Epigenetic Drug Treatment Induces Presentation of New Class of Non-Exonic, Cryptic Neoantigens in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a 5-year overall survival rate of less than 30% which causes over 10,000 deaths per year in the United States. Treatment options for this disease increasingly include epigenetic drugs, such as hypomethylating agents (e.g. decitabine) or histone deacetylase (HDAC) inhibitors (e.g. pracinostat) which can function via direct cytotoxic mechanisms and also through altered differentiation of AML blasts; immunomodulatory effects like reactivation and presentation of cancer testis antigens in context of human leukocyte antigen (HLA) complexes have been reported as well, which may result in clearance of cells via the adaptive immune system. However, the landscape of immunogenic T cell epitopes induced by these drugs might be even broader than reported since standard analyses only consider exonic protein sequences and do not take into account typically untranslated genomic regions.

Recently, it has been shown that single and combination treatment of decitabine and pracinostat can induce cryptic transcription start sites in generally epigenetically repressed solitary long-terminal repeats (LTRs) of the LTR12C family which give rise to novel mRNAs and resulting protein variants. We hypothesized that the intronic parts of these gene products might provide a source of cryptic T cell epitopes with high immunogenic potential, which are induced through epigenetic drug treatment.

To test this hypothesis, we treated 5 different AML cell lines (HL-60, U937, OCI-AML02, MOLM13, AML14) with (1) DMSO, (2) 500 nM decitabine or (3) a combination of 500 nM decitabine and 100 nM pracinostat for 72 hours to induce transcription of non-annotated transcription start sites. Subsequently, HLA class I complexes were immunopurified and peptides presented by these complexes isolated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The activation of silenced genes by epigenetic drug treatment with either decitabine alone or the combination treatment yielded increases of about two-fold in the identified unique HLA ligands. This increase in peptide identifications also led to improved detection of cancer testis antigen-derived epitopes, as has been reported before. Intriguingly, by adding LTR12C derived sequences stretching from the published GATA2 specific binding site until the next genomic exon to the peptide search analyses we were able to identify several cryptic peptides from 4 out of 5 AML cell lines derived from these usually untranscribed genomic regions. The identifications were exclusively dependent on previous treatment with either decitabine alone or in combination with pracinostat. Though the immunogenicity of these HLA ligands has not been determined yet, we assume that due to their genetically repressed state in untreated cells, these new peptide sequences represent a new class of neoepitopes, with potential to be novel targets of existing T cells within patients or after augmentation by other immunotherapies.

In summary, we demonstrated for the first time the induced presentation of epitopes from normally untranscribed LTR12C regions through epigenetic drug treatment and therefore provide a previously undescribed source of potential targets for immunotherapy in AML.