The S100A9/Fto Axis Induces Formation of RNA:DNA Hybrids That Serve As Damps Initiating Myeloid Skewing & Genetic Injury in MDS

We previously reported that S100A9 activates the NLRP3 inflammasome to promote pyroptosis, ineffective hematopoiesis and the development of MDS in a feed forward fashion. The critical role of S100A8/9 was recently confirmed by others, demonstrating its importance as a key pathogenic driver of disease. Nonetheless, the precise mechanism by which S100A9 may foster DNA damage in MDS is unclear. We recently showed that S100A9 directs overexpression of the fat-mass and obesity-associated gene (FTO) encoding an m⁶A RNA demethylase, which leads to nuclear exclusion of SRSF2. Removal of SRSF2 from its functional domain in the nucleosome leads to stalling of RNA polymerase II and formation of the nucleic acid R-loops, comprising DNA:RNA hybrids and the associated non-template single-stranded DNA. S100A9/FTO axis activation leads to SRSF2 deregulation through suppression of its main nuclear transport protein RanBP2, thereby stalling transcription with resulting accumulation of nuclear R-loops and cytosolic/extracellular RNA:DNA hybrids. Persistent R-loops induce DNA damage while also compromising DNA repair. Here we identify an S100A9/FTO-regulated pathway responsible for induction of genomic instability through the accumulation of cytoplasmic RNA:DNA hybrids. While accumulation of these damage associated molecular pattern (DAMP) molecules coincides with the formation of double stranded breaks (DSB), their precise role in the age-induced inflammatory process initiated by S100A9 in MDS pathogenesis remains unknown. We first investigated which components of the S100A9/FTO axis are critical to hematopoiesis and those that are important for both the development of RNA:DNA hybrids and γH2AX activation. We analyzed the contribution of RanBP2 and the effects of elimination of R-loop formation via overexpression of RNAse H1, an enzyme that removes stalled R-loops by degrading DNA-hybridized RNA, thereby reducing accumulation of cytoplasmic RNA:DNA hybrids. CRISPR knock-down of RanBP2 showed that the protein is critical for accumulation of DSB as evidenced by upregulation of γH2AX foci and RNA:DNA hybrid accumulation. Importantly, overexpression of RNAse H1 degraded R-loops and restored colony-forming capacity, indicating that RNA:DNA hybrids induced by S100A9/FTO have profound effects on hematopoietic potential. We hypothesized that the RNA:DNA hybrid DAMPs have an important role in the pathogenesis of MDS through engagement and activation of its two principal receptors, TLR9 and NLRP3. Both are upregulated in MDS, while the NLRP3 inflammasome plays a critical role in MDS pathobiology through the induction of pyroptosis, self-renewal and cell swelling (Basiorka S, et. al. Blood 2016). CRISPR knock-downof each receptor in MDS bone marrow (BM) mononuclear cells (MNC) had distinct effects: (1) proportional reduction in γH2AX with TLR9 knock-down, and (2) reduction in RNA:DNA hybrids with NLRP3 suppression. Using synthetic RNA:DNA hybrids, transfection (to simulate internal DAMPs) of healthy BM MNC or passive uptake (to simulate their extracellular effect) induced myeloid skewing and reduced colony formation indicating that S100A9/FTO-induced RNA:DNA hybrids are critical intracellular DAMPs that contribute to disease pathobiology. These findings were validated by flow cytometric analysis showing that exposure to synthetic RNA:DNA hybrids directly induced the accumulation of γH2AX in normal hematopoietic stem cells. We conclude that S100A9/FTO-induced RNA:DNA-hybrids lead to genomic instability, representing a previously uncharacterized mechanism contributing to MDS pathogenesis. Our studies provide evidence that targeting this cascade offers significant potential for development of novel, biologically rational therapeutics for MDS.