L1 mapping, where different laboratories have houses approximately 750 TB of raw sequencing data for their reached different conclusions on the extent of somatic insertions approximately 2,500 sequenced cases. in neuronal development, is especially needed. Experimentally, there is a need for more disease-relevant in vitro and in vivo model Summary systems to interrogate mobile elements in cell-specific contexts, in the germline and CNS, as well as in a variety of normal and Mobile DNAs give rise to interspersed repeats, sequences that malignant tissues. Finally, a better understanding of how host comprise the majority of our genomes. These sequences have been systems restrict L1 and other active retrotransposons, and what historically understudied, both because their significance is results when those cellular systems fail, will provide important unknown and because high copy number repeats can pose exper- perspectives on cancer biology. We expect that these efforts will imental challenges to high-throughput genomic analyses. As increase our knowledge of the biology of mobile DNAs and their enabling technologies are maturing at a fast pace, neither imped- contributions in cancer. iment now seems steadfast, and interest in defining the functional mobilome in health and diseases, such as cancer, has never been greater. Disclosure of Potential Conflicts of Interest fi In particular, the intersection of the L1 eld and cancer biology No potential conflicts of interest were disclosed. has recently been burgeoning with intriguing questions. New sequencing technologies and bioinformatics tools, as well as new Received December 15, 2015; revised April 12, 2016; accepted April 13, 2016; reagents for detecting L1 ORF1p, have revealed L1 expression and published OnlineFirst July 18, 2016.

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OF4 Cancer Res; 76(15) August 1, 2016 Cancer Research

Meeting Report: The Role of the Mobilome in Cancer

Daniel Ardeljan, Martin S. Taylor, Kathleen H. Burns, et al.

Cancer Res Published OnlineFirst July 18, 2016.

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