Identificação de novas vulnerabilidades das células tumorais através do código m6A do RNA

In DeCode, our central aim is to fully unlock the relevance of m6A modifications and its molecular effectors in GC to reveal new biomarkers and therapeutic options that may be used to treat this disease. The current proposal will leverage on our extensive prior characterization of the m6A machinery and its function in GC (Preliminary data, manuscript submitted for publication) to take the leap from global m6A modifications to the role of single, site-specific ones in relevant transcripts in GC, while filling the gap between the in vitro and the clinical contexts. Our central hypothesis is that the m6A modification in RNA plays central and multifunctional roles in GC progression, metastasis and drug resistance and that targeting it provides novel therapeutic opportunities. We have recently demonstrated that YTHDF3, a protein that binds to the most prevalent modification of RNA - N6-methyladenosine (m6A), is highly expressed in GC tumors and cell lines and is associated with poor patient survival (Figure 1). To study the function of YTHDF3 in GC, we used CRISPR-Cas9 technology to generate YTHDF3 knockout (KO) cell lines that resulted in a conspicuous phenotype with disturbed cell morphology, with disorganized actin cytoskeleton, decreased cell motility in vitro and decreased metastization in vivo (Figure 2). These results indicated that m6A modifications impact bona fide cancer cell phenotypes in GC. To track down the molecular and signaling events involved, we performed RNA-seq that indicated significant gene expression alterations between YTHDF3 KO and wild-type cells. Gene ontology analysis revealed that among the top ten pathways with most significant alterations were motility and migration pathways, which was consistent with the phenotype observed. Moreover, we have identified Ezrin (EZR) as a highly downregulated transcript and protein in the KO cell lines (Figure 3 and 4). EZR is part of the ezrin/radixin/moesin (ERM) family of proteins that link the actin cytoskeleton to the cellular membrane, participating in various cellular processes such as signal transduction, cell proliferation, cell adhesion, membrane projections and cell motility, among others (Arpin et al,2011). The role of EZR in cancer cell migration and metastization is well established in multiple cancer types; pharmacological inhibition of EZR is possible and has already been tested in clinical trials (Buenaventura et al,2023). However, that EZR expression and function could be modulated by chemical modifications of its transcript is completely new and opens unexplored paths towards cancer treatment. We confirmed, using RNA immunoprecipitation with an anti-m6A antibody, that EZR transcript is methylated (Figure 3) and we further showed that impaired EZR led to mitotic spindle misorientation as a consequence of the cytoskeleton disorganization (Kschonsak et al,2018). This is an unexplored EZR function, which may have important clinical applications, since it led to increased sensitivity to paclitaxel, a chemotherapeutic drug that targets the mitotic spindle (Weaver,2014) a link that we were the first to reveal (Figure 4). In this proposal, to unlock the full potential of these findings, while uncovering new functions and m6A targets in GC, we will: 1. Uncover the role of site-specific m6A modifications in GC (Task1 and Task2). 2. Assess the role of m6A targets in GC using organoids (Task3 and Task4). 3. Profile the m6A signatures in GC clinical samples (Task5).