B18-04 Runx1 Inhibition Mitigates Sox17 Deficiency in Pulmonary Arterial Hypertension

Abstract Rationale Numerous genetic mutations are associated with an increased risk for developing pulmonary arterial hypertension (PAH). Genome-wide association studies have identified 2 independent risk variant-containing signals (SOX17-signal 1 and 2) upstream of the SOX17 promoter. Fifty-nine percent of PAH patients are homozygous for the risk allele of both signals versus 46% of control subjects. In addition, rare deleterious variants in SOX17 are associated with severe PAH. Transgenic mice with deletion of signal 1 (SOX17eKO) are more susceptible to developing Sugen/hypoxia pulmonary hypertension (SuHx-PH) than wild-type mice. Notably, SOX17 directly suppresses RUNX1 gene expression by binding to its promoter. RUNX1 functions as a key regulator of myeloid differentiation, aberrant angiogenesis and adverse cardiac remodeling. We hypothesize that SOX17 dysregulation leads to RUNX1 upregulation and pulmonary endothelial dysfunction, that can be reversed through RUNX1 inhibition. Methods Human pulmonary artery endothelial cells (HPAECs) with stable SOX17 CRISPR/Cas9 knockout or RUNX1 overexpression were generated and examined for endothelial gene expression, proliferation, migration, apoptosis, and in vitro angiogenesis. Lymphoblastoid cell lines (LCLs) from PAH patients with SOX17 rare mutations and age- and sex-matched healthy controls were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into ECs. To induce SuHx-PH, triple transgenic VE-Cdh5-CreERT2;Runx1(flox/flox);SOX17eKO mice were bred and treated with low dose Sugen (5mg/kg) and mild hypoxia (12.5% oxygen) for 3 weeks; rats were injected subcutaneously with Sugen (25mg/kg) and placed in hypoxia (10.5% oxygen) for 3 weeks followed by 2 weeks of normoxic recovery. A small molecule RUNX 1 inhibitor Ro5-3335 (20mg/kg) was used for RUNX1 inhibition in vivo. Results Plasma RUNX1 gene expression levels were elevated in PAH patients lacking detectable SOX17 expression. SOX17 deletion or RUNX1 overexpression in HPAECs similarly downregulated endothelial markers and led to enhanced proliferation and migration, defective angiogenesis, as well as decreased apoptosis. RUNX1 siRNA knockdown or RUNX1 inhibition by Ro5-3335 in HPAECs with defective SOX17 expression partially restored the endothelial characteristics. Furthermore, compared with ECs differentiated from healthy individual iPSCs, ECs differentiated from SOX17 rare mutant PAH patient iPSCs exhibited upregulated RUNX1 expression and the loss of endothelial properties. In addition, inducible endothelial specific deletion of RUNX1 in vivo rescued the susceptibility of SOX17eKO mice to SuHx-PH and RUNX1 inhibitor Ro5-3335 attenuated SuHx-PH development in both SOX17eKO mice and in wild-type rats. Conclusions RUNX1 inhibition mitigates SOX17 deficiency in PAH. Small molecule RUNX1 inhibitors may be used to treat PAH in patients with or without SOX17 mutations. This abstract is funded by: National Institutes of Health