Identification and functional characterisation of M6A71: a novel YTH inhibitor targeting YTHDF2 in Acute Myeloid Leukaemia models

Acute myeloid leukaemia (AML) is a rapidly evolving blood tumour characterised by the uncontrolled proliferation of myeloid haematopoietic precursor cells. Current therapeutic interventions are predominantly focused on intensive chemotherapy and allogeneic bone marrow transplantation. However, it is important to note that not all patients diagnosed with AML are eligible for these treatments. Recent studies have demonstrated a positive correlation between the development of AML and the dysregulation of the YT-521-b Homology Domain Family protein 2 (YTHDF2). YTHDF2 is an RNA-binding protein that recognises N6-methyladenosine (m6A) modifications on mRNA molecules. m6A is the most abundant epitranscriptomic modification in eukaryotic cells, and new frontiers in drug discovery are targeting proteins and RNAs involved in its regulatory machinery. Indeed, m6A is a key regulator of cell biology and plays pivotal roles in several pathologies, including cancer. To identify new small molecules that interfere with YTHDF2 activity for potential AML treatment, I performed in silico molecular docking on 1.5 million small molecules. I identified M6A71 as a novel YTH inhibitor through Homogeneous Time-Resolved Fluorescence (HTRF) high-throughput screening of the recombinant YTHDF2 domain. I evaluated the biological effects of M6A71 on YTHDF2 in acute myeloid leukaemia (AML) cell lines, in which it plays a central role in regulating disease progression and cell proliferation. M6A71 showed significant cytotoxic effects across the tested cell lines, resulting in a marked decrease in overall cell viability, reduced proliferation, and induction of apoptosis. Transcriptome-wide analysis using the enhanced Crosslinking Immunoprecipitation (eCLIP) assay in the THP-1 AML cell line revealed that YTHDF2 specificity was partially lost after treatment with M6A71, favouring AU-rich DRACH-containing sequences. This evidence supports the conclusion that M6A71 acts at the m6A-binding site, thereby modifying its reading activity. Moreover, comparative transcriptomic and proteomic analyses revealed a significant association between enriched transcripts and upregulated proteins following M6A71 treatment. The collective evidence suggests that M6A71 exerts its effect by directly binding to the YTH-pocket, thereby modulating the YTHDF2 recognition motif, altering its specificity, and inducing apoptosis while reducing cell proliferation in the AML cell model.