The small activating RNA AW1-51 induces locus-specific demethylation and CEBPA protein early upregulation Free

Aberrant DNA methylation is a well-known feature of hematological malignancies, frequently leading to tumor suppressor genes silencing. The CCAAT enhancer binding protein alpha (CEBPA) gene, a critical regulator of myeloid differentiation and a known tumor suppressor, is often epigenetically repressed in leukemias by DNA methylation (Hackanson et al., 2007). Small activating RNAs (saRNAs) are a novel class of therapeutic agents capable of upregulating gene expression, although their mechanisms of action remain incompletely understood (Ghanbarian et al., 2021).

Here, we studied the saRNA AW1-51, which targets the CEBPA locus and is currently in clinical trials for advanced liver cancer (ClinicalTrials.gov: NCT02716012; EudraCT 2021-005431-23) (Sarker et al., 2020) and advanced solid tumors (ClinicalTrials.gov: NCT04105335) (Plummer et al., 2025), to provide deeper insight into its molecular mechanism of action. Specifically, we assessed its ability to induce transcriptional activation of CEBPA through epigenetic modulation, since the CEBPA locus is methylation sensitive (Di Ruscio et al., 2013).

Our study was developed using K562 chronic myeloid leukemia cells. Notably, K562 cells do not express detectable CEBPA protein (Perrotti et al., 2002), which provides a suitable system for investigating reactivation mechanisms specifically at the transcriptional level. To complement this model, since CEBPA is also a known tumor suppressor in lung cancer (Halmos et al., 2002), we also used A549 lung adenocarcinoma cells. A549 cells express basal CEBPA protein, as a surrogate system to study post-transcriptional events such as protein dynamics and mRNA stability in a context including CEBPA protein restoration.

Upon AW1-51 transfection, we observed a specific demethylation of the CEBPA locus promoter region and a significant upregulation of CEBPA transcripts in both models. Remarkably, in A549 cells this was accompanied by an early CEBPA protein upregulation that preceded its mRNA upregulation, as detected by western blot and immunofluorescence. The described effects occurred in the absence of global changes in the genome DNA methylation profile. We are currently exploring the mechanisms underlying this early protein induction through AW1-51 pull-down mass spectrometry and mRNA stability assays.

In conclusion, our findings reveal a novel locus-specific demethylation mechanism mediated by AW1-51, coupled with early protein upregulation, providing new insights into saRNA molecular mechanisms. These results may be also relevant for the development of saRNA-based therapies targeting silenced key tumor suppressor genes in hematologic malignancies, such as myeloid leukemias, where their restoration represents a promising therapeutic strategy.