N6-methyladenosine (m6A) is a common modification of mRNA, with potential roles in fine-tuning the RNA life-cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them, WTAP, METTL14 and KIAA1429, are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps, and their classification into WTAP-dependent and independent sites. WTAP-dependent sites are located at internal positions in transcripts, are topologically static across a variety of systems we surveyed, and are inversely correlated with mRNA stability, consistent with a role in establishing ‘basal’ degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure, and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data sheds new light on proteomic and transcriptional underpinnings of this epitranscriptomic modification.

Although the roles of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here we show that the IKK-related kinases TBK1 and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and IL-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 and MEK inhibitor therapy induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, JAK, and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS.