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Insights into the role of the tumor suppressor pVHL in oxygen sensing motivated the testing of drugs that target the transcription factor HIF or HIF-responsive growth factors, such as VEGF, for the treatment of cancers caused by VHL inactivation, such as clear-cell renal cell carcinoma (ccRCC). Multiple VEGF inhibitors are now approved for the treatment of ccRCC, and a HIF2α inhibitor has advanced to phase 3 development for this disease. These inhibitors are now also increasingly combined with immune-checkpoint blockers. In this Perspective, we describe the understanding of the mechanisms of oxygen sensing and hypoxia signaling that resulted in the development of HIF2α-targeted therapies for patients with VHL-associated tumors. We also present future directions for extending the use of these therapies to other cancers. Anticancer therapies that target the HIF oxygen-sensing pathways are moving into the clinic, in particular in kidney 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 a 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 HIFa 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.

Key Points Scientific robustness refers to the ability of a finding to withstand experimental variation. Results that are reproducible, but only under an extremely narrow set of conditions, are unlikely to make predictions that will be true (robust) under real-world conditions, such as in the clinic. Whether an elevated level of a particular protein is associated with a poor prognosis in a given cancer provides very little information as to whether that protein would be a good target in that cancer. Being associated with a poor prognosis is neither necessary nor sufficient to be a good cancer target. The fact that A correlates with B and that it is biologically plausible that A causes B does not formally prove that A causes B. For example, observing that high expression of a gene correlates with poor survival in cancer patients and knowing that that gene regulates malignant cell behaviour would not formally prove that the high expression of that gene is responsible for the poor survival. Similarly, observing that a drug is having its expected pharmacodynamic effect on its intended target and knowing that its intended target is important for cancer cell survival would not formally prove that the cytotoxicity of the drug is on-target. Most of the cellular assays used in cancer pharmacology are 'down' rather than 'up' assays, which is problematic because there are far more uninteresting ways to make a complex system, such as a cell, perform less well than there are to make it work better. Cellular phenotypes caused by a chemical or genetic perturbant should be considered to be off-target until proved otherwise, especially when the phenotypes were detected in a down assay and therefore could reflect a nonspecific loss of cellular fitness. It is only by performing rescue experiments that one can formally address whether the effects of a perturbant are on-target. The basis for the therapeutic indices of the currently available cancer drugs, including cytotoxic and targeted agents, is still poorly understood. Most successful drugs do not inhibit their targets completely and continuously at their therapeutically useful doses and accurately predicting, a priori, the therapeutic index for inhibition of a new cancer target is virtually impossible. This Perspective discusses some of the causes of the robustness and reproducibility problem in preclinical cancer research and suggests solutions. An alarming number of papers from laboratories nominating new cancer drug targets contain findings that cannot be reproduced by others or are simply not robust enough to justify drug discovery efforts. This problem probably has many causes, including an underappreciation of the danger of being misled by off-target effects when using pharmacological or genetic perturbants in complex biological assays. This danger is particularly acute when, as is often the case in cancer pharmacology, the biological phenotype being measured is a 'down' readout (such as decreased proliferation, decreased viability or decreased tumour growth) that could simply reflect a nonspecific loss of cellular fitness. These problems are compounded by multiple hypothesis testing, such as when candidate targets emerge from high-throughput screens that interrogate multiple targets in parallel, and by a publication and promotion system that preferentially rewards positive findings. In this Perspective, I outline some of the common pitfalls in preclinical cancer target identification and some potential approaches to mitigate them.

2-Oxoglutarate-dependent dioxygenases (2OGDDs) are a superfamily of enzymes that play diverse roles in many biological processes, including regulation of hypoxia-inducible factor-mediated adaptation to hypoxia, extracellular matrix formation, epigenetic regulation of gene transcription and the reprogramming of cellular metabolism. 2OGDDs all require oxygen, reduced iron and 2-oxoglutarate (also known as α-ketoglutarate) to function, although their affinities for each of these co-substrates, and hence their sensitivity to depletion of specific co-substrates, varies widely. Numerous 2OGDDs are recurrently dysregulated in cancer. Moreover, cancer-specific metabolic changes, such as those that occur subsequent to mutations in the genes encoding succinate dehydrogenase, fumarate hydratase or isocitrate dehydrogenase, can dysregulate specific 2OGDDs. This latter observation suggests that the role of 2OGDDs in cancer extends beyond cancers that harbour mutations in the genes encoding members of the 2OGDD superfamily. Herein, we review the regulation of 2OGDDs in normal cells and how that regulation is corrupted in cancer.This Review discusses the metabolic regulation of 2-oxoglutarate-dependent dioxygenases (2OGDDs) and how dysregulation of 2OGDDs in cancer, by genetic aberrations or environmental factors including hypoxia and/or the action of oncometabolites, can contribute to tumour development and growth.

The ( R )-enantiomer of 2-hydroxyglutarate, which is produced when IDH is mutated in human tumours, is shown to stimulate the activity of the EGLN prolyl 4-hydroxylases, leading to diminished levels of HIF and enhanced human astrocyte proliferation. Cancer induction by isocitrate dehydrogenase mutation Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 have been identified in gliomas, the most common form of brain tumour, and in other cancers including leukaemias. The mutated enzymes produce 2-hydroxyglutarate (2HG), which is a potential oncometabolite. Three papers in this issue of Nature examine the mechanisms through which IDH mutations promote cancers. Lu et al . show that 2HG-producing IDH mutants can prevent the histone demethylation that is required for progenitor cells to differentiate, potentially contributing to tumour-cell accumulation. Turcan et al . show that IDH1 mutation in primary human astrocytes induces DNA hypermethylation and reshapes the methylome to resemble that of the CIMP phenotype, a common feature of gliomas and other solid tumours. Koivunen et al . show that the ( R )-enantiomer of 2HG (but not the ( S )-enantiomer) can stimulate the activity of the EGLN prolyl 4-hydroxylases, leading to diminished levels of hypoxia-inducible factor (HIF), which in turn can enhance cell proliferation. These papers establish a framework for understanding gliomagenesis and highlight the interplay between genomic and epigenomic changes in human cancers. The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation 1 . Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the ( R )-enantiomer of 2-hydroxyglutarate (( R )-2HG) 2 , 3 . Here we show that ( R )-2HG, but not ( S )-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the ( R )-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.

Key Points Germline mutations that inactivate the VHL tumour suppressor gene cause a variety of tumours including clear cell renal carcinomas, haemangioblastomas and pheochromocytomas. VHL mutations are also common in sporadic clear cell renal carcinomas and haemangioblastomas. The product of VHL has multiple functions, including directing the polyubiquitylation of hypoxia-inducible factor-α (HIFα). Recognition by VHL requires that the HIFα subunit be modified by O 2 -dependent prolyl hydroxylase (Phd) family members. Inappropriate accumulation of HIFα, and especially the HIF2α subunit, has a causal role in VHL −/− renal carcinomas and its involvement is suspected in VHL −/− haemangioblastomas. Genotype–phenotype correlations suggest that these two tumours differ with respect to the level of HIFα activation required for tumorigenesis. Hypomorphic VHL , hypomorphic PHD2 and hypermorphic HIF2 α mutations have been linked to familial polycythaemia. VHL binds to microtubules and is required for maintenance of a specialized structure called the primary cilium. Loss of this activity probably contributes to the development of visceral cysts in VHL disease. Pheochromocytomas are intra-adrenal paragangliomas (sympathetic nervous system tumours). The genes linked to familial paraganglioma, including VHL , NF1 , RET and succinate dehydrogenase subunit genes, encode proteins that regulate neuronal apoptosis in response to loss of growth factors such as nerve growth factor. Deregulation of hypoxia-inducible factor (HIF) is an established feature of tumours that develop in patients with von Hippel–Lindau disease, caused by inactivating germline mutations of the VHL tumour suppressor gene. However, HIF-independent activities of VHL also seem to be important for the pathogenesis of the disease. The von Hippel–Lindau disease is caused by inactivating germline mutations of the VHL tumour suppressor gene and is associated with an increased risk of a variety of tumours in an allele-specific manner. The role of the heterodimeric transcription factor hypoxia-inducible factor (HIF) in the pathogenesis of VHL-defective tumours has been more firmly established during the past 5 years. In addition, there is now a greater appreciation of HIF-independent VHL functions that are relevant to tumour development, including maintenance of the primary cilium, regulation of extracellular matrix formation and turnover, and modulation of cell death in certain cell types following growth factor withdrawal or in response to other forms of stress.

Key Points Many chemicals kill cancer cells but their toxicity to normal cells limits their usefulness as anticancer drugs. Epigenetic and genetic alterations within cancer cells, as well as changes in their microenvironment, might increase their requirement for a particular molecular target (or targets) relative to normal cells, creating an opportunity for selectivity. Two genes are synthetic lethal if mutation of either gene alone is compatible with viability but mutation of both leads to death. Inhibiting the products of genes that are synthetic lethal to cancer-causing mutations should, by definition, kill cells that harbour such mutations, while sparing normal cells. Most drugs induce a loss-of-function phenotype. High-throughput screens using matched cell-line pairs and chemical libraries allow the identification of chemicals that inhibit or kill cells in a genotype-specific manner. The challenge in this setting is to identify the relevant target (or targets) of compounds that score positively. Genome-wide RNA-interference screens can now be used to identify synthetic lethal interactions in cells that are derived from higher eukaryotes, including humans. Gene–gene interactions, including synthetic lethal interactions that are discovered in cell-culture experiments, will ultimately need to be validated in vivo . It seems likely that some gene–gene interactions will be highly robust, whereas others might be valid only in specific cells or under specific experimental conditions. Two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death. So, targeting a gene that is synthetic lethal to a cancer-relevant mutation should kill only cancer cells and spare normal cells. Synthetic lethality therefore provides a conceptual framework for the development of cancer-specific cytotoxic agents. This paradigm has not been exploited in the past because there were no robust methods for systematically identifying synthetic lethal genes. This is changing as a result of the increased availability of chemical and genetic tools for perturbing gene function in somatic cells.

Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG–independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.

Realizing the full potential of si/shRNA technology requires more sophisticated approaches to address the pitfalls. For decades, scientists studying mammalian cells could only marvel at the genetic tools available to scientists who studied organisms such as yeasts or drosophila. However, many genes linked to human diseases do not have orthologs in such model organisms, or they require an appropriate cellular context to display their disease-relevant phenotypes. The wish for a facile method to disrupt gene function in somatic mammalian cells appeared to be granted with the discovery of RNA interference (RNAi) and small interfering RNA (siRNA) (or short hairpin RNA, shRNA), which brought with them great promise—particularly for discovering novel drug targets through the use of genetic screens ( 1 – 3 ). However, the honeymoon is now over, and although some new discoveries have been made, the yield has fallen far short of expectations. Many drug targets identified by means of si/shRNA technology in academic laboratories are not robust when tested in industrial laboratories ( 4 , 5 ). Avoiding this fate requires a more sophisticated interpretation of si/shRNA results, especially in the context of high-throughput screens.

Thalidomide-like drugs such as lenalidomide are clinically important treatments for multiple myeloma and show promise for other B cell malignancies. The biochemical mechanisms underlying their antitumor activity are unknown. Thalidomide was recently shown to bind to, and inhibit, the cereblon ubiquitin ligase. Cereblon loss in zebrafish causes fin defects reminiscent of the limb defects seen in children exposed to thalidomide in utero. Here we show that lenalidomide-bound cereblon acquires the ability to target for proteasomal degradation two specific B cell transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3). Analysis of myeloma cell lines revealed that loss of IKZF1 and IKZF3 is both necessary and sufficient for lenalidomide's therapeutic effect, suggesting that the antitumor and teratogenic activities of thalidomide-like drugs are dissociable.