Abstract 4141912: Targeting NEK2 to Induce Senescence and DNA Damage in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and vascular remodeling of the distal pulmonary arteries (PAs) leading to progressive elevation of PA pressure, right heart failure and death. Extensive proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs) is perhaps the most prominent feature of PAH accounting for the histopathological changes seen in this disease. With the recognition of this, direct targeting of vascular remodeling may represent a promising strategy. Goal: To illuminate new actionable targets involved in pulmonary vascular remodeling in PAH. Methods: Molecular, biochemical and pharmacological approaches were used to identify Never In Mitosis A-Related Kinase 2 (NEK2) and study its role in pulmonary vascular remodeling in PAH Results: We performed a comparative RNA-sequencing analysis between control and PAH-PASMCs and then integrated our dataset with publicly available datasets conducted on comparable samples. Among the genes that were concordantly up-regulated in PAH-PASMCs, we identified NEK2, a kinase and druggable target implicated in the progression of mitosis. Using Western blot (WB) and immunofluorescence (IF), we confirmed that NEK2 is upregulated in PASMCs and distal PAs of PAH patients and animal models (Sugen/Hypoxia- and monocrotaline-exposed rats). Pharmacological (NCL0017509) and molecular (siNEK2) inhibition of NEK2 reduced PAH-PASMCs proliferation, as assessed by IF (Ki67 labeling, EdU incorporation, p<0.05) and qPCR (MCM2, PLK1 and Survivin , p<0.05). Further analysis revealed that pharmacological inhibition of NEK2 blocks cell cycle progression in the G2/M phase (flow cytometry, p<0.01) and increases DNA damage (comet assay p<0.001, WB of pDNAPK/DNAPK and RAD51, p<0.05). Moreover, we found that PAH-PASMCs subjected to NEK2 inhibition acquire a senescent phenotype (WB of p16, p21 and p53, p<0.05). Conclusion: Our results suggest that NEK2 contributes to the pro-proliferative phenotype of PAH-PASMC and may represent a new target in PAH.