# Cancer Immunotherapy: How to Exploit Transposable Elements?

Sophie Lanciano, Gael Cristofari

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## To cite this version:

Sophie Lanciano, Gael Cristofari. Cancer Immunotherapy: How to Exploit Transposable Elements?. Clinical Chemistry, 2023, 10.1093/clinchem/hvad091. inserm-04180892

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Submitted on 14 Aug 2023

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# Cancer immunotherapy: how to exploit transposable elements?

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Sophie Lanciano* and Gael Cristofari*

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University Cote d'Azur, Inserm, CNRS, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, France

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* Co-corresponding authors. Correspondence should be addressed to:

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Sophie.Lanciano@univ-cotedazur.fr or Gael.Cristofari@univ-cotedazur.fr

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Immunotherapy is one of the most promising advances in the fight against cancer and is beginning to join more conventional treatment approaches such as surgery, radiation therapy or chemotherapy. A set of strategies consists of ensuring that the immune cells recognize tumor-specific antigens (TSAs) exposed at the surface of tumor cells and eliminate the tumor. TSAs can be processed within tumor cells and presented by human leukocyte antigen (HLA) molecules at the cell membrane, where they can be recognized by cytotoxic T lymphocytes. Additionally, the external region of membrane proteins can act as TSAs independently of HLA presentation. They can be recognized by antibodies, including therapeutic monoclonal antibodies, and thus mark tumor cells for elimination by another set of immune cells. Membrane proteins are also potential targets for chimeric antigen receptor (CAR) T-cells, which are patient-derived T-cells engineered ex vivo to recognize a specific antigen in an HLA-independent manner, then expanded and reinjected into the patient. Discovering TSAs with the strongest immunogenicity and specificity is essential for the development of efficient and safe immunotherapies. Moreover, the identification of TSAs that are shared by many tumor types and cases could enable the development of universal and off-the-shelf cancer vaccines or therapies, as opposed to the current state of more personalized but expensive and time-consuming therapies.

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Currently, the prevailing approach to immunotherapy is to target neoantigens resulting from somatic mutations in canonical coding genomic regions, with a preference for recurrent mutations. However, recent findings suggest that the presumed non-coding part of the genome, particularly transposable
element (TE)-derived sequences, may represent a predominant source of highly immunogenic and recurrent TSAs. TEs are repeated sequences that can disperse and replicate through a cut-and-paste or copy-and-paste mechanism and comprise nearly half of the human genome (1). Some of the TEs from the L1, Alu or SVA families are able to replicate in modern humans and are major drivers of genome evolution, active in the germ line or the early embryo but silenced in most somatic tissues. Nevertheless, they are reactivated in many cancers, presumably because of the profound epigenetic remodeling that occurs in tumors, such as the global alteration of DNA methylation. Consequently, cis-regulatory sequences carried over by TEs, particularly their embedded promoters, become derepressed. As TEs are frequently inserted in introns or close to genes and often contain splice sites, TEs thereby reactivated can function as alternative promoters and first exons for neighboring genes, resulting in the synthesis of chimeric transcripts subsequently translated into noncanonical proteins. Of note, this phenomenon not only involves replication-competent TEs, but also fossil sequences that have lost their capacity for mobilization for millions of years.

A first hint that TSAs could originate from TEs came from an unbiased proteomic approach based on the identification of HLA-bound peptides by mass spectrometry in murine cell lines and in a handful of human primary lung tumors or leukemic cells (2). However, a recent study by Ting Wang and colleagues has now considerably extended the breadth of this initial observation (3). They developed a computational tool to systematically identify tumor-specific chimeric transcripts and applied it to more than 10,000 tumor samples across 33 cancer types from The Cancer Genome Atlas (TCGA) datasets, and to 675 cancer cell lines (Figure 1, a). To ensure tumor-specificity, candidate chimeric transcripts were filtered out using normal samples from TCGA, or from other comprehensive catalogues of normal tissue expression such as GTEx or FANTOM. Ultimately, they identified ~2,300 tumor-specific TE-chimeric transcripts for which nearly half were predicted to encode chimeric or out-of-frame proteins. Going further, they examined tumor mass spectrometry data available from the Clinical Proteomic Tumor Analysis Consortium and show that some of the predicted peptides are indeed translated in tumors. They also performed HLA-precipitation mass spectrometry experiments in 4 cancer cell lines,
53 validating between 1 and 4 TE-derived tumor-specific peptides as TSAs with strong predicted affinities
54 for HLA molecules, per cell line (Figure 1, b).

55 With the same idea in mind, Sebastian Amigorena, Joshua Waterfall, Marianne Burbage, and colleagues
56 have recently focused on peptides derived from TE-exons junctions (JETs) as potential TSAs in humans
57 and mouse models (4,5). While their study was focused on a particular type of cancer – non-small cell
58 lung cancer – they too have shown the expression of TE-derived TSAs that are presented by HLA at the
59 surface of primary lung tumors and can be shared by different patients. Notably, they also provided
60 functional evidence of immunogenicity ex vivo, as well as the presence of specific T cells in tumors and
61 lymph-nodes of patients, indicative of an immune response directed against the tumor and mediated
62 by TE-derived TSAs. In mouse models, both prophylactic and therapeutic immunization with TE-derived
63 TSAs could also delay tumor growth and improve survival (5).

64 Interestingly, most analyzed tumors (94%) express at least one such chimeric transcript, independently
65 of the tumor type (3). Some tumor-specific TE-chimeric transcripts are recurrently detected in a wide
66 range of tumors. For example, the *L1P2_STK31* chimeric transcript is present in 15% of the samples
67 across all cancer types (3). Others are found in more than 30% of lung adenocarcinoma or squamous
68 cell carcinoma cases (4). Altogether, combining 20 TE-derived TSAs was predicted to be found in up to
69 two-thirds of the TCGA samples across all cancer types (3), opening the possibility of large-spectrum
70 and even universal pan-cancer vaccines.

71 Finally, in addition to HLA-bound peptides, Ting Wang and colleagues also spotted tumor-specific TE
72 chimeric transcripts encoding membrane proteins that could be targeted by CAR-T cells or monoclonal
73 antibodies (Figure 1, c-d) (3). This finding parallels the recent discovery that some TEs (endogenous
74 retroviruses or ERV) encode envelope glycoproteins exposed at the membrane of tumor cells, which
75 can elicit a B-cell response with antitumoral activity and are particularly important in achieving immune
76 checkpoint blockade (see Supplemental Information).
A key aspect of these efforts is to ensure the tumor specificity of the discovered epitopes. Given the repeated, interspersed – and sometimes polymorphic – nature of TEs, some TE-derived epitopes may originate from multiple loci and be redundant (1), which could complicate detection in healthy tissues. With respect to this issue, the focus on peptides corresponding to TE-exon junctions (4,5) represents a clear benefit, but also narrows the range of possibilities. Recent work showing the expression of some TEs in normal somatic tissues, particularly epithelia from the gastro-intestinal tract (see Supplemental Information), will also help with defining the most tumor-specific antigens to limit toxicity in normal tissues. Different classes and many families of TEs were found capable to drive TSA synthesis. Despite their repeated nature, individual copies have acquired unique reactivation capacities in tumors, with modalities depending on the cancer type. In mouse models, ablation of Setdb1, a histone 3-lysine 9 (H3K9me3) methyltransferase which controls heterochromatin formation, broadens the spectrum of immunogenic TE-exon junctions expressed in tumor cells, suggesting that carefully designed epigenetic therapies could be combined with TE-based immunotherapies (Figure 1, g) (5). Therefore, a deeper understanding of the mechanisms behind the silencing of individual TE loci is essential to fully deliver the promises of TE-based biomarkers and immunotherapies, while maintaining a high-level of specificity and safety. Dedicated approaches are emerging to identify expressed TE loci, to explore the full diversity of TE transcripts (unit-length TE transcripts, chimeric gene-TE transcripts, or pervasive transcription), and to study their regulation (1).

Beyond providing an unsuspected reservoir of recurrent and immunogenic tumor-specific antigens, with multiple applications towards the development of specific immunotherapies (Figure 1, h; universal cancer vaccines, new CAR-T cells, response to immune checkpoint blockade), TE expression in tumors can also trigger a viral mimicry state mediated by innate immune responses through double-stranded RNA and cytosolic DNA sensing (see Supplemental Information, and Figure 1, e-f). Thus, altogether, TE activity in tumors is becoming a promising and actionable Trojan horse for antitumoral therapies, acting both through innate and acquired immunity. Combining expertise in cancer biology,
102 immunology, genomics, and bioinformatics will be pivotal to understand the role of these elements in
cancer and fully uncover how they can be leveraged to create new vulnerabilities in tumors.
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104 ACKNOWLEDGMENTS

105 This work was supported by the Agence Nationale de la Recherche (Labex SIGNALIFE, ANR-11-LABX-0028; Idex UCAJEDI, ANR-15-IDEX-01; ImpacTE, ANR-19-CE12-0032; ActiveLINE, ANR-21-CE12-0001),
106 the Institut National du Cancer (INCa PLBIO 2020-095), the Canceropôle PACA, INCa and the Région
107 Sud (Projet Emergence), Inserm (GOLD cross-cutting programme on genomic variability), CNRS (GDR
108 3546). This work was funded, in whole or in part, by the Agence Nationale de la Recherche, Grant
109 ANR-21-CE12-0001. A CC-BY public copyright license has been applied by the authors to the present
110 document and will be applied to all subsequent versions up to the Author Accepted Manuscript
111 arising from this submission, in accordance with the grant's open access conditions.
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113 REFERENCES

1. Lanciano S, Cristofari G. Measuring and interpreting transposable element expression. Nat Rev Genet 2020;21:721-36

115 2. Laumont CM, Vincent K, Hesnard L, Audemard É, Bonneil É, Laverdure J-P, et al. Noncoding regions are the main source of targetable tumor-specific antigens. Sci Transl Med 2018;10:eaau5516

118 3. Shah NM, Jang HJ, Liang Y, Maeng JH, Tzeng S-C, Wu A, et al. Pan-cancer analysis identifies tumor-specific antigens derived from transposable elements. Nat Genet 2023;55:631-9

120 4. Merlotti A, Sadacca B, Arribas YA, Ngoma M, Burbage M, Goudot C, et al. Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer. Sci Immunol 2023;8:eabm6359

123 5. Burbage M, Rocañín-Arjó A, Baudon B, Arribas YA, Merlotti A, Rookhuizen DC, et al. Epigenetically controlled tumor antigens derived from splice junctions between exons and transposable elements. Sci Immunol 2023;8:eabm6360

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<figure><img src="image_3.png" /><figcaption>**Figure 1.** Transposable elements can be harnessed for anticancer immunotherapy through different pathways, which encompass both acquired and innate immunity. (a) A recent study has achieved a pan-cancer screen for tumor-specific antigens (TSAs) derived from transposable element (TE, light brown) chimeric transcripts (3). TE-derived transcripts (including TE chimeric transcripts and unit-length TE transcripts) can code for: (b) tumor-specific antigens (TSAs) either bound by HLAs and recognized by cytotoxic T-cells, or (c-d) directly exposed at the cell surface as membrane proteins. The latter can be targeted by CAR-T cells (c) or marked by</figcaption></figure>

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antibodies for subsequent elimination by the immune system (d). In addition, TE-derived nucleic acids can
induce a viral mimicry state. TE-derived single-stranded DNA (ssDNA) molecules are produced by endogenous
reverse transcriptases (RT), presumably encoded by LINE-1 retrotransposons (e). Such ssDNA species can
accumulate in the cytosol and activate the cGAS-STING pathway (not shown). Moreover, double-stranded RNA
(dsRNA) species, predominantly formed by inverted TEs (i.e. Alu retrotransposons) inserted in intronic regions
can activate RNA sensors such as MDA5 or RIG-I (f). Epigenetic therapies could reverse TE silencing, thereby
promoting TE expression and TE-mediated immunotherapy efficacy (g) (4,5). In the near future, TE-derived TSAs
could serve for the development of specific antitumoral immunotherapies such as pan-cancer vaccines, new
CAR-T cells, or monoclonal antibodies (h).