Deciphering functions of the ATR-METTL3-BRCA1 Axis in genome instability and Tumorigenesis

PROJECT SUMMARY /ABSTRACT A defect in DNA damage repair triggers genome instability as a hallmark of cancer that is exploited by treatments such as ionizing radiation, platinum chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPi). Pathogenic germline and somatic variants in the DNA repair gene BRCA1 are frequently detected in triple negative breast cancer (TNBC) and BRCA1-mutated TNBC can be targeted with PARPi to achieve a clinical response. Elucidating the mechanism(s) by which BRCA1 and its associated partners resolve DNA lesions will provide new insights into how genome instability promotes cellular transformation and identify new therapeutic targets. In this proposal, we will investigate functions of the ATR-METTL3-BRCA1 axis in R-loop associated DNA damage to sustain stable genome. Persistent and unscheduled R-loops create structural barriers when they collide with collapsed replication forks or double strand breaks (DSB) to elicit genome instability. Our preliminary data found that METTL3, a protein promoting RNA m6A modification when R-loops form during transcription, is recruited to repair DNA lesions at active transcription sites. METTL3 is phosphorylated by ATR, a key kinase in R-loop associated DNA damage signaling, and this phosphorylation promotes association with BRCA1. We found that BRCA1 is both necessary and sufficient for METTL3 recruitment to DNA damage sites. We identified several breast cancer associated METTL3 mutations and found that two catalytically inactive mutations impede HR repair by abolishing the association with BRCA1. Our proteomic analysis has identified a DNA/RNA helicase that resolves R-loops as the effector of the ATR-METTL3-BRCA1 axis. Furthermore, inhibition of METTL3 catalytic activity with a small molecule induces a defect in homologous recombination and enhances PARPi sensitivity in TNBC cell lines. We hypothesize that the ATR-METTL3-BRCA1 axis is critical for the R-loop- associated DNA damage repair and breast cancer development and offers a new therapeutic target for TNBC. We will test this hypothesis in the following specific aims. In Aim 1, we will elucidate mechanisms by which METTL3 Serine 43 phosphorylation promotes DNA/RNA helicase recruitment to resolve R-loop associated DNA damage. In Aim 2, we will determine the roles of the ATR-METTL3-BRCA1 axis in breast cancer development with mouse models. In Aim 3, we will target the ATR-METTL3-BRCA1 axis with a METTL3 inhibitor in TNBC. Our studies will unravel a novel function for METTL3 in DNA repair and identify METTL3 as a promising therapeutic target in TNBC.