Abstract B126: A small molecule inhibitor of the RNA m6A writer METTL3 inhibits the development of acute myeloid leukemia (AML) in vivo
Authors: Albertella Mark , Blackaby Wesley , Fosbeary Richard , Hendrick Alan , Leggate Dan , Ofir-Rosenfeld Yaara , Sapetschnig Alexandra , Tzelepis Konstantinos , Yankova Eliza , Kouzarides Tony , Rausch Oliver 
Published: March 2020

Abstract
METTL3 is an RNA methyltransferase which is responsible for the deposition of N-6-methyladenosine (m6A) on mRNA targets such as SP1, to modulate their stability and expression. METTL3 was identified as an essential gene for the growth of AML cells and proposed as a novel target for cancer therapy (Barbieri 2017). We present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3, which recapitulate the genetic validation of METTL3 as a novel cancer target using a pharmacological audit trail. Small molecule inhibitors from 2 distinct chemical series were identified and optimised using a structure-guided medicinal chemistry platform. Compounds 1 and 2 are from different series, and both showed biochemical inhibition of METTL3 enzyme with single digit nanomolar potency. Direct binding to METTL3 was confirmed by SPR with comparable potency. Compound 3 is an inactive analog which was confirmed inactive in the enzyme assay (IC50 &gt;50microM). Compounds 1-2 are selective for METTL3 and did not inhibit a panel of other RNA, DNA or protein methyltransferases tested (&gt;10microM IC50). Cellular target engagement was confirmed by the demonstration that compounds 1 and 2 inhibited SP1 and Brd4 protein expression with submicromolar potency, whereas the inactive analog compound 3 had no effect. Compounds 1 and 2 treatment of MOLM13 cells inhibited their proliferation which correlated with SP1 inhibition, and compound 3 had no effect, demonstrating that their activity was METTL3-dependent. Compound 1 has excellent oral bioavailability with good dose-proportional exposure in mice and a half-life of 3.5 hrs, and was well-tolerated with no body weight loss or clinical signs. Compound 1 was evaluated for anti-tumor effects in an MLL-AF9 driven primary murine AML model. 30 mg/kg daily oral dosing of compound 1 gave a significant reduction in AML expansion and a reduction in spleen weight compared to vehicle control, indicating a pronounced anti-tumor effect in vivo. Target engagement was confirmed in bone marrow and spleen as measured by reduction of METTL3-dependent m6A targets. We have described the comprehensive characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrated their activity and utility using biochemical, cellular and in vivo systems. We have demonstrated that inhibition of METTL3 by small molecules in vivo leads to a pronounced anti-tumor effect in a physiologically relevant model of acute myeloid leukemia. To our knowledge, this is the first demonstration of in vivo activity of inhibitors of an RNA methyltransferase and providing proof of concept that RNA modifying enzymes are a new target class for the development of novel cancer therapeutics.
Citation Format: Mark Albertella, Wesley Blackaby, Richard Fosbeary, Alan Hendrick, Dan Leggate, Yaara Ofir-Rosenfeld, Alexandra Sapetschnig, Konstantinos Tzelepis, Eliza Yankova, Tony Kouzarides, Oliver Rausch. A small molecule inhibitor of the RNA m6A writer METTL3 inhibits the development of acute myeloid leukemia (AML) in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B126. doi:10.1158/1535-7163.TARG-19-B126