Clear cell Renal Cell Carcinoma (ccRCC) is characterized by VHL inactivation1,2. Because no other gene is mutated as frequently, and VHL mutations are truncal3, VHL inactivation is regarded as the governing event4. VHL loss activates HIF-2, and constitutive HIF-2 restores tumorigenesis in VHL-reconstituted ccRCC cells5. HIF-2 is implicated in angiogenesis and multiple other processes6–9, but angiogenesis is the main target of drugs like sunitinib10. HIF-2, a transcription factor, has been regarded as undruggable11. A structure-based design approach identified a selective HIF-2 antagonist (PT2399) that we evaluate using a tumorgraft (TG)/PDX platform12,13. PT2399 dissociated HIF-2 (an obligatory heterodimer [HIF-2α/HIF-1β])14 in human ccRCC suppressing tumorigenesis in 56% (10/18) lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumors, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant. Resistance occurred despite HIF-2 dissociation in tumors 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 tumors. Illustrating drug specificity, gene expression was largely unaffected by PT2399 in resistant tumors. Sensitive tumors exhibited a distinguishing gene expression signature, and generally higher HIF-2α levels. Prolonged PT2399 treatment led to resistance. We identified a binding site and second site suppressor mutation in HIF-2α and HIF-1β respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient with a sensitive TG had disease control for >11 months with the close analogue PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCC are, unexpectedly, HIF-2 independent, and set the stage for biomarker-driven clinical trials.

By leveraging tumorgraft (patient-derived xenograft) RNA-sequencing data, we developed an empirical approach, DisHet, to dissect the tumor microenvironment (eTME). We found that 65% of previously defined immune signature genes are not abundantly expressed in renal cell carcinoma (RCC) and identified 610 novel immune/stromal transcripts. Using eTME, genomics, pathology, and medical record data involving >1,000 patients, we established an inflamed pan-RCC subtype (IS) enriched for regulatory T cells, natural killer cells, T1 cells, neutrophils, macrophages, B cells, and CD8 T cells. IS is enriched for aggressive RCCs, including -deficient clear-cell and type 2 papillary tumors. The IS subtype correlated with systemic manifestations of inflammation such as thrombocytosis and anemia, which are enigmatic predictors of poor prognosis. Furthermore, IS was a strong predictor of poor survival. Our analyses suggest that tumor cells drive the stromal immune response. These data provide a missing link between tumor cells, the TME, and systemic factors. We undertook a novel empirical approach to dissect the renal cell carcinoma TME by leveraging tumorgrafts. The dissection and downstream analyses uncovered missing links between tumor cells, the TME, systemic manifestations of inflammation, and poor prognosis. .

Purpose: The heterodimeric transcription factor HIF-2 is arguably the most important driver of clear cell renal cell carcinoma (ccRCC). Although considered undruggable, structural analyses at the University of Texas Southwestern Medical Center (UTSW, Dallas, TX) identified a vulnerability in the a subunit, which heterodimerizes with HIF1b, ultimately leading to the development of PT2385, a first-in-class inhibitor. PT2385 was safe and active in a first-in-human phase I clinical trial of patients with extensively pretreated ccRCC at UTSW and elsewhere. There were no dose-limiting toxicities, and disease control !4 months was achieved in 42% of patients.Patients and Methods: We conducted a prospective companion substudy involving a subset of patients enrolled in the phase I clinical trial at UTSW (n ¼ 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNAsequencing studies.Results: PT2385 inhibited HIF-2 in nontumor tissues, as determined by a reduction in erythropoietin levels (a pharmacodynamic marker), in all but one patient, who had the lowest drug concentrations. PT2385 dissociated HIF-2 complexes in ccRCC metastases, and inhibited HIF-2 target gene expression. In contrast, HIF-1 complexes were unaffected. Prolonged PT2385 treatment resulted in the acquisition of resistance, and we identified a gatekeeper mutation (G323E) in HIF2a, which interferes with drug binding and precluded HIF-2 complex dissociation. In addition, we identified an acquired TP53 mutation elsewhere, suggesting a possible alternate mechanism of resistance.Conclusions: These findings demonstrate a core dependency on HIF-2 in metastatic ccRCC and establish PT2385 as a highly specific HIF-2 inhibitor in humans. New approaches will be required to target mutant HIF-2 beyond PT2385 or the closely related PT2977 (MK-6482).

Recent experiments have demonstrated that O6-alkylguanine is rapidly removed from hepatocyte DNA following continuous exposure to 1,2-dimethylhydrazine or diethylnitrosamine. In contrast, O4-ethyldeoxythymidine accumulates to concentrations more than 50 times greater than O6-ethyldexyguanosine. Studies on the formation and persistence of O4-methyldeoxythymidine in vivo have not been reported. This study reports the development of sensitive radioimmune assays to O4-methyldeoxythymidine and O4-ethyldeoxythymidine. Utilizing this method, the accumulation and removal of O4-methyldeoxythymidine and O4-ethyldeoxythymidine in liver DNA from rats exposed to 1,2-dimethylhydrazine or diethylnitrosamine were measured. The results demonstrated that O4-methyldeoxythymidine was formed at an O6-methylguanine/O4-methyldeoxythymidine ratio of approximately 100/1 and was repaired with a half-time of approximately 20 h. In contrast, O4-ethyldeoxythymidine removal was 13 times slower with a t 1/2 of approximately 11 days after both pulse dose and cessation of continuous DEN administration. Combined with previously reported data, results presented here suggest that (i) despite a lower rate of formation, O4-methyldeoxythymidine becomes nearly equal in importance to O6-methylguanine as a promutagenic adduct in hepatocytes from continuously exposed rats and (ii) differential repair of O4-alkylthymidine adducts provides a mechanism that may explain in part the superior ability of ethylating versus methylating agents to induce hepatocellular carcinomas in the rat.

Highlights d Generation of a large PDX library from a diverse population d The PDX library is characterized by next-generation sequencing (exome and RNA-seq) d Interactive tool for selecting TG lines representative of RCC molecular subtypes d Precision diagnostics and therapeutic applications illustrated