Pharmacological Inhibition of the RNA m6a Writer METTL3 As a Novel Therapeutic Strategy for Acute Myeloid Leukemia
Authors: Tzelepis Konstantinos , De Braekeleer Etienne , Yankova Eliza , Rak Justyna , Aspris Demetrios , Domingues Ana Filipa , Fosbeary Richard , Hendrick Alan , Leggate Dan , Ofir-Rosenfeld Yaara , Sapetschnig Alexandra , Pina Cristina , Albertella Mark , Blackaby Wesley , Rausch Oliver , Vassiliou George S. , Kouzarides Tony 
Published: February 2020

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. New evidence has revealed an important role for RNA modifications in cancer development and maintenance via the catalytic function of RNA-modifying enzymes. We and others have recently shown that METTL3, the RNA methyltransferase responsible for the deposition of N-6-methyl groups on adenosine (m6A) in mRNA, is a promising therapeutic target for AML1,2. Here we present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3 as an effective therapeutic strategy in AML.
Recently, we generated a comprehensive catalogue of RNA-modifying enzymes that are essential for AML cells using CRISPR-Cas9 recessive screens and characterised METTL3 as a novel therapeutic candidate through its effects on mRNA translational efficiency of key leukemia oncogenes1. Using a structure-guided medicinal chemistry platform we developed and optimised small molecule inhibitors of METTL3 from 2 distinct chemical series. Here we demonstrate that compounds 1 and 2 show biochemical inhibition of METTL3 enzyme with single digit nanomolar potency, while direct binding to METTL3 was confirmed by Surface Plasmon Resonance (SPR) analysis with comparable potency between compounds. Additionally, we developed compound 3 as an inactive analog which was confirmed inactive in enzyme assays (&gt;50 µM IC50). Importantly, we verified that compounds 1 and 2 are selective for METTL3 and do not inhibit a panel of other RNA, DNA or protein methyltransferases tested (&gt;10 µM IC50).
Cellular target engagement was confirmed by demonstrating that compounds 1 and 2 reduced m6A levels and inhibited the protein expression of METTL3-dependent m6A substrates in mouse and human AML models, including SP1, with nanomolar potency. Furthermore, treatment of MOLM13 cells with compounds 1 and 2 inhibited their proliferation with comparable potency to SP1 inhibition. The same anti-proliferative effect was observed using a large panel of human AML cell lines. In addition, polyribosome profiling in MOLM13 cells treated with compounds 1 and 2 revealed enhanced blocking of mRNA translation, mirroring the effects derived from the genetic inhibition of METTL3. Notably, all of the above effects were not observed when the inactive analog (compound 3) was used, further highlighting the specificity and sensitivity of our active candidates.
We subsequently performed in vivo characterisation of compound 1. This compound exhibited excellent bioavailability after oral or intraperitoneal administration with good dose-proportional exposure in mice and a half-life of 3.5 hours. It also appeared to be well-tolerated with no body weight loss or clinical signs of toxicity. We also evaluated its anti-tumor effects in patient derived xenotransplantation experiments (PDX) as well as transplantation experiments using an MLL-AF9 driven primary murine AML model. Daily dosing of 30 mg/kg significantly inhibited AML expansion and reduced 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 the reduction of METTL3-dependent m6A targets. Importantly, we went on to demonstrate that, while the pharmacological inhibition of METTL3 is required for AML cell survival, it was dispensable for normal hematopoiesis.
Collectively, we describe the detailed characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrate their activity and utility using biochemical, cellular and in vivo systems. We show that inhibition of METTL3 by small molecules in vivo leads to strong anti-tumor effects in physiologically and clinically relevant models of AML. To our knowledge, this is the first study demonstrating in vivo activity of inhibitors of an RNA methyltransferase, hence providing proof of concept that RNA modifying enzymes represent a new target class for anti-cancer therapeutics.
References
Barbieri, I. et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature552, 126-131, doi:10.1038/nature24678 (2017).
Vu, L. P. et al. The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med23, 1369-1376, doi:10.1038/nm.4416 (2017).

Disclosures
Yankova: STORM THERAPEUTICS: Employment. Fosbeary:STORM THERAPEUTICS: Employment. Hendrick:STORM THERAPEUTICS: Employment. Leggate:STORM THERAPEUTICS: Employment. Ofir-Rosenfeld:STORM THERAPEUTICS: Employment. Sapetschnig:STORM THERAPEUTICS: Employment. Albertella:STORM THERAPEUTICS: Employment. Blackaby:STORM THERAPEUTICS: Employment. Rausch:STORM THERAPEUTICS: Employment. Vassiliou:Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding. Kouzarides:STORM THERAPEUTICS: Equity Ownership.