Genetic and hypoxic control of a lncRNA axis orchestrates endothelial reprogramming in pulmonary hypertension

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

Background and Hypothesis: Pulmonary hypertension (PH) is a deadly disease, where Group 1 PAH and Group 3 PH are driven by hypoxia, HIF-2, and non-coding RNAs. We found that the lncRNA KMT2E-AS1 is up-regulated in Groups 1/3 PH and is induced by HIF-2. This lncRNA gene neighbors KMT2E, a gene controlling histone 3 lysine 4 trimethylation (H3K4me3) and chromatin remodeling. In pulmonary endothelial cells (ECs), KMT2E-AS1 stabilizes KMT2E to increase H3K4me3, thus driving HIF-2-specific metabolic and pathogenic alterations. The G-allele of single nucleotide variant (SNV) rs73184087 within KMT2E is associated with risk of developing Group 1 PAH (in discovery/validation cohorts and a meta-analysis of 2,181 PAH vs. 10,060 controls). rs73184087 also displays more avid allele (G)-specific association with HIF-2 leading to induction of this lncRNA-KMT2E pair. A mouse deficient in the conserved lncRNA sequence is protected against Groups 1/3 PH; this is phenocopied by inhibition of histone methylation in PAH rats. We postulate that the KMT2E-AS1/KMT2E axis is a central lynchpin in pathogenic reprogramming in ECs, promoting PH. Aim 1) Define the allele-specific role of the KMT2E SNV rs73184087 in controlling HIF-2-dependent EC lncRNA-KMT2E expression and PH pathophenotypes. Using ECs derived from genome-edited inducible pluripotent stem cells (iPSC) as well as primary lung ECs carrying rs73184087 A and G alleles, we will determine if (G) increases lncRNA-KMT2E by more HIF-2 binding and drives more severe EC phenotypes. We will also pursue expression quantitative trait loci (eQTL) analysis in blood samples from PAH patients (discovery/validation cohorts) and PAH lung tissues carrying A and G alleles of rs73184087. Aim 2) Define the role of this lncRNA-KMT2E axis and H3K4me3 in promoting PH in vivo. We will quantify Groups 1/3 PH severity in rodents after EC-specific knockdown of this lncRNA vs. lncRNA+KMT2E and after AAV-driven EC- specific expression of lncRNA vs. lncRNA+KMTE2. We will also determine if MM-589, a specific H3K4me3 inhibitor, reverses PAH in rats. Thus, we aim to determine if lncRNA+KMT2E together are necessary and sufficient to drive Group 1/3 PH and if PAH is dependent upon H3K4me3 activity, thus offering a new epigenetic PH therapy. Aim 3) Define the causative role of the G allele of rs73184087 on pulmonary vascular remodeling and PH in vivo. Culturing human precision cut lung slices, we will determine if the rs73184087 G allele drives vascular remodeling via regulation of the lncRNA-KMT2E axis and H3K4me3. We have also inserted the human rs73184087 G vs. A allele in mice and will use these “humanized” mice to study these alleles in vivo. With these 2 unique platforms, we will determine if the G allele drives HIF-2-specific EC phenotypes and PH. Significance: We plan to shift paradigms of lncRNA biology in PH, via defining the links of hypoxia to epigenetics and metabolism and by introducing new epigenetic therapies. By establishing the causative role of rs73184087 in PH, we are poised to leverage functional genomics to gain mechanistic insight in PH specifically for humans.