Therapeutic inhibition of HIF-2α reverses polycythemia and pulmonary hypertension in murine models of human diseases

Abstract

Polycythemia and pulmonary hypertension are 2 human diseases for which better therapies are needed. Upregulation of hypoxia-inducible factor-2α (HIF-2α) and its target genes, erythropoietin (EPO) and endothelin-1, causes polycythemia and pulmonary hypertension in patients with Chuvash polycythemia who are homozygous for the R200W mutation in the von Hippel Lindau (VHL) gene and in a murine mouse model of Chuvash polycythemia that bears the same homozygous VhlR200W mutation. Moreover, the aged VhlR200W mice developed pulmonary fibrosis, most likely due to the increased expression of Cxcl-12, another Hif-2α target. Patients with mutations in iron regulatory protein 1 (IRP1) also develop polycythemia, and Irp1-knockout (Irp1-KO) mice exhibit polycythemia, pulmonary hypertension, and cardiac fibrosis attributable to translational derepression of Hif-2α, and the resultant high expression of the Hif-2α targets EPO, endothelin-1, and Cxcl-12. In this study, we inactivated Hif-2α with the second-generation allosteric HIF-2α inhibitor MK-6482 in VhlR200W, Irp1-KO, and double-mutant VhlR200W;Irp1-KO mice. MK-6482 treatment decreased EPO production and reversed polycythemia in all 3 mouse models. Drug treatment also decreased right ventricular pressure and mitigated pulmonary hypertension in VhlR200W, Irp1-KO, and VhlR200W;Irp1-KO mice to near normal wild-type levels and normalized the movement of the cardiac interventricular septum in VhlR200Wmice. MK-6482 treatment reduced the increased expression of Cxcl-12, which, in association with CXCR4, mediates fibrocyte influx into the lungs, potentially causing pulmonary fibrosis. Our results suggest that oral intake of MK-6482 could represent a new approach to treatment of patients with polycythemia, pulmonary hypertension, pulmonary fibrosis, and complications caused by elevated expression of HIF-2α.

Graphical abstract

Figure 1.

Structures of PT2385 and MK-6482 (also known as PT2977), specific inhibitors of HIF-2α. Structure of the originally designed HIF-2α inhibitor PT2385, which contains the difluoro group in the geminal position, was modified to enhance the pharmacokinetics and efficacy of MK-6482 in which the difluoro group is in the vicinal position.

Figure 2.

MK-6482 reversed polycythemia in Vhl^R200W, Irp1-KO, and double-mutant Vhl^R200W;Irp1-KO mice. Hematocrit levels, as determined by the capillary centrifugation method (A-B), hemoglobin (C), RBC (D), MCV (E), and MCH (F) levels of 6- to 11-month-old WT, Vhl^R200W, Irp1-KO, and double-mutant Vhl^R200W;Irp1-KO mice treated with vehicle or the Hif-2α inhibitor MK-6482. Oral administration of MK-6482 significantly decreased the elevated hematocrit, hemoglobin, and RBC levels, and ameliorated polycythemia in all 3 mutant models. MK-6482 also decreased MCV and MCH values in the mutant mice. Notably, MCV and MCH levels were decreased in the Irp1-KO mice, suggesting that IRP1 deficiency induces mild iron deficiency that is reversed by Vhl^R200W mutation. ***P < .001, by ordinary 1-way ANOVA (multiple comparisons). D-mutant, double-mutant Vhl^R200W;Irp1-KO mouse.

Figure 3.

MK-6482 attenuated polycythemia by reducing EPO levels in Vhl^R200W and Irp1-KO mice and in hypoxic Hep3b cells. (A) Serum EPO levels measured by ELISA were significantly increased in Vhl^R200W and Irp1-KO mice, but on treatment with the drug, the elevated EPO levels in Vhl^R200W and Irp1-KO mice decreased to normal WT levels. (B) Epo mRNA levels of Hep3b cells were more than 50-fold higher when grown at 2.5% oxygen concentration than levels grown at atmospheric oxygen concentrations, and the drug treatment significantly reduced the Epo expression levels. *P < .05; **P < .01; ***P < .001, by ordinary 1-way ANOVA (multiple comparisons). ns, not significant.

Figure 4.

MK-6482 treatment reduced pulmonary hypertension in Vhl^R200W, Irp1-KO, and double-mutant Vhl^R200W;Irp1-KO mice and diminished elevated endothelin-1 levels in all 3 mouse models. (A) RVPs measured by catheterization of lightly anesthetized mice showed significantly elevated pressures in Vhl^R200W, Irp1-KO, and Vhl^R200W;Irp1-KO mice in comparison with WT mice, and MK-6482 treatment significantly decreased the RVPs, not only in Vhl^R200W and Irp1-KO mice, but also in double-mutant Vhl^R200W;Irp1-KO mice. (B) Transthoracic echocardiography showed systolic flattening of the IVS in vehicle-treated Vhl^R200W mice, as indicated by the appearance of an abnormal D-like structure (arrows) compared with the normal convex appearance of the IVS in the WT mice. The D-like structure disappeared and the normal convex shape of the IVS reappeared upon treatment with MK-6482. Representative images from 2 animals of each group are shown. Five mice from each group were tested, except for the drug-treated Vhl^R200W;Irp1-KO group, in which 3 mice were tested. (C) Serum endothelin-1 levels were elevated in Vhl^R200W, Irp1-KO, and Vhl^R200W;Irp1-KO mice compared with the WT mice. When treated with MK-6482, the endothelin-1 levels of the mutant and double-mutant mice reverted to normal WT levels. ns, not significant, *P < .05; ***P < .001 by ordinary 1-way ANOVA (multiple comparisons). D-mutant, double-mutant Vhl^R200W;Irp1-KO mouse.

Figure 5.

MK-6482 decreased the elevated expression of Cxcl-12 in Vhl^R200W mice. (A) Cxcl-12 protein levels were significantly increased in the serum of Vhl^R200W mice. When treated with MK-6482, serum Cxcl-12 protein reverted to WT levels. Cxcl-12 mRNA expression levels in lung (B) and heart (C) were elevated in the Vhl^R200W mice. Drug treatment significantly reduced the Cxcl12 mRNA levels. **P < .01; ***P < .001, by ordinary 1-way ANOVA (multiple comparisons). D-mutant, double-mutant Vhl^R200W;Irp1-KO mouse.

Figure 6.

Model for the therapeutic action of MK-6482 in Vhl^R200W, Irp1-KO, and double-mutant Vhl^R200W;Irp1-KO mice. The expression of Hif-2α protein is regulated at multiple levels. Irp1, which is most likely the predominant IRE-binding protein in renal interstitial fibroblast and pulmonary endothelial cells, binds to the Hif-2α-IRE at the 5′ UTR and thus represses Hif-2α translation. Thus, Irp1 deficiency results in an increased Hif-2α expression through derepression of Hif-2α translation. In contrast, the Vhl protein promotes degradation of Hif-2α (as well as Hif-1α) under normoxic conditions. However, Vhl^R200W does not mediate normal Hif-2α degradation. Thus, either Irp1 deletion or the Vhl^R200W mutation leads to high levels of Hif-2α and augmented levels of Hif-2α target genes, including EPO, endothelin-1 (ET-1), and Cxcl-12. MK-6482, the second-generation, specific inhibitor of Hif-2α, binds to the internal cavity in the PAS-B domain of Hif-2α, distorting its structure and disrupting the formation of Hif-2α-Hif-1β dimers, thus inhibiting Hif-2α–dependent transcriptional activity and thereby reducing the elevated expression levels of Hif-2α targets in Vhl^R200W, Irp1-KO, and double-mutant Vhl^R200W;Irp1-KO mice.