Tofacitinib restores psoriatic arthritis fibroblast-like synoviocytes function via autophagy and mitochondrial quality control modulation.

OBJECTIVES: To evaluate the in vitro effect of tofacitinib on autophagy activity of psoriatic arthritis (PsA) fibroblast-like synoviocytes (FLS), and to confirm its activity on inflammatory and invasive properties of FLS and synovial cells, deepening the impact on mitochondrial function. METHODS: FLS, peripheral blood mononuclear cells (PBMCs), and synovial cells from active PsA patients were cultured with tofacitinib 1 µM or vehicle control for 24 h. Autophagy was measured by Western blot and by fluorescence microscopy. Chemokines/cytokines released into culture supernatants were quantified by ELISA, while invasive properties of FLS by migration assays. Specific mitochondrial probes were adopted to measure intracellular reactive oxygen species (ROS), mitochondrial potential, morphology, turnover and mitophagy. Oxygen consumption rate (OCR), reflecting oxidative phosphorylation, was quantified using the Seahorse technology. Differences were determined by adopting the non-parametric Wilcoxon signed rank test. RESULTS: 18 patients with moderately-to-severely active PsA were enrolled. Tofacitinib significantly increased the levels of the autophagy markers LC3-II and ATG7 in PsA FLS compared to vehicle control, suggesting an increase in spontaneous autophagy activity; no effect was highlighted in PBMCs and synovial cells cultures. Tofacitinib reduced migration properties of PsA FLS, and reduced MCP-1 and IL-6 release into FLS and synovial cells cultures supernatants. Furthermore, tofacitinib decreased intracellular ROS production, increased basal OCR, ATP production and maximal respiratory capacity, and enhanced mitophagy and mitochondrial turnover. CONCLUSIONS: The JAK inhibitor tofacitinib reduces the pro-invasive and pro-inflammatory properties of PsA FLS. Autophagy induction and mitochondrial quality control modulation by tofacitinib might contribute to FLS function restoration.

Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth.

Although mitochondria play a multifunctional role in cancer progression and Ca2+ signaling is remodeled in a wide variety of tumors, the underlying mechanisms that link mitochondrial Ca2+ homeostasis with malignant tumor formation and growth remain elusive. Here, we show that phosphorylation at the N-terminal region of the mitochondrial calcium uniporter (MCU) regulatory subunit MICU1 leads to a notable increase in the basal mitochondrial Ca2+ levels. A pool of active Akt in the mitochondria is responsible for MICU1 phosphorylation, and mitochondrion-targeted Akt strongly regulates the mitochondrial Ca2+ content. The Akt-mediated phosphorylation impairs MICU1 processing and stability, culminating in reactive oxygen species (ROS) production and tumor progression. Thus, our data reveal the crucial role of the Akt-MICU1 axis in cancer and underscore the strategic importance of the association between aberrant mitochondrial Ca2+ levels and tumor development.

PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumour growth.

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the 'quasisynaptical' feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca2+ release results in mitochondrial Ca2+ overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca2+-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca2+ influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca2+ release from the endoplasmic reticulum and sensitization to Ca2+-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten-/- mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca2+ mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca2+-dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca2+ overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.

Evaluation of the Synovial Effects of Biological and Targeted Synthetic DMARDs in Patients with Psoriatic Arthritis: A Systematic Literature Review and Meta-Analysis.

The aims of this systematic literature review (SLR) were to identify the effects of approved biological and targeted synthetic disease modifying antirheumatic drugs (b/tsDMARDs) on synovial membrane of psoriatic arthritis (PsA) patients, and to determine the existence of histological/molecular biomarkers of response to therapy. A search was conducted on MEDLINE, Embase, Scopus, and Cochrane Library (PROSPERO:CRD42022304986) to retrieve data on longitudinal change of biomarkers in paired synovial biopsies and in vitro studies. A meta-analysis was conducted by adopting the standardized mean difference (SMD) as a measure of the effect. Twenty-two studies were included (19 longitudinal, 3 in vitro). In longitudinal studies, TNF inhibitors were the most used drugs, while, for in vitro studies, JAK inhibitors or adalimumab/secukinumab were assessed. The main technique used was immunohistochemistry (longitudinal studies). The meta-analysis showed a significant reduction in both CD3+ lymphocytes (SMD -0.85 [95% CI -1.23; -0.47]) and CD68+ macrophages (sublining, sl) (SMD -0.74 [-1.16; -0.32]) in synovial biopsies from patients treated for 4-12 weeks with bDMARDs. Reduction in CD3+ mostly correlated with clinical response. Despite heterogeneity among the biomarkers evaluated, the reduction in CD3+/CD68+sl cells during the first 3 months of treatment with TNF inhibitors represents the most consistent variation reported in the literature.

Synthesis and NLRP3-Inflammasome Inhibitory Activity of the Naturally Occurring Velutone F and of Its Non-Natural Regioisomeric Chalconoids.

Plant-derived remedies rich in chalcone-based compounds have been known for centuries in the treatment of specific diseases, and nowadays, the fascinating chalcone framework is considered a useful and, above all, abundant natural chemotype. Velutone F, a new chalconoid from Millettia velutina, exhibits a potent effect as an NLRP3-inflammasome inhibitor; the search for new natural/non-natural lead compounds as NLRP3 inhibitors is a current topical subject in medicinal chemistry. The details of our work toward the synthesis of velutone F and the unknown non-natural regioisomers are herein reported. We used different synthetic strategies both for the construction of the distinctive benzofuran nucleus (BF) and for the key phenylpropenone system (PhP). Importantly, we have disclosed a facile entry to the velutone F via synthetic routes that can also be useful for preparing non-natural analogs, a prerequisite for extensive SAR studies on the new flavonoid class of NLRP3-inhibitors.

Regulation of PKCß levels and autophagy by PML is essential for high-glucose-dependent mesenchymal stem cell adipogenesis.

BACKGROUND/OBJECTIVES: Obesity is a complex disease characterized by the accumulation of excess body fat, which is caused by an increase in adipose cell size and number. The major source of adipocytes comes from mesenchymal stem cells (MSCs), although their roles in obesity remain unclear. An understanding of the mechanisms, regulation, and outcomes of adipogenesis is crucial for the development of new treatments for obesity-related diseases. Recently an unexpected role for the tumor suppressor promyelocytic leukemia protein (PML) in hematopoietic stem cell biology and metabolism regulation has come to light, but its role in MSC biology remains unknown. Here, we investigated the molecular pathway underlying the role of PML in the control of adipogenic MSC differentiation. SUBJECTS/METHODS: Muscle-derived stem cells (MDSCs) and adipose-derived stem cells (ADSCs) obtained from mice and voluntary patients (as a source of MSCs) were cultured in the presence of high glucose (HG) concentration, a nutrient stress condition known to promote MSCs differentiation into mature adipocytes and the adipogenic potential of PML was assessed. RESULTS: PML is essential for a correct HG-dependent adipogenic differentiation, and the enhancement of PML levels is fundamental during adipogenesis. Increased PML expression enables the upregulation of protein kinase Cß (PKCß), which, in turn, by controlling autophagy levels permits an increase in peroxisome proliferator-activated receptor γ (PPARγ) that leads the adipogenic differentiation. Therefore, genetic and pharmacological depletion of PML prevents PKCß expression, and by increasing autophagy levels, impairs the MSCs adipogenic differentiation. Human ADSCs isolated from overweight patients displayed increased PML and PKCß levels compared to those found in normal weight individuals, indicating that the PML-PKCß pathway is directly involved in the enhancement of adipogenesis and human metabolism. CONCLUSIONS: The new link found among PML, PKCß, and autophagy opens new therapeutic avenues for diseases characterized by an imbalance in the MSCs differentiation process, such as metabolic syndromes and cancer.

Endoplasmic reticulum-mitochondria Ca2+ crosstalk in the control of the tumor cell fate.

Mitochondria-associated membranes are juxtaposed between the endoplasmic reticulum and mitochondria and have been identified as a critical hub in the regulation of apoptosis and tumor growth. One key function of mitochondria-associated membranes is to provide asylum to a number of proteins with tumor suppressor and oncogenic properties. In this review, we discuss how Ca2+ flux manipulation represents the primary mechanism underlying the action of several oncogenes and tumor-suppressor genes and how these networks might be manipulated to provide novel therapies for cancer. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner.

The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca(2+)) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca(2+) homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca(2+)-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca(2+) load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca(2+) overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca(2+) signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca(2+) signal-dependent apoptosis.

Endoplasmic Reticulum-Mitochondria Communication Through Ca2+ Signaling: The Importance of Mitochondria-Associated Membranes (MAMs).

The execution of proper Ca2+ signaling requires close apposition between the endoplasmic reticulum (ER) and mitochondria. Hence, Ca2+ released from the ER is "quasi-synaptically" transferred to mitochondrial matrix, where Ca2+ stimulates mitochondrial ATP synthesis by activating the tricarboxylic acid (TCA) cycle. However, when the Ca2+ transfer is excessive and sustained, mitochondrial Ca2+ overload induces apoptosis by opening the mitochondrial permeability transition pore. A large number of regulatory proteins reside at mitochondria-associated ER membranes (MAMs) to maintain the optimal distance between the organelles and to coordinate the functionality of both ER and mitochondrial Ca2+ transporters or channels. In this chapter, we discuss the different pathways involved in the regulation of ER-mitochondria Ca2+ flux and describe the activities of the various Ca2+ players based on their primary intra-organelle localization.

Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.

Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to ß-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.

Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis.

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) emerged to play critical roles in numerous cellular processes, and their dysregulation has been associated to neurodegenerative disorders. Mutations in the SPG4 gene coding for spastin are among the main causes of hereditary spastic paraplegia (HSP). Spastin binds and severs microtubules, and the long isoform of this protein, namely M1, spans the outer leaflet of ER membrane where it interacts with other ER-HSP proteins. Here, we showed that overexpressed M1 spastin localizes in ER-mitochondria intersections and that endogenous spastin accumulates in MERCs. We demonstrated in different cellular models that downregulation of spastin enhances the number of MERCs, alters mitochondrial morphology, and impairs ER and mitochondrial calcium homeostasis. These effects are associated with reduced mitochondrial membrane potential, oxygen species levels, and oxidative metabolism. These findings extend our knowledge on the role of spastin in the ER and suggest MERCs deregulation as potential causes of SPG4-HSP disease.

ER-mitochondria association negatively affects wound healing by regulating NLRP3 activation.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most common causative agent of acute bacterial skin and skin-structure infections (ABSSSI), one of the major challenges to the health system worldwide. Although the use of antibiotics as the first line of intervention for MRSA-infected wounds is recommended, important side effects could occur, including cytotoxicity or immune dysregulation, thus affecting the repair process. Here, we show that the oxazolidinone antibiotic linezolid (LZD) impairs wound healing by aberrantly increasing interleukin 1 ß (IL-1ß) production in keratinocytes. Mechanistically, LZD triggers a reactive oxygen species (ROS)-independent mitochondrial damage that culminates in increased tethering between the endoplasmic reticulum (ER) and mitochondria, which in turn activates the NLR family pyrin domain-containing 3 (NLRP3) inflammasome complex by promoting its assembly to the mitochondrial surface. Downregulation of ER-mitochondria contact formation is sufficient to inhibit the LZD-driven NLRP3 inflammasome activation and IL-1ß production, restoring wound closure. These results identify the ER-mitochondria association as a key factor for NLRP3 activation and reveal a new mechanism in the regulation of the wound healing process that might be clinically relevant.

The NLRP3 Inflammasome in Neurodegenerative Disorders: Insights from Epileptic Models.

Neuroinflammation represents a dynamic process of defense and protection against the harmful action of infectious agents or other detrimental stimuli in the central nervous system (CNS). However, the uncontrolled regulation of this physiological process is strongly associated with serious dysfunctional neuronal issues linked to the progression of CNS disorders. Moreover, it has been widely demonstrated that neuroinflammation is linked to epilepsy, one of the most prevalent and serious brain disorders worldwide. Indeed, NLRP3, one of the most well-studied inflammasomes, is involved in the generation of epileptic seizures, events that characterize this pathological condition. In this context, several pieces of evidence have shown that the NLRP3 inflammasome plays a central role in the pathophysiology of mesial temporal lobe epilepsy (mTLE). Based on an extensive review of the literature on the role of NLRP3-dependent inflammation in epilepsy, in this review we discuss our current understanding of the connection between NLRP3 inflammasome activation and progressive neurodegeneration in epilepsy. The goal of the review is to cover as many of the various known epilepsy models as possible, providing a broad overview of the current literature. Lastly, we also propose some of the present therapeutic strategies targeting NLRP3, aiming to provide potential insights for future studies.

Palmitoylation and PDE6δ regulate membrane-compartment-specific substrate ubiquitylation and degradation.

Substrate degradation by the ubiquitin proteasome system (UPS) in specific membrane compartments remains elusive. Here, we show that the interplay of two lipid modifications and PDE6δ regulates compartmental substrate targeting via the SCFFBXL2. FBXL2 is palmitoylated in a prenylation-dependent manner on cysteines 417 and 419 juxtaposed to the CaaX motif. Palmitoylation/depalmitoylation regulates its subcellular trafficking for substrate engagement and degradation. To control its subcellular distribution, lipid-modified FBXL2 interacts with PDE6δ. Perturbing the equilibrium between FBXL2 and PDE6δ disrupts the delivery of FBXL2 to all membrane compartments, whereas depalmitoylated FBXL2 is enriched on the endoplasmic reticulum (ER). Depalmitoylated FBXL2(C417S/C419S) promotes the degradation of IP3R3 at the ER, inhibits IP3R3-dependent mitochondrial calcium overload, and counteracts calcium-dependent cell death upon oxidative stress. In contrast, disrupting the PDE6δ-FBXL2 equilibrium has the opposite effect. These findings describe a mechanism underlying spatially-restricted substrate degradation and suggest that inhibition of FBXL2 palmitoylation and/or binding to PDE6δ may offer therapeutic benefits.

Novel Aryl Sulfonamide Derivatives as NLRP3 Inflammasome Inhibitors for the Potential Treatment of Cancer.

The NLRP3 inflammasome is a critical component of innate immunity that senses diverse pathogen- and host-derived molecules. However, its aberrant activation has been associated with the pathogenesis of multiple diseases, including cancer. In this study, we designed and synthesized a series of aryl sulfonamide derivatives (ASDs) to inhibit the NLRP3 inflammasome. Among these, compounds 6c, 7n, and 10 specifically inhibited NLRP3 activation at nanomolar concentrations without affecting the activation of the NLRC4 and AIM2 inflammasomes. Furthermore, we demonstrated that these compounds reduce interleukin-1ß (IL-1ß) production in vivo and attenuate melanoma tumor growth. Moreover, metabolic stability in liver microsomes of 6c, 7n, and 10 was studied along with plasma exposure in mice of the most interesting compound 6c. Therefore, we generated potent NLRP3 inflammasome inhibitors, which can be considered in future medicinal chemistry and pharmacological studies aimed at developing a new therapeutic approach for NLRP3 inflammasome-driven cancer.

PML at mitochondria-associated membranes governs a trimeric complex with NLRP3 and P2X7R that modulates the tumor immune microenvironment.

Uncontrolled inflammatory response arising from the tumor microenvironment (TME) significantly contributes to cancer progression, prompting an investigation and careful evaluation of counter-regulatory mechanisms. We identified a trimeric complex at the mitochondria-associated membranes (MAMs), in which the purinergic P2X7 receptor - NLRP3 inflammasome liaison is fine-tuned by the tumor suppressor PML. PML downregulation drives an exacerbated immune response due to a loss of P2X7R-NLRP3 restraint that boosts tumor growth. PML mislocalization from MAMs elicits an uncontrolled NLRP3 activation, and consequent cytokines blast fueling cancer and worsening the tumor prognosis in different human cancers. New mechanistic insights are provided for the PML-P2X7R-NLRP3 axis to govern the TME in human carcinogenesis, fostering new targeted therapeutic approaches.

ATP synthesis and storage.

Since 1929, when it was discovered that ATP is a substrate for muscle contraction, the knowledge about this purine nucleotide has been greatly expanded. Many aspects of cell metabolism revolve around ATP production and consumption. It is important to understand the concepts of glucose and oxygen consumption in aerobic and anaerobic life and to link bioenergetics with the vast amount of reactions occurring within cells. ATP is universally seen as the energy exchange factor that connects anabolism and catabolism but also fuels processes such as motile contraction, phosphorylations, and active transport. It is also a signalling molecule in the purinergic signalling mechanisms. In this review, we will discuss all the main mechanisms of ATP production linked to ADP phosphorylation as well the regulation of these mechanisms during stress conditions and in connection with calcium signalling events. Recent advances regarding ATP storage and its special significance for purinergic signalling will also be reviewed.

Mitochondria-associated membranes (MAMs) as hotspot Ca(2+) signaling units.

The tight interplay between endoplasmic reticulum (ER) and mitochondria is a key determinant of cell function and survival through the control of intracellular calcium (Ca(2+)) signaling. The specific sites of physical association between ER and mitochondria are known as mitochondria-associated membranes (MAMs). It has recently become clear that MAMs are crucial for highly efficient transmission of Ca(2+) from the ER to mitochondria, thus controlling fundamental processes involved in energy production and also determining cell fate by triggering or preventing apoptosis. In this contribution, we summarize the main features of the Ca(2+)-signaling toolkit, covering also the latest breakthroughs in the field, such as the identification of novel candidate proteins implicated in mitochondrial Ca(2+) transport and the recent direct characterization of the high-Ca(2+) microdomains between ER and mitochondria. We review the main functions of these two organelles, with special emphasis on Ca(2+) handling and on the structural and molecular foundations of the signaling contacts between them. Additionally, we provide important examples of the physiopathological role of this cross-talk, briefly describing the key role played by MAMs proteins in many diseases, and shedding light on the essential role of mitochondria-ER interactions in the maintenance of cellular homeostasis and the determination of cell fate.

The LRRC8C-STING-p53 axis in T cells: A Ca2+ affair.

Up to now, no role has been associated with VRAC channels in T cells. In a recent paper published in Nature Immunology, LRRC8C has been described as an essential component of VRAC in T cells. These data raise the intriguing possibility that the LRRC8C-STING-p53 signaling axis may represent a new inhibitory pathway in T cells that controls their function and adaptive immunity.

Outcomes of Locally Advanced Rectal Cancer Patients Treated with Total Neoadjuvant Treatment: A Meta-Anaysis of Randomized Controlled Trials.

BACKGROUND & AIMS: Determining outcomes using the total neoadjuvant therapy (TNT) in patients with local advanced rectal cancer is important for stratifying patients according to expected outcomes in future studies in the era of treatment combination. The present meta-analysis estimated the pathological complete response, disease-free survival, and overall survival probabilities of rectal cancer patients and identified predictors of outcomes. METHODS: Studies reporting pathological complete response rate and time-dependent outcomes (progression or death) after total neoadjuvant treatment of locally advanced rectal cancer (LARC) were identified in MEDLINE through January 2022. Three independent observers extracted data on patient populations and outcomes and combined the data using a distribution-free summary survival curve. The primary outcomes were actuarial probabilities of recurrence and survival. RESULTS: Fourteen RCTs, including 18 TNT arms, met the inclusion criteria. The pooled estimate of pathological complete response (pCR) probability was 23.6%, with moderate heterogeneity between studies. The pooled estimates of actuarial disease-free survival rate were 70.6% at 3 years and 65.4% at 5 years. The pooled estimates of actuarial survival rates were 93% at 3 years and 81.6% at 5 years. In both these outcomes, heterogeneity between studies was highly significant. CONCLUSION: This meta-analysis showed that Total Neoadjuvant Therapy is an optimal approach for LARC patients. The results provide a useful benchmark for future comparisons of the benefits of combinations of other drug families as target therapies or immunotherapies.