3Derepression of human endogenous retroviruses in ccRCC: implications for immunotherapy

Abstract Background Clear cell renal cell carcinoma (ccRCC) often responds to immune checkpoint inhibitors (ICIs) despite having a relatively low burden of non-synonymous mutations, distinguishing it from other cancers like melanoma or mismatch repair-deficient colorectal cancer that are typically responsive to ICIs. ccRCC has historically been considered an immunogenic tumor due to characteristics such as spontaneous remissions, response to high-dose interleukin-2 (IL-2), and abundant T-cell infiltration. However, paradoxically, high T-cell infiltration in ccRCC correlates with poor prognosis and inconsistent ICI responses. Over 70% of ccRCCs harbor inactivated pVHL tumor suppressor, which leads to the stabilization of HIF2α, a transcription factor critical for tumor growth. Recent studies suggest that HIF2α regulates the expression of endogenous retroviruses (ERVs)—ancient viral elements embedded in the human genome—whose expression is typically suppressed by DNA methylation. Notably, ccRCC has been found to express high levels of ERVs, and this correlates with better responses to ICIs. One specific ERV, ERVE-4, has been shown to produce an HLA-bound peptide recognized by donor-derived T cells in a patient who achieved complete remission after allogeneic stem cell transplantation (allo-SCT). Since ERVE-4 is transcriptionally activated by HIF2α and expressed in ccRCC but not normal tissues, it, along with other HIF2-regulated ERVs, could serve as potential tumor-specific antigens. This raises the possibility of targeting ERV-derived peptides as a novel immunotherapy strategy for ccRCC. Methods To assess whether ERVs in ccRCC are transcribed, translated, and presented by HLA complexes, a multi-omics approach was employed. RNA-seq, ChIP-seq, PRO-seq, single-cell RNA-seq and ­single-cell ATAC-seq were used to profile ERV transcription and regulatory mechanisms. Polysome-seq was conducted to identify ERV transcripts actively undergoing translation, while HLA immunoprecipitation followed by mass spectrometry (HLA-IP/MS) was employed to detect HLA-bound ERV-derived peptides. The immunogenicity of these peptides was evaluated using IFNγ ELISpot assays in both humanized mouse models and primary immune cells derived from ccRCC patients. Results RNA-seq and RT-qPCR analyses identified several HIF2α-responsive ERVs in ccRCC cell lines, such as ERVE-4, ERV3.2, and ERV4700, which were upregulated by active HIF2α and downregulated upon its inhibition. Additionally, treatment with the DNA methyltransferase inhibitor GSK3685032 induced the expression of ERV4700, suggesting that epigenetic mechanisms also regulate ERV activation. Polysome-seq confirmed that these ERVs, including ERVE-4 and ERV3.2, were actively translated, and HLA-IP/MS identified 22 ERV-derived peptides presented by HLA complexes, including several that were tumor-specific. Single-cell RNA-seq and scATAC-seq further demonstrated that these ERVs were primarily expressed in tumor cells, indicating their specificity to ccRCC. To assess the immunogenicity of these ERV-derived peptides, humanized HLA-A∗11:01 mice were immunized with a mix of these peptides, and several of the predicted strong binders elicited T-cell responses, confirming their potential immunogenicity. Next, we incubated these peptides with immune cells from patients who had responded to immunotherapies. Peripheral blood mononuclear cells (PBMCs) from kidney cancer patients revealed reactivity to multiple ERV-derived peptides both before and after allo-SCT. The T-cell response increased in complete responders (CRs) post-transplant, further supporting the idea that ERV-derived peptides can trigger immune responses. Additionally, the T-Scan platform identified ERV-derived peptides recognized by T cells from patients with tumors harboring VHL mutations, reinforcing the hypothesis that HIF2-responsive, ERV-derived peptides presented on ccRCC cells are immunogenic. Conclusions This study uncovers a novel mechanism by which HIF2α enhances the immunogenicity of ccRCC through the transcriptional activation of ERVs. It provides compelling evidence that ERV-derived peptides can be translated, presented by HLA complexes, and recognized by T cells, positioning them as promising tumor-specific antigens. These findings support the potential of targeting ERVs for the development of next-generation immunotherapies in ccRCC.