Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity [QuantSeq 3' mRNA-Seq]

Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl­transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti–PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti–PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole­cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. Overall design: CaOV3 cells were treated with 500 nM STM3006 or 0.3% DMSO in triplicate for 48 hours. For total RNA extraction, cells were homogenized in 700 µl Trizol (Thermo Fisher Scientific) followed by Chloroform addition and centrifugation. The aqueous phase was transferred to new tubes and RNA was precipitated using equal volumes of 100% Isopropanol followed by 2 washes with 80% Ethanol. RNA pellets were air-dried and resuspended in 25 µl nuclease-free water. RNA concentration was determined by Qubit RNA broad-range assay kit (Thermo Fisher Scientific, Q10211). QuantSeq 3' mRNA-Seq Library preparation was performed by Lexogen GmbH (Vienna, Austria) according to manufacturer's instructions. Sequencing was performed using the Illumina NextSeq single-end 75 bp High Output V2 chemistry.