Targeting PRMT3 impairs methylation and oligomerization of HSP60 to boost anti-tumor immunity by activating cGAS/STING signaling.

Immune checkpoint blockade (ICB) has emerged as a promising therapeutic option for hepatocellular carcinoma (HCC), but resistance to ICB occurs and patient responses vary. Here, we uncover protein arginine methyltransferase 3 (PRMT3) as a driver for immunotherapy resistance in HCC. We show that PRMT3 expression is induced by ICB-activated T cells via an interferon-gamma (IFNγ)-STAT1 signaling pathway, and higher PRMT3 expression levels correlate with reduced numbers of tumor-infiltrating CD8+ T cells and poorer response to ICB. Genetic depletion or pharmacological inhibition of PRMT3 elicits an influx of T cells into tumors and reduces tumor size in HCC mouse models. Mechanistically, PRMT3 methylates HSP60 at R446 to induce HSP60 oligomerization and maintain mitochondrial homeostasis. Targeting PRMT3-dependent HSP60 methylation disrupts mitochondrial integrity and increases mitochondrial DNA (mtDNA) leakage, which results in cGAS/STING-mediated anti-tumor immunity. Lastly, blocking PRMT3 functions synergize with PD-1 blockade in HCC mouse models. Our study thus identifies PRMT3 as a potential biomarker and therapeutic target to overcome immunotherapy resistance in HCC.

An Immunometabolic Route for Activating cGAS/STING to Drive Anticancer Immunity.

The cGAS/STING pathway is a crucial immune activator in cancer biology, triggering innate immunosurveillance against tumors by sensing and reacting to endogenous mitochondrial DNA (mtDNA). In this issue of Cancer Research, research by Saha and colleagues highlights the significant impact of serine deprivation on this pathway, thereby unveiling its potential for anticancer therapy. Serine is essential for cellular metabolism and influences tumor growth and immune responses. Depriving cells of serine caused mitochondrial dysfunction and the release of mtDNA into the cytosol, activating the cGAS/STING pathway and inducing type I IFN responses. In mouse models, serine deprivation enhanced antitumor immunity, with increased tumoral immune infiltration, including CD4+/CD8+ T cells and type I IFN responses. Clinically, a genetic signature indicative of lower serine enrichment in colorectal cancer patients correlated with immune activation and improved survival. Furthermore, combining serine deprivation with PD1 blockade significantly reduced tumor volume and led to long-term immunity in mice, suggesting that serine depletion enhances the efficacy of immune checkpoint blockade. These findings propose serine deprivation as a promising strategy to boost antitumor immunity and improve cancer patient outcomes. See related article by Saha et al., p. 2645.

Mitochondrial DNA Programs Lactylation of cGAS to Induce IFN Responses in Patients with Systemic Lupus Erythematosus.

Mitochondrial DNA (mtDNA) is frequently released from mitochondria, activating cGAS-STING signaling and inducing type I IFNs (IFN-Is) in systemic lupus erythematosus (SLE). Meanwhile, whether and how the glycolytic pathway was involved in such IFN-I responses in human SLE remain unclear. In this study, we found that monocytes from SLE patients exerted robust IFN-I generation and elevated level of cytosolic mtDNA. Transfection of mtDNA into THP-1 macrophages was efficient in inducing IFN-I responses, together with the strong glycolytic pathway that promoted lactate production, mimicking the SLE phenotype. Blockade of lactate generation abrogated such IFN-I responses and, vice versa, exogenous lactate enhanced the IFN-I generation. Mechanistically, lactate promoted the lactylation of cGAS, which inhibited its binding to E3 ubiquitination ligase MARCHF5, blocking cGAS degradation and leading to strong IFN-I responses. In accordance, targeting lactate generation alleviated disease development in humanized SLE chimeras. Collectively, cytosolic mtDNA drives metabolic adaption toward the glycolytic pathway, promoting lactylation of cGAS for licensing IFN-I responses in human SLE and thereby assigning the glycolytic pathway as a promising therapeutic target for SLE.

Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers.

Low migratory dendritic cell (DC) levels pose a challenge in cancer immune surveillance, yet their impact on tumor immune status and immunotherapy responses remains unclear. We present clinical evidence linking reduced migratory DC levels to immune-cold tumor status, resulting in poor patient outcomes. To address this, we develop an autologous DC-based nanovaccination strategy using patient-derived organoid or cancer cell lysate-pulsed cationic nanoparticles (cNPs) to load immunogenic DC-derived microvesicles (cNPcancer cell@MVDC). This approach transforms immune-cold tumors, increases migratory DCs, activates T cells and natural killer cells, reduces tumor growth, and enhances survival in orthotopic pancreatic and lung cancer models, surpassing conventional methods. In vivo imaging reveals superior cNPcancer cell@MVDC accumulation in tumors and lymph nodes, promoting immune cell infiltration. Mechanistically, cNPs enrich mitochondrial DNA, enhancing cGAS-STING-mediated DC activation and migration. Our strategy shifts cold tumors to a hot state, enhancing antitumor immunity for potential personalized cancer treatments.

Serine Depletion Promotes Antitumor Immunity by Activating Mitochondrial DNA-Mediated cGAS-STING Signaling.

Serine is critical for supporting cancer metabolism, and depriving malignant cells of this nonessential amino acid exerts antineoplastic effects, in large part, through disrupting metabolic pathways. Given the intricate relationship between cancer metabolism and the immune system, the metabolic defects imposed by serine deprivation might impact tumor-targeting immunity. In this study, we demonstrated that restricting endogenous and exogenous sources of serine in colorectal cancer cells results in mitochondrial dysfunction, leading to mitochondrial DNA (mtDNA) accumulation in the cytosol and consequent cGAS-STING1-driven type I IFN secretion. Depleting mtDNA or blocking its release attenuated cGAS-STING1 activation during serine deprivation. In vivo studies revealed that serine deprivation limits tumor growth, accompanied by enhanced type I IFN signaling and intratumoral infiltration of immune effector cells. Notably, the tumor-suppressive and immune-enhancing effects of serine restriction were impaired by T-cell depletion and IFN receptor blockade. Moreover, disrupting cGAS-STING1 signaling in colorectal cancer cells limited the immunostimulatory and tumor-suppressive effects of serine deprivation. Lastly, serine depletion increased the sensitivity of tumors to an immune checkpoint inhibitor targeting PD-1. Taken together, these findings reveal a role for serine as a suppressor of antitumor immunity, suggesting that serine deprivation may be employed to enhance tumor immunogenicity and improve responsiveness to immune checkpoint inhibitors. Significance: Depriving cancer cells of serine provokes mitochondrial perturbations that induce cytosolic mitochondrial DNA accumulation and subsequent activation of cGAS-STING signaling, stimulating tumor-targeting immune responses that can be enhanced with PD-1 targeted therapy. See related commentary by Borges and Garg, p. 2569.

Mitochondrial DNA release and sensing in innate immune responses.

Mitochondria are pleiotropic organelles central to an array of cellular pathways including metabolism, signal transduction, and programmed cell death. Mitochondria are also key drivers of mammalian immune responses, functioning as scaffolds for innate immune signaling, governing metabolic switches required for immune cell activation, and releasing agonists that promote inflammation. Mitochondrial DNA (mtDNA) is a potent immunostimulatory agonist, triggering pro-inflammatory and type I interferon responses in a host of mammalian cell types. Here we review recent advances in how mtDNA is detected by nucleic acid sensors of the innate immune system upon release into the cytoplasm and extracellular space. We also discuss how the interplay between mtDNA release and sensing impacts cellular innate immune endpoints relevant to health and disease.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Warburg Effect Reshapes Tumor Immunogenicity.

Tumor cells rewire their metabolism to fulfill the demands of highly proliferative cells. This changes cellular metabolism to adapt to fuel and oxygen availability for energy production and to increase the synthesis capacity of building blocks for cell division and growth. In addition, the metabolic shift modulates the immunogenicity of the tumor cells. Recently, Mahmood and colleagues reported a connection between mitochondrial DNA mutations in cancer cells and their response to immunotherapy in a mouse model of melanoma.

Therapeutic landscape in systemic lupus erythematosus: mtDNA activation of the cGAS-STING pathway.

Mitochondrial DNA (mtDNA) serves as a pivotal immune stimulus in the immune response. During stress, mitochondria release mtDNA into the cytoplasm, where it is recognized by the cytoplasmic DNA receptor cGAS. This activation initiates the cGAS-STING-IRF3 pathway, culminating in an inflammatory response. The cGAS-STING pathway has emerged as a critical mediator of inflammatory responses in microbial infections, stress, autoimmune diseases, chronic illnesses, and tissue injuries. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by connective tissue involvement across various bodily systems. Its hallmark is the production of numerous autoantibodies, which prompt the immune system to target and damage the body's own tissues, resulting in organ and tissue damage. Increasing evidence implicates the cGAS-STING pathway as a significant contributor to SLE pathogenesis. This article aims to explore the role of the mtDNA-triggered cGAS-STING pathway and its mechanisms in SLE, with the goal of providing novel insights for clinical interventions.

Telomere length and mitochondrial DNA copy number in multidrug-resistant tuberculosis.

Multidrug resistant tuberculosis (MDR-TB) is a severe disease that requires prolonged chemotherapy and is associated with an increased probability of treatment failure and death. MDR-TB is a state of heightened oxidative stress and inflammation, which could be related to the aging-related processes and immunosenescence. We, therefore, tested the hypothesis that MDR-TB is associated with alterations in aging biomarkers in peripheral blood cells. We investigated 51 MDR-TB patients and 57 healthy individuals and carried out an analysis of covariance to assess the possible impact of different variables on biomarker perturbations. The results showed that MDR-TB patients had significantly reduced telomere length (TL) and increased mitochondrial DNA copy number (mtDNA CN) (P < 0.05) in comparison to the controls, and MDR-TB infection was the main influencing factor. Male sex and extrapulmonary TB strongly influenced mtDNA CN increment, and MDR-TB patients with normal weight had longer telomeres than those who were underweight (P < 0.05). In conclusion, the evidence for shorter telomeres and higher mtDNA CN in the peripheral blood cells of MDR-TB patients was obtained indicating the connection between MDR-TB and aging biomarkers. The observed associations highlight a complicated interplay between MDR-TB and immunosenescence, thus further studies are required to achieve full understanding.

Mitochondrial Nucleic Acid as a Driver of Pathogenic Type I Interferon Induction in Mendelian Disease.

The immune response to viral infection involves the recognition of pathogen-derived nucleic acids by intracellular sensors, leading to type I interferon (IFN), and downstream IFN-stimulated gene, induction. Ineffective discrimination of self from non-self nucleic acid can lead to autoinflammation, a phenomenon implicated in an increasing number of disease states, and well highlighted by the group of rare genetic disorders referred to as the type I interferonopathies. To understand the pathogenesis of these monogenic disorders, and polyfactorial diseases associated with pathogenic IFN upregulation, such as systemic lupus erythematosus and dermatomyositis, it is important to define the self-derived nucleic acid species responsible for such abnormal IFN induction. Recently, attention has focused on mitochondria as a novel source of immunogenic self nucleic acid. Best appreciated for their function in oxidative phosphorylation, metabolism and apoptosis, mitochondria are double membrane-bound organelles that represent vestigial bacteria in the cytosol of eukaryotic cells, containing their own DNA and RNA enclosed within the inner mitochondrial membrane. There is increasing recognition that a loss of mitochondrial integrity and compartmentalization can allow the release of mitochondrial nucleic acid into the cytosol, leading to IFN induction. Here, we provide recent insights into the potential of mitochondrial-derived DNA and RNA to drive IFN production in Mendelian disease. Specifically, we summarize current understanding of how nucleic acids are detected as foreign when released into the cytosol, and then consider the findings implicating mitochondrial nucleic acid in type I interferonopathy disease states. Finally, we discuss the potential for IFN-driven pathology in primary mitochondrial disorders.

Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse.

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

Mitochondria-Induced Immune Response as a Trigger for Neurodegeneration: A Pathogen from Within.

Symbiosis between the mitochondrion and the ancestor of the eukaryotic cell allowed cellular complexity and supported life. Mitochondria have specialized in many key functions ensuring cell homeostasis and survival. Thus, proper communication between mitochondria and cell nucleus is paramount for cellular health. However, due to their archaebacterial origin, mitochondria possess a high immunogenic potential. Indeed, mitochondria have been identified as an intracellular source of molecules that can elicit cellular responses to pathogens. Compromised mitochondrial integrity leads to release of mitochondrial content into the cytosol, which triggers an unwanted cellular immune response. Mitochondrial nucleic acids (mtDNA and mtRNA) can interact with the same cytoplasmic sensors that are specialized in recognizing genetic material from pathogens. High-energy demanding cells, such as neurons, are highly affected by deficits in mitochondrial function. Notably, mitochondrial dysfunction, neurodegeneration, and chronic inflammation are concurrent events in many severe debilitating disorders. Interestingly in this context of pathology, increasing number of studies have detected immune-activating mtDNA and mtRNA that induce an aberrant production of pro-inflammatory cytokines and interferon effectors. Thus, this review provides new insights on mitochondria-driven inflammation as a potential therapeutic target for neurodegenerative and primary mitochondrial diseases.

Circulating Mitochondrial DNA Stimulates Innate Immune Signaling Pathways to Mediate Acute Kidney Injury.

Mitochondrial dysfunction is increasingly considered as a critical contributor to the occurrence and progression of acute kidney injury (AKI). However, the mechanisms by which damaged mitochondria mediate AKI progression are multifactorial and complicated. Mitochondrial DNA (mtDNA) released from damaged mitochondria could serve as a danger-associated molecular pattern (DAMP) and activate the innate immune system through STING, TLR9, NLRP3, and some other adaptors, and further mediate tubular cell inflammation and apoptosis. Accumulating evidence has demonstrated the important role of circulating mtDNA and its related pathways in the progression of AKI, and regulating the proteins involved in these pathways may be an effective strategy to reduce renal tubular injury and alleviate AKI. Here, we aim to provide a comprehensive overview of recent studies on mtDNA-mediated renal pathological events to provide new insights in the setting of AKI.

Cell Death and Inflammation: The Role of Mitochondria in Health and Disease.

Mitochondria serve as a hub for a multitude of vital cellular processes. To ensure an efficient deployment of mitochondrial tasks, organelle homeostasis needs to be preserved. Mitochondrial quality control (MQC) mechanisms (i.e., mitochondrial dynamics, biogenesis, proteostasis, and autophagy) are in place to safeguard organelle integrity and functionality. Defective MQC has been reported in several conditions characterized by chronic low-grade inflammation. In this context, the displacement of mitochondrial components, including mitochondrial DNA (mtDNA), into the extracellular compartment is a possible factor eliciting an innate immune response. The presence of bacterial-like CpG islands in mtDNA makes this molecule recognized as a damaged-associated molecular pattern by the innate immune system. Following cell death-triggering stressors, mtDNA can be released from the cell and ignite inflammation via several pathways. Crosstalk between autophagy and apoptosis has emerged as a pivotal factor for the regulation of mtDNA release, cell's fate, and inflammation. The repression of mtDNA-mediated interferon production, a powerful driver of immunological cell death, is also regulated by autophagy-apoptosis crosstalk. Interferon production during mtDNA-mediated inflammation may be exploited for the elimination of dying cells and their conversion into elements driving anti-tumor immunity.

Stem cell-derived mitochondria transplantation: A promising therapy for mitochondrial encephalomyopathy.

Mitochondrial encephalomyopathies are disorders caused by mitochondrial and nuclear DNA mutations which affect the nervous and muscular systems. Current therapies for mitochondrial encephalomyopathies are inadequate and mostly palliative. However, stem cell-derived mitochondria transplantation has been demonstrated to play an key part in metabolic rescue, which offers great promise for mitochondrial encephalomyopathies. Here, we summarize the present status of stem cell therapy for mitochondrial encephalomyopathy and discuss mitochondrial transfer routes and the protection mechanisms of stem cells. We also identify and summarize future perspectives and challenges for the treatment of these intractable disorders based on the concept of mitochondrial transfer from stem cells.

Mitochondria: Powering the Innate Immune Response to Mycobacterium tuberculosis Infection.

Within the last decade, we have learned that damaged mitochondria activate many of the same innate immune pathways that evolved to sense and respond to intracellular pathogens. These shared responses include cytosolic nucleic acid sensing and type I interferon (IFN) expression, inflammasome activation that leads to pyroptosis, and selective autophagy (called mitophagy when mitochondria are the cargo). Because mitochondria were once bacteria, parallels between how cells respond to mitochondrial and bacterial ligands are not altogether surprising. However, the potential for cross talk or synergy between bacterium- and mitochondrion-driven innate immune responses during infection remains poorly understood. This interplay is particularly striking, and intriguing, in the context of infection with the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Multiple studies point to a role for Mtb infection and/or specific Mtb virulence factors in disrupting the mitochondrial network in macrophages, leading to metabolic changes and triggering potent innate immune responses. Research from our laboratories and others argues that mutations in mitochondrial genes can exacerbate mycobacterial disease severity by hyperactivating innate responses or activating them at the wrong time. Indeed, growing evidence supports a model whereby different mitochondrial defects or mutations alter Mtb infection outcomes in distinct ways. By synthesizing the current literature in this minireview, we hope to gain insight into the molecular mechanisms driving, and consequences of, mitochondrion-dependent immune polarization so that we might better predict tuberculosis patient outcomes and develop host-directed therapeutics designed to correct these imbalances.

Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance.

Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function1. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown2. Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation5 mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I-MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents.

Deciphering the genetic and epidemiological landscape of mitochondrial DNA abundance.

Mitochondrial (MT) dysfunction is a hallmark of aging and has been associated with most aging-related diseases as well as immunological processes. However, little is known about aging, lifestyle and genetic factors influencing mitochondrial DNA (mtDNA) abundance. In this study, mtDNA abundance was estimated from the weighted intensities of probes mapping to the MT genome in 295,150 participants from the UK Biobank. We found that the abundance of mtDNA was significantly elevated in women compared to men, was negatively correlated with advanced age, higher smoking exposure, greater body-mass index, higher frailty index as well as elevated red and white blood cell count and lower mortality. In addition, several biochemistry markers in blood-related to cholesterol metabolism, ion homeostasis and kidney function were found to be significantly associated with mtDNA abundance. By performing a genome-wide association study, we identified 50 independent regions genome-wide significantly associated with mtDNA abundance which harbour multiple genes involved in the immune system, cancer as well as mitochondrial function. Using mixed effects models, we estimated the SNP-heritability of mtDNA abundance to be around 8%. To investigate the consequence of altered mtDNA abundance, we performed a phenome-wide association study and found that mtDNA abundance is involved in risk for leukaemia, hematologic diseases as well as hypertension. Thus, estimating mtDNA abundance from genotyping arrays has the potential to provide novel insights into age- and disease-relevant processes, particularly those related to immunity and established mitochondrial functions.