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Small activating RNA AW1-51 (CEBPA-51) elicits targeted DNA demethylation to promote gene activation [RNA-seq]

ABSTRACT: Small activating RNAs (saRNAs), are short double-stranded RNAs designed to upregulate transcription of target genes. By this virtue, they can be used to restore expression of genes frequently silenced in cancer. AW1-51 (also referred to as CEBPA-51), the first saRNA therapeutic to enter clinical evaluation, has demonstrated biological activity and safety in Phase II trials for hepatocellular carcinoma, both as monotherapy and in combination with sorafenib, and in Phase 1a/1b in combination with pembrolizumab for patients with advanced solid tumors. It targets the master regulator CCAAT enhancer-binding protein alpha (CEBPA), abnormally silenced by DNA methylation in a wide range of hematological and not hematological malignancies. However, the molecular events enabling this mechanism are only partially elucidated. In this study, we uncovered the molecular basis for AW1-51-induced transcriptional reactivation of CEBPA demonstrating that by directly promoting DNA demethylation of CEBPA promoter restores CEBPA expression, protein synthesis, and consequently cell differentiation. These findings unveil AW1-51 as a prototype for RNA-based precision medicine enabling conditional expression of CEBPA in diseases characterized by aberrant gene silencing and extending its potential therapeutic impact beyond cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE305099 | GEO | 2026/08/01

REPOSITORIES: GEO

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Project description:Human leukocyte antigen (HLA) - B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are two strong predisposing genetic factors to Behçet's disease (BD). Previous studies have focused on two subgroups of HLA-B*51 peptidome containing Proline (Pro) or Alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel sub-peptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by Mass Spectrometry. The characteristics of non-Pro/Ala2, Pro2 and Ala2 peptides, and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Proline or Alanine at P2. This unconventional group of HLA-B*51:01-bound peptides was enriched for 8-mers (with fewer 9-mers) compared to Pro2 and Ala2 sub-peptidomes, and had similar N-terminal and C-terminal residue usages to Ala2 peptides with the exception of the less abundant Leucine at position Ω. Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 to approximately 40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of Leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell type - dependent manner. The HLA-B*51:01 peptidome includes a surprisingly high proportion of unconventional non-Pro/Ala2 peptides which are increased by ERAP1 silencing, mimicking the loss-of-function BD risk variant.

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Small activating RNA AW1-51 (CEBPA-51) elicits targeted DNA demethylation to promote gene activation [RNA-seq]

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