Acquired drug resistance prevents cancer therapies from achieving stable and complete responses.1 Emerging evidence implicates a key role for nonmutational drug resistance mechanisms underlying the survival of residual cancer “persister” cells.2-4 The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse.5 In an earlier report, we found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival.6 Here, we describe the discovery that a similar therapy-resistant cell state underlies the behavior of persister cells derived from a wide range of cancers and drug treatments. Consequently, we show that persister cells acquire a dependency on GPX4. We demonstrate that loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in vivo. These findings support targeting GPX4 as a therapeutic strategy to prevent acquired drug resistance.

Known protein coding gene exons compose less than 3% of the human genome. The remaining 97% is largely uncharted territory, with only a small fraction characterized. The recent observation of transcription in this intergenic territory has stimulated debate about the extent of intergenic transcription and whether these intergenic RNAs are functional. Here we directly observed with a large set of RNA-seq data covering a wide array of human tissue types that the majority of the genome is indeed transcribed, corroborating recent observations by the ENCODE project. Furthermore, using de novo transcriptome assembly of this RNA-seq data, we found that intergenic regions encode far more long intergenic noncoding RNAs (lincRNAs) than previously described, helping to resolve the discrepancy between the vast amount of observed intergenic transcription and the limited number of previously known lincRNAs. In total, we identified tens of thousands of putative lincRNAs expressed at a minimum of one copy per cell, significantly expanding upon prior lincRNA annotation sets. These lincRNAs are specifically regulated and conserved rather than being the product of transcriptional noise. In addition, lincRNAs are strongly enriched for trait-associated SNPs suggesting a new mechanism by which intergenic trait-associated regions may function. These findings will enable the discovery and interrogation of novel intergenic functional elements.

Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

The visualization of biomolecules as they function in living systems is important for understanding complex biological processes. Here, we applied the bioorthogonal chemical reporter technique to image cell surface glycans using multiple metabolic labels. We introduced two different chemical reporters into sialic acid and N-acetylgalactosamine (GalNAc) residues and then simultaneously imaged their associated cell surface glycans with fluorescent probes.

Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.

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Fluorogenic probes activated by bioorthogonal chemical reactions can enable biomolecule imaging in situations where it is not possible to wash away unbound probe. One challenge for the development of such probes is the a priori identification of structures that will undergo a dramatic fluorescence enhancement by virtue of the chemical transformation. With the aid of density functional theory calculations reported previously by Nagano and coworkers, we identified azidofluorescein derivatives that were predicted to undergo an increase in fluorescence quantum yield upon Cu-catalyzed or Cu-free cycloaddition with linear or cyclic alkynes, respectively. Four derivatives were experimentally verified in model reactions, and one, a 4-azidonaphthyl fluorescein analog, was further shown to label alkyne-functionalized proteins in vitro and glycoproteins on cells with excellent selectivity. The azidofluorescein derivative also enabled cell imaging under no-wash conditions with good signal above background. This work establishes a platform for the rational design of fluorogenic azide probes with spectral properties tailored for biological imaging.

Azide imaging: A fluorogenic phosphine based on a FRET‐quenching mechanism allows for live‐cell imaging of azido sugars by the Staudinger ligation. This design strategy can accommodate numerous fluorophores and complementary quenchers, enabling extension to multicolor imaging. In the image shown, the nuclei are blue while the cell surfaces and Golgi are green.