Toward a CRISPR-based mouse model of Vhl-deficient clear cell kidney cancer: Initial experience and lessons learned.

CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models. Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective. Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1, and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard-of-care agent, axitinib. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs, are HIF2 independent.

Total loss of VHL gene function impairs neuroendocrine cancer cell fitness due to excessive HIF2α activity.

Loss-of-function germline von Hippel-Lindau (VHL) tumor suppressor mutations cause VHL disease, which predisposes individuals to kidney cancer, hemangioblastomas, and paragangliomas. The risk that a given VHL disease family will manifest some or all these tumor types is profoundly influenced by the VHL allele it carries. For example, almost all VHL disease families that develop paraganglioma have missense VHL mutations. VHL families with null VHL alleles develop kidney cancer and hemangioblastomas without a high risk of paraganglioma. The latter is surprising because the VHL gene product, pVHL, suppresses the HIF2 transcription factor and gain-of-function HIF2 mutations are also linked to paraganglioma. Paragangliomas arise from the sympathetic or parasympathetic nervous system. Given the lack of human paraganglioma cell lines, we studied the effects of inactivating VHL in neuroblastoma cell lines, which also arise from the sympathetic nervous system. We found that total loss of pVHL function profoundly impairs the fitness of neuroblastoma cell lines in a HIF2-dependent manner both ex vivo and in vivo. This fitness defect can be rescued by pVHL variants linked to paraganglioma, but not by pVHL variants associated with a low risk of paraganglioma. These findings suggest that HIF2 activity above a critical threshold prevents the development of paraganglioma.

Induction of DEPP1 by HIF Mediates Multiple Hallmarks of Ischemic Cardiomyopathy.

BACKGROUND: HIF (hypoxia inducible factor) regulates many aspects of cardiac function. We and others previously showed that chronic HIF activation in the heart in mouse models phenocopies multiple features of ischemic cardiomyopathy in humans, including mitochondrial loss, lipid accumulation, and systolic cardiac dysfunction. In some settings, HIF also causes the loss of peroxisomes. How, mechanistically, HIF promotes cardiac dysfunction is an open question. METHODS: We used mice lacking cardiac pVHL (von Hippel-Lindau protein) to investigate how chronic HIF activation causes multiple features of ischemic cardiomyopathy, such as autophagy induction and lipid accumulation. We performed immunoblot assays, RNA sequencing, mitochondrial and peroxisomal autophagy flux measurements, and live cell imaging on isolated cardiomyocytes. We used CRISPR-Cas9 gene editing in mice to validate a novel mediator of cardiac dysfunction in the setting of chronic HIF activation. RESULTS: We identify a previously unknown pathway by which cardiac HIF activation promotes the loss of mitochondria and peroxisomes. We found that DEPP1 (decidual protein induced by progesterone 1) is induced under hypoxia in a HIF-dependent manner and localizes inside mitochondria. DEPP1 is both necessary and sufficient for hypoxia-induced autophagy and triglyceride accumulation in cardiomyocytes ex vivo. DEPP1 loss increases cardiomyocyte survival in the setting of chronic HIF activation ex vivo, and whole-body Depp1 loss decreases cardiac dysfunction in hearts with chronic HIF activation caused by VHL loss in vivo. CONCLUSIONS: Our findings identify DEPP1 as a key component in the cardiac remodeling that occurs with chronic ischemia.

Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.

Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.

von-Hippel Lindau and Hypoxia-Inducible Factor at the Center of Renal Cell Carcinoma Biology.

The most common form of kidney cancer is clear cell renal cell carcinoma (ccRCC). Biallelic VHL tumor suppressor gene inactivation is the usual initiating event in both hereditary (VHL Disease) and sporadic ccRCCs. The VHL protein, pVHL, earmarks the alpha subunits of the HIF transcription factor for destruction in an oxygen-dependent manner. Deregulation of HIF2 drives ccRCC pathogenesis. Drugs inhibiting the HIF2-responsive growth factor VEGF are now mainstays of ccRCC treatment. A first-in-class allosteric HIF2 inhibitor was recently approved for treating VHL Disease-associated neoplasms and appears active against sporadic ccRCC in early clinical trials.

Protocol to establish a genetically engineered mouse model of IDH1-mutant astrocytoma.

Lower-grade gliomas exhibit a high prevalence of isocitrate dehydrogenase 1 (IDH1) mutations, but faithful models for studying these tumors are lacking. Here, we present a protocol to establish a genetically engineered mouse (GEM) model of grade 3 astrocytoma driven by the Idh1R132H oncogene. We describe steps for breeding compound transgenic mice and intracranially delivering adeno-associated virus particles, followed by post-surgical surveillance via magnetic resonance imaging. This protocol enables the generation and use of a GEM to study lower-grade IDH-mutant gliomas. For complete details on the use and execution of this protocol, please refer to Shi et al. (2022).1.

Isocitrate dehydrogenase (IDH) mutant gliomas: A Society for Neuro-Oncology (SNO) consensus review on diagnosis, management, and future directions.

Isocitrate dehydrogenase (IDH) mutant gliomas are the most common adult, malignant primary brain tumors diagnosed in patients younger than 50, constituting an important cause of morbidity and mortality. In recent years, there has been significant progress in understanding the molecular pathogenesis and biology of these tumors, sparking multiple efforts to improve their diagnosis and treatment. In this consensus review from the Society for Neuro-Oncology (SNO), the current diagnosis and management of IDH-mutant gliomas will be discussed. In addition, novel therapies, such as targeted molecular therapies and immunotherapies, will be reviewed. Current challenges and future directions for research will be discussed.

THE JEREMIAH METZGER LECTURE:VON HIPPEL-LINDAU DISEASE: INSIGHTS INTO OXYGEN SENSING, CANCER AND DRUGGING THE UNDRUGGABLE.

Germline VHL mutations predispose to hemangioblastomas of the retina, cerebellum, and spinal cord; clear cell renal cell carcinomas (ccRCCs); and paragangliomas. Consistent with the Knudson two-hit model, somatic biallelic VHL mutations are common in sporadic ccRCCs. The VHL gene product nucleates an ubiquitin ligase that targets the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for proteasomal degradation when oxygen is plentiful. The recognition of HIF↑ by pVHL requires that HIF↑ be hydroxylated on one (or both) of two conserved prolyl residues by the oxygen-dependent EglN (also called PHD) prolyl hydroxylases. HIF↑, bound to HIF↓ (also called ARNT), transcriptionally activates genes that promote adaptation to hypoxia such as VEGF and EPO. Deregulation of HIF, and particularly HIF2, drives pVHL-defective tumorigenesis. EglN inhibitors are being developed for the treatment of anemia and ischemic diseases, whereas HIF2 inhibitors are being developed for the treatment of pVHL-defective tumors. The thalidomide-like drugs ("IMiDs") bind to cereblon, which is the substrate recognition subunit of another ubiquitin ligase that loosely resembles the pVHL ubiquitin ligase. The IMiDs kill multiple myeloma cells by reprogramming the cereblon ligase to earmark the transcription factors IKZF1 and IKZF3 for destruction. This discovery has galvanized interest in developing drugs that degrade otherwise undruggable proteins.

Von Hippel-Lindau disease: insights into oxygen sensing, protein degradation, and cancer.

Germline loss-of-function mutations of the VHL tumor suppressor gene cause von Hippel-Lindau disease, which is associated with an increased risk of hemangioblastomas, clear cell renal cell carcinomas (ccRCCs), and paragangliomas. This Review describes mechanisms involving the VHL gene product in oxygen sensing, protein degradation, and tumor development and current therapeutic strategies targeting these mechanisms. The VHL gene product is the substrate recognition subunit of a ubiquitin ligase that targets the α subunit of the heterodimeric hypoxia-inducible factor (HIF) transcription factor for proteasomal degradation when oxygen is present. This oxygen dependence stems from the requirement that HIFα be prolyl-hydroxylated on one (or both) of two conserved prolyl residues by members of the EglN (also called PHD) prolyl hydroxylase family. Deregulation of HIF, and particularly HIF2, drives the growth of VHL-defective ccRCCs. Drugs that inhibit the HIF-responsive gene product VEGF are now mainstays of ccRCC treatment. An allosteric HIF2 inhibitor was recently approved for the treatment of ccRCCs arising in the setting of VHL disease and has advanced to phase III testing for sporadic ccRCCs based on promising phase I/II data. Orally available EglN inhibitors are being tested for the treatment of anemia and ischemia. Five of these agents have been approved for the treatment of anemia in the setting of chronic kidney disease in various countries around the world.

Mitochondrial remodeling and ischemic protection by G protein-coupled receptor 35 agonists.

Kynurenic acid (KynA) is tissue protective in cardiac, cerebral, renal, and retinal ischemia models, but the mechanism is unknown. KynA can bind to multiple receptors, including the aryl hydrocarbon receptor, the a7 nicotinic acetylcholine receptor (a7nAChR), multiple ionotropic glutamate receptors, and the orphan G protein-coupled receptor GPR35. Here, we show that GPR35 activation was necessary and sufficient for ischemic protection by KynA. When bound by KynA, GPR35 activated Gi- and G12/13-coupled signaling and trafficked to the outer mitochondria membrane, where it bound, apparantly indirectly, to ATP synthase inhibitory factor subunit 1 (ATPIF1). Activated GPR35, in an ATPIF1-dependent and pertussis toxin-sensitive manner, induced ATP synthase dimerization, which prevented ATP loss upon ischemia. These findings provide a rationale for the development of specific GPR35 agonists for the treatment of ischemic diseases.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma.

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

A Mesenchymal Tumor Cell State Confers Increased Dependency on the BCL-XL Antiapoptotic Protein in Kidney Cancer.

PURPOSE: Advanced/metastatic forms of clear-cell renal cell carcinomas (ccRCC) have limited therapeutic options. Genome-wide genetic screens have identified cellular dependencies in many cancers. Using the Broad Institute/Novartis combined short hairpin RNA (shRNA) dataset, and cross-validation with the CRISPR/Cas9 DepMap (21Q3) dataset, we sought therapeutically actionable dependencies in kidney lineage cancers. EXPERIMENTAL DESIGN: We identified preferential genetic dependencies in kidney cancer cells versus other lineages. BCL2L1, which encodes the BCL-XL antiapoptotic protein, scored as the top actionable dependency. We validated this finding using genetic and pharmacologic tools in a panel of ccRCC cell lines. Select BCL-XL-dependent (versus independent) cell lines were then transcriptionally profiled to identify biomarkers and mechanistic drivers of BCL-XL dependence. Cell-based studies (in vitro and in vivo) and clinical validations were used to address physiologic relevance. RESULTS: Inactivation of BCL-XL, but not BCL-2, led to fitness defects in renal cancer cells, and sensitized them to chemotherapeutics. Transcriptomic profiling identified a "BCL-XL dependency" signature, including an elevated mesenchymal gene signature. A mesenchymal state was both necessary and sufficient to confer increased BCL-XL dependence. The "BCL-XL dependency" signature was observed in approximately 30% of human ccRCCs, which were also associated with worse clinical outcomes. Finally, an orally bioavailable BCL-XL inhibitor, A-1331852, showed antitumor efficacy in vivo. CONCLUSIONS: Our studies uncovered an unexpected link between cell state and BCL-XL dependence in ccRCC. Therapeutic agents that specifically target BCL-XL are available. Our work justifies testing the utility of BCL-XL blockade to target, likely, a clinically aggressive subset of human kidney cancers. See related commentary by Wang et al., p. 4600.

Sensitivity of VHL mutant kidney cancers to HIF2 inhibitors does not require an intact p53 pathway.

Inactivation of the VHL tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), which is the most common form of kidney cancer. The VHL tumor suppressor protein marks hypoxia-inducible factor 1 (HIF1) and HIF2 for proteasomal degradation when oxygen is present. The inappropriate accumulation of HIF2 drives tumor formation by VHL tumor suppressor protein (pVHL)­defective ccRCC. Belzutifan, a first-in-class allosteric HIF2 inhibitor, has advanced to phase 3 testing for advanced ccRCC and is approved for ccRCCs arising in patients with VHL disease, which is caused by germline VHL mutations. HIF2 can suppress p53 function in some settings and preliminary data suggested that an intact p53 pathway, as measured by activation in response to DNA damage, was necessary for HIF2 dependence. Here, we correlated HIF2 dependence and p53 status across a broader collection of ccRCC cell lines. We also genetically manipulated p53 function in ccRCC lines that were or were not previously HIF2-dependent and then assessed their subsequent sensitivity to HIF2 ablation using CRISPR-Cas9 or the HIF2 inhibitor PT2399, which is closely related to belzutifan. From these studies, we conclude that p53 status does not dictate HIF2 dependence, at least in preclinical models, and thus is unlikely to be a useful biomarker for predicting which ccRCC patients will respond to HIF2 inhibitors.

From Basic Science to Clinical Translation in Kidney Cancer: A Report from the Second Kidney Cancer Research Summit.

The second Kidney Cancer Research Summit was held virtually in October 2020. The meeting gathered worldwide experts in the field of kidney cancer, including basic, translational, and clinical scientists as well as patient advocates. Novel studies were presented, addressing areas of unmet need related to different topics. These include novel metabolic targets, promising immunotherapeutic regimens, predictive genomic and transcriptomic biomarkers, and variant histologies of renal cell carcinoma (RCC). With the development of pioneering technologies, and an unprecedented commitment to kidney cancer research, the field has tremendously evolved. This perspective aims to summarize the different sessions of the conference, outline major advances in the understanding of RCC and discuss current challenges faced by the field.

Belzutifan, a Potent HIF2α Inhibitor, in the Pacak-Zhuang Syndrome.

The integration of genomic testing into clinical care enables the use of individualized approaches to the management of rare diseases. We describe the use of belzutifan, a potent and selective small-molecule inhibitor of the protein hypoxia-inducible factor 2α (HIF2α), in a patient with polycythemia and multiple paragangliomas (the Pacak-Zhuang syndrome). The syndrome was caused in this patient by somatic mosaicism for an activating mutation in EPAS1. Treatment with belzutifan led to a rapid and sustained tumor response along with resolution of hypertension, headaches, and long-standing polycythemia. This case shows the application of a targeted therapy for the treatment of a patient with a rare tumor-predisposition syndrome. (Funded by the Morin Family Fund for Pediatric Cancer and Alex's Lemonade Stand Foundation.).

Targeting oncoproteins with a positive selection assay for protein degraders.

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.