CHCHD4-TRIAP1 regulation of innate immune signaling mediates skeletal muscle adaptation to exercise.

Exercise training can stimulate the formation of fatty-acid-oxidizing slow-twitch skeletal muscle fibers, which are inversely correlated with obesity, but the molecular mechanism underlying this transformation requires further elucidation. Here, we report that the downregulation of the mitochondrial disulfide relay carrier CHCHD4 by exercise training decreases the import of TP53-regulated inhibitor of apoptosis 1 (TRIAP1) into mitochondria, which can reduce cardiolipin levels and promote VDAC oligomerization in skeletal muscle. VDAC oligomerization, known to facilitate mtDNA release, can activate cGAS-STING/NFKB innate immune signaling and downregulate MyoD in skeletal muscle, thereby promoting the formation of oxidative slow-twitch fibers. In mice, CHCHD4 haploinsufficiency is sufficient to activate this pathway, leading to increased oxidative muscle fibers and decreased fat accumulation with aging. The identification of a specific mediator regulating muscle fiber transformation provides an opportunity to understand further the molecular underpinnings of complex metabolic conditions such as obesity and could have therapeutic implications.

WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by various disabling symptoms including exercise intolerance and is diagnosed in the absence of a specific cause, making its clinical management challenging. A better understanding of the molecular mechanism underlying this apparent bioenergetic deficiency state may reveal insights for developing targeted treatment strategies. We report that overexpression of Wiskott-Aldrich Syndrome Protein Family Member 3 (WASF3), here identified in a 38-y-old woman suffering from long-standing fatigue and exercise intolerance, can disrupt mitochondrial respiratory supercomplex formation and is associated with endoplasmic reticulum (ER) stress. Increased expression of WASF3 in transgenic mice markedly decreased their treadmill running capacity with concomitantly impaired respiratory supercomplex assembly and reduced complex IV levels in skeletal muscle mitochondria. WASF3 induction by ER stress using endotoxin, well known to be associated with fatigue in humans, also decreased skeletal muscle complex IV levels in mice, while decreasing WASF3 levels by pharmacologic inhibition of ER stress improved mitochondrial function in the cells of the patient with chronic fatigue. Expanding on our findings, skeletal muscle biopsy samples obtained from a cohort of patients with ME/CFS showed increased WASF3 protein levels and aberrant ER stress activation. In addition to revealing a potential mechanism for the bioenergetic deficiency in ME/CFS, our study may also provide insights into other disorders associated with fatigue such as rheumatic diseases and long COVID.

Li-Fraumeni Syndrome-Associated Dimer-Forming Mutant p53 Promotes Transactivation-Independent Mitochondrial Cell Death.

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li-Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. SIGNIFICANCE: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027.

Mitochondrial respiration reduces exposure of the nucleus to oxygen.

The endosymbiotic theory posits that ancient eukaryotic cells engulfed O2-consuming prokaryotes, which protected them against O2 toxicity. Previous studies have shown that cells lacking cytochrome c oxidase (COX), required for respiration, have increased DNA damage and reduced proliferation, which could be improved by reducing O2 exposure. With recently developed fluorescence lifetime microscopy-based probes demonstrating that the mitochondrion has lower [O2] than the cytosol, we hypothesized that the perinuclear distribution of mitochondria in cells may create a barrier for O2 to access the nuclear core, potentially affecting cellular physiology and maintaining genomic integrity. To test this hypothesis, we utilized myoglobin-mCherry fluorescence lifetime microscopy O2 sensors without subcellular targeting ("cytosol") or with targeting to the mitochondrion or nucleus for measuring their localized O2 homeostasis. Our results showed that, similar to the mitochondria, the nuclear [O2] was reduced by ∼20 to 40% compared with the cytosol under imposed O2 levels of ∼0.5 to 18.6%. Pharmacologically inhibiting respiration increased nuclear O2 levels, and reconstituting O2 consumption by COX reversed this increase. Similarly, genetic disruption of respiration by deleting SCO2, a gene essential for COX assembly, or restoring COX activity in SCO2-/- cells by transducing with SCO2 cDNA replicated these changes in nuclear O2 levels. The results were further supported by the expression of genes known to be affected by cellular O2 availability. Our study reveals the potential for dynamic regulation of nuclear O2 levels by mitochondrial respiratory activity, which in turn could affect oxidative stress and cellular processes such as neurodegeneration and aging.

Fluorescence lifetime imaging of metMyoglobin formation due to nitric oxide stress.

Myoglobin is a protein that is expressed quite unevenly among different cell types. Nevertheless, it has been widely acknowledged that the Fe3+ state of myoglobin, metmyoglobin (metMb) has a broad functional role in metabolism, oxidative/nitrative regulation and gene networks. Accordingly, real-time monitoring of oxygenated, deoxygenated and metMb proportions- or, more broadly, of the mechanisms by which metMb is formed, presents a promising line of research. We had previously introduced a Förster resonance energy transfer (FRET) method to read out the deoxygenation/oxygenation states of myoglobin, by creating the targetable oxygen (O2) sensor Myoglobin-mCherry. In this sensor, changes in myoglobin absorbance features that occur with lost O2 occupancy -or upon metMb production- control the FRET rate from the fluorescent protein to myoglobin. When O2 is bound, mCherry fluorescence is only slightly quenched, but if either O2 is released or met is produced, FRET will increase- and this rate competing with emission reduces both emission yield and lifetime. Nitric oxide (NO) is an important signal (but also a toxic molecule) that can oxidize myoglobin to metMb with absorbance increases in the red visible range. mCherry thus senses both met and deoxygenated myoglobin, which cannot be easily separated at hypoxia. In order to dissect this, we treat cells with NO and investigate how the Myoglobin-mCherry lifetime is affected by generating metMb. More discriminatory power is then achieved when the fluorescent protein EYFP is added to Myoglobin-mCherry, creating a sandwich probe whose lifetime can selectively respond to metMb while being indifferent to O2 occupancy.

Intracellular imaging of metmyoglobin and oxygen using new dual purpose probe EYFP-Myoglobin-mCherry.

The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both [O2 ] and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular [O2 ] sensor Myoglobin-mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and [O2 ]. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making [O2 ] sensing available to a wider group of laboratories.

Mitochondria and oxygen homeostasis.

Molecular oxygen possesses a dual nature due to its highly reactive free radical property: it is capable of oxidizing metabolic substrates to generate cellular energy, but can also serve as a substrate for genotoxic reactive oxygen species generation. As a labile substance upon which aerobic life depends, the mechanisms for handling cellular oxygen have been fine-tuned and orchestrated in evolution. Protection from atmospheric oxygen toxicity as originally posited by the Endosymbiotic Theory of the Mitochondrion is likely to be one basic principle underlying oxygen homeostasis. We briefly review the literature on oxygen homeostasis both in vitro and in vivo with a focus on the role of the mitochondrion where the majority of cellular oxygen is consumed. The insights gleaned from these basic mechanisms are likely to be important for understanding disease pathogenesis and developing strategies for maintaining health.

Cardiotoxicity of Cancer Treatments: Focus on Anthracycline Cardiomyopathy.

Significant progress has been made in developing new treatments and refining the use of preexisting ones against cancer. Their successful use and the longer survival of cancer patients have been associated with reports of new cardiotoxicities and the better characterization of the previously known cardiac complications. Immunotherapies with monoclonal antibodies against specific cancer-promoting genes, chimeric antigen receptor T cells, and immune checkpoint inhibitors have been developed to fight cancer cells, but they can also show off-target effects on the heart. Some of these cardiotoxicities are thought to be due to nonspecific immune activation and inflammatory damage. Unlike immunotherapy-associated cardiotoxicities which are relatively new entities, there is extensive literature on anthracycline-induced cardiomyopathy. Here, we provide a brief overview of the cardiotoxicities of immunotherapies for the purpose of distinguishing them from anthracycline cardiomyopathy. This is especially relevant as the expansion of oncological treatments presents greater diagnostic challenges in determining the cause of cardiac dysfunction in cancer survivors with a history of multiple cancer treatments including anthracyclines and immunotherapies administered concurrently or serially over time. We then provide a focused review of the mechanisms proposed to underlie the development of anthracycline cardiomyopathy based on experimental data mostly in mouse models. Insights into its pathogenesis may stimulate the development of new strategies to identify patients who are susceptible to anthracycline cardiomyopathy while permitting low cardiac risk patients to receive optimal treatment for their cancer.

Extracellular Acidity Reprograms Macrophage Metabolism and Innate Responsiveness.

Although organ hypofunction and immunosuppression are life-threatening features of severe sepsis, the hypofunctioning organs and immune cells usually regain normal functionality if patients survive. Because tissue interstitial fluid can become acidic during the septic response, we tested the hypothesis that low extracellular pH (pHe) can induce reversible metabolic and functional changes in peritoneal macrophages from C57BL/6J mice. When compared with macrophages cultured at normal pHe, macrophages living in an acidic medium used less glucose and exogenous fatty acid to produce ATP. Lactate, glutamine, and de novo-synthesized fatty acids supported ATP production by mitochondria that gained greater mass, maximal oxygen consumption rate, and spare respiratory capacity. The cells transitioned to an M2-like state, with altered immune responses to LPS and slightly decreased phagocytic ability, yet they regained basal energy production, normal mitochondrial function, and proinflammatory responsiveness when neutral pHe was restored. Low pHe induces changes that support macrophage survival while rendering the cells less proinflammatory (more "tolerant") and less able to phagocytose bacteria. Macrophage responses to low interstitial pH may contribute to the reversible organ hypofunction and immunoparalysis noted in many patients with sepsis.

Reducing Fatty Acid Oxidation Improves Cancer-free Survival in a Mouse Model of Li-Fraumeni Syndrome.

Germline mutations of TP53, which cause the cancer predisposition disorder Li-Fraumeni syndrome (LFS), can increase mitochondrial activity as well as fatty acid ß-oxidation (FAO) in mice. Increased fatty acid metabolism can promote cancer malignancy, but its specific contribution to tumorigenesis in LFS remains unclear. To investigate this, we crossed LFS mice carrying the p53 R172H knock-in mutation (p53172H/H , homolog of the human TP53 R175H LFS mutation) with myoglobin-knockout (MB-/- ) mice known to have decreased FAO. MB-/- p53172H/H double-mutant mice also showed mildly reduced FAO in thymus, a common site of T lymphoma development in LFS mice, in association with an approximately 40% improvement in cancer-free survival time. RNA sequencing profiling revealed that the p53 R172H mutation promotes mitochondrial metabolism and ribosome biogenesis, both of which are suppressed by the disruption of MB. The activation of ribosomal protein S6, involved in protein translation and implicated in cancer promotion, was also inhibited in the absence of MB. To further confirm the role of FAO in lymphomagenesis, mitochondrial FAO enzyme, carnitine palmitoyltransferase 2 (CPT2), was specifically disrupted in T cells of p53172H/H mice using a Cre-loxP-mediated strategy. The heterozygous knockout of CPT2 resulted in thymus FAO haploinsufficiency and an approximately 30% improvement in survival time, paralleling the antiproliferative signaling observed with MB disruption. Thus, this study demonstrates that moderating FAO in LFS can suppress tumorigenesis and improve cancer-free survival with potential implications for cancer prevention. PREVENTION RELEVANCE: Mildly inhibiting the increased fatty acid oxidation observed in a mouse model of Li-Fraumeni syndrome, a cancer predisposition disorder caused by inherited mutations of TP53, dampens aberrant pro-tumorigenic cell signaling and improves the survival time of these mice, thereby revealing a potential strategy for cancer prevention in patients.

Tumour predisposition and cancer syndromes as models to study gene-environment interactions.

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.

Protective role of p53 in doxorubicin-induced cardiomyopathy as a mitochondrial disease.

Doxorubicin is widely used against cancer but carries the risk of a progressive cardiomyopathy associated with mitochondrial loss. Using genetic models, our recent study demonstrates that mitochondrial genomic DNA regulation by tumor protein p53 (TP53, best known as p53) prevents the cardiotoxicity of low dose doxorubicin which does not activate the p53-dependent cell death pathway.

A Mouse Homolog of a Human TP53 Germline Mutation Reveals a Lipolytic Activity of p53.

The physiological effects of the many germline mutations of TP53, encoding the tumor suppressor protein p53, are poorly understood. Here we report generating a p53 R178C knockin mouse modeling the human TP53 R181C mutation, which is notable for its prevalence and prior molecular characterization. Consistent with its weak cancer penetrance in humans, homozygous p53178C/C mice show a modest increase in tumorigenesis but, surprisingly, are lean with decreased body fat content. They display evidence of increased lipolysis and upregulation of fatty acid metabolism in their inguinal white adipose tissue (iWAT). Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses show that the mutant p53 bound and transactivated Beta-3-Adrenergic Receptor (ADRB3), a gene that is known to promote lipolysis and is associated with obesity. This study reveals that a germline mutation of p53 can affect fat metabolism, which has been implicated in cancer development.

Pilot Study Assessing Tolerability and Metabolic Effects of Metformin in Patients With Li-Fraumeni Syndrome.

BACKGROUND: Li-Fraumeni syndrome (LFS) is a highly penetrant autosomal dominant cancer predisposition disorder caused by germline TP53 pathogenic variants. Patients with LFS have increased oxidative phosphorylation capacity in skeletal muscle and oxidative stress in blood. Metformin inhibits oxidative phosphorylation, reducing available energy for cancer cell proliferation and decreasing production of reactive oxygen species that cause DNA damage. Thus, metformin may provide pharmacologic risk reduction for cancer in patients with LFS, but its safety in nondiabetic patients with germline TP53 pathogenic variants has not been documented. METHODS: This study assessed safety and tolerability of metformin in nondiabetic LFS patients and measured changes in metabolic profiles. Adult patients with LFS and germline TP53 variant received 14 weeks of metformin. Blood samples were obtained for measurement of serum insulin-like growth factor-1, insulin, and insulin-like growth factor binding protein 3. Hepatic mitochondrial function was assessed with fasting exhaled CO2 after ingestion of 13C-labeled methionine. Changes in serum metabolome were measured. All statistical tests were 2-sided. RESULTS: We enrolled 26 participants: 20 females and 6 males. The most common adverse events were diarrhea (50.0%) and nausea (46.2%). Lactic acidosis did not occur, and there were no changes in fasting glucose. Cumulative mean 13C exhalation was statistically significantly suppressed by metformin (P = .001). Mean levels of insulin-like growth factor binding protein 3 and insulin-like growth factor-1 were statistically significantly lowered (P = .02). Lipid metabolites and branched-chain amino acids accumulated. CONCLUSIONS: Metformin was safe and tolerable in patients with LFS. It suppressed hepatic mitochondrial function as expected in these individuals. This study adds to the rationale for development of a pharmacologic risk-reduction clinical trial of metformin in LFS.

p53 prevents doxorubicin cardiotoxicity independently of its prototypical tumor suppressor activities.

Doxorubicin is a widely used chemotherapeutic agent that causes dose-dependent cardiotoxicity in a subset of treated patients, but the genetic determinants of this susceptibility are poorly understood. Here, we report that a noncanonical tumor suppressor activity of p53 prevents cardiac dysfunction in a mouse model induced by doxorubicin administered in divided low doses as in the clinics. While relatively preserved in wild-type (p53+/+ ) state, mice deficient in p53 (p53-/- ) developed left ventricular (LV) systolic dysfunction after doxorubicin treatment. This functional decline in p53-/- mice was associated with decreases in cardiac oxidative metabolism, mitochondrial mass, and mitochondrial genomic DNA (mtDNA) homeostasis. Notably, mice with homozygous knockin of the p53 R172H (p53172H/H ) mutation, which like p53-/- state lacks the prototypical tumor suppressor activities of p53 such as apoptosis but retains its mitochondrial biogenesis capacity, showed preservation of LV function and mitochondria after doxorubicin treatment. In contrast to p53-null state, wild-type and mutant p53 displayed distinct mechanisms of transactivating mitochondrial transcription factor A (TFAM) and p53-inducible ribonucleotide reductase 2 (p53R2), which are involved in mtDNA transcription and maintenance. Importantly, supplementing mice with a precursor of NAD+ prevented the mtDNA depletion and cardiac dysfunction. These findings suggest that loss of mtDNA contributes to cardiomyopathy pathogenesis induced by doxorubicin administered on a schedule simulating that in the clinics. Given a similar mtDNA protection role of p53 in doxorubicin-treated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, the mitochondrial markers associated with cardiomyopathy development observed in blood and skeletal muscle cells may have prognostic utility.

Hypoxia-tropic Protein Nanocages for Modulation of Tumor- and Chemotherapy-Associated Hypoxia.

Despite its central role in tumor progression and treatment resistance, poor vascularization that necessitates penetration of therapeutics through tumor extracellular matrix (ECM) constitutes a significant challenge to managing tumor hypoxia via conventional systemic treatment regimens. In addition, methods to target hypoxic tumor cells are lacking. Here, we discovered that human ferritin nanocages (FTn) possess an intrinsic ability to preferentially engage with hypoxic tumor tissues, in addition to normoxic tumor areas. We also developed a simple method of endowing FTn with spatially controlled "mosaic" surface poly(ethylene glycol) (PEG) coatings that facilitate deep penetration of FTn through ECM to reach hypoxic tumor tissues while retaining its inherent hypoxia-tropic property. Hypoxia-inhibiting agents systemically delivered via this surface-PEGylated FTn were readily accumulated in hypoxic tumor tissues, thereby providing significantly enhanced therapeutic benefits compared to the identical agents delivered in solution as a stand-alone therapy or an adjuvant to restore efficacy of conventional systemic chemotherapy.

Mouse Homolog of the Human TP53 R337H Mutation Reveals Its Role in Tumorigenesis.

Inheritance of germline mutations in the tumor suppressor gene TP53 causes Li-Fraumeni syndrome (LFS), a cancer predisposition disorder. The arginine to histidine substitution at amino acid position 337 of p53 (R337H) is a founder mutation highly prevalent in southern and southeastern Brazil and is considered an LFS mutation. Although this mutation is of significant clinical interest, its role in tumorigenesis using animal models has not been described. Here, we generate a knockin mouse model containing the homologous R337H mutation (mouse R334H). De novo tumorigenesis was not significantly increased in either heterozygous (p53334R/H ) or homozygous (p53334H/H ) p53 R334H knockin mice compared with wild-type mice. However, susceptibility to diethylnitrosamine (DEN)-induced liver carcinogenesis was increased in a mutant allele dose-dependent manner. In parallel, p53334H/H mice exposed to DEN exhibited increased DNA damage but decreased cell-cycle regulation in the liver. Oligomerization of p53, which is required for transactivation of target genes, was reduced in R334H liver, consistent with its decreased nuclear activity compared with wild-type. By modeling a TP53 mutation in mice that has relatively weak cancer penetrance, this study provides in vivo evidence that the human R337H mutation can compromise p53 activity and promote tumorigenesis.Significance: A germline mutation in the oligomerization domain of p53 decreases its transactivation potential and renders mice susceptible to carcinogen-induced liver tumorigenesis. Cancer Res; 78(18); 5375-83. ©2018 AACR.

Inhibiting mitochondrial respiration prevents cancer in a mouse model of Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is a cancer predisposition disorder caused by germline mutations in TP53 that can lead to increased mitochondrial metabolism in patients. However, the implications of altered mitochondrial function for tumorigenesis in LFS are unclear. Here, we have reported that genetic or pharmacologic disruption of mitochondrial respiration improves cancer-free survival in a mouse model of LFS that expresses mutant p53. Mechanistically, inhibition of mitochondrial function increased autophagy and decreased the aberrant proliferation signaling caused by mutant p53. In a pilot study, LFS patients treated with metformin exhibited decreases in mitochondrial activity concomitant with activation of antiproliferation signaling, thus reproducing the effects of disrupting mitochondrial function observed in LFS mice. These observations indicate that a commonly prescribed diabetic medicine can restrain mitochondrial metabolism and tumorigenesis in an LFS model, supporting its further consideration for cancer prevention in LFS patients.