METTL3 inhibition disrupts R-loop homeostasis and activates the cgas-sting pathway in Acute Myeloid Leukemia Free

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. Heterogeneity within its drivers and low cure rate especially in the aging population underscore the need for novel therapeutics. The N6-methyladenosine (m6A) RNA writer Methyltransferase 3 (METTL3) has been reported to be overexpressed in AML and knockdown or inhibition of METTL3 results in leukemic blast differentiation and improved survival in in vivo mouse models. The fate of m6A-modified RNA is determined by recognition by several readers of the YT521-B homology (YTH) and Insulin-like Growth Factor 2 mRNA-Binding Protein (IGF2BP) binding protein families.

STM3675 (STORM Therapeutics) is a highly specific and potent inhibitor of METTL3 (Mayday et al., Blood 2025) with an IC50 of 0.289 µM in MOLM13 AML cells. METTL3 inhibition (METTL3i) induced differential gene expression with 3162 up-regulated and 1970 down-regulated genes in MOLM13 cells. Gene Set Enrichment Analysis (GSEA) of the down-regulated genes revealed enrichment in transcripts related to DNA damage repair and cell cycle, leading us to hypothesize that METTL3 inhibition could lead to accumulation of DNA damage. We observed an increase in DNA double-strand breaks (DSBs) as indicated by an increase in γH2AX foci detected by immunofluorescence.

m6A RNA modification has been shown to play an important role in resolution of R-loops, three-stranded nucleic acid structures containing a DNA-RNA hybrid and a displaced strand of DNA, which form during transcription. Accumulation of R-loops can lead to loss of genome integrity by creating ssDNA regions vulnerable to mutation and by collisions during DNA replication leading to DSBs. To determine whether METTL3i induces R-loops, we treated MOLM13 cells with STM3675 over a 48-hour time course and examined R-loop dynamics. Using the S9.6 antibody, which specifically detects DNA-RNA hybrids, in a dot blot assay, we found that hybrids accumulated to a maximum level at 24 hours of METTL3i. To confirm that R-loop accumulation induced DSBs, we performed the Proximity Ligation Assay (PLA) against S9.6 and γH2AX confirming R-loop-proximal DNA damage foci in response to METTL3i. To determine whether R-loop formation contributes to METTL3i-induced killing of leukemia cells, we overexpressed the RNAaseH1 nuclease, which specifically degrades R-loops, or a catalytically inactive form. Expression of wild type RNAseH1 (but not the catalytically dead form) rescued MOLM13 cell survival in response to METTL3i. To identify specific genomic loci affected by R-loop accumulation and DSB induction, we are currently performing DNA-RNA Immunoprecipitation (DRIP) and CUT&Tag using the S9.6 and an antibody recognizing the DNA repair factor RAD51, respectively.

To further validate R-loop accumulation in METTL3i-treated cells, we utilized expression of a catalytically dead form of RNAseH1 conjugated with GFP to reveal DNA-RNA hybrids. Using this tool, we observed the presence of cytoplasmic R-loops that were lost upon inhibition of the nuclear export factor Crm1 (Exportin-1), suggesting that cytoplasmic DNA-RNA hybrids are actively transported to the cytoplasm. We are currently in the process of identifying the presence and specific sequences of cytoplasmic hybrids using the Cyto-DRIP assay. Cytoplasmic DNA can activate a critical innate immune response via the cGAS-STING pathway. We have previously shown that METTL3 deletion and inhibition result in aberrant endogenous dsRNA formation and activation of an antiviral innate immune response. GSEA of our RNA-seq data confirms activation of an innate immune pathway. In ongoing studies, we are now dissecting the specific contributions of a dsRNA versus a cytoplasmic DNA-RNA hybrid induced innate immune response. Our studies will allow full understanding of METTL3 multifaceted contributions to cellular processes, and guidance of treatment of leukemia and cancer with METTL3 inhibitors.