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Genomic profiling often fails to predict therapeutic outcomes in cancer. This failure is, in part, due to a myriad of genetic alterations and the plasticity of cancer signaling networks. Functional profiling, which ascertains signaling dynamics, is an alternative method to anticipate drug responses. It is unclear whether integrating genomic and functional features of solid tumours can provide unique insight into therapeutic vulnerabilities. We perform combined molecular and functional characterization, via BH3 profiling of the intrinsic apoptotic machinery, in glioma patient samples and derivative models. We identify that standard-of-care therapy rapidly rewires apoptotic signaling in a genotype-specific manner, revealing targetable apoptotic vulnerabilities in gliomas containing specific molecular features (e.g., TP53 WT). However, integration of BH3 profiling reveals high mitochondrial priming is also required to induce glioma apoptosis. Accordingly, a machine-learning approach identifies a composite molecular and functional signature that best predicts responses of diverse intracranial glioma models to standard-of-care therapies combined with ABBV-155, a clinical drug targeting intrinsic apoptosis. This work demonstrates how complementary functional and molecular data can robustly predict therapy-induced cell death.

Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates.

The selenoprotein glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid peroxides into nontoxic lipid alcohols. GPX4 has emerged as a promising therapeutic target for cancer treatment, but some cancer cells are resistant to ferroptosis triggered by GPX4 inhibition. Using a chemical-genetic screen, we identify LRP8 (also known as ApoER2) as a ferroptosis resistance factor that is upregulated in cancer. Loss of LRP8 decreases cellular selenium levels and the expression of a subset of selenoproteins. Counter to the canonical hierarchical selenoprotein regulatory program, GPX4 levels are strongly reduced due to impaired translation. Mechanistically, low selenium levels result in ribosome stalling at the inefficiently decoded GPX4 selenocysteine UGA codon, leading to ribosome collisions, early translation termination and proteasomal clearance of the N-terminal GPX4 fragment. These findings reveal rewiring of the selenoprotein hierarchy in cancer cells and identify ribosome stalling and collisions during GPX4 translation as ferroptosis vulnerabilities in cancer.

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.

Ferroptosis is an oxidative form of nonapoptotic cell death whose transcriptional regulation is poorly understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2 (NRF2) are important regulators of oxidative stress responses. NFE2L1 abundance and function are regulated posttranslationally by N-glycosylation. Functional maturation of NFE2L1 requires deglycosylation by cytosolic peptide:N-glycanase 1 (NGLY1). We find that NGLY1 and NFE2L1 work in a common pathway to enhance ferroptosis resistance, independent of NFE2L2, by promoting expression of the key antiferroptotic enzyme glutathione peroxidase 4 (GPX4). Enhanced ferroptosis sensitivity in NFE2L1-knockout cells can be reverted by expression of an NFE2L1 mutant containing eight asparagine-to-aspartate protein sequence substitutions, which mimic NGLY1-catalyzed protein sequence editing. These results suggest that ferroptosis sensitivity may be regulated by NGLY1-catalyzed NFE2L1 deglycosylation. These results highlight a role for the disease-associated NGLY1/NFE2L1 pathway in ferroptosis regulation. [ABSTRACT FROM AUTHOR]

The goal of cancer chemotherapy is to induce homogeneous cell death within the population of targeted cancer cells. However, no two cells are exactly alike at the molecular level, and sensitivity to drug-induced cell death, therefore, varies within a population. Genetic alterations can contribute to this variability and lead to selection for drug resistant clones. However, there is a growing appreciation for the role of non-genetic variation in producing drug-tolerant cellular states that exhibit reduced sensitivity to cell death for extended periods of time, from hours to weeks. These cellular states may result from individual variation in epigenetics, gene expression, metabolism, and other processes that impact drug mechanism of action or the execution of cell death. Such population-level non-genetic heterogeneity may contribute to treatment failure and provide a cellular “substrate” for the emergence of genetic alterations that confer frank drug resistance. [ABSTRACT FROM PUBLISHER]

Summary The tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38 , leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity. Graphical Abstract Highlights • FACS-based screening can identify genetic regulators of glutathione abundance • MRP1 effluxes glutathione, which accelerates ferroptosis • NAA38 and KEAP1 regulate expression of NRF2 and also MRP1 • High MRP1 expression promotes collateral sensitivity to ferroptosis-inducing agents Glutathione suppresses the non-apoptotic cell death process of ferroptosis. Using genome-wide human haploid cell mutagenesis and FACS-based detection, Cao et al. identify negative regulators of intracellular glutathione abundance that affect glutathione efflux and NRF2 protein levels, altering ferroptosis sensitivity. [ABSTRACT FROM AUTHOR]

Ferroptosis is a regulated form of cell death associated with the iron-dependent accumulation of phospholipid hydroperoxides. Inducing ferroptosis is a promising approach to treat therapy-resistant cancer. Ferroptosis suppressor protein 1 (FSP1) promotes ferroptosis resistance in cancer by generating the antioxidant form of coenzyme Q10 (CoQ). Despite the important role of FSP1, few molecular tools exist that target the CoQ-FSP1 pathway. Through a series of chemical screens, we identify several structurally diverse FSP1 inhibitors. The most potent of these compounds, ferroptosis sensitizer 1 (FSEN1), is an uncompetitive inhibitor that acts selectively through on-target inhibition of FSP1 to sensitize cancer cells to ferroptosis. Furthermore, a synthetic lethality screen reveals that FSEN1 synergizes with endoperoxide-containing ferroptosis inducers, including dihydroartemisinin, to trigger ferroptosis. These results provide new tools that catalyze the exploration of FSP1 as a therapeutic target and highlight the value of combinatorial therapeutic regimes targeting FSP1 and additional ferroptosis defense pathways. [Display omitted] • Identification of structurally diverse small molecule inhibitors of FSP1 • FSEN1 is an uncompetitive FSP1 inhibitor that sensitizes cancer cells to ferroptosis • FSEN1 synergizes with GPX4 inhibitors and endoperoxide-based ferroptosis inducers Hendricks et al. identify a set of structurally diverse small molecules that inhibit FSP1, a CoQ oxidoreductase that suppresses ferroptosis. The most potent FSP1 inhibitor, ferroptosis sensitizer 1 (FSEN1) sensitizes cancer cells to ferroptosis and synergizes with several ferroptosis inducers, including GPX4 inhibitors and endoperoxide-containing molecules such as DHA. [ABSTRACT FROM AUTHOR]

Cancer cells need a steady supply of nutrients to evade cell death and proliferate. Depriving cancer cells of the amino acid cystine can trigger the non-apoptotic cell death process of ferroptosis. Here, we report that cancer cells can evade cystine deprivation-induced ferroptosis by uptake and catabolism of the cysteine-rich extracellular protein albumin. This protective mechanism is enhanced by mTORC1 inhibition and involves albumin degradation in the lysosome, predominantly by cathepsin B (CTSB). CTSB-dependent albumin breakdown followed by export of cystine from the lysosome via the transporter cystinosin fuels the synthesis of glutathione, which suppresses lethal lipid peroxidation. When cancer cells are grown under non-adherent conditions as spheroids, mTORC1 pathway activity is reduced, and albumin supplementation alone affords considerable protection against ferroptosis. These results identify the catabolism of extracellular protein within the lysosome as a mechanism that can inhibit ferroptosis in cancer cells. [Display omitted] • Extracellular albumin can block ferroptosis caused by cystine deprivation • Albumin is cysteine rich and, when catabolized, can replenish GSH stores • Lysosomal albumin catabolism requires cathepsin B (CTSB) • Extracellular albumin suppresses ferroptosis in spheroid cell models Mechanisms that regulate ferroptosis sensitivity and resistance remain poorly defined. Armenta et al. demonstrate that uptake and lysosomal catabolism of extracellular protein can prevent ferroptosis in cystine-deprived cells by maintaining intracellular levels of cysteine and glutathione. This compensatory mechanism may limit the effectiveness of cancer therapies that target cystine uptake. [ABSTRACT FROM AUTHOR]

Ferroptosis is an important mediator of pathophysiological cell death and an emerging target for cancer therapy. Whether ferroptosis sensitivity is governed by a single regulatory mechanism is unclear. Here, based on the integration of 24 published chemical genetic screens combined with targeted follow-up experimentation, we find that the genetic regulation of ferroptosis sensitivity is highly variable and context-dependent. For example, the lipid metabolic gene acyl-coenzyme A (CoA) synthetase long chain family member 4 (ACSL4) appears far more essential for ferroptosis triggered by direct inhibition of the lipid hydroperoxidase glutathione peroxidase 4 (GPX4) than by cystine deprivation. Despite this, distinct pro-ferroptotic stimuli converge upon a common lethal effector mechanism: accumulation of lipid peroxides at the plasma membrane. These results indicate that distinct genetic mechanisms regulate ferroptosis sensitivity, with implications for the initiation and analysis of this process in vivo. [Display omitted] • An integrated genetic network reveals high variability in ferroptosis regulation • ACSL4 is most important for ferroptosis in response to direct GPX4 inhibition • AGPS is not required for ferroptosis in all contexts • Ferroptotic stimuli converge on plasma membrane lipid peroxidation The genetic regulation of ferroptosis remains poorly understood. Magtanong et al. demonstrate that ACSL4 , AGPS , and other lipid metabolic genes are context-dependent ferroptosis regulators that are not universally essential for this process. Results suggest that there may not exist a single ferroptosis genetic regulatory mechanism in all cells and conditions. [ABSTRACT FROM AUTHOR]