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Background: Natural killer (NK) cell impairment is a feature of pulmonary arterial hypertension (PAH) and contributes to vascular remodeling in animal models of disease. Although mutations in BMPR2 , the gene encoding the BMP (bone morphogenetic protein) type-II receptor, are strongly associated with PAH, the contribution of BMPR2 loss to NK cell impairment remains unknown. We explored the impairment of IL (interleukin)-15 signaling, a central mediator of NK cell homeostasis, as both a downstream target of BMPR2 loss and a contributor to the pathogenesis of PAH. Methods: The expression, trafficking, and secretion of IL-15 and IL-15Rα (interleukin 15 α-type receptor) were assessed in human pulmonary artery endothelial cells, with or without BMPR2 silencing. NK cell development and IL-15/IL-15Rα levels were quantified in mice bearing a heterozygous knock-in of the R899X-BMPR2 mutation ( bmpr2 +/R899X ). NK-deficient Il15 −/− rats were exposed to the Sugen/hypoxia and monocrotaline models of PAH to assess the impact of impaired IL-15 signaling on disease severity. Results: BMPR2 loss reduced IL-15Rα surface presentation and secretion in human pulmonary artery endothelial cells via impaired trafficking through the trans-Golgi network. bmpr2 +/R899X mice exhibited a decrease in NK cells, which was not attributable to impaired hematopoietic development but was instead associated with reduced IL-15/IL-15Rα levels in these animals. Il15 −/− rats of both sexes exhibited enhanced disease severity in the Sugen/hypoxia model, with only male Il15 −/− rats developing more severe PAH in response to monocrotaline. Conclusions: This work identifies the loss of IL-15 signaling as a novel BMPR2 -dependent contributor to NK cell impairment and pulmonary vascular disease.

Background. AXL receptor tyrosine kinase (AXL) is a transmembrane protein that is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer and stromal cells and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL by its ligand, growth arrest specific protein 6 (Gas6), or ligand-independent dimerization facilitates AXL phosphorylation, initiates signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the discovery and characterization of a novel, highly potent and selective novel AXL inhibitor. Materials and Methods. The potency and specificity of the novel Arcus inhibitor against AXL and other kinases was determined using a panel of HTRF KinEASE-TK assays. Intracellular target engagement was evaluated by monitoring displacement of a competitive fluorescent tracer using an AXL NanoBRETTM intracellular kinase assay. To further assess the inhibitory effect of the novel Arcus inhibitor on AXL kinase activity, AXL autophosphorylation induced SH2 domain translocation was measured in cells using a PathHunterÒ U2OS AXL Functional assay system. Inhibition of AXL phosphorylation in cancer cells was evaluated by Western blot and levels of soluble and surface AXL were assessed by ELISA and flow cytometry, respectively. Pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were evaluated in murine models. Results. A novel, potent, reversible, and highly selective AXL kinase inhibitor has been generated by Arcus. The novel inhibitor exhibits single-digit nanomolar potency in both biochemical and cell-based assays, retains significant activity in 100% human serum and does not show significant inhibition of the major CYP450 isoforms nor the hERG potassium channel. The novel Arcus molecule inhibits AXL phosphorylation mediated by both ligand-dependent Gas6 stimulation as well as ligand-independent autophosphorylation. AXL phosphorylation and subsequent signaling leads to receptor internalization, thereby decreasing both surface AXL expression and soluble AXL levels. AXL activity is inhibited in a concentration dependent manner significantly increasing both surface AXL expression and soluble AXL levels. More importantly, significant anti-tumor efficacy is observed in combination with targeted therapies in several in vivo models. Furthermore, AXL inhibition significantly reduces tumor growth after relapse to single-agent targeted therapy. Conclusions. A novel selective inhibitor of AXL tyrosine kinase activity has been developed that demonstrates single-digit nanomolar potency and inhibition of both ligand-dependent and ligand-independent AXL phosphorylation. Significant anti-tumor activity is observed in combination with targeted therapy and upon acquired resistance in xenograft models. Selective AXL inhibition is a promising therapeutic strategy to overcome resistance to chemotherapy, targeted therapy, and/or immunotherapy. Citation Format: Susan L. Paprcka, Subhasree Sridhar, Irene M. Luu, Salema Jafri, Dillon H. Miles, Suan Liu, Ruben Flores, Shiwei Qu, Manjunath Lamani, Sriivas Paladugu, Cesar Meleza, James Wu, Hema Singh, Yu Chen, Sean Cho, Akshata Udyavar, Angelo Kaplan, Enzo Stargnaro, Xiaoning Zhao, Lixia Jin, Manmohan R. Leleti, Stephen W. Young, Jay P. Powers, Matthew J. Walters, Ester Fernandez-Salas. Potent and selective AXL tyrosine kinase inhibition demonstrates significant anti-tumor efficacy in combination with standard of care therapeutics in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P253.

Systemic hypertension, preeclampsia, and pulmonary arterial hypertension (PAH) are diseases of high blood pressure in the systemic or pulmonary circulation. Beyond the well-defined contribution of more traditional pathophysiological mechanisms, such as changes in the renin–angiotensin–aldosterone system, to the development of these hypertensive disorders, there is substantial clinical evidence supporting an important role for inflammation and immunity in the pathogenesis of each of these three conditions. Over the last decade, work in small animal models, bearing targeted deficiencies in specific cytokines or immune cell subsets, has begun to clarify the immune-mediated mechanisms that drive changes in vascular structure and tone in hypertensive disease. By summarizing the clinical and experimental evidence supporting a contribution of the immune system to systemic hypertension, preeclampsia, and PAH, the current review highlights the cellular and molecular pathways that are common to all three hypertensive disorders. These mechanisms are centered on an imbalance in CD4+ helper T cell populations, defined by excessive Th17 responses and impaired Treg activity, as well as the excessive activation or impairment of additional immune cell types, including macrophages, dendritic cells, CD8+ T cells, B cells, and natural killer cells. The identification of common immune mechanisms in systemic hypertension, preeclampsia, and PAH raises the possibility of new therapeutic strategies that target the immune component of hypertension across multiple disorders.