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Targeting renal cell carcinoma with a HIF-2 antagonist


Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL)1,2. Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal3, VHL inactivation is regarded as the governing event4. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells5. HIF-2 has been implicated in angiogenesis and multiple other processes6,7,8,9, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib10. HIF-2 has been regarded as undruggable11. Here we use a tumourgraft/patient-derived xenograft platform12,13 to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α–HIF-1β)14 in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target15 and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

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Figure 1: Evaluation of PT2399 in RCC tumourgraft-bearing mice.
Figure 2: PT2399 dissociates HIF-2 complexes in sensitive and resistant RCCs and induces changes in gene expression in sensitive tumours.
Figure 3: Sensitive and resistant tumours can be distinguished by HIF-2α levels and gene expression signature.
Figure 4: Acquired resistance following prolonged PT2399 exposure.

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We thank the patients who generously provided tissues and participated in our studies. PT2399 was provided by Peloton Therapeutics, Inc. Funding was provided by Peloton Therapeutics, Inc. (OTD-105466), CPRIT (RP160440) and philanthropy, including the Tom Green memorial. W.C. is supported by grants from the National Natural Science Foundation of China (No. 811011934) and the Science and Technology Program of Guangzhou, China (No. 2012J5100031). M.S.K. and H.Z. are supported by a grant from CPRIT (RP150596). I.P. is supported by grants from the NIH (R01CA154475, P50CA196516). X.S. is supported by a grant from CPRIT (RP110771). J.B. is a Virginia Murchison Linthicum endowed scholar and is supported by grants from the NIH (R01CA175754, P50CA196516, P30CA142543) and CPRIT (RP130603). R.K.B. is the Michael L. Rosenberg Scholar in Medical Research and was supported by CPRIT (RP130513). Histology equipment was purchased with funding from the National Center for Advancing Translational Sciences (Center for Translational Medicine UL1TR001105).

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Authors and Affiliations



W.C. designed and performed biochemical experiments; H.H., E.H., A.P.-J., Q.Y., and A.J. performed tumourgraft experiments; A.C. performed extensive statistical analyses with the supervision of X.-J.X.; M.S.K. performed RNA-seq analyses under the supervision of T.H.H. and Y.X., who also supervised H.Z. on sequencing analysis; Y.M. and N.P. performed experiments; F.H. and P.Y. performed histological analyses under the supervision of P.K.; G.H. performed PET studies under the supervision of X.S.; I.P. performed patient imaging analyses; H.G. and C.R. performed RNA-seq validation studies at the New York Genome Center; J.C. is the clinical research coordinator of the PT2385 phase 1 trial overseen by K.C. and N.Z.; K.H.G., R.K.B., and E.F. participated in discussions; T.W., J.P.R., E.M.W., and J.A.J. oversaw the development and characterization of PT2399 and provided the drug; R.M.M. assisted with manuscript preparation, writing and submission; and J.B. conceived and supervised the project, and wrote the manuscript with input from R.M.M. and the other authors.

Corresponding author

Correspondence to James Brugarolas.

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Competing interests

T.W., J.P.R., E.M.W., N.Z. and J.A.J. are employees and own equity in Peloton Therapeutics, Inc. and K.H.G. and R.K.B. have licensed intellectual property, consult for and own equity in Peloton Therapeutics, Inc. M.S.K., T.H.H, Y.X. and J.B. are authors on a filed patent pertaining to a biomarker of PT2399. J.B. is a member of the advisory board for Bethyl Laboratories.

Additional information

RNAseq was released to NCBI Sequence Read Archive (SRA). ID: SRP073253.

Extended data figures and tables

Extended Data Figure 1 Effects of PT2399 on human RCC-bearing mice.

a, Platelet, white blood cell, neutrophil, and lymphocyte counts from tumourgraft-bearing mice treated with vehicle (n = 52), PT2399 (n = 58), or sunitinib (n = 53) at the end of the drug trial period (~28 days). Low lymphocyte levels throughout are consistent with expected levels in age- and sex-matched NOD/SCID mice. b, Tumour growth trend lines for sensitive, intermediate, and resistant groups after controlling for baseline tumour volume (refer to Fig. 1d for individual curves). c, Representative gross images of tumours from sensitive (XP164 and XP373; green) and resistant (XP169 and XP490; red) lines at the end of the drug trial. d, Representative haematoxylin and eosin-stained images illustrating different effects of PT2399 on sensitive tumours including patchy intercellular fibrosis and hyalinization (open arrow heads), reduced tumour necrosis (red arrows), decreased tumour cell density (XP164 and XP469), reduced nuclear-to-cytoplasmic ratio (XP469), cell ballooning (filled arrow), and dystrophic calcification (blue stars). Scale bars, 50 μm. e, Summary of histopathological changes induced by PT2399 in 10 sensitive tumourgraft lines represented as number of tumours (n) compared to the total or as mean ± s.e. in 28 vehicle-treated tumours compared to 31 PT2399-treated tumours. MVD, microvessel density per mm2; MLA, mean lumen area (μm2). PT2399 collapsed tumour vasculature without decreasing the number of CD31-expressing endothelial cells. f, Top, IHC for Ki67 in tumours harvested from sensitive (XP144 and XP373) or resistant (XP530 and XP506) tumours following treatment with vehicle or PT2399. Bottom, haematoxylin and eosin staining and IHC for CD31 in sensitive tumours (XP373 and XP469) treated with vehicle or PT2399. Scale bars, 100 μm. g, Representative [18F]FLT-PET/CT images of mice with subcutaneous tumourgrafts treated with either vehicle or PT2399. Yellow arrows point to tumours. h, Representative [18F]FLT-PET/CT images of XP144 mice with orthotopic tumours before and after treatment with PT2399 for 10 days. Yellow arrowheads, kidney tumours. White asterisks, intestine. FLT uptake in tumour compared to normal kidney reduced by 19% after 10-day treatment (n = 3; paired t-test, P = 0.001). i, Human and mouse VEGF levels in plasma as determined by ELISA in different treatment groups (vehicle: n = 63; PT2399: n = 74; sunitinib: n = 61). a, i, Tests completed using a mixed model analysis with compound symmetrical covariance structure for mice in the same tumourgraft line using vehicle as the reference group. b, Trend lines were obtained from a mixed model analysis for each response group using an autoregressive (1) covariance structure for the longitudinal measurements on each mouse, compound symmetry for mice within the same tumourgraft line, and controlled for baseline volume. e, Continuous measures were analysed using a mixed model with compound symmetrical covariance structure for mice in the same tumourgraft line and using vehicle treatment as the reference group. Specifically for categorical variables, a binomial test was used to test whether the proportion of tumours affected by PT2399 compared to vehicle was different from 10%. hVEGF and mVEGF levels were Box-Cox transformed; raw values depicted in all graphs. All boxplots have median centre values. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Extended Data Figure 2 Evaluation of the effects of PT2399 on tumours progressing on sunitinib.

a, Tumour volumes in mice from sensitive lines (XP374 or XP144) switched from vehicle or sunitinib to PT2399 as indicated (bottom black arrows). b, Circulating tumour-produced hVEGF levels in mice treated with vehicle, sunitinib, or sunitinib followed by PT2399. The Wilcoxon rank-sum test was used to determine whether sunitinib (n = 4) or sunitinib followed by PT2399 (n = 6) were different from vehicle (n = 4). *P < 0.05. Boxplots have median centre values. c, Representative images of haematoxylin and eosin and Ki67 staining of tumours from mice (XP144) treated with vehicle or sunitinib (left) and from tumours following a switch to PT2399 (right). Scale bars, 100 μm.

Extended Data Figure 3 RNA-seq analyses of vehicle and PT2399-treated tumourgrafts.

a, Unsupervised clustering analyses of all tumourgraft samples (sensitive (S) and resistant (R), both vehicle (V)- and PT2399 (P)-treated) showing clustering by tumourgraft line. b, RNA sequencing in sensitive tumourgrafts evaluating the effects of PT2399 on selected HIF-2 target genes. All tests completed using mixed model analysis with compound symmetrical covariance structure for mice in the same tumourgraft line. Values were log2-transformed for analysis; raw values depicted in all graphs as individual bars.

Extended Data Figure 4 HIF-2α and HIF-1α levels in sensitive and resistant tumourgrafts.

a, HIF-2α and HIF-1α IHC. 786-O cells, which express high levels of HIF-2α, shown as controls. Scale bars, 100 μm. b, Western blot analyses showing heterogeneity within tumours but with overall similar results (compare to Fig. 3c). Green, sensitive; red, resistant. Asterisks, underloaded samples. c, Heatmap from RNA-seq analysis showing differentially expressed genes in sensitive (S) versus resistant (R) tumourgrafts based on uniform cutoff (see Extended Data Table 3). See Supplementary Fig. 1 for gel source images.

Extended Data Figure 5 Evaluation of imaging characteristics of tumours in patients corresponding to sensitive, intermediate, and resistant tumourgrafts.

CT scan images from patient tumours that gave rise to tumourgrafts according to tumourgraft sensitivity to PT2399. Tumours were classified into masses with peripheral hypervascularity and a central non-enhancing area (blue outline), focally infiltrating (brown outline) and diffuse infiltrating (yellow outline). Three of the seven resistant tumours presented as non-mass-like, infiltrative neoplasms (red arrows) whereas another tumour presented with both a largely necrotic renal mass and retroperitoneal lymph nodes (black outline; white arrows).

Extended Data Figure 6 Prolonged disease control in heavily pretreated patient with metastatic ccRCC with sensitive (XP165) tumourgraft.

CT images of selected lesions in patient treated with highly related HIF-2 inhibitor (PT2385) in phase 1 clinical trial showing overall stability in the size of lesions over time. Start of treatment, day 0.

Extended Data Table 1 Tumourgraft features
Extended Data Table 2 RNA sequencing read data
Extended Data Table 3 Number of differentially regulated RNAs across tumourgraft groups by RNA-seq analysis
Extended Data Table 4 Top 15 downregulated and top 15 upregulated pathways in sensitive tumours treated with PT2399

Supplementary information

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This file contains Supplementary Figure 1(gel source data) and Supplementary Figure 2 (Tumor dimensions). (PDF 2694 kb)

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Chen, W., Hill, H., Christie, A. et al. Targeting renal cell carcinoma with a HIF-2 antagonist. Nature 539, 112–117 (2016).

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