Translatome Profiling Research Articles

Translatome and Transcriptome Profiling of Hypoxic-Induced Rat Cardiomyocytes

Adult cardiac hypoxia as a crucial pathogenesis factor can induce detrimental effects on cardiac injury and dysfunction. The global transcriptome and translatome reflecting the cellular response to hypoxia have not yet been extensively studied in myocardium. In this study, we conducted RNA sequencing (RNA-seq) and ribosome profiling technique (polyribo-seq) in rat heart tissues and H9C2 cells exposed to different periods of hypoxia stress in vivo and in vitro. The temporal gene-expression profiling displayed the distinction of transcriptome and translatome, which were mainly concentrated in cell apoptosis, autophagy, DNA repair, angiogenesis, vascular process, and cardiac cell proliferation and differentiation. A large number of genes such as GNAI3, SEPT4, FANCL, BNIP3, TBX3, ESR2, PTGS2, KLF4, and ADRB2, whose transcript and translation levels are closely correlated, were identified to own a common RNA motif “5′-GAAGCUGCC-3′” in 5′ UTR. NCBP3 was further determined to recognize this RNA motif and facilitate translational process in myocardium under hypoxia stress. Taken together, our data show the close connection between alterations of transcriptome and translatome after hypoxia exposure, emphasizing the significance of translational regulation in related studies. The profiled molecular responses in current study may be valuable resources for advanced understanding of the mechanisms underlying hypoxia-induced effect on heart diseases.

Abstract B32: A specialized post-transcriptional program in chemoresistant, quiescent cancer cells

Abstract Quiescent (G0) cells are a clinically relevant fraction in cancers, which include dormant cancer stem cells, and resist clinical therapy. G0 cells reveal extensive changes in gene expression at the protein and translation levels. We previously identified that the translation mechanism is altered in G0 cancer cells. MicroRNAs, noncoding RNAs that target distinct mRNAs to alter gene expression, were found to associate with a key RNA-binding protein and enable specialized functions in G0, where they recruit noncanonical translation factors to regulate specific mRNA translation. We find that G0 leukemic cells show similar proteome and translatome to cells isolated post-chemotherapy. These data suggest that specialized post-transcriptional mechanisms in G0 leukemic cells regulate a distinct translatome to mediate chemoresistance. To understand the role of post-transcriptional regulation in chemoresistance, we compared global transcriptome, translatome and proteome profiling in chemoresistant G0 acute monocytic leukemic (AML) cells. We find that chemotherapy or G0 induction leads to DNA damage responsive ATM and stress signaling, which alter post-transcriptional and translational mechanisms. ATM and stress-activated p38 MAPK/MK2 increase AU-rich-element (ARE) bearing proinflammatory cytokine and immune gene mRNAs, by regulating a key ARE RNA binding protein and modifying canonical translation. AREs are present on 3'UTRs of tightly regulated oncogenes and cytokines, to post-transcriptionally control their expression. Both rate-limiting steps—mRNA cap recognition and tRNA recruitment—in canonical translation are altered. These signaling pathways lead to low mTOR activity in G0, which activates the cap complex inhibitor, eIF4EBP to impair canonical translation, leading to noncanonical translation of specific mRNAs with specialized cap binding and ribosome recruitment factors. In addition, stress and STAT1/interferon signaling are activated to reduce the canonical tRNA recruitment mechanism, enabling noncanonical translation of specific mRNAs. These changes permit translation of ARE bearing proinflammatory cytokine TNFa, and immune and cell-migration modulators that promote survival. Co-inhibiting p38 MAPK and TNFa that promote antiapoptosis—prior to or along with chemotherapy—decreases chemoresistance in AML cells, in vivo, and in patient samples without affecting normal cells. Our studies reveal a proinflammatory subpopulation in AML that mediates resistance, enabled by DNA damage- and stress-regulated post-transcriptional and translational mechanisms that are mediated by AU-rich-elements and a critical ARE RNA binding protein. Disrupting ARE regulation reduces TNFα and chemoresistance, revealing AREs, an important ARE RNA binding protein and noncanonical translation as regulators of chemoresistance. These studies reveal the significance of post-transcriptional regulation of proinflammatory and immune gene-mediated chemoresistance. Citation Format: Sooncheol Lee, Syed I.A. Bukhari, Samuel S. Truesdell, Swapna Kollu, Richard D. Mortensen, Myriam Boukhali, Esha Jain, Dongjun Lee, Maria Mazzola, Radhika Raheja, Adam Langenbucher, Nicholas Haradhwala, Akiko Yanagiya, Michael Lawrence, Roopali Gandhi, Ruslan Sadreyev, David Sweetser, Wilhelm Haas, Shobha Vasudevan. A specialized post-transcriptional program in chemoresistant, quiescent cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B32.

Age-related differences in the translational landscape of mammalian oocytes.

Increasing maternal age in mammals is associated with poorer oocyte quality, involving higher aneuploidy rates and decreased developmental competence. Prior to resumption of meiosis, fully developed mammalian oocytes become transcriptionally silent until the onset of zygotic genome activation. Therefore, meiotic progression and early embryogenesis are driven largely by translational utilization of previously synthesized mRNAs. We report that genome‐wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show that a number of aberrantly translated mRNAs in oocytes from aged females are associated with cell cycle. Indeed, we demonstrate that four specific maternal age‐related transcripts (Sgk1, Castor1, Aire and Eg5) with differential translation rates encode factors that are associated with the newly forming meiotic spindle. Moreover, we report substantial defects in chromosome alignment and cytokinesis in the oocytes of young females, in which candidate CASTOR1 and SGK1 protein levels or activity are experimentally altered. Our findings indicate that improper translation of specific proteins at the onset of meiosis contributes to increased chromosome segregation problems associated with female ageing.

Translatome Profiling of Plum Pox Virus-Infected Leaves in European Plum Reveals Temporal and Spatial Coordination of Defense Responses in Phloem Tissues.

Plum pox virus (PPV) is the causative agent of sharka, a devastating disease of stone fruits including peaches, apricots, and plums. PPV infection levels and associated disease symptoms can vary greatly, depending upon the virus strain, host species, or cultivar as well as developmental age of the infected tissues. For example, peaches often exhibit mild symptoms in leaves and fruit while European plums typically display severe chlorotic rings. Systemic virus spread into all host tissues occurs via the phloem, a process that is poorly understood in perennial plant species that undergo a period of dormancy and must annually renew phloem tissues. Currently, little is known about how phloem tissues respond to virus infection. Here, we used translating ribosome affinity purification followed by RNA sequencing to identify phloem- and nonphloem-specific gene responses to PPV infection during leaf development in European plum (Prunus domestica L.). Results showed that, during secondary leaf morphogenesis (4- and 6-week-old leaves), the phloem had a disproportionate response to PPV infection with two- to sixfold more differentially expressed genes (DEGs) in phloem than nonphloem tissues, despite similar levels of viral transcripts. In contrast, in mature 12-week-old leaves, virus transcript levels dropped significantly in phloem tissues but not in nonphloem tissues. This drop in virus transcripts correlated with an 18-fold drop in phloem-specific DEGs. Furthermore, genes associated with defense responses including RNA silencing were spatially coordinated in response to PPV accumulation and were specifically induced in phloem tissues at 4 to 6 weeks. Combined, these findings highlight the temporal and spatial dynamics of leaf tissue responses to virus infection and reveal the importance of phloem responses within a perennial host.

Cell type-specific translatome profiling of cortical neural progenitors in the developing brain

Cell type-specific translatome profiling of cortical neural progenitors in the developing brain

Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies.

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain macrophages, we report here a side-by-side comparison of classical cell sort-based transcriptome sequencing and the ‘RiboTag’ method that avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole cell microglia transcriptomes were found to be significantly tainted by artifacts induced by tissue-dissociation, cargo contaminations and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling to assess the accuracy of Cre transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sort-based profiling strategies.

Global translational reprogramming is a fundamental layer of immune regulation in plants.

In the absence of specialized immune cells, the need for plants to reprogram transcription to transition from growth-related activities to defence is well understood1, 2. However, little is known about translational changes that occur during immune induction. Using ribosome footprinting (RF), we performed global translatome profiling on Arabidopsis exposed to the microbe-associated molecular pattern (MAMP) elf18. We found that during this pattern-triggered immunity (PTI), translation was tightly regulated and poorly correlated with transcription. Identification of genes with altered translational efficiency (TE) led to the discovery of novel regulators of this immune response. Further investigation of these genes showed that mRNA sequence features are major determinants of the observed TE changes. In the 5′ leader sequences of transcripts with increased TE, we found a highly enriched mRNA consensus sequence, R-motif, consisting of mostly purines. We showed that R-motif regulates translation in response to PTI induction through interaction with poly(A)-binding proteins. Therefore, this study provides not only strong evidence, but also a molecular mechanism for global translational reprogramming during PTI in plants.

Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism.

5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in colorectal cancer. Previous studies showed that 5-FU modulates RNA metabolism and mRNA expression. In addition, it has been reported that 5-FU incorporates into the RNAs constituting the translational machinery and that 5-FU affects the amount of some mRNAs associated with ribosomes. However, the impact of 5-FU on translational regulation remains unclear. Using translatome profiling, we report that a clinically relevant dose of 5-FU induces a translational reprogramming in colorectal cancer cell lines. Comparison of mRNA distribution between polysomal and non-polysomal fractions in response to 5-FU treatment using microarray quantification identified 313 genes whose translation was selectively regulated. These regulations were mostly stimulatory (91%). Among these genes, we showed that 5-FU increases the mRNA translation of HIVEP2, which encodes a transcription factor whose translation in normal condition is known to be inhibited by mir-155. In response to 5-FU, the expression of mir-155 decreases thus stimulating the translation of HIVEP2 mRNA. Interestingly, the 5-FU-induced increase in specific mRNA translation was associated with reduction of global protein synthesis. Altogether, these findings indicate that 5-FU promotes a translational reprogramming leading to the increased translation of a subset of mRNAs that involves at least for some of them, miRNA-dependent mechanisms. This study supports a still poorly evaluated role of translational control in drug response.

Integrative Analysis of RNA-Interactome and Translatome Reveal Functional Targets of MSI2 in Myeloid Leukemia

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate of several cell types including hematopoietic stem and progenitor cells. MSI2 was recently shown to be transcriptionally up-regulated in aggressive myeloid leukemia including chronic myelogenous leukemia in blast crisis (CML-BC) and acute myeloid leukemia (AML). Loss-of-function studies have confirmed an important role for MSI2 in the aggressive biology of these leukemia, but the precise molecular mechanisms remain undefined. MSI2 is thought to bind to the 3' untranslated region (3'UTR) of target mRNA in a sequence-specific fashion and inhibit their translation, but functional targets of MSI2 are not known. We implemented two transcriptome-wide techniques to define these targets: iCLIP (individual nucleotide-resolution cross-link and immunoprecipitation) and polysome profiling. We first defined the RNA interactome of MSI2 in a CML-BC cell line (K562) by iCLIP of FLAG-tagged MSI2. iCLIP relies on the ability of ultraviolet (UV) radiation to cross-link RBPs to interacting RNA allowing for stringent immunoprecipitation of RBP-RNA complexes and subsequent analysis by next generation sequencing. iCLIP analysis revealed direct binding of MSI2 to transcripts of 5036 genes, specifically enriched in the 3'UTR. These iCLIP peaks were also enriched in UAG, the minimal sequence motif of MSI2 previously defined through biochemical assays. Given that MSI2 is ubiquitously expressed, but has very specific cellular functions, we hypothesized that only a small fraction of the direct targets demonstrable by iCLIP are functionally relevant. To determine those target mRNA, we performed translatome profiling of K562 cells (MSI2 knock-down and control cells) with polysome profiling. Polyribosomes or polysomes are aggregates of ribosomes assembled on efficiently translated transcripts. Comparing change in abundance of polysome-associated transcripts (polysome profiling) allows determination of quantitative changes to translation. Using polysome profiling, we found that transcripts from only 413 genes underwent translational up-regulation (without a corresponding change in total transcript abundance) in response to MSI2 knock-down, of which 319 were deemed to be direct MSI2 targets from iCLIP analysis. Translational targets thus defined were highly enriched in biological pathways such as differentiation, cell-cycling, DNA damage response and apoptosis. Specific transcripts thus identified included transcription factors critical to myeloid and leukemia biology (cJUN, MYC and SP1) and cell-cycle regulators (CDK1, CDKN1B, RAD21 and RB1). Using expression profiles of 91 CML patient samples (reported in Radich JP et al PNAS 2006 Feb 21;103(8)) we tested if changes in gene-expression during disease progression (from chronic phase to blast crisis) reflect the change in translation of transcription factors as predicted by the translatome analysis. Accordingly, we found that transcripts down-regulated in CML-BC were highly enriched in targets of JUN and SP1, confirming a primary role for MSI2 targets in progression of CML. In summary, our novel approach that integrates two transcriptome-wide techniques show that while thousands of transcripts are directly bound by MSI2, only a small proportion of these undergo translational repression. Functional targets thus identified include transcription factors and cell-cycle regulators critical to progression of myeloid leukemia. Our results will form the basis of further studies to explore therapeutic targeting of MSI2 in myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Cellular Recycling of Proteins in Seed Dormancy Alleviation and Germination.

Each step of the seed-to-seed cycle of plant development including seed germination is characterized by a specific set of proteins. The continual renewal and/or replacement of these biomolecules are crucial for optimal plant adaptation. As proteins are the main effectors inside the cells, their levels need to be tightly regulated. This is partially achieved by specific proteolytic pathways via multicatalytic protease complexes defined as 20S and 26S proteasomes. In plants, the 20S proteasome is responsible for degradation of carbonylated proteins, while the 26S being a part of ubiquitin-proteasome pathway is known to be involved in proteolysis of phytohormone signaling regulators. On the other hand, the role of translational control of plant development is also well-documented, especially in the context of pollen tube growth and light signaling. Despite the current progress that has been made in seed biology, the sequence of cellular events that determine if the seed can germinate or not are still far from complete understanding. The role and mechanisms of regulation of proteome composition during processes occurring in the plant’s photosynthetic tissues have been well-characterized since many years, but in non-photosynthetic seeds it has emerged as a tempting research task only since the last decade. This review discusses the recent discoveries providing insights into the role of protein turnover in seed dormancy alleviation, and germination, with a focus on the control of translation and proteasomal proteolysis. The presented novel data of translatome profiling in seeds highlighted that post-transcriptional regulation of germination results from a timely regulated initiation of translation. In addition, the importance of 26S proteasome in the degradation of regulatory elements of cellular signaling and that of the 20S complex in proteolysis of specific carbonylated proteins in hormonal- and light-dependent processes occurring in seeds is discussed. Based on the current knowledge the model of cellular recycling of proteins in germinating seeds is also proposed.

Translational downregulation of HSP90 expression by iron chelators in neuroblastoma cells.

Iron is an essential cellular nutrient, being a critical cofactor of several proteins involved in cell growth and replication. Compared with normal cells, neoplastic cells have been shown to require a greater amount of iron, thus laying the basis for the promising anticancer activity of iron chelators. In this work, we evaluated the effects of molecules with iron chelation activity on neuroblastoma (NB) cell lines. Of the 17 iron chelators tested, six reduced cell viability of two NB cell lines with an inhibition of growth of 50% below 10 µM; four of the six molecules-ciclopirox olamine (CPX), piroctone, 8-hydroxyquinoline, and deferasirox-were also shown to efficiently chelate intracellular iron within minutes after addition. Effects on cell viability of one of the compounds, CPX, were indeed dependent on chelation of intracellular iron and mediated by both G0/G1 cell cycle block and induction of apoptosis. By combined transcriptome and translatome profiling we identified early translational downregulation of several members of the heat shock protein group as a specific effect of CPX treatment. We functionally confirmed iron-dependent depletion of HSP90 and its client proteins at pharmacologically achievable concentrations of CPX, and we extended this effect to piroctone, 8-hydroxyquinoline, and deferasirox. Given the documented sensitivity of NB cells to HSP90 inhibition, we propose CPX and other iron chelators as investigational antitumor agents in NB therapy.

Translatome profiling: methods for genome-scale analysis of mRNA translation.

During the past decade, there has been a rapidly increased appreciation of the role of translation as a key regulatory node in gene expression. Thereby, the development of methods to infer the translatome, which refers to the entirety of mRNAs associated with ribosomes for protein synthesis, has facilitated the discovery of new principles and mechanisms of translation and expanded our view of the underlying logic of protein synthesis. Here, we review the three main methodologies for translatome analysis, and we highlight some of the recent discoveries made using each technique. We first discuss polysomal profiling, a classical technique that involves the separation of mRNAs depending on the number of bound ribosomes using a sucrose gradient, and which has been combined with global analysis tools such as DNA microarrays or high-throughput RNA sequencing to identify the RNAs in polysomal fractions. We then introduce ribosomal profiling, a recently established technique that enables the mapping of ribosomes along mRNAs at near-nucleotide resolution on a global scale. We finally refer to ribosome affinity purification techniques that are based on the cell-type-specific expression of tagged ribosomal proteins, allowing the capture of translatomes from specialized cells in organisms. We discuss the advantages and disadvantages of these three main techniques in the pursuit of defining the translatome, and we speculate about future developments.

How to discover new proteins—translatome profiling

How to discover new proteins—translatome profiling

28S rRNA is inducibly pseudouridylated by the mTOR pathway translational control in CHO cell cultures

The mTOR pathway is a conserved master regulator of translational activity that influences the fate of industrially relevant CHO cell cultures, yet its molecular mechanisms remain unclear. Interestingly, rapamycin specific inhibition of the mTOR pathway in CHO cells was found to down-regulate the small nucleolar RNA U19 (snoRNA U19) by 2-fold via translatome profiling. snoRNA U19 guides the two most conserved pseudouridylation modifications on 28S ribosomal RNA (rRNA) that are important for the biogenesis and proper function of ribosomes. In order to further understand the role of snoRNA U19 as a potential player in the mTOR pathway, we measured 28S rRNA pseudouridylation upon rapamycin treatments and/or snoRNA U19 overexpression conditions, thereby characterizing the subsequent effects on ribosome efficiency and global translation by polysome profiling. We showed that 28S rRNA pseudouridylation was increased by rapamycin treatment and/or overexpression of snoRNA U19, but only the latter condition improved ribosome efficiency toward higher global translation, thus implying that the mTOR pathway induces pseudouridylation at different sites along the 28S rRNA possibly with either positive or negative effects on the cellular phenotype. This discovery of snoRNA U19 as a new downstream effector of the mTOR pathway suggests that cell engineering of snoRNAs can be used to regulate translation and improve cellular growth in CHO cell cultures in the future.

PUNCH-P for global translatome profiling: Methodology, insights and comparison to other techniques.

Regulation of mRNA translation is a major modulator of gene expression, allowing cells to fine tune protein levels during growth and differentiation and in response to physiological signals and environmental changes. Mass-spectrometry and RNA-sequencing methods now enable global profiling of the translatome, but these still involve significant analytical and economical limitations. We developed a novel system-wide proteomic approach for direct monitoring of translation, termed PUromycin-associated Nascent CHain Proteomics (PUNCH-P), which is based on the recovery of ribosome-nascent chain complexes from cells or tissues followed by incorporation of biotinylated puromycin into newly-synthesized proteins. Biotinylated proteins are then purified by streptavidin and analyzed by mass-spectrometry. Here we present an overview of PUNCH-P, describe other methodologies for global translatome profiling (pSILAC, BONCAT, TRAP/Ribo-tag, Ribo-seq) and provide conceptual comparisons between these methods. We also show how PUNCH-P data can be combined with mRNA measurements to determine relative translation efficiency for specific mRNAs.

A versatile method for cell-specific profiling of translated mRNAs in Drosophila.

In Drosophila melanogaster few methods exist to perform rapid cell-type or tissue-specific expression profiling. A translating ribosome affinity purification (TRAP) method to profile actively translated mRNAs has been developed for use in a number of multicellular organisms although it has only been implemented to examine limited sets of cell- or tissue-types in these organisms. We have adapted the TRAP method for use in the versatile GAL4/UAS system of Drosophila allowing profiling of almost any tissue/cell-type with a single genetic cross. We created transgenic strains expressing a GFP-tagged ribosomal protein, RpL10A, under the control of the UAS promoter to perform cell-type specific translatome profiling. The GFP::RpL10A fusion protein incorporates efficiently into ribosomes and polysomes. Polysome affinity purification strongly enriches mRNAs from expected genes in the targeted tissues with sufficient sensitivity to analyze expression in small cell populations. This method can be used to determine the unique translatome profiles in different cell-types under varied physiological, pharmacological and pathological conditions.

Cell‐type specific analysis of translating RNAs in developing flowers reveals new levels of control

Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.

Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis

Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion cells of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels.

Propagation protocol for crimsoneyed rosemallowHibiscus moscheutosL. (Malvaceae)

Iron is an essential cellular nutrient, being a critical cofactor of several proteins involved in cell growth and replication. Compared with normal cells, neoplastic cells have been shown to require a greater amount of iron, thus laying the basis for the promising anticancer activity of iron chelators. In this work, we evaluated the effects of molecules with iron chelation activity on neuroblastoma (NB) cell lines. Of the 17 iron chelators tested, six reduced cell viability of two NB cell lines with an inhibition of growth of 50% below 10 <i>µ</i>M; four of the six molecules—ciclopirox olamine (CPX), piroctone, 8-hydroxyquinoline, and deferasirox—were also shown to efficiently chelate intracellular iron within minutes after addition. Effects on cell viability of one of the compounds, CPX, were indeed dependent on chelation of intracellular iron and mediated by both G₀/G₁ cell cycle block and induction of apoptosis. By combined transcriptome and translatome profiling we identified early translational downregulation of several members of the heat shock protein group as a specific effect of CPX treatment. We functionally confirmed iron-dependent depletion of HSP90 and its client proteins at pharmacologically achievable concentrations of CPX, and we extended this effect to piroctone, 8-hydroxyquinoline, and deferasirox. Given the documented sensitivity of NB cells to HSP90 inhibition, we propose CPX and other iron chelators as investigational antitumor agents in NB therapy.