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Acknowledgements
We thank J. Massague for 786-M1A and M2A cells. Supported by grants from the NIH. W.G.K. is an HHMI Investigator. R.K.B. is the Michael L. Rosenberg Scholar in Medical Research and was supported by the Cancer Prevention and Research Institute of Texas (RP130513).
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Authors and Affiliations
Department of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, 02215, Massachusetts, USA
Hyejin Cho,Ella Liberzon,Abhishek A. Chakraborty,Wenhua Gao,Ingrid Carvo,Sabina Signoretti&William G. Kaelin
Peloton Therapeutics, Inc., Dallas, 75235, Texas, USA
Xinlin Du,James P. Rizzi,John A. Josey&Eli M. Wallace
Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, 02115, Massachusetts, USA
Ingrid Carvo&Sabina Signoretti
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, 75235, Texas, USA
Richard K. Bruick
Howard Hughes Medical Institute, Chevy Chase, 20815, Maryland, USA
William G. Kaelin
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- Hyejin Cho
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- Xinlin Du
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- John A. Josey
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- Eli M. Wallace
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Contributions
W.G.K. conceived the study, analysed data and, with H.C., wrote the manuscript; H.C. designed and did all of the experiments, except for the structural, isothermal titration calorimetry, and pharmaco*kinetic studies and the experiments for Extended Data Figs 1c, e; X.D. generated proteins, performed ITC experiments and collected and solved the HIF-2–PT2399 co-crystal structure; J.P.R., J.A.J. and E.M.W. coordinated discovery, synthesis and characterization of PT2399; J.A.J. and E.M.W. designed the PDX experiment; E.L. assisted with lung colonization assays; A.A.C. carried out bioinformatic analyses; W.G. designed and generated CRISPR reagents; I.C. and S.S. performed and analysed immunohistochemistry; R.B. designed the PT2399-resistant HIF-2α.
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Correspondence to Eli M. Wallace or William G. Kaelin.
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Competing interests
X.D., J.P.R., R.B., J.A.J., E.M.W. and W.G.K. own equity in Peloton as Peloton employees (X.D., J.P.R., J.A.J. and E.M.W.), licensors (R.B.) or advisors (R.B. and W.G.K.).
Extended data figures and tables
Extended Data Figure 1 Binding of PT2399 to PAS-B domain of human HIF-2α as determined by X-ray co-crystal structure.
a, X-ray co-crystal of PT2399 (magenta) bound to complex formed by HIF-2α and ARNT PAS-B domains (ARNT removed for clarity). b, X-ray co-crystal of PT2399 (magenta) with HIF-2α and ARNT PAS-B domains (zoomed in on HIF-2α PAS-B pocket). c, Immunoblots of anti-ARNT1 immunoprecipitates (IP) of Hep3B cells treated with PT2399 or DMSO. d, Immunoblot of 786-O cells expressing shRNA against HIF-2α (3806) or control shRNA. e, HIF-2α-specific gene regulation in Hep3B cells; n = 3 biological replicates. f, Immunoblot analysis (top) and quantification (bottom) of HIF-2α in 786-O cells treated with DMSO or PT2399 for 16 h and then exposed to cycloheximide for the indicated time periods; n = 3 biological replicates. g, Enrichment plots for representative gene sets previously linked to HIF, hypoxia, or c-Myc. h, i, Plasma PT2399 levels after administration of a single dose of PT2399 to CD-1 mice; n = 3 per time point from one experiment.
Extended Data Figure 2 Inhibition of cell proliferation by PT2399 ex vivo.
a, b, Immunoblot analysis of 786-O cells after CRISPR-based gene editing with control sgRNA or HIF-2α sgRNA (guides 4 and 6). In b, cells were also infected with an empty vector (EV) or a virus expressing an HIF-2α sgRNA guide 6-resistant HIF-2α cDNA. c–f, Cell proliferation of parental 786-O cells (c) and 786-O clones subjected to CRISPR-based gene editing with a control sgRNA (d) or in which endogenous HIF-2α was successfully inactivated using two different HIF-2α sgRNAs (guides 4 and 6) (e, f); n = 3 biological replicates. g–j, Proliferation curves for 786-M1A (g), UMRC-2 (h), Caki-1 (i), and Caki-2 cells (j) treated with the indicated concentrations of PT2399. k, Immunoblot analysis of the indicated cell lines. l, m, Proliferation curves for MDA-MB-231 (l) and A549 cells (m) treated with the indicated concentrations of PT2399; n = 3 biological replicates. Data shown as mean ± s.d. (c–j, l, m).
Extended Data Figure 3 Effects of PT2399 on soft agar growth.
a–c, Soft agar colonies formed by 786-O cells (a), A498 cells (b), and the indicated cell lines (c) in the presence of PT2399 at the indicated concentrations for 21 days; n = 3 biological replicates. d, Soft agar colonies formed by the indicated polyclonal cell line populations after CRISPR-based gene editing with control sgRNA or HIF-2α sgRNA (guides 4 and 6); n = 3 biological replicates. The reason for the differential sensitivity of RCC10 cells to PT2399 and the HIF-2α sgRNAs is not yet clear. e, Immunoblot analysis of the cells used in d. For SLR21 cells, 1 mM DMOG was added for 16 h to detect HIF-2α. f, g, Quantification of soft agar colonies formed in a–d and Fig. 5h, j, respectively; n = 3. Data shown as mean ± s.e.m. *P < 0.01 by two-tailed Student’s t-tests (f, g).
Extended Data Figure 4 Pharmacodynamic effects of PT2399 in vivo.
a, Levels of the indicated mRNAs, normalized to ACTB, in 786-O orthotopic tumours treated with PT2399 (30 mg kg−1) (n = 3 mice from two independent experiments) or vehicle (n = 3 mice from two independent experiments) twice daily for two days in vivo. b, Immunoblot analysis of 786-O orthotopic tumours treated with PT2399 (30 mg kg−1) or vehicle twice daily for two days in vivo; vehicle, n = 3 mice from two independent experiments; PT2399, n = 3 mice from two independent experiments. c, Quantification of Ki-67 staining (vehicle, n = 3; PT2399, n = 3 mice from two independent experiments). d, Immunohistochemistry of representative 786-O orthotopic tumours treated with PT2399 (30 mg kg−1) or vehicle twice daily for two days in vivo; for vehicle, n = 3 and PT2399, n = 3. Scale bars, 50 μm. e, Microvessel density (vehicle, n = 5; PT2399, n = 3 mice from two independent experiments) from tumours as in d. f, g, Representative tumours at necropsy (f) and serum VEGF concentrations (vehicle, n = 10; PT2399, n = 11 mice from three independent experiments) (g) from mice as in Fig. 4a–c just before necropsy. h, Representative immunohistochemical staining of 786-O tumours treated as in Fig. 4a–c (vehicle, n = 4; PT2399, n = 5); Scale bars, 50 μm. Data shown as median with range (a, c, e, g). Statistical significance was assessed by using Mann–Whitney test (e) or unpaired t-test (g). NS, P > 0.05.
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Extended Data Figure 5 Antitumour activity of PT2399 in lung colonization and PDX models.
a, BLI of lung colonies formed after tail vein injection of 9,000 786-M2A cells treated with PT2399 (30 mg kg−1) or vehicle twice daily by oral gavage. Treatment began at week 1. b, Quantification of BLI values as in a. Data shown as median with range (vehicle, n = 2 and PT2399, n = 3 mice from one experiment). c, Partial rescue of PT2399 pharmacodynamic effect by HIF-2α S304M; n = 3 biological replicates. Levels of the indicated mRNAs, normalized to ACTB mRNA and then to DMSO treatment, in cells from Fig. 4g treated with PT2399 at the indicated concentrations for 48 h; n = 3. Data shown as mean ± s.e.m. *P < 0.05 by two-tailed Student’s t-tests. Note that rescue is only partial, perhaps because these cells still produce endogenous wild-type HIF-2α in addition to exogenous HIF-2α S304M. d, Subcutaneous PDX measurements in mice randomized to the indicated treatments, including the FDA-approved ccRCC drug sunitinib, when the tumours reached 200–300 mm3. P < 0.05 for difference between PT2399 and vehicle (n = 8, unpaired t-test). e, Immunohistochemistry of PDX in d before treatment. Scale bars, 100 μm. Data shown as mean ± s.e.m. (d).
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Extended Data Figure 6 Antitumour activity of PT2399 using A498 cells.
a, b, Representative BLI (a) and quantification of BLI measurements (b) of orthotopic tumours formed by A498 cells expressing firefly luciferase under the control of a CMV promoter before and after (30 days) treatment with PT2399 (30 mg kg−1) or vehicle twice daily by oral gavage (vehicle, n = 10; PT2399, n = 10 mice from two independent experiments) c, d, Representative tumours (c) and tumour masses (d) at necropsy from mice treated as in a (vehicle, n = 10; PT2399, n = 10 mice). e, Serum VEGF concentrations from mice treated as in a at time of necropsy (vehicle, n = 4; PT2399, n = 4 mice from two independent experiments). f, Immunoblot of representative tumours from a; vehicle, n = 4; PT2399, n = 3. g, Levels of the indicated mRNAs, normalized to ACTB, in A498 orthotopic tumours (vehicle, n = 4; PT2399, n = 3 mice from two independent experiments) treated as in a. Data shown as median with range (b, d, e, g). Statistical significance was assessed by using two-tailed Student’s t-tests with Welch’s correction (b, d) or Mann–Whitney test (e).
Source data
Extended Data Figure 7 Elimination of HIF-1α does not render UMRC-2 cells sensitive to PT2399 in soft agar assays.
a–c, Firefly luciferase activity in the indicated cell lines after infection with a virus containing firefly luciferase under the control of a HIF-responsive (HRE-Luc) promoter (a, c) or CMV promoter (b) and treatment with the indicated concentrations of PT2399 for 16 h relative to DMSO-treated controls; n = 3 biological replicates. d, Immunoblot analysis of HRE-Luc-expressing UMRC-2 cells after CRISPR-based gene editing with control sgRNA or HIF-1α sgRNA (guides 2 and 3). Note that deletion of HIF-1α in c and d was used to eliminate the contribution of HIF-1α in a. e, f, Immunoblot (e) and mRNA levels (f) of UMRC-2 cells after CRISPR-based gene editing with control sgRNA or HIF-1α sgRNA (guides 2 and 3). In f mRNA levels were normalized to ACTB and then to the corresponding control sgRNA value; n = 3 biological replicates. g, Soft agar assays of the cells analysed in e and f in the presence of the indicated concentrations of PT2399; n = 3 biological replicates. Data shown as mean ± s.e.m. (a–c, f).
Extended Data Figure 8 Variable sensitivity of ccRCC lines to PT2399 and pVHL.
a, Levels of the indicated mRNAs, normalized to ACTB, in UMRC-2 orthotopic tumours treated with PT2399 (30 mg kg−1) or vehicle twice daily for 1 month; vehicle, n = 2; PT2399, n = 2 mice from one experiment. b, c, Downregulation of HIF-responsive mRNAs by PT2399 in indicated cell lines. For each cell line the mRNA levels were normalized to ACTB mRNA (c) and then normalized to the untreated value for that cell line (b); n = 3 biological replicates. Data shown as median with range (a) and mean ± s.d. (b, c). d, e, Variable suppression of HIF target genes by PT2399 across a panel of ccRCC cell lines. Downregulation of HIF-responsive VEGFA, CCND1 and SLC2A1 mRNAs by PT2399 in the indicated cell lines. For each cell line the mRNA levels were normalized to ACTB (e) and then normalized to the untreated value for that cell line (d); n = 3 biological replicates. VHL+/+ SLR21 renal carcinoma cells were included for comparison. Data shown as mean ± s.d.
Source data
Extended Data Figure 9 HIF-2 dependence of RCC10 cells.
a, Immunoblot analysis of anti-ARNT1 immunoprecipitates (IP) and whole cell extracts (input) prepared from RCC10 cells treated with increasing amounts of PT2399 or DMSO. C, control IP without ARNT1 antibody. b, Levels of the indicated mRNAs, normalized to ACTB, in 786-O, A498 and RCC10 cells treated with PT2399 at the indicated concentrations for 24 h or an effective HIF-2α sgRNA (sgHIF-2α-6), and then normalized to cells treated with DMSO or a control sgRNA, respectively. Data shown as mean ± s.d.; n = 3 biological replicates. c, Immunoblot analysis of RCC10 cells after CRISPR-based editing with HIF-2α sgRNAs or control sgRNA. d, e, soft agar colonies formed by RCC10 cells as in c; n = 3 biological replicates. In e cells were engineered to express an exogenous sgRNA-resistant HIF-2α or empty vector (EV); n = 3. f, Soft agar colony counts as in e using ImageJ software. Colonies were counted using the following criteria: circularity range from 0.5 to 1.0 and size (pixels2) from 200 to infinity. Data shown as mean ± s.e.m. Statistical significance was assessed by using two-tailed Student’s t-tests (f). *P < 0.05. g–k, p53 pathway status in ccRCC lines. g, j, Immunoblot analysis of the indicated cell lines treated for 16 h with etoposide or vehicle. Note overproduction of p53 in RCC10 cells and off-size p53 band in UMRC-2 cells. SLR21 cells are VHL+/+. Red, PT2399 sensitive in soft agar assays. Blue, PT2399 insensitive. RCC4 cells do not form soft agar colonies and are therefore indeterminate. h, Immunoblot analysis of 786-O cells that were infected with an empty lentivirus conferring puromycin resistance and then later found to have spontaneously acquired a p53 mutation (R248W) compared to cells that retained wild-type p53. Cells were treated with PT2399 for 48 h or with nutlin-3 (30 μM) or etoposide (20 μM) for 16 h. i, Soft agar colony formation from cells in h treated with PT2399; n = 3 biological replicates. k, Immunoblot analysis of parental 786-O cells that underwent CRISPR-based gene editing with a control sgRNA or HIF-2α sgRNA (guide 6) (as in Extended Data Fig. 2a) and were then treated with PT2399 for 58 h or treated with nutlin-3 (30 μM) or etoposide (20 μM) for 10 h.
Extended Data Figure 10 Loss of HIF-2α does not suppress UMRC-2 orthotopic tumour growth.
a–c, Tumours (a), tumour weights (b), and tumour immunoblots (c) at necropsy from mice after orthotopic injection of UMRC-2 cells that had undergone CRISPR-based editing with control sgRNA or sgHIF-2α-6 as in Fig. 5g; sgCon, n = 5; sgHIF-2α, n = 5 mice from two independent experiments. The reason for the variable HIF-2α levels in c is unknown but could reflect, at least partly, variable numbers of host-derived cells in the tumour samples. d, Levels of the indicated mRNAs, normalized to ACTB, in tumours from a–c. Data shown as median with range (b, d). Statistical significance was assessed by using two-tailed Student’s t-tests (b) or Mann–Whitney test (d). Loss of HIF-2α did suppress subcutaneous tumour growth (data not shown).
Source data
Supplementary information
Supplementary Information
This file contains Supplementary Methods, Text and Data, additional references and Supplementary Tables 1-3. (PDF 1165 kb)
Supplementary Data
This file contains the Western Blots for Figures 1, 2, 4 and Extended Data Figures 1, 2, 3, 4, 6, 7, 9 and 10. (PDF 2167 kb)
Supplementary Table 4
This file contains the Enzyme assay. (XLSX 43 kb)
Supplementary Table 5
This file contains the GSEA analysis. (XLSX 85 kb)
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Cho, H., Du, X., Rizzi, J. et al. On-target efficacy of a HIF-2α antagonist in preclinical kidney cancer models. Nature 539, 107–111 (2016). https://doi.org/10.1038/nature19795
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DOI: https://doi.org/10.1038/nature19795
