Oral antimicrobial therapy for cellulitis versus outpatient parenteral antimicrobial therapy: a single-centre audit of cellulitis outcomes.

BACKGROUND: Cellulitis is a common acute skin and soft tissue infection that causes substantial morbidity and healthcare costs. AIMS: To audit the impact on cellulitis management, regimen tolerability and outcomes of switching from outpatient parenteral antimicrobial therapy (OPAT) using intravenous (i.v.) cefazolin once daily plus probenecid to oral beta-lactam therapy (OBLT) using oral flucloxacillin plus probenecid. METHODS: We undertook a retrospective audit on cellulitis management, regimen tolerability and outcomes at the Dunedin Public Hospital Emergency Department (ED) before and after a change of the local outpatient cellulitis treatment pathway from OPAT using i.v. cefazolin once daily plus probenecid to OBLT using oral flucloxacillin plus probenecid. RESULTS: OPAT was used in 97/123 (78.9%) patients with cellulitis before compared to 1/70 (1.4%) after the pathway change (odds ratio (OR), 0.04, P < 0.01). OBLT was used in 26/123 (21.1%) patients with cellulitis before and 69/70 (98.6%) after (OR, 218.8, P < 0.01). Antimicrobial change due to intolerance occurred in 4/123 (3.2%) patients with cellulitis before and 4/70 (5.7%) after (OR, 1.8, P, not significant (NS)) the pathway change. Inpatient admission within 28 days occurred in 15/123 (12.2%) cellulitis patients before and 9/70 (12.9%) after (OR, 1.1, P, NS) the pathway change. CONCLUSIONS: Implementation of a change in outpatient cellulitis treatment pathway resulted in a significant change in prescribing practice. Our findings suggest that OBLT was both tolerable and had similar outcomes to OPAT.

HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development.

The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cell-based quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.

Tumour-induced osteomalacia due to residual benign glomangioma.

Tumour-induced osteomalacia (TIO) is a rare paraneoplastic syndrome. The constellation of findings of unprovoked fractures, hypophosphataemia, urinary phosphate wasting and a negative genetic evaluation suggest a TIO diagnosis. Tumours leading to TIO are often small and difficult to localise using standard imaging studies. The 68Ga-DOTATATE CT/positron emission tomography, a somatostatin receptor imaging modality, is the radiographical study of choice for localisation. It is highly sensitive and specific since tumours that cause oncogenic osteomalacia have been shown to express somatostatin receptors. Complete surgical resection is the treatment of choice; however, it may not always be feasible. Burosumab, a human anti-fibroblast growth factor-23 monoclonal antibody, is a therapeutic option in cases of unresectable TIO to normalise phosphorus levels and improve fracture healing. Our patient was initiated on burosumab, which led to healing of his fractures and profound symptomatic improvement of his pain. TIO is often undiagnosed for many years, leading to significant patient morbidity.

Upregulation of Receptor Tyrosine Kinase Activity and Stemness as Resistance Mechanisms to Akt Inhibitors in Breast Cancer.

The PI3K/Akt pathway is frequently deregulated in human cancers, and multiple Akt inhibitors are currently under clinical evaluation. Based on the experience from other molecular targeted therapies, however, it is likely that acquired resistance will be developed in patients treated with Akt inhibitors. We established breast cancer models of acquired resistance by prolonged treatment of cells with allosteric or ATP-competitive Akt inhibitors. Phospho-Receptor tyrosine kinase (Phospho-RTK) arrays revealed hyper-phosphorylation of multiple RTKS, including EGFR, Her2, HFGR, EhpB3 and ROR1, in Akt-inhibitor-resistant cells. Importantly, resistance can be overcome by treatment with an EGFR inhibitor. We further showed that cancer stem cells (CSCs) are enriched in breast tumor cells that have developed resistance to Akt inhibitors. Several candidates of CSC regulators, such as ID4, are identified by RNA sequencing. Cosmic analysis indicated that sensitivity of tumor cells to Akt inhibitors can be predicted by ID4 and stem cell/epithelial-mesenchymal transition pathway targets. These findings indicate the potential of targeting the EGFR pathway and CSC program to circumvent Akt inhibitor resistance in breast cancer.

One Health security lessons from a year-long webinar series on international COVID-19 response.

Following the principles outlined by the Global Outbreak Alert and Response Network, the Federal Bureau of Investigation's International Biosecurity and Prevention Forum, the European Commission's Joint Research Centre, and the Middlebury Institute of International Studies' James Martin Center for Nonproliferation Studies cohosted a webinar series from April 2020 to January 2021 on COVID-19 management across Africa, Europe, and North America. We provide here an overview of the webinar series and discuss how lessons learned during the COVID-19 pandemic and debated during the webinars can be used to bridge One Health with biological threat-driven health security. This report can be used to inform recommendations for future One Health security approaches to strengthen global capacity and multidisciplinary cooperation.

Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy.

The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.

Copper Modulates the Catalytic Activity of Protein Kinase CK2.

Casein kinase 2 (CK2) is an evolutionarily conserved serine/threonine kinase implicated in a wide range of cellular functions and known to be dysregulated in various diseases such as cancer. Compared to most other kinases, CK2 exhibits several unusual properties, including dual co-substrate specificity and a high degree of promiscuity with hundreds of substrates described to date. Most paradoxical, however, is its apparent constitutive activity: no definitive mode of catalytic regulation has thus far been identified. Here we demonstrate that copper enhances the enzymatic activity of CK2 both in vitro and in vivo. We show that copper binds directly to CK2, and we identify specific residues in the catalytic subunit of the enzyme that are critical for copper-binding. We further demonstrate that increased levels of intracellular copper result in enhanced CK2 kinase activity, while decreased copper import results in reduced CK2 activity. Taken together, these findings establish CK2 as a copper-regulated kinase and indicate that copper is a key modulator of CK2-dependent signaling pathways.

BRAFV600E-Driven Lung Adenocarcinoma Requires Copper to Sustain Autophagic Signaling and Processing.

The transition metal copper (Cu) is an essential micronutrient required for development and proliferation, but the molecular mechanisms by which Cu contributes to these processes is not fully understood. Although traditionally studied as a static cofactor critical for the function of Cu-dependent enzymes, an expanding role for Cu is emerging to include its novel function as a dynamic mediator of signaling processes through the direct control of protein kinase activity. We now appreciate that Cu directly binds to and influences MEK1/2 and ULK1/2 kinase activity, and show here that reductions in MAPK and autophagic signaling are associated with dampened growth and survival of oncogenic BRAF-driven lung adenocarcinoma cells upon loss of Ctr1. Efficient autophagy, clonogenic survival, and tumorigenesis of BRAF-mutant cells required ULK1 Cu-binding. Although treatment with canonical MAPK inhibitors resulted in the upregulation of protective autophagy, mechanistically, the Cu chelator tetrathiomolybdate (TTM) was sufficient to target both autophagic and MAPK signaling as a means to blunt BRAF-driven tumorigenic properties. These findings support leveraging Cu chelation with TTM as an alternative therapeutic strategy to impair autophagy and MAPK signaling. As traditional MAPK monotherapies initiate autophagy signaling and promote cancer cell survival. IMPLICATIONS: We establish that copper chelation therapy inhibits both autophagy and MAPK signaling in BRAFV600E-driven lung adenocarcinoma, thus overcoming the upregulation of protective autophagy elicited by canonical MAPK pathway inhibitors.

Loss of p19Arf promotes fibroblast survival during leucine deprivation.

Fibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated, however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here, we show that prolonged leucine deprivation results in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation. This article has an associated First Person interview with the first author of the paper.

Glutamine deprivation triggers NAGK-dependent hexosamine salvage.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

Copper biology.

Metals are vital for life as they are necessary for essential biological processes. Traditionally, metals are categorized as either dynamic signals or static cofactors. Redox-inactive metals such as calcium (Ca), potassium (K), sodium (Na), and zinc (Zn) signal through large fluctuations in their metal-ion pools. In contrast, redox-active transition metals such as copper (Cu) and iron (Fe) drive catalysis and are largely characterized as static cofactors that must be buried and protected within the active sites of proteins, due to their ability to generate damaging reactive-oxygen species through Fenton chemistry. Cu has largely been studied as a static cofactor in fundamental processes from cellular respiration to pigmentation, working through cytochrome c oxidase and tyrosinase, respectively. However, within the last decade, a new paradigm in nutrient sensing and protein regulation - termed 'metalloallostery' - has emerged, expanding the repertoire of Cu beyond the catalytic proteins to dynamic signaling molecules essential for cellular processes that impact normal physiology and disease states. In this Primer we introduce both the 'traditional' and emerging roles for Cu in biology and the many ways in which Cu intersects with human health.

Tiny Earth: A Big Idea for STEM Education and Antibiotic Discovery.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery.

Inverse Association between Serotonin 2A Receptor Antagonist Medication Use and Mortality in Severe COVID-19 Infection.

Advanced age and medical co-morbidity are strong predictors of mortality in COVID-19 infection. Yet few studies (to date) have specifically addressed risk factors associated with COVID-19 mortality in a high-risk subgroup of older US adults having one or more chronic diseases. Our hypothesis is that medications having 'off-target' anti-inflammatory effects may play a role in modulating the immune response in COVID-19 infection. We analyzed baseline risk factors associated with respiratory failure or death in 55 older adult US military veterans hospitalized for COVID-19 infection during (March-June 2020) the peak of the pandemic in New Jersey. Fifty-three percent (29/55) of patients experienced respiratory failure and thirty-one percent (17/55) died. In adjusted logistic regression analysis, baseline neutrophil to lymphocyte ratio (NLR) (P=0.0035) and body mass index (P=0.03) were significant predictors of the risk for respiratory failure. Age (P=0.05) and non-use (vs. use) of psychotropic medications having serotonin 2A receptor antagonist properties (odds ratio 5.06; 95% confidence intervals 1.18-21.7; P= 0.029) was each a significant predictor of an increased risk of death. There was a significant interaction effect of age and non-use (vs.. use) of psychotropic serotonin 2A receptor antagonist medications on the odds ratio (OR) for death (P=0.011). In selected, ventilator-dependent COVID-19 pneumonia patients treated with psychotropic serotonin 2A receptor antagonist medications to control agitation and ICU delirium, there was an apparent positive association between medication use and significant rise in the absolute lymphocyte count and decrease in the neutrophil: lymphocyte ratio. Taken together, these data are the first to suggest that certain psychotropic medications used in the treatment of chronic psychiatric illness and/or for acute delirium are inversely associated with mortality in severe COVID-19 infection by unknown mechanism which may involve (in part) immunomodulatory effects.

Mining and visualizing high-order directional drug interaction effects using the FAERS database.

BACKGROUND: Adverse drug events (ADEs) often occur as a result of drug-drug interactions (DDIs). The use of data mining for detecting effects of drug combinations on ADE has attracted growing attention and interest, however, most studies focused on analyzing pairwise DDIs. Recent efforts have been made to explore the directional relationships among high-dimensional drug combinations and have shown effectiveness on prediction of ADE risk. However, the existing approaches become inefficient from both computational and illustrative perspectives when considering more than three drugs. METHODS: We proposed an efficient approach to estimate the directional effects of high-order DDIs through frequent itemset mining, and further developed a novel visualization method to organize and present the high-order directional DDI effects involving more than three drugs in an interactive, concise and comprehensive manner. We demonstrated its performance by mining the directional DDIs associated with myopathy using a publicly available FAERS dataset. RESULTS: Directional effects of DDIs involving up to seven drugs were reported. Our analysis confirmed previously reported myopathy associated DDIs including interactions between fusidic acid with simvastatin and atorvastatin. Furthermore, we uncovered a number of novel DDIs leading to increased risk for myopathy, such as the co-administration of zoledronate with different types of drugs including antibiotics (ciprofloxacin, levofloxacin) and analgesics (acetaminophen, fentanyl, gabapentin, oxycodone). Finally, we visualized directional DDI findings via the proposed tool, which allows one to interactively select any drug combination as the baseline and zoom in/out to obtain both detailed and overall picture of interested drugs. CONCLUSIONS: We developed a more efficient data mining strategy to identify high-order directional DDIs, and designed a scalable tool to visualize high-order DDI findings. The proposed method and tool have the potential to contribute to the drug interaction research and ultimately impact patient health care. AVAILABILITY AND IMPLEMENTATION: http://lishenlab.com/d3i/explorer.html.

Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma.

Although the transition metal copper (Cu) is an essential nutrient that is conventionally viewed as a static cofactor within enzyme active sites, a non-traditional role for Cu as a modulator of kinase signalling is emerging. Here, we found that Cu is required for the activity of the autophagic kinases ULK1 and ULK2 (ULK1/2) through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt the binding of Cu reduced ULK1/2-dependent signalling and the formation of autophagosome complexes. Increased levels of intracellular Cu are associated with starvation-induced autophagy and are sufficient to enhance ULK1 kinase activity and, in turn, autophagic flux. The growth and survival of lung tumours driven by KRASG12D is diminished in the absence of Ctr1, is dependent on ULK1 Cu binding and is associated with reduced levels of autophagy and signalling. These findings suggest a molecular basis for exploiting Cu-chelation therapy to prevent autophagy signalling to limit proliferation and improve patient survival in cancer.

Inhibition of BCL2 Family Members Increases the Efficacy of Copper Chelation in BRAFV600E-Driven Melanoma.

The principal unmet need in BRAFV600E-positive melanoma is lack of an adequate therapeutic strategy capable of overcoming resistance to clinically approved targeted therapies against oncogenic BRAF and/or the downstream MEK1/2 kinases. We previously discovered that copper (Cu) is required for MEK1 and MEK2 activity through a direct Cu-MEK1/2 interaction. Repurposing the clinical Cu chelator tetrathiomolybdate (TTM) is supported by efficacy in BRAFV600E-driven melanoma models, due in part to inhibition of MEK1/2 kinase activity. However, the antineoplastic activity of Cu chelators is cytostatic. Here, we performed high-throughput small-molecule screens to identify bioactive compounds that synergize with TTM in BRAFV600E-driven melanoma cells. Genetic perturbation or pharmacologic inhibition of specific members of the BCL2 family of antiapoptotic proteins (BCL-W, BCL-XL, and MCL1) selectively reduced cell viability when combined with a Cu chelator and induced CASPASE-dependent cell death. Further, in BRAFV600E-positive melanoma cells evolved to be resistant to BRAF and/or MEK1/2 inhibitors, combined treatment with TTM and the clinically evaluated BCL2 inhibitor, ABT-263, restored tumor growth suppression and induced apoptosis. These findings further support Cu chelation as a therapeutic strategy to target oncogene-dependent tumor cell growth and survival by enhancing Cu chelator efficacy with chemical inducers of apoptosis, especially in the context of refractory or relapsed BRAFV600E-driven melanoma. SIGNIFICANCE: This study unveils a novel collateral drug sensitivity elicited by combining copper chelators and BH3 mimetics for treatment of BRAFV600E mutation-positive melanoma.

A Genome-wide CRISPR Screen Identifies ZCCHC14 as a Host Factor Required for Hepatitis B Surface Antigen Production.

A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.

Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism.

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

Copper chaperone ATOX1 is required for MAPK signaling and growth in BRAF mutation-positive melanoma.

Copper (Cu) is a tightly regulated micronutrient that functions as a structural or catalytic cofactor for specific proteins essential for a diverse array of biological processes. While the study of the extremely rare genetic diseases, Menkes and Wilson, has highlighted the requirement for proper Cu acquisition and elimination in biological systems for cellular growth and proliferation, the importance of dedicated Cu transport systems, like the Cu chaperones ATOX1 and CCS, in the pathophysiology of cancer is not well defined. We found that ATOX1 was significantly overexpressed in human blood, breast, and skin cancer samples, while CCS was significantly altered in human brain, liver, ovarian, and prostate cancer when compared to normal tissue. Further analysis of genetic expression data in Cancer Cell Line Encyclopedia (CCLE) revealed that ATOX1 is highly expressed in melanoma cell lines over other cancer cell lines. We previously found that Cu is required for BRAFV600E-driven MAPK signaling and melanomagenesis. Here we show that genetic loss of ATOX1 decreased BRAFV600E-dependent growth and signaling in human melanoma cell lines. Pharmacological inhibition of ATOX1 with a small molecule, DCAC50, decreased the phosphorylation of ERK1/2 and reduced the growth of BRAF mutation-positive melanoma cell lines in a dose-dependent manner. Taken together, these results suggest that targeting the Cu chaperone ATOX1 as a novel therapeutic angle in BRAFV600E-driven melanomas.