Inflammaging-Associated Metabolic Alterations Foster Development of the MDS Genotype (2015)

Abstract Recent studies suggest that aging-associated inflammation, or "inflammaging", contributes to genetic instability and MDS predisposition. Although innumerable somatic genetic events have been annotated in recent years, including many that are not unique to MDS, they are not sufficient for disease initiation. The precise underlying mechanisms conducive to the emergence of these genetic events also remain to be delineated. We reported that bone marrow (BM) myeloid derived suppressor cells (MDSC) activated by the damage associated molecular pattern (DAMP) protein S100A9, promote ineffective hematopoiesis and the development of MDS. Inflammaging associated alterations in metabolism have been implicated in predisposition to cancer development with age. Here we report that S100A9 and ROS-induced inflammaging are associated with insulin resistance and hyperglycemia in the BM microenvironment that triggers activation of adaptive oncogenic pathways and genomic instability in HSPC. Glucose concentrations were markedly elevated in MDS BM plasma vs. age-matched controls, and directly correlated with S100A9 concentration (r=0.513, P=0.003, n=41). The magnitude of BM-glucose elevation significantly exceeded that in the peripheral blood and negatively correlated with the proportion of HSPC while directly correlating with BM MDSC percentage. S100A9 transgenic (Tg) mice displayed age-dependent elevation of glucose in peripheral blood and BM when compared to wild type mice accompanied by accumulation of somatic mutations (SM) by sequencing in aged S100A9-Tg compared to younger counterparts. NGS of 38 primary MDS BM specimens showed that SM common to MDS, such as those involving ASXL1, U2AF1 and DNMT3A, we re present only in high glucose stratified MDS-BM specimens (glucose &gt; 110 ug/ml). Furthermore, there was a strong correlation between the cellular ROS/nuclear-β-catenin to DNA damage (γH2AX+ cells) linking S100A9-induced ROS accumulation to genetic instability. Elevation in BM plasma glucose was specifically associated with upregulation of the fat mass and obesity associated (FTO) transcript and protein in both MDS BM and S100A9Tg mice. FTO is a risk factor for type 2 diabetes, and encodes an α-ketoglutarate-dependent dioxygenase that functions as an RNA demethylase specific for N 6-methyladenosine (m6A) residues, targeted by splicing factors. Both human and murine MDS specimens displayed decreased m6A mRNA methylation compared to controls. FTO knockdown with CRISPR increased mRNA m6A methylation in MDS primary specimens, whereas overexpression of FTO led to a corresponding decrease that was enhanced by S100A9 stimulation. Moreover, S100A9 treatment induced FTO and demethylation of m6A mRNA in human and murine BM cells. These effects were accompanied by disruption of spliceosomes in the nucleus, as demonstrated by delocalization of SRSF2 from nuclear speckles into the cytoplasm where they colocalize with FTO in MDS patients. Interestingly, from the S100A9Tg mouse sequencing studies, we discovered that the mutation hotspots were in locations specific for histone H3K27 acetylation which have been previously linked to genomic instability, as well as splicing regulation, and regulated by histone deacetylase 1 (HDAC1). Reduced HDAC1 levels are known to make cells hypersensitive to DNA-damaging insults, such as those inducing ROS, similar to what we observed with treatment of S100A9 in healthy human bone marrow, MDS patient samples and S100A9Tg mice. Furthermore, this correlated with increased levels of DNA damage in our patient samples and in our murine S100A9Tg model as measured by phosphorylated γH2AX histones. Our studies provide a biological rationale for the initiation of DNA-genetic damage under inflammatory senescence conditions in MDS. These findings demonstrate that S100A9-induced inflammation activates a signaling cascade that enhances development of splicing variants and somatic gene mutations critical to MDS pathogenesis. Disclosures List: Celgene Corporation: Honoraria, Research Funding.