HIF2 Inactivation and Tumor Suppression with a Tumor-Directed RNA-Silencing Drug in Mice and Humans.

PURPOSE: HIF2α is a key driver of kidney cancer. Using a belzutifan analogue (PT2399), we previously showed in tumorgrafts (TG) that ∼50% of clear cell renal cell carcinomas (ccRCC) are HIF2α dependent. However, prolonged treatment induced resistance mutations, which we also identified in humans. Here, we evaluated a tumor-directed, systemically delivered, siRNA drug (siHIF2) active against wild-type and resistant-mutant HIF2α. EXPERIMENTAL DESIGN: Using our credentialed TG platform, we performed pharmacokinetic and pharmacodynamic analyses evaluating uptake, HIF2α silencing, target gene inactivation, and antitumor activity. Orthogonal RNA-sequencing studies of siHIF2 and PT2399 were pursued to define the HIF2 transcriptome. Analyses were extended to a TG line generated from a study biopsy of a siHIF2 phase I clinical trial (NCT04169711) participant and the corresponding patient, an extensively pretreated individual with rapidly progressive ccRCC and paraneoplastic polycythemia likely evidencing a HIF2 dependency. RESULTS: siHIF2 was taken up by ccRCC TGs, effectively depleted HIF2α, deactivated orthogonally defined effector pathways (including Myc and novel E2F pathways), downregulated cell cycle genes, and inhibited tumor growth. Effects on the study subject TG mimicked those in the patient, where HIF2α was silenced in tumor biopsies, circulating erythropoietin was downregulated, polycythemia was suppressed, and a partial response was induced. CONCLUSIONS: To our knowledge, this is the first example of functional inactivation of an oncoprotein and tumor suppression with a systemic, tumor-directed, RNA-silencing drug. These studies provide a proof-of-principle of HIF2α inhibition by RNA-targeting drugs in ccRCC and establish a paradigm for tumor-directed RNA-based therapeutics in cancer.

ImmunoPET Imaging with 89Zr-Labeled Atezolizumab Enables In Vivo Evaluation of PD-L1 in Tumorgraft Models of Renal Cell Carcinoma.

PURPOSE: Immune checkpoint inhibitors (ICI) targeting the programmed cell death protein 1 and its ligand (PD-1/PD-L1) have transformed the treatment paradigm for metastatic renal cell carcinoma (RCC). However, response rates to ICIs as single agents or in combination vary widely and predictive biomarkers are lacking. Possibly related to the heterogeneity and dynamic nature of PD-L1 expression, tissue-based methods have shown limited value. Immuno-positron emission tomography (immunoPET) may enable noninvasive, comprehensive, and real-time PD-L1 detection. Herein, we systematically examined the performance of immunoPET for PD-L1 detection relative to IHC in an RCC patient-derived tumorgraft (TG) platform. EXPERIMENTAL DESIGN: Eight independent RCC TGs with a wide range of PD-L1 expression (0%-85%) were evaluated by immunoPET. Uptake of 89Zr-labeled atezolizumab ([89Zr]Zr-DFO-ATZ) was compared with PD-L1 expression in tumors by IHC through double-blind analyses. Clinical outcomes of ICI-treated patients whose TGs were examined were analyzed to evaluate the clinical role of immunoPET in RCC. RESULTS: ImmunoPET with [89Zr]Zr-DFO-ATZ (day 6/7 postinjection) revealed a statistically significant association with PD-L1 IHC assays (P = 0.0014; correlation ρXY = 0.78). Furthermore, immunoPET can be used to assess the heterogeneous distribution of PD-L1 expression. Finally, studies in the corresponding patients (n = 4) suggest that PD-L1 signal may influence ICI responsiveness. CONCLUSIONS: ImmunoPET with [89Zr]Zr-DFO-ATZ may enable a thorough and dynamic assessment of PD-L1 across sites of disease. The power of immunoPET to predict ICI response in RCC is being explored in an ongoing clinical trial (NCT04006522).

A renal cell carcinoma tumorgraft platform to advance precision medicine.

Renal cell carcinoma (RCC) encompasses a heterogenous group of tumors, but representative preclinical models are lacking. We previously showed that patient-derived tumorgraft (TG) models recapitulate the biology and treatment responsiveness. Through systematic orthotopic implantation of tumor samples from 926 ethnically diverse individuals into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we generate a resource comprising 172 independently derived, stably engrafted TG lines from 148 individuals. TG lines are characterized histologically and genomically (whole-exome [n = 97] and RNA [n = 102] sequencing). The platform features a variety of histological and oncogenotypes, including TCGA clades further corroborated through orthogonal metabolomic analyses. We illustrate how it enables a deeper understanding of RCC biology; enables the development of tissue- and imaging-based molecular probes; and supports advances in drug development.

Determinants of renal cell carcinoma invasion and metastatic competence.

Metastasis is the principal cause of cancer related deaths. Tumor invasion is essential for metastatic spread. However, determinants of invasion are poorly understood. We addressed this knowledge gap by leveraging a unique attribute of kidney cancer. Renal tumors invade into large vessels forming tumor thrombi (TT) that migrate extending sometimes into the heart. Over a decade, we prospectively enrolled 83 ethnically-diverse patients undergoing surgical resection for grossly invasive tumors at UT Southwestern Kidney Cancer Program. In this study, we perform comprehensive histological analyses, integrate multi-region genomic studies, generate in vivo models, and execute functional studies to define tumor invasion and metastatic competence. We find that invasion is not always associated with the most aggressive clone. Driven by immediate early genes, invasion appears to be an opportunistic trait attained by subclones with diverse oncogenomic status in geospatial proximity to vasculature. We show that not all invasive tumors metastasize and identify determinants of metastatic competency. TT associated with metastases are characterized by higher grade, mTOR activation and a particular immune contexture. Moreover, TT grade is a better predictor of metastasis than overall tumor grade, which may have implications for clinical practice.

Complement as Prognostic Biomarker and Potential Therapeutic Target in Renal Cell Carcinoma.

Preclinical studies demonstrated that complement promotes tumor growth. Therefore, we sought to determine the best target for complement-based therapy among common human malignancies. High expression of 11 complement genes was linked to unfavorable prognosis in renal cell carcinoma. Complement protein expression or deposition was observed mainly in stroma, leukocytes, and tumor vasculature, corresponding to a role of complement in regulating the tumor microenvironment. Complement abundance in tumors correlated with a high nuclear grade. Complement genes clustered within an aggressive inflammatory subtype of renal cancer characterized by poor prognosis, markers of T cell dysfunction, and alternatively activated macrophages. Plasma levels of complement proteins correlated with response to immune checkpoint inhibitors. Corroborating human data, complement deficiencies and blockade reduced tumor growth by enhancing antitumor immunity and seemingly reducing angiogenesis in a mouse model of kidney cancer resistant to PD-1 blockade. Overall, this study implicates complement in the immune landscape of renal cell carcinoma, and notwithstanding cohort size and preclinical model limitations, the data suggest that tumors resistant to immune checkpoint inhibitors might be suitable targets for complement-based therapy.

HIF-2 Complex Dissociation, Target Inhibition, and Acquired Resistance with PT2385, a First-in-Class HIF-2 Inhibitor, in Patients with Clear Cell Renal Cell Carcinoma.

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 α subunit, which heterodimerizes with HIF1ß, 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 = 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNA-sequencing 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 HIF2α, 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).

PD-L1 detection using 89Zr-atezolizumab immuno-PET in renal cell carcinoma tumorgrafts from a patient with favorable nivolumab response.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression in metastatic renal cell carcinoma (RCC) correlates with a worse prognosis, but whether it also predicts responsiveness to anti-PD-1/PD-L1 therapy remains unclear. Most studies of PD-L1 are limited by evaluation in primary rather than metastatic sites, and in biopsy samples, which may not be representative. These limitations may be overcome with immuno-positron emission tomography (iPET), an emerging tool allowing the detection of cell surface proteins with radiolabeled antibodies. Here, we report iPET studies of PD-L1 in a preclinical tumorgraft model of clear cell RCC (ccRCC) from a patient who had a favorable response to anti-PD-1 therapy. CASE PRESENTATION: A 49-year-old man underwent a cytoreductive nephrectomy in 2017 of a right kidney tumor invading into the adrenal gland that was metastatic to the lungs and a rib. Histological analyses revealed a ccRCC of ISUP grade 4 with extensive sarcomatoid features. IMDC risk group was poor. Within two hours of surgery, a tumor sample was implanted orthotopically into NOD/SCID mice. Consistent with an aggressive tumor, a renal mass was detected 18 days post-implantation. Histologically, the tumorgraft showed sarcomatoid differentiation and high levels of PD-L1, similar to the patient's tumor. PD-L1 was evaluated in subsequently transplanted mice using iPET and the results were compared to control mice implanted with a PD-L1-negative tumor. We labeled atezolizumab, an anti-PD-L1 antibody with a mutant Fc, with zirconium-89. iPET revealed significantly higher 89Zr-atezolizumab uptake in index than control tumorgrafts. The patient was treated with high-dose IL2 initially, and subsequently with pazopanib, with rapidly progressive disease, but had a durable response with nivolumab. CONCLUSIONS: To our knowledge, this is the first report of non-invasive detection of PD-L1 in renal cancer using molecular imaging. This study supports clinical evaluation of iPET to identify RCC patients with tumors deploying the PD-L1 checkpoint pathway who may be most likely to benefit from PD-1/PD-L1 disrupting drugs.