RESUMEN
Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.
Asunto(s)
Antineoplásicos , Especies Reactivas de Oxígeno , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Núcleo Celular/metabolismo , HumanosRESUMEN
Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.
Asunto(s)
Gasderminas , Piroptosis , Proteínas de Neoplasias/metabolismo , Cardiolipinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Inflamasomas/metabolismoRESUMEN
The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Proteoma/análisis , Transcriptoma , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Cisteína/metabolismo , Receptor Nuclear Huérfano DAX-1/metabolismo , Redes Reguladoras de Genes , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ligandos , Neoplasias Pulmonares/metabolismoRESUMEN
The mTOR Complex 1 (mTORC1) pathway regulates cell growth in response to numerous cues, including amino acids, which promote mTORC1 translocation to the lysosomal surface, its site of activation. The heterodimeric RagA/B-RagC/D GTPases, the Ragulator complex that tethers the Rags to the lysosome, and the v-ATPase form a signaling system that is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of guanosine triphosphate to RagA and RagB but the factors that regulate Rag nucleotide loading are unknown. Here, we identify HBXIP and C7orf59 as two additional Ragulator components that are required for mTORC1 activation by amino acids. The expanded Ragulator has nucleotide exchange activity toward RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, we provide mechanistic insight into how mTORC1 senses amino acids by identifying Ragulator as a guanine nucleotide exchange factor (GEF) for the Rag GTPases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminoácidos/metabolismo , Drosophila/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Animales , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Datos de Secuencia Molecular , Complejos Multiproteicos , Serina-Treonina Quinasas TORRESUMEN
NRF2 (nuclear factor erythroid-2-related factor 2) is a key regulator of genes involved in the cell's protective response to oxidative stress. Upon activation by disturbed redox homeostasis, NRF2 promotes the expression of metabolic enzymes to eliminate reactive oxygen species (ROS). Cell internalization of peroxisome-like artificial organelles that harbor redox-regulating enzymes was previously shown to reduce ROS-induced stress and thus cell death. However, if and to which extent ROS degradation by such nanocompartments interferes with redox signaling pathways is largely unknown. Here, we advance the design of H2O2-degrading artificial nano-organelles (AnOs) that exposed surface-attached cell penetrating peptides (CPP) for enhanced uptake and were equipped with a fluorescent moiety for rapid visualization within cells. To investigate how such AnOs integrate in cellular redox signaling, we engineered leukemic K562 cells that report on NRF2 activation by increased mCherry expression. Once internalized, ROS-metabolizing AnOs dampen intracellular NRF2 signaling upon oxidative injury by degrading H2O2. Moreover, intracellular AnOs conferred protection against ROSinduced cell death in conditions when endogenous ROS-protection mechanisms have been compromised by depletion of glutathione or knockdown of NRF2. We demonstrate CPP-facilitated AnO uptake and AnO-mediated protection against ROS insults also in the T lymphocyte population of primary peripheral blood mononuclear cells from healthy donors. Overall, our data suggest that intracellular AnOs alleviated cellular stress by the on-site reduction of ROS.
Asunto(s)
Peróxido de Hidrógeno , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Especies Reactivas de Oxígeno , Transducción de Señal , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células K562 , Especies Reactivas de Oxígeno/metabolismo , Oxidación-Reducción , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Orgánulos/metabolismoRESUMEN
The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in disease. The Rag GTPases interact with mTORC1 and are proposed to activate it in response to amino acids by promoting mTORC1 translocation to a membrane-bound compartment that contains the mTORC1 activator, Rheb. We show that amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. A complex encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, which we term Ragulator, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation. Constitutive targeting of mTORC1 to the lysosomal surface is sufficient to render the mTORC1 pathway amino acid insensitive and independent of Rag and Ragulator, but not Rheb, function. Thus, Rag-Ragulator-mediated translocation of mTORC1 to lysosomal membranes is the key event in amino acid signaling to mTORC1.
Asunto(s)
Lisosomas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminoácidos/metabolismo , Animales , Línea Celular , Drosophila , Humanos , Membranas Intracelulares/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/metabolismo , Sistema de Señalización de MAP Quinasas , Diana Mecanicista del Complejo 1 de la Rapamicina , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos , Mutación , Neuropéptidos/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Reguladora Asociada a mTOR , Transducción de Señal , Serina-Treonina Quinasas TOR , Factores de Transcripción/genéticaRESUMEN
MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proliferación Celular/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , Proteínas Represoras/metabolismo , Aptámeros de Nucleótidos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Proteína 9 Asociada a CRISPR/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Regiones Promotoras Genéticas , Dominios Proteicos , ARN Guía de Kinetoplastida , ARN Largo no Codificante/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
For the past five decades, the lysosome has been characterized as an unglamorous cellular recycling center. This notion has undergone a radical shift in the last 10 years, with new research revealing that this organelle serves as a major hub for metabolic signaling pathways. The discovery that master growth regulators, including the protein kinase mTOR (mechanistic target of rapamycin), make their home at the lysosomal surface has generated intense interest in the lysosome's key role in nutrient sensing and cellular homeostasis. The transcriptional networks required for lysosomal maintenance and function are just being unraveled and their connection to lysosome-based signaling pathways revealed. The catabolic and anabolic pathways that converge on the lysosome connect this organelle with multiple facets of cellular function; when these pathways are deregulated they underlie multiple human diseases, and promote cellular and organismal aging. Thus, understanding how lysosome-based signaling pathways function will not only illuminate the fascinating biology of this organelle but will also be critical in unlocking its therapeutic potentials.
Asunto(s)
Envejecimiento/metabolismo , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Transducción de Señal , Envejecimiento/genética , Animales , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Lisosomas/genéticaRESUMEN
Phenotypic screening has identified small-molecule modulators of aging, but the mechanism of compound action often remains opaque due to the complexities of mapping protein targets in whole organisms. Here, we combine a library of covalent inhibitors with activity-based protein profiling to coordinately discover bioactive compounds and protein targets that extend lifespan in Caenorhabditis elegans. We identify JZL184-an inhibitor of the mammalian endocannabinoid (eCB) hydrolase monoacylglycerol lipase (MAGL or MGLL)-as a potent inducer of longevity, a result that was initially perplexing as C. elegans does not possess an MAGL ortholog. We instead identify FAAH-4 as a principal target of JZL184 and show that this enzyme, despite lacking homology with MAGL, performs the equivalent metabolic function of degrading eCB-related monoacylglycerides in C. elegans. Small-molecule phenotypic screening thus illuminates pure pharmacological connections marking convergent metabolic functions in distantly related organisms, implicating the FAAH-4/monoacylglyceride pathway as a regulator of lifespan in C. elegans.
Asunto(s)
Benzodioxoles/farmacología , Caenorhabditis elegans/efectos de los fármacos , Endocannabinoides/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Longevidad/efectos de los fármacos , Monoacilglicerol Lipasas/antagonistas & inhibidores , Piperidinas/farmacología , Animales , Benzodioxoles/química , Caenorhabditis elegans/metabolismo , Endocannabinoides/metabolismo , Inhibidores Enzimáticos/química , Estructura Molecular , Monoacilglicerol Lipasas/metabolismo , Piperidinas/químicaRESUMEN
The mTORC1 kinase is a master growth regulator that senses numerous environmental cues, including amino acids. The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by promoting the translocation of mTORC1 to the lysosomal surface, its site of activation. The Rags are unusual GTPases in that they function as obligate heterodimers, which consist of RagA or B bound to RagC or D. While the loading of RagA/B with GTP initiates amino acid signaling to mTORC1, the role of RagC/D is unknown. Here, we show that RagC/D is a key regulator of the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D must be GDP bound for the interaction to occur. We identify FLCN and its binding partners, FNIP1/2, as Rag-interacting proteins with GAP activity for RagC/D, but not RagA/B. Thus, we reveal a role for RagC/D in mTORC1 activation and a molecular function for the FLCN tumor suppressor.
Asunto(s)
Aminoácidos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/fisiología , Proteínas Portadoras/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Unión Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
PHARC (polyneuropathy, hearing loss, cerebellar ataxia, retinitis pigmentosa, and cataract) is a human neurological disorder caused by deleterious mutations in the ABHD12 gene, which encodes an integral membrane lyso-phosphatidylserine (lyso-PS) lipase. Pharmacological or genetic disruption of ABHD12 leads to higher levels of lyso-PS lipids in human cells and the central nervous system (CNS) of mice. ABHD12 loss also causes rapid rewiring of PS content, resulting in selective increases in the level of arachidonoyl (C20:4) PS and decreases in the levels of other PS species. The biochemical basis for ABHD12-dependent PS remodeling and its pathophysiological significance remain unknown. Here, we show that genetic deletion of the lysophospholipid acyltransferase LPCAT3 blocks accumulation of brain C20:4 PS in mice lacking ABHD12 and concurrently produces hyper-increases in the level of lyso-PS in these animals. These lipid changes correlate with exacerbated auditory dysfunction and brain microgliosis in mice lacking both ABHD12 and LPCAT3. Taken together, our findings reveal that ABHD12 and LPCAT3 coordinately regulate lyso-PS and C20:4 PS content in the CNS and point to lyso-PS lipids as the likely bioactive metabolites contributing to PHARC-related neuropathologies.
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1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Monoacilglicerol Lipasas/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Fosfatidilserinas/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/deficiencia , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Animales , Ratones , Ratones Noqueados , Estructura Molecular , Monoacilglicerol Lipasas/deficiencia , Monoacilglicerol Lipasas/genéticaRESUMEN
Cellular metabolism encompasses a complex array of interconnected biochemical pathways that are required for cellular homeostasis. When dysregulated, metabolism underlies multiple human pathologies. At the heart of metabolic networks are enzymes that have been historically studied through a reductionist lens, and more recently, using high throughput approaches including genomics and proteomics. Merging these two divergent viewpoints are chemical proteomic technologies, including activity-based protein profiling, which combines chemical probes specific to distinct enzyme families or amino acid residues with proteomic analysis. This enables the study of metabolism at the network level with the precision of powerful biochemical approaches. Herein, we provide a primer on how chemical proteomic technologies custom-built for studying metabolism have unearthed fundamental principles in metabolic control. In parallel, these technologies have leap-frogged drug discovery through identification of novel targets and drug specificity. Collectively, chemical proteomics technologies appear to do the impossible: uniting systematic analysis with a reductionist approach.
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Genómica , Proteómica , HumanosRESUMEN
Redox imbalance is defined by disruption in oxidative and reductive pathways and has a central role in cancer initiation, development, and treatment. Although redox imbalance has traditionally been characterized by high levels of oxidative stress, emerging evidence suggests that an overly reductive environment is just as detrimental to cancer proliferation. Reductive stress is defined by heightened levels of antioxidants, including glutathione and elevated NADH, compared with oxidized NAD, which disrupts central biochemical pathways required for proliferation. With the advent of new technologies that measure and manipulate reductive stress, the sensors and drivers of this overlooked metabolic stress are beginning to be revealed. In certain genetically defined cancers, targeting reductive stress pathways may be an effective strategy. Redox-based pathways are gaining recognition as essential 'regulatory hubs,' and a broader understanding of reductive stress signaling promises not only to reveal new insights into metabolic homeostasis but also potentially to transform therapeutic options in cancer.
Asunto(s)
Neoplasias , Estrés Oxidativo , Humanos , Antioxidantes/uso terapéutico , Oxidación-ReducciónRESUMEN
Translocation renal cell carcinoma (tRCC) is an aggressive subtype of kidney cancer driven by TFE3 gene fusions, which act via poorly characterized downstream mechanisms. Here we report that TFE3 fusions transcriptionally rewire tRCCs toward oxidative phosphorylation (OXPHOS), contrasting with the highly glycolytic metabolism of most other renal cancers. This TFE3 fusion-driven OXPHOS program, together with heightened glutathione levels found in renal cancers, renders tRCCs sensitive to reductive stress - a metabolic stress state induced by an imbalance of reducing equivalents. Genome-scale CRISPR screening identifies tRCC-selective vulnerabilities linked to this metabolic state, including EGLN1, which hydroxylates HIF-1α and targets it for proteolysis. Inhibition of EGLN1 compromises tRCC cell growth by stabilizing HIF-1a and promoting metabolic reprogramming away from OXPHOS, thus representing a vulnerability to OXPHOS-dependent tRCC cells. Our study defines a distinctive tRCC-essential metabolic program driven by TFE3 fusions and nominates EGLN1 inhibition as a therapeutic strategy to counteract fusion-induced metabolic rewiring.
RESUMEN
UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.
RESUMEN
Heightened concentrations of reactive metabolites, including reactive oxygen species (ROS), can damage all macromolecules leading to the erosion of cellular fidelity. In this regard, the control of ROS in the nuclues is essential for cellular homeostasis, and dysregulation of nuclear ROS has been attributed to multiple pathologies and the mechanism of action of certain chemotherapies. How nuclear ROS is generated, detoxified and sensed is poorly understood, and stems in part, from a historical lack of tools that allow for its precise generation and detection. Here, we summarize the latest advances in chemical biology inspired approaches that have been developed to study nuclear ROS and highlight how these tools have led to major breakthroughs in understanding its regulation. The continued development and application of chemical biology approaches to understand nuclear ROS promises to unlock fundamental insights into human physiology and disease.
Asunto(s)
Estrés Oxidativo , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Epithelial ovarian cancer (EOC) is treated in the first-line setting with combined platinum and taxane chemotherapy, often followed by a maintenance poly (ADP-ribose) polymerase inhibitor (PARPi). Responses to first-line treatment are frequent. For many patients, however, responses are suboptimal or short-lived. Over the last several years, multiple new classes of agents targeting DNA damage response (DDR) mechanisms have advanced through clinical development. In this review, we explore the preclinical rationale for the use of ATR inhibitors, CHK1 inhibitors, and WEE1 inhibitors, emphasizing their application to chemotherapy-resistant and PARPi-resistant ovarian cancer. We also present an overview of the clinical development of the leading drugs in each of these classes, emphasizing the rationale for monotherapy and combination therapy approaches.
RESUMEN
Multiple cancers regulate oxidative stress by activating the transcription factor NRF2 through mutation of its negative regulator, KEAP1. NRF2 has been studied extensively in KEAP1-mutant cancers; however, the role of this pathway in cancers with wild-type KEAP1 remains poorly understood. To answer this question, we induced NRF2 via pharmacological inactivation of KEAP1 in a panel of 50+ non-small cell lung cancer cell lines. Unexpectedly, marked decreases in viability were observed in >13% of the cell lines-an effect that was rescued by NRF2 ablation. Genome-wide and targeted CRISPR screens revealed that NRF2 induces NADH-reductive stress, through the upregulation of the NAD+-consuming enzyme ALDH3A1. Leveraging these findings, we show that cells treated with KEAP1 inhibitors or those with endogenous KEAP1 mutations are selectively vulnerable to Complex I inhibition, which impairs NADH oxidation capacity and potentiates reductive stress. Thus, we identify reductive stress as a metabolic vulnerability in NRF2-activated lung cancers.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Factor 2 Relacionado con NF-E2 , Humanos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , NAD/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/genética , Transducción de SeñalRESUMEN
Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, it's unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H 2 O 2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H 2 O 2 . Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H 2 O 2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.