ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia.

Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.

Stromal changes in the aged lung induce an emergence from melanoma dormancy.

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.

An Interleukin-23-Interleukin-22 Axis Regulates Intestinal Microbial Homeostasis to Protect from Diet-Induced Atherosclerosis.

Although commensal flora is involved in the regulation of immunity, the interplay between cytokine signaling and microbiota in atherosclerosis remains unknown. We found that interleukin (IL)-23 and its downstream target IL-22 restricted atherosclerosis by repressing pro-atherogenic microbiota. Inactivation of IL-23-IL-22 signaling led to deterioration of the intestinal barrier, dysbiosis, and expansion of pathogenic bacteria with distinct biosynthetic and metabolic properties, causing systemic increase in pro-atherogenic metabolites such as lipopolysaccharide (LPS) and trimethylamine N-oxide (TMAO). Augmented disease in the absence of the IL-23-IL-22 pathway was mediated in part by pro-atherogenic osteopontin, controlled by microbial metabolites. Microbiota transfer from IL-23-deficient mice accelerated atherosclerosis, whereas microbial depletion or IL-22 supplementation reduced inflammation and ameliorated disease. Our work uncovers the IL-23-IL-22 signaling as a regulator of atherosclerosis that restrains expansion of pro-atherogenic microbiota and argues for informed use of cytokine blockers to avoid cardiovascular side effects driven by microbiota and inflammation.

IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4-6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death.

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth in vitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers ex vivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Regulation of EBNA1 protein stability and DNA replication activity by PLOD1 lysine hydroxylase.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

DLBCL-associated NOTCH2 mutations escape ubiquitin-dependent degradation and promote chemoresistance.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.

Global Analysis of Post-Translational Side-Chain Arginylation Using Pan-Arginylation Antibodies.

Arginylation is a post-translational modification mediated by the arginyltransferase 1 (ATE1), which transfers the amino acid arginine to a protein or peptide substrate from a tRNA molecule. Initially, arginylation was thought to occur only on N-terminally exposed acidic residues, and its function was thought to be limited to targeting proteins for degradation. However, more recent data have shown that ATE1 can arginylate side chains of internal acidic residues in a protein without necessarily affecting metabolic stability. This greatly expands the potential targets and functions of arginylation, but tools for studying this process have remained limited. Here, we report the first global screen specifically for side-chain arginylation. We generate and validate "pan-arginylation" antibodies, which are designed to detect side-chain arginylation in any amino acid sequence context. We use these antibodies for immunoaffinity enrichment of side-chain arginylated proteins from wildtype and Ate1 knockout cell lysates. In this way, we identify a limited set of proteins that likely undergo ATE1-dependent side-chain arginylation and that are enriched in specific cellular roles, including translation, splicing, and the cytoskeleton.

Ghost mitochondria drive metastasis through adaptive GCN2/Akt therapeutic vulnerability.

Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.

Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression.

Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and ß1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.

Adaptation of the heart to Frataxin depletion: Evidence that integrated stress response can predominate over mTORC1 activation.

Friedreich's ataxia (FRDA) is an inherited disorder caused by depletion of frataxin (FXN), a mitochondrial protein required for iron-sulfur cluster (ISC) biogenesis. Cardiac dysfunction is the main cause of death. Yet pathogenesis, and, more generally, how the heart adapts to FXN loss, remain poorly understood, though are expected to be linked to an energy deficit. We modified a transgenic (TG) mouse model of inducible FXN depletion that permits phenotypic evaluation of the heart at different FXN levels, and focused on substrate-specific bioenergetics and stress signaling. When FXN protein in the TG heart was 17% of normal, bioenergetics and signaling were not different from control. When, 8 weeks later, FXN was ~ 97% depleted in the heart, TG heart mass and cardiomyocyte cross-sectional area were less, without evidence of fibrosis or apoptosis. mTORC1 signaling was activated, as was the integrated stress response, evidenced by greater phosphorylation of eIF2α relative to total eIF2α, and decreased protein translation. We interpret these results to suggest that, in TG hearts, an anabolic stimulus was constrained by eIF2α phosphorylation. Cardiac contractility was maintained in the 97%-FXN-depleted hearts, possibly contributed by an unexpected preservation of ß-oxidation, though pyruvate oxidation was lower. Bioenergetics alterations were matched by changes in the mitochondrial proteome, including a non-uniform decrease in abundance of ISC-containing proteins. Altogether, these findings suggest that the FXN depleted heart can suppress a major ATP demanding process such as protein translation, which, together with some preservation of ß-oxidation, could be adaptive, at least in the short term.

EBNA2 driven enhancer switching at the CIITA-DEXI locus suppresses HLA class II gene expression during EBV infection of B-lymphocytes.

Viruses suppress immune recognition through diverse mechanisms. Epstein-Barr Virus (EBV) establishes latent infection in memory B-lymphocytes and B-cell malignancies where it impacts B-cell immune function. We show here that EBV primary infection of naïve B-cells results in a robust down-regulation of HLA genes. We found that the viral encoded transcriptional regulatory factor EBNA2 bound to multiple regulatory regions in the HLA locus. Conditional expression of EBNA2 correlated with the down regulation of HLA class II transcription. EBNA2 down-regulation of HLA transcription was found to be dependent on CIITA, the major transcriptional activator of HLA class II gene transcription. We identified a major EBNA2 binding site downstream of the CIITA gene and upstream of DEXI, a dexamethasone inducible gene that is oriented head-to-head with CIITA gene transcripts. CRISPR/Cas9 deletion of the EBNA2 site upstream of DEXI attenuated CIITA transcriptional repression. EBNA2 caused an increase in DEXI transcription and a graded change in histone modifications with activation mark H3K27ac near the DEXI locus, and a loss of activation marks at the CIITA locus. A prominent CTCF binding site between CIITA and DEXI enhancers was mutated and further diminished the effects of EBNA2 on CIITA. Analysis of HiC data indicate that DEXI and CIITA enhancers are situated in different chromosome topological associated domains (TADs). These findings suggest that EBNA2 down regulates HLA-II genes through the down regulation of CIITA, and that this down regulation is an indirect consequence of EBNA2 enhancer formation at a neighboring TAD. We propose that enhancer competition between these neighboring chromosome domains represents a novel mechanism for gene regulation demonstrated by EBNA2.

A multi-omics approach to Epstein-Barr virus immortalization of B-cells reveals EBNA1 chromatin pioneering activities targeting nucleotide metabolism.

Epstein-Barr virus (EBV) immortalizes resting B-lymphocytes through a highly orchestrated reprogramming of host chromatin structure, transcription and metabolism. Here, we use a multi-omics-based approach to investigate these underlying mechanisms. ATAC-seq analysis of cellular chromatin showed that EBV alters over a third of accessible chromatin during the infection time course, with many of these sites overlapping transcription factors such as PU.1, Interferon Regulatory Factors (IRFs), and CTCF. Integration of RNA-seq analysis identified a complex transcriptional response and associations with EBV nuclear antigens (EBNAs). Focusing on EBNA1 revealed enhancer-binding activity at gene targets involved in nucleotide metabolism, supported by metabolomic analysis which indicated that adenosine and purine metabolism are significantly altered by EBV immortalization. We further validated that adenosine deaminase (ADA) is a direct and critical target of the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA may be useful therapeutic targets for EBV-driven lymphoid cancers.

Selective inhibition of Ph-positive ALL cell growth through kinase-dependent and -independent effects by CDK6-specific PROTACs.

Expression of the cell cycle regulatory gene CDK6 is required for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cell growth, whereas expression of the closely related CDK4 protein is dispensable. Moreover, CDK6 silencing is more effective than treatment with the dual CDK4/6 inhibitor palbociclib in suppressing Ph+ ALL in mice, suggesting that the growth-promoting effects of CDK6 are, in part, kinase-independent in Ph+ ALL. Accordingly, we developed CDK4/6-targeted proteolysis-targeting chimeras (PROTACs) that inhibit CDK6 enzymatic activity in vitro, promote the rapid and preferential degradation of CDK6 over CDK4 in Ph+ ALL cells, and markedly suppress S-phase cells concomitant with inhibition of CDK6-regulated phospho-RB and FOXM1 expression. No such effects were observed in CD34+ normal hematopoietic progenitors, although CDK6 was efficiently degraded. Treatment with the CDK6-degrading PROTAC YX-2-107 markedly suppressed leukemia burden in mice injected with de novo or tyrosine kinase inhibitor-resistant primary Ph+ ALL cells, and this effect was comparable or superior to that of the CDK4/6 enzymatic inhibitor palbociclib. These studies provide "proof of principle" that targeting CDK6 with PROTACs that inhibit its enzymatic activity and promote its degradation represents an effective strategy to exploit the "CDK6 dependence" of Ph+ ALL and, perhaps, of other hematologic malignancies. Moreover, they suggest that treatment of Ph+ ALL with CDK6-selective PROTACs would spare a high proportion of normal hematopoietic progenitors, preventing the neutropenia induced by treatment with dual CDK4/6 inhibitors.

sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance.

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in ß-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine-Thiol Displacement Reaction.

We have developed a novel bioorthogonal reaction that can selectively displace fluorine substitutions alpha to amide bonds. This fluorine-thiol displacement reaction (FTDR) allows for fluorinated cofactors or precursors to be utilized as chemical reporters, hijacking acetyltransferase-mediated acetylation both in vitro and in live cells, which cannot be achieved with azide- or alkyne-based chemical reporters. The fluoroacetamide labels can be further converted to biotin or fluorophore tags using FTDR, enabling the general detection and imaging of acetyl substrates. This strategy may lead to a steric-free labeling platform for substrate proteins, expanding our chemical toolbox for functional annotation of post-translational modifications in a systematic manner.

Leronlimab, a humanized monoclonal antibody to CCR5, blocks breast cancer cellular metastasis and enhances cell death induced by DNA damaging chemotherapy.

BACKGROUND: Triple-negative breast cancer (BCa) (TNBC) is a deadly form of human BCa with limited treatment options and poor prognosis. In our prior analysis of over 2200 breast cancer samples, the G protein-coupled receptor CCR5 was expressed in > 95% of TNBC samples. A humanized monoclonal antibody to CCR5 (leronlimab), used in the treatment of HIV-infected patients, has shown minimal side effects in large patient populations. METHODS: A humanized monoclonal antibody to CCR5, leronlimab, was used for the first time in tissue culture and in mice to determine binding characteristics to human breast cancer cells, intracellular signaling, and impact on (i) metastasis prevention and (ii) impact on established metastasis. RESULTS: Herein, leronlimab was shown to bind CCR5 in multiple breast cancer cell lines. Binding of leronlimab to CCR5 reduced ligand-induced Ca+ 2 signaling, invasion of TNBC into Matrigel, and transwell migration. Leronlimab enhanced the BCa cell killing of the BCa chemotherapy reagent, doxorubicin. In xenografts conducted with Nu/Nu mice, leronlimab reduced lung metastasis of the TNBC cell line, MB-MDA-231, by > 98% at 6 weeks. Treatment with leronlimab reduced the metastatic tumor burden of established TNBC lung metastasis. CONCLUSIONS: The safety profile of leronlimab, together with strong preclinical evidence to both prevent and reduce established breast cancer metastasis herein, suggests studies of clinical efficacy may be warranted.

Uncovering Thousands of New Peptides with Sequence-Mask-Search Hybrid De Novo Peptide Sequencing Framework.

Typical analyses of mass spectrometry data only identify amino acid sequences that exist in reference databases. This restricts the possibility of discovering new peptides such as those that contain uncharacterized mutations or originate from unexpected processing of RNAs and proteins. De novo peptide sequencing approaches address this limitation but often suffer from low accuracy and require extensive validation by experts. Here, we develop SMSNet, a deep learning-based de novo peptide sequencing framework that achieves >95% amino acid accuracy while retaining good identification coverage. Applications of SMSNet on landmark proteomics and peptidomics studies reveal over 10,000 previously uncharacterized HLA antigens and phosphopeptides, and in conjunction with database-search methods, expand the coverage of peptide identification by almost 30%. The power to accurately identify new peptides of SMSNet would make it an invaluable tool for any future proteomics and peptidomics studies, including tumor neoantigen discovery, antibody sequencing, and proteome characterization of non-model organisms.

Myc-mediated transcriptional regulation of the mitochondrial chaperone TRAP1 controls primary and metastatic tumor growth.

The role of mitochondria in cancer continues to be debated, and whether exploitation of mitochondrial functions is a general hallmark of malignancy or a tumor- or context-specific response is still unknown. Using a variety of cancer cell lines and several technical approaches, including siRNA-mediated gene silencing, ChIP assays, global metabolomics and focused metabolite analyses, bioenergetics, and cell viability assays, we show that two oncogenic Myc proteins, c-Myc and N-Myc, transcriptionally control the expression of the mitochondrial chaperone TNFR-associated protein-1 (TRAP1) in cancer. In turn, this Myc-mediated regulation preserved the folding and function of mitochondrial oxidative phosphorylation (OXPHOS) complex II and IV subunits, dampened reactive oxygen species production, and enabled oxidative bioenergetics in tumor cells. Of note, we found that genetic or pharmacological targeting of this pathway shuts off tumor cell motility and invasion, kills Myc-expressing cells in a TRAP1-dependent manner, and suppresses primary and metastatic tumor growth in vivo We conclude that exploitation of mitochondrial functions is a general trait of tumorigenesis and that this reliance of cancer cells on mitochondrial OXPHOS pathways could offer an actionable therapeutic target in the clinic.