More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1a and HIF2a, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2a that allosterically blocks its dimerization with the

Calcitriol (1, 25-dihydroxycholecalciferol), the major active form of vitamin D, is anti-proliferative in tumor cells and tumor-derived endothelial cells (TDEC). These actions of calcitriol are mediated at least in part by vitamin D receptor (VDR), which is expressed in many tissues including endothelial cells. To investigate the role of VDR in calcitriol effects on tumor vasculature, we established TRAMP-2 tumors subcutaneously into either VDR wild type (WT) or knockout (KO) mice. Within 30 days post inoculation, tumors in KO mice were larger than those in WT (P<0.001). TDEC from WT expressed VDR and were able to transactivate a reporter gene whereas TDEC from KO mice were not. Treatment with calcitriol resulted in growth inhibition in TDEC expressing VDR. However, TDEC from KO mice were relatively resistant, suggesting that calcitriol-mediated growth inhibition on TDEC is VDR-dependent. Further analysis of the TRAMP-C2 tumor sections revealed that the vessels in KO mice were enlarged and had less pericyte coverage compared to WT (P<0.001). Contrast-enhanced MRI demonstrated an increase in vascular volume of TRAMP tumors grown in VDR KO mice compared to WT mice (P<0.001) and FITC-dextran permeability assay suggested a higher extent of vascular leakage in tumors from KO mice. Using ELISA and Western blot analysis, there was an increase of HIF-1 alpha, VEGF, Ang1 and PDGF-BB levels observed in tumors from KO mice. These results indicate that calcitriol-mediated anti-proliferative effects on TDEC are VDR dependent and loss of VDR can lead to abnormal tumor angiogenesis.

Recent evidence demonstrates that the androgen receptor (AR) continues to influence prostate cancer growth despite medical therapies that reduce circulating androgen ligands to castrate levels and͞or block ligand binding. Whereas the mutation, amplification, overexpression of AR, or cross-talk between AR and other growth factor pathways may explain the failure of androgen ablation therapies in some cases, there is little evidence supporting a causal role between AR and prostate cancer. In this study, we functionally and directly address the role whereby AR contributes to spontaneous cancer progression by generating transgenic mice expressing (i) AR-WT to recapitulate increased AR levels and ligand sensitivity, (ii) AR-T857A to represent a promiscuous AR ligand response, and (iii) AR-E231G to model altered AR function. Whereas transgenes encoding either AR-WT or AR-T857A did not cause prostate cancer when expressed at equivalent levels, expression of AR-E231G, which carries a mutation in the most highly conserved signature motif of the NH 2-terminal domain that also influences interactions with cellular coregulators, caused rapid development of prostatic intraepithelial neoplasia that progressed to invasive and metastatic disease in 100% of mice examined. Taken together, our data now demonstrate the oncogenic potential of steroid receptors and implicate altered AR function and receptor coregulator interaction as critical determinants of prostate cancer initiation, invasion, and metastasis.

We have used the autochthonous transgenic adenocarcinoma of mouse prostate (TRAMP) model to investigate the relationship between somatic mutation in the androgen receptor (AR) and the emergence of androgenindependent prostate cancer. Here we report the identification, isolation, and characterization of distinct classes of AR variants from spontaneous prostate tumors in the TRAMP model. Using cDNA cloning, single stranded conformation polymorphism and sequencing strategies, 15 unique somatic mutations in the AR were identified in prostate tumors obtained from eight TRAMP mice between 24 and 29 weeks of age. At least one mutation was isolated from each mouse. All mutations were single base substitutions, 10 were missense and 5 were silent. Nine mutations in the AR were identified in tumors of four mice that were castrated at 12 weeks of age. Interestingly, the majority of mutations (seven out of nine, 78%) identified in the androgen-independent tumors colocalized in the AR transactivation domain. The remaining mutations colocalized in the AR ligand binding domain. In general, the AR variants demonstrated promoter-, cell-, and cofactor-specific activities in response to various hormones. All AR variants isolated in this study maintained strong sensitivity for androgens, and four AR variants isolated from castrated mice demonstrated increased activities in the absence of ligand. The K638M and F677S variants demonstrated increased activities in response to androgen, and K638M also demonstrated increased response to estradiol. In the presence of AR coactivator ARA70 the E231G variant demonstrated increased activity in response to both androgen and estradiol. However, in the presence of AR coactivator ARA160 the E231G variant was selectively responsive to androgen. Collectively these analyses not only indicate that somatic mutations in the AR gene occur spontaneously in TRAMP tumors but also how changes in the hormonal environment may drive the selection of spontaneous somatic mutations that provide a growth advantage.

HIF-2α, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the overaccumulation of HIF-2α protein, often by inactivation of the E3 ligase VHL (von Hippel−Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2α antagonist to enter clinical trials. Highlights include the use of a putative n → π* Ar interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC 50 = 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.

The hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α inhibitor PT2385 demonstrated promising proof of concept clinical activity in heavily pretreated advanced ccRCC patients. However, PT2385 was restricted by variable and dose-limited pharmacokinetics resulting from extensive metabolism of PT2385 to its glucuronide metabolite. Herein we describe the discovery of second-generation HIF-2α inhibitor PT2977 with increased potency and improved pharmacokinetic profile achieved by reduction of phase 2 metabolism. Structural modification by changing the geminal difluoro group in PT2385 to a vicinal difluoro group resulted in enhanced potency, decreased lipophilicity, and significantly improved pharmacokinetic properties. In a phase 1 dose-escalation study, the clinical pharmacokinetics for PT2977 supports the hypothesis that attenuating the rate of glucuronidation would improve exposure and reduce variability in patients. Early evidence of clinical activity shows promise for PT2977 in the treatment of ccRCC.

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The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.