Haploid genetic screening identifies a novel regulator of BMPR2

Pulmonary arterial hypertension (PAH), is characterised by profound remodelling of small pulmonary arteries, leading to increased pulmonary arterial pressures and premature death by right heart failure. Heterozygous germ-line mutations in the bone morphogenetic protein type II receptor (BMPR2) cause ~70% of familial PAH and ~20% of idiopathic PAH cases. The majority of mutations lead to haploinsufficiency but crucially, regardless of the presence of mutation, lung BMPR2 expression is reduced in all forms of PAH. Therefore, restoration of BMPR2 levels is an important therapeutic target. We previously showed that BMPR2 ubiquitination results in lysosomal degradation and can be partially inhibited by the lysosomotropic drug, chloroquine. However, the factors involved in regulating BMPR2 turnover remain poorly understood. We employed a haploid forward genetic screen to identify potential targets involved in controlling BMPR2 expression. Next-generation sequencing identified ~800 “inactivated” genes that induced “high” BMPR2 expression. Novel genes were selected based upon functional relevance in lysosomal degradation or ubiquitination, including the E2 ligase, UBE2E2. UBE2E2 knockdown in pulmonary artery endothelial cells significantly increased BMPR2 protein expression and reduced proliferation and apoptosis. Also, downstream signalling was enhanced in UBE2E2 siRNA targeted cells with phospho-Smad1/5 and ID2 increased in pulmonary endothelial cells. Moreover, UBE2E2 was highly expressed in remodelled vascular lesions in lungs of PAH patients. These observations highlight a new approach to the discovery of novel pathways regulating BMPR2 protein expression which might lead to the discovery of novel therapeutics in PAH.