AbstractImmunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.