Cellular neonatal Fc receptor recycling efficiencies can differentiate target-independent clearance mechanisms of monoclonal antibodies.

In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain ß2-microglobulin. A series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.

Liver-specific mitochondrial amidoxime-reducing component 1 (Mtarc1) knockdown protects the liver from diet-induced MASH in multiple mouse models.

BACKGROUND: Human genetic studies have identified several mitochondrial amidoxime-reducing component 1 (MTARC1) variants as protective against metabolic dysfunction-associated steatotic liver disease. The MTARC1 variants are associated with decreased plasma lipids and liver enzymes and reduced liver-related mortality. However, the role of mARC1 in fatty liver disease is still unclear. METHODS: Given that mARC1 is mainly expressed in hepatocytes, we developed an N-acetylgalactosamine-conjugated mouse Mtarc1 siRNA, applying it in multiple in vivo models to investigate the role of mARC1 using multiomic techniques. RESULTS: In ob/ob mice, knockdown of Mtarc1 in mouse hepatocytes resulted in decreased serum liver enzymes, LDL-cholesterol, and liver triglycerides. Reduction of mARC1 also reduced liver weight, improved lipid profiles, and attenuated liver pathological changes in 2 diet-induced metabolic dysfunction-associated steatohepatitis mouse models. A comprehensive analysis of mARC1-deficient liver from a metabolic dysfunction-associated steatohepatitis mouse model by metabolomics, proteomics, and lipidomics showed that Mtarc1 knockdown partially restored metabolites and lipids altered by diet. CONCLUSIONS: Taken together, reducing mARC1 expression in hepatocytes protects against metabolic dysfunction-associated steatohepatitis in multiple murine models, suggesting a potential therapeutic approach for this chronic liver disease.

Context-dependent regulation of lipid accumulation in adipocytes by a HIF1α-PPARγ feedback network.

Hypoxia-induced upregulation of HIF1α triggers adipose tissue dysfunction and insulin resistance in obese patients. HIF1α closely interacts with PPARγ, the master regulator of adipocyte differentiation and lipid accumulation, but there are conflicting results regarding how this interaction controls the excessive lipid accumulation that drives adipocyte dysfunction. To directly address these conflicts, we established a differentiation system that recapitulated prior seemingly opposing observations made across different experimental settings. Using single-cell imaging and coarse-grained mathematical modeling, we show how HIF1α can both promote and repress lipid accumulation during adipogenesis. Our model predicted and our experiments confirmed that the opposing roles of HIF1α are isolated from each other by the positive-feedback-mediated upregulation of PPARγ that drives adipocyte differentiation. Finally, we identify three factors: strength of the differentiation cue, timing of hypoxic perturbation, and strength of HIF1α expression changes that, when considered together, provide an explanation for many of the previous conflicting reports.

ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, possesses characteristic alterations to multiple metabolic pathways, including the accumulation of cytosolic lipid droplets. However, the pathways that drive lipid droplet accumulation in ccRCC cells and their importance to cancer biology remain poorly understood. METHODS: We sought to identify the carbon sources necessary for lipid droplet accumulation using Oil red O staining and isotope-tracing lipidomics. The role of the acyl-CoA synthetase (ACSL) family members, an important group of lipid metabolic enzymes, was investigated using siRNA and drug mediated inhibition. CTB and XTT assays were performed to determine the effect of ACSL3 knockdown and lipid starvation on ccRCC cell viability and shRNA was used to study the effect of ACSL3 in an orthotopic mouse model. The relationship between ferroptosis susceptibility of ccRCC and ACSL3 controlled lipid metabolism was examined using CTB and FACS-based assays. The importance of 5-LOX in ferroptosis susceptibility in ccRCC was shown with XTT survival assays, and the expression level and predictive value of 5-LOX in TCGA ccRCC data was assessed. RESULTS: We found that ccRCC cells obtain the necessary substrates for lipid droplet accumulation by metabolizing exogenous serum derived lipids and not through de novo lipogenesis. We show that this metabolism of exogenous fatty acids into lipid droplets requires the enzyme acyl-CoA synthetase 3 (ACSL3) and not other ACSL family proteins. Importantly, genetic or pharmacologic suppression of ACSL3 is cytotoxic to ccRCC cells in vitro and causes a reduction of tumor weight in an orthotopic mouse model. Conversely, ACSL3 inhibition decreases the susceptibility of ccRCC cells to ferroptosis, a non-apoptotic form of cell death involving lipid peroxidation. The sensitivity of ccRCC to ferroptosis is also highly dependent on the composition of exogenous fatty acids and on 5-lipoxygenase (5-LOX), a leukotriene producing enzyme which produces lipid peroxides that have been implicated in other cancers but not in ccRCC. CONCLUSIONS: ACSL3 regulates the accumulation of lipid droplets in ccRCC and is essential for tumor growth. In addition, ACSL3 also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both functions of ACSL3 could be exploited for ccRCC therapy.

KLF15 cistromes reveal a hepatocyte pathway governing plasma corticosteroid transport and systemic inflammation.

Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6, which encodes corticosteroid-binding globulin (CBG). Klf15-deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf153xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6, where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

The HIF target MAFF promotes tumor invasion and metastasis through IL11 and STAT3 signaling.

Hypoxia plays a critical role in tumor progression including invasion and metastasis. To determine critical genes regulated by hypoxia that promote invasion and metastasis, we screen fifty hypoxia inducible genes for their effects on invasion. In this study, we identify v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (MAFF) as a potent regulator of tumor invasion without affecting cell viability. MAFF expression is elevated in metastatic breast cancer patients and is specifically correlated with hypoxic tumors. Combined ChIP- and RNA-sequencing identifies IL11 as a direct transcriptional target of the heterodimer between MAFF and BACH1, which leads to activation of STAT3 signaling. Inhibition of IL11 results in similar levels of metastatic suppression as inhibition of MAFF. This study demonstrates the oncogenic role of MAFF as an activator of the IL11/STAT3 pathways in breast cancer.

Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.

Mitochondrial copper depletion suppresses triple-negative breast cancer in mice.

Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

An Activatable NIR Fluorescent Rosol for Selectively Imaging Nitroreductase Activity.

Hypoxia (pO2 ≤ ~1.5%) is an important characteristic of tumor microenvironments that directly correlates with resistance against first-line therapies and tumor proliferation/infiltration. The ability to accurately identify hypoxic tumor cells/tissue could afford tailored therapeutic regimens for personalized treatment, development of more-effective therapies, and discerning the mechanisms underlying disease progression. Fluorogenic constructs identifying aforesaid cells/tissue operate by targeting the bioreductive activity of primarily nitroreductases (NTRs), but collectively present photophysical and/or physicochemical shortcomings that could limit effectiveness. To overcome these limitations, we present the rational design, development, and evaluation of the first activatable ultracompact xanthene core-based molecular probe (NO 2 -Rosol) for selectively imaging NTR activity that affords an "OFF-ON" near-infrared (NIR) fluorescence response (> 700 nm) alongside a remarkable Stokes shift (> 150 nm) via NTR activity-facilitated modulation to its energetics whose resultant interplay discontinues an intramolecular d-PET fluorescence-quenching mechanism transpiring between directly-linked electronically-uncoupled π-systems comprising its components. DFT calculations guided selection of a suitable fluorogenic scaffold and nitroaromatic moiety candidate that when adjoined could (i) afford such photophysical response upon bioreduction by upregulated NTR activity in hypoxic tumor cells/tissue and (ii) employ a retention mechanism strategy that capitalizes on an inherent physical property of the NIR fluorogenic scaffold for achieving signal amplification. NO 2 -Rosol demonstrated 705 nm NIR fluorescence emission and 157 nm Stokes shift, selectivity for NTR over relevant bioanalytes, and a 28-/12-fold fluorescence enhancement in solution and between cells cultured under different oxic conditions, respectively. In establishing feasibility for NO 2 -Rosol to provide favorable contrast levels in solutio/vitro, we anticipate NO 2 -Rosol doing so in preclinical studies.

Acetate supplementation restores chromatin accessibility and promotes tumor cell differentiation under hypoxia.

Despite the fact that Otto H. Warburg discovered the Warburg effect almost one hundred years ago, why cancer cells waste most of the glucose carbon as lactate remains an enigma. Warburg proposed a connection between the Warburg effect and cell dedifferentiation. Hypoxia is a common tumor microenvironmental stress that induces the Warburg effect and blocks tumor cell differentiation. The underlying mechanism by which this occurs is poorly understood, and no effective therapeutic strategy has been developed to overcome this resistance to differentiation. Using a neuroblastoma differentiation model, we discovered that hypoxia repressed cell differentiation through reducing cellular acetyl-CoA levels, leading to reduction of global histone acetylation and chromatin accessibility. The metabolic switch triggering this global histone hypoacetylation was the induction of pyruvate dehydrogenase kinases (PDK1 and PDK3). Inhibition of PDKs using dichloroacetate (DCA) restored acetyl-CoA generation and histone acetylation under hypoxia. Knocking down PDK1 induced neuroblastoma cell differentiation, highlighting the critical role of PDK1 in cell fate control. Importantly, acetate or glycerol triacetate (GTA) supplementation restored differentiation markers expression and neuron differentiation under hypoxia. Moreover, ATAC-Seq analysis demonstrated that hypoxia treatment significantly reduced chromatin accessibility at RAR/RXR binding sites, which can be restored by acetate supplementation. In addition, hypoxia-induced histone hypermethylation by increasing 2-hydroxyglutarate (2HG) and reducing α-ketoglutarate (αKG). αKG supplementation reduced histone hypermethylation upon hypoxia, but did not restore histone acetylation or differentiation markers expression. Together, these findings suggest that diverting pyruvate flux away from acetyl-CoA generation to lactate production is the key mechanism that Warburg effect drives dedifferentiation and tumorigenesis. We propose that combining differentiation therapy with acetate/GTA supplementation might represent an effective therapy against neuroblastoma.

Suppressing Mitochondrial Respiration Is Critical for Hypoxia Tolerance in the Fetal Growth Plate.

Oxygen (O2) is both an indispensable metabolic substrate and a regulatory signal that controls the activity of Hypoxia-Inducible Factor 1α (Hif1a), a mediator of the cellular adaptation to low O2 tension (hypoxia). Hypoxic cells require Hif1a to survive. Additionally, Hif1a is an inhibitor of mitochondrial respiration. Hence, we hypothesized that enhancing mitochondrial respiration is detrimental to the survival of hypoxic cells in vivo. We tested this hypothesis in the fetal growth plate, which is hypoxic. Our findings show that mitochondrial respiration is dispensable for survival of growth plate chondrocytes. Furthermore, its impairment prevents the extreme hypoxia and the massive chondrocyte death observed in growth plates lacking Hif1a. Consequently, augmenting mitochondrial respiration affects the survival of hypoxic chondrocytes by, at least in part, increasing intracellular hypoxia. We thus propose that partial suppression of mitochondrial respiration is crucial during development to protect the tissues that are physiologically hypoxic from lethal intracellular anoxia.

Generation of Stable Expression Mammalian Cell Lines Using Lentivirus.

Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines. The advantage of using the 3rd generation lentivirus are that are very safe and they are replication incompetent.

Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy.

Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx4cv/mTRG2 mouse model with "humanized" telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/ß leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

The ever-expanding role of HIF in tumour and stromal biology.

Low oxygen tension (hypoxia) is a hallmark of cancer that influences cancer cell function, but is also an important component of the tumour microenvironment as it alters the extracellular matrix, modulates the tumour immune response and increases angiogenesis. Here we discuss the regulation and role of hypoxia and its key transcriptional mediators, the hypoxia-inducible factor (HIF) family of transcription factors, in the tumour microenvironment and stromal compartments.

Suppression of PGC-1α Is Critical for Reprogramming Oxidative Metabolism in Renal Cell Carcinoma.

Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC), frequent activation of HIF signaling induces a metabolic switch that promotes tumorigenesis. Here, we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild-type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC.

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin.

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

Analysis of p53 transactivation domain mutants reveals Acad11 as a metabolic target important for p53 pro-survival function.

The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.