Normally induced by hypoxia, hypoxia-inducible factor 2 alpha (HIF2a) is arguably the most important driver of kidney cancer. HIF2a is constitutively activated following von Hippel-Lindau (VHL) gene inactivation, which is the signature event of the most common type of kidney cancer, clear cell renal cell carcinoma (ccRCC). HIF2a functions as a heterodimeric transcription factor in partnership with the constitutive HIF1b subunit and regulates a program of gene expression that promotes cell proliferation, stemness, and angiogenesis. While as a transcription factor HIF2a had escaped drug targeting, structural studies revealed an unusual cavity, which became the foundation for the development of small molecule inhibitors such as PT2385 (a first-in-class drug), or the related PT2399 tool compound and the recently FDA-approved PT2977 (also called belzutifan). PT drugs bind a small pocket in the PAS-B domain of HIF2a inducing a conformational change that triggers dissociation from its obligatory partner HIF1b. PT drugs are highly specific - they do not bind the close paralog HIF1a and do not induce changes in gene expression in cells devoid of HIF2a. Using an extensive library of patient-derived xenografts (PDXs), we previously showed that PT drugs have activity against 50% of ccRCCs implanted in mice, and similar observations were made in the clinic. Perhaps unsurprisingly, sensitive tumors showed higher HIF2a levels. Here, we leverage the specificity of PT2385 to develop a HIF2a tracer for positron emission tomography (PET). By substituting a native fluorine atom for 18F, we generated [18F]PT2385. [18F]PT2385 was able to discriminate HIF2a-expressing ccRCCs from tumors that did not express HIF2a in mice simultaneously implanted with both. These data set the foundation for an investigator new drug (IND) approval from the FDA, and a clinical trial that is currently accruing patients (NCT04989959). [18F]PT2385 PET may have applications in identifying kidney cancer patients most likely to respond to HIF2a-targeted therapies, the identification of other tumors relying on HIF2a, and beyond oncology. Reporting on a hypoxia sensor, a HIF2a radiotracer may be a useful ischemia probe. In summary, we report the development of a novel radiotracer with extensive potential applications currently being evaluated in humans. Citation Format: Sashi Debnath, Christina Stevens, Olivia Brandenburg, Justin Sovich, Paulina Gonzalez, Qian (Janie) Qin, Sydney Haldeman, Vanina Toffessi Tcheuyap, Alana Christie, Pawan Thapa, Ning Zhou, Aditi Mulgaonkar, Guiyang Hao, Jeffrey Miyata, Deyssy Carrillo, Jeffrey Cadeddu, Payal Kapur, Jon Anderson, Ivan Pedrosa, Marianna Dakanali, Orhan Oz, Xiankai Sun, James Brugarolas. Development of a novel HIF2a PET tracer: From proof of concept to a clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2478.

Hypoxia-inducible factor 2 alpha (HIF2a) is arguably the most important driver of kidney cancer. HIF2a is constitutively activated following von Hippel-Lindau (VHL) gene inactivation, the signature event of the most common type of kidney cancer, clear cell renal cell carcinoma (ccRCC). HIF2a functions as a heterodimeric transcription factor and regulates a program of gene expression that promotes cell proliferation, stemness, and angiogenesis. Using a highly specific inhibitor designed to target a structural vulnerability in HIF2a (PT2399), we previously showed that approximately 50% of ccRCCs are dependent on HIF2a. However, prolonged drug exposure results in resistance and the acquisition of gatekeeper mutations, which we reported first in patient-derived xenografts (PDXs) and subsequently in humans. Using the same PDX platform that previously validated PT2399, we show that HIF2a can be effectively inhibited using a tumor-directed siRNA (siHIF2). Referring herein to both first- and second-generation (ARO-HIF2) siRNA drugs, siHIF2 is specifically taken up by human ccRCC tumors transplanted in mice, where it depletes HIF2a inhibiting target gene expression and tumor growth. Through orthogonal RNA-seq studies integrating both PT2399 and siHIF2 in PDXs, we provide unprecedented detail on the HIF2a effector transcriptome, which we further dissect by incorporating ChIP-seq. A PDX line was generated from a ccRCC patient who had paraneoplastic polycythemia (a HIF2a dependent syndrome due to erythropoietin [Epo] secretion by the tumor) and participated in the phase I trial of ARO-HIF2 (NCT04169711). We show that siHIF2 effectively depleted HIF2a in both the PDX as well as in the patient, that it normalized Epo (and hemoglobin), and that it inhibited tumor growth. siHIF2 has activity against both wild-type and drug-resistant mutant HIF2a and is expected to be active in patients progressing on PT2977 (belzutifan), a PT2399-related drug recently approved by the FDA. To our knowledge, this is the first example of functional inactivation of an oncoprotein with a tumor-directed siRNA in humans. In summary, these studies provide unique insight into HIF2a (the only known core dependency in ccRCC), illustrate how it can be effectively inhibited by an siRNA drug, and establish a paradigm for the development of tumor directed siRNA-based therapeutics. Citation Format: Yuanqing Ma, Christina Stevens, Olivia Brandenburg, Vanina Toffessi Tcheuyap, Quyen N. Do, Faeze Saatchi, Tanner Hardy, Oluwatomilade Fatunde, Alyssa Macchiaroli, Jeffrey Miyata, Deyssy Carrillo, Thomas Schluep, So Wong, Alana Christie, Payal Kapur, Ivan Pedrosa, James Hamilton, James Brugarolas. Targeting HIF2a with siRNA: From preclinical models to the clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6304.