Myelodysplastic Syndromes-Associated Gene Mutations Lead to Pseudohypoxia Condition and Epigenome Hyper-Methylation in Mouse Genetic Models

Myelodysplastic syndromes (MDS) are heterogeneous diseases caused by a complex combination of various gene mutations. Patients develop anemia, pancytopenia, and uni-/multi-lineage dysplasia, which represent ineffective hematopoiesis and also potential for leukemic transformation. Recent advances in next generation sequencing identified a variety of gene mutations involved in transcriptional and epigenetic regulation, RNA splicing, or metabolic enzymes. However, the underlying mechanism of those commonly shared MDS phenotypes caused by these heterogenous genetic mutations has not been fully elucidated yet.

We recently have identified that HIF1A activation caused by metabolic rewiring and pathobiological pseudohypoxia are critical for the pathogenesis of MDS, and HIF1A is a potential novel therapeutic target in MDS (Cancer Discovery2018). This unique metabolic rewiring is associated with activation of glycolysis, suppression of mitochondrial TCA cycle and oxidative phosphorylation (OXPHOS), accumulation of the intermediate metabolites in TCA cycle, which suppress a-KG-dependent dioxygenases including prolyl hydroxylase domain (PHD), aspartate hydroxylase (FIH1), DNA demethylases (TETs), and RNA demethylases (FTO/ALKBH5), and lysine demethylases (KDMs). This pseudohypoxia condition also leads to HIF1A stabilization and hypermethylation of DNA/RNA/histones (epigenome). Interestingly, we found that mitochondrial complex II, succinate dehydrogenase (SDH), is downregulated in hematopoietic stem/progenitor cells (HSPCs) of Mll-PTD (partial tandem duplication) knocked-in (Mll^PTD/WT) mice, which is one of the MDS-associated mutations found in patients. We confirmed that Mll^PTD/WTmice indeed exhibited activated glycolysis and suppressed TCA cycle and OXPHOS in NMR-based Stable Isotope-Resolved Metabolomics (SIRM)analysis with labeled glucose (¹³C-glucose). Based on these early findings, we further investigated the impact of the major MDS-associated mutations on pseudohypoxia in several genetic mouse models in this study.

We first established multiple mouse genetic MDS models mimicking co-occurrence of gene mutations found in MDS patients. MLL-PTDis not sufficient for MDS onset in humans and mice, and is frequently accompanied by RUNX1 mutations in MDS as well as AML. MLL-PTD has been identified in healthy populations, which reflects the clonal hematopoiesis of indeterminant potential (CHIP) condition. Indeed, HSPCs from Mll^PTD/WTknocked-in mice have a clonal advantage after bone marrow (BM) transplantation, but does not induce an MDS phenotype in mice (Blood2012). In contrast, Vav1-Cre-driven Runx1 knockout in Mll^PTD/WT background induces MDS phenotypes: pancytopenia, macrocytic anemia, thrombocytopenia, and multi-lineage dysplasia. We further examined HSPCs of Mll^PTD/WT/Vav1-Cre/Runx1^Flox/Flox mice and confirmed that there are increases of methylation levels of DNA, RNA, and histones. HIF1A protein was also accumulated in HSPCs of these mice. Gene mutations in epigenetic modifiers, TET2, DNMT3A, and ASXL1, are often found in the CHIP condition as well as in MDS, and some patients have double or triple mutations of these genes. Thus, we developed Vav1-Cre-driven Tet2, Dnmt3a, and Asxl1 knockout mice. The triple conditional knockout mice exhibit anemia, thrombocytopenia, leukocytosis, and expansion of HSPCs. HSPCs from this strain exhibited elevated DNA, RNA, and histone methylation levels, which were more obvious compared to Mll^PTD/WT/Runx1^Δ/Δ, and also high HIF1A protein expression. These results suggest that pseudohypoxia is commonly generated by MDS-associated gene mutations and may contribute to the pathophysiology of MDS.

Collectively, in this study we found that the combinations of MDS-associated mutations are sufficient for generating pseudohypoxia condition, HIF1A activation and hypermethylation of epigenomes, which recapitulates the conditions in MDS patients. We are currently investigating the detailed mechanism of pseudohypoxia and the metabolic rewiring, which are caused by the individual or the multiple combinations of gene mutations found in MDS. We believe that our research on pseudohypoxia may provide a better understanding for the pathophysiology of MDS and can also help us develop novel therapeutic strategies targeting the vulnerabilities of these unique metabolic and epigenetic statuses in MDS.