High resolution evaluation of the N6-methyladenosine RNA modification pathway as a driver of tumor proliferation and drug resistance

The methylation of RNA adenosines into N6-methyladenosine (m6A) is the most abundant internal mRNA modification in mammalian cells. It is regulated by writers (e.g., methyltransferase-like 3 (METTL3)) and erasers, while readers mediate its molecular effects at the level of different biological processes (e.g., RNA translation and decay). Consequently, m6A affects multiple cellular functions like cell proliferation. Indeed, downregulation of METTL3 significantly reduces CRC cell proliferation, and CRISPR- knock-out (KO) screenings identified m6A effectors (writers, erasers and readers) as essential in acute myeloid leukaemia (AML). This opens new questions regarding the mechanisms by which these effectors contribute to the function of m6A in cancer, which may be investigated with CRISPR gene editing approach. In particular, CRISPR-cytosine base editing (CBE) and -KO can create single nucleotide changes (C>T) and deletions of the targeted genes respectively. The aim of this project is to study m6A in cancer cell proliferation by means of a pooled CRISPR screening. We will develop a CRISPR-base editing and KO platform for the high-density mutagenesis of the genes that are active in the m6A-pathway and enrichment analysis of the sgRNAs will determine the association between specific single-nucleotide mutations, and gene deletions, and the proliferation phenotype. In details, we prepared an in-silico sgRNA library targeting m6A and control genes (essential and non-essential genes, non-targeting and intergenic sequences). We applied a tiling strategy (i.e., multiple sgRNAs targeting different gene positions) to study the targeted sequences in HT-29 (CRC) and AML (MOLM-13, NOMO-1) cell lines. We are working on the generation of cell lines stably expressing CBE or Cas9, which will be then infected with the lentiviral sgRNA library. Upon puromycin selection, the following proliferation screening will result in the NGS of sgRNAs at T1 vs T0. The identification of the significantly depleted sgRNAs by data analysis highlights domains associated with proliferation phenotype, and that might represent druggable sites; this dataset may be useful for assessing the function of variants of uncertain significance (VUS). The study will investigate the role of m6A writers, erasers and readers in CRC and AML proliferation and expect to provide molecular insights for the development of novel drug treatments.