Dual mTOR1/2 Inhibitor Sapanisertib (FTH-003/TAK-228) in Combination With Weekly Paclitaxel in Patients With Previously Treated Metastatic Urothelial Carcinoma: A Phase II Open-Label Study.

BACKGROUND: The PI3K/AKT/mTOR pathway is frequently altered at genomic level in metastatic urothelial carcinoma (mUC). Since mTOR is the last protein in the PI3K signaling cascade, it may have the largest impact on the pathway and has been a focus of targeted therapies. Sapanisertib (FTH-003/TAK-228) is an oral highly selective mTOR1 and mTOR2 inhibitor. NFE2L2 mutations have been described as predictive biomarkers of response in patients with advanced squamous cell lung cancer treated with sapanisertib. PATIENTS AND METHODS: This was an open-label, investigator-initiated phase II study evaluating safety and efficacy of sapanisertib plus paclitaxel in patients with mUC who had progressed to prior platinum therapy, and the correlation with NFE2L2 mutations in responders. Primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. Patients were treated with weekly paclitaxel at dose of 80 mg/m2 on days 1, 8, and 15 in combination with sapanisertib 4 mg administered orally 3 days per week on days 2-4, 9-11, 16-18, and 23-25 of a 28-day cycle. NFE2L2 mutations were analyzed by Sanger sequencing in responders. RESULTS: 22 patients were enrolled from May 2018 to April 2020; the trial was halted early due to slow accrual and the COVID-19 pandemic. ORR was 18.2% (n = 4). Disease control rate was 50% (7 SD and 4 PR). Median PFS was 3.4 months (95% CI: 1.8-6.1) and median OS was 6.1 months (95% CI: 1.8-13.4). Adverse events (AE) of grade 3-4 were seen in 86% of patients, but no patients discontinued treatment due to AEs. NFE2L2 mutations were not found in responders. CONCLUSIONS: Although the primary endpoint was no met, sapanisertib and paclitaxel combination demonstrated clinical activity in a heavily pretreated population of mUC. This trial generates insight for future combination of sapaniserib with immunotherapy and/or antibody drug conjugates.

C/EBPα mediates the maturation and antitumor functions of macrophages in human hepatocellular carcinoma.

Recent studies have suggested that therapeutic upregulation of CCAAT/enhancer binding protein α (C/EBPα) prevents hepatocellular carcinoma (HCC) progression. However, the mechanisms underlying this outcome are not fully understood. In this study, we investigated the expression and functional roles of C/EBPα in human HCC, with a focus on monocytes/macrophages (Mφs). Paraffin-embedded tissues were used to visualize C/EBPα expression and analyze the prognostic value of C/EBPα+ monocytes/Mφs in HCC patients. The underlying regulatory mechanisms were examined using human monocyte-derived Mφs. The results showed that the expression of C/EBPα on monocytes/Mφs was significantly decreased in intra-tumor tissues compared to the corresponding peri-tumor tissues. C/EBPα+ monocytes/Mφs displayed well-differentiation and antitumor capacities, and the accumulation of these cells in tissue was associated with antitumor immune responses and predicted longer overall survival (OS) of HCC patients. Mechanistic studies demonstrated that C/EBPα was required for Mφ maturation and HLA-DR, CD169 and CD86 expression, which initiates antitumor cytotoxic T-cell responses; however, these effects were inhibited by monocyte autocrine IL-6- and IL-1ß-induced suppression of mTOR1 signaling. Reprogramming Mφs via the upregulation of C/EBPα may provide a novel strategy for cancer immunotherapy in patients with HCC.

Research progress in the role and mechanism of Leucine in regulating animal growth and development.

Leucine, a branched-chain amino acid, is essential in regulating animal growth and development. Recent research has uncovered the mechanisms underlying Leucine's anabolic effects on muscle and other tissues, including its ability to stimulate protein synthesis by activating the mTORC1 signaling pathway. The co-ingestion of carbohydrates and essential amino acids enhances Leucine's anabolic effects. Moreover, Leucine has been shown to benefit lipid metabolism, and insulin sensitivity, making it a promising strategy for preventing and treating metabolic diseases, including type 2 diabetes and obesity. While emerging evidence indicates that epigenetic mechanisms may mediate Leucine's effects on growth and development, more research is needed to elucidate its mechanisms of action fully. Specific studies have demonstrated that Leucine promotes muscle growth and metabolic health in animals and humans, making it a promising therapeutic agent. However, it is essential to note that Leucine supplementation may cause digestive issues or interact with certain medications, and More study is required to determine definitively optimal dosages. Therefore, it is important to understand how Leucine interacts with other nutrients, dietary factors, and lifestyle habits to maximize its benefits. Overall, Leucine's importance in human nutrition is far-reaching, and its potential to prevent muscle loss and enhance athletic performance warrants further investigation.

Inhibition of the mTORC1 pathway alleviates adipose tissue fibrosis.

Background: Adipose fibrosis is a major factor of adipose dysfunction, which causes metabolic dysfunction during obesity, but its molecular mechanisms are poorly understood. This study investigated the role and potential mechanisms of mTORC1 in obesity-induced adipose fibrosis. Methods: ob/ob mice were injected with rapamycin or the same volume of normal saline. The level of fibrosis in epididymal adipose tissue (EAT) was detected by observing aberrant deposition of extracellular matrix. Expression of fibrotic related genes was analysed using RNA-seq. 3T3-L1 preadipocytes were treated with cobalt chloride (CoCl2) and TGF-ß1 to induce preadipocyte fibrosis. The fibrosis-related gene expression and protein levels were determined by RT-PCR, WB, and immunofluorescence in two types of fibrotic preadipocytes with or without rapamycin. Results: Compared with vehicle treatment, EAT fibrosis-related aberrant deposition of extracellular matrix proteins and fibrotic gene expression were reduced in ob/ob mice treated with rapamycin. Both CoCl2-induced hypoxia and TGF-ß1 successfully promoted adipocyte fibrosis, and the upregulated fibrosis-related genes expression was inhibited after the mTORC1 pathway was inhibited by rapamycin. Conclusion: Inhibition of the mTORC1 pathway ameliorates adipose fibrosis by suppressing fibrosis-related genes in hypoxia- and TGF-ß-induced fibrotic preadipocytes.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) negatively regulates the immunity and resistance to Vibrio alginolyticus of white shrimp, Penaeus vannamei.

Shrimp immunology is vital in establishing prophylactic and therapeutic strategies for controlling pathological problems that threaten shrimp production. Apart from dietary treatments, the adenosine 5'-monophosphate-activated protein kinase (AMPK), an important regulatory enzyme that restores cellular energy balance during metabolic and physiological stress, is known to have therapeutic potential to improve shrimp's defense mechanism. Despite this, studies targeting the AMPK pathway in shrimp exposed to stressful conditions are vastly limited. In this study, AMPK was knocked down to assess the immunological changes and white shrimp, Penaeus vannamei resistance to Vibrio alginolyticus infection. Shrimps were injected individually and simultaneously with dsRNA targeting specific genes such as AMPK, Rheb, and TOR, after which the hepatopancreas was analyzed for the different gene expressions. The gene expressions of AMPK, Rheb, and TOR were effectively suppressed after being treated with dsRNAs. The Western blot analysis further confirmed a reduction in the protein concentration of AMPK and Rheb in the hepatopancreas. The suppression of AMPK gene led to a robust increase in the shrimp's resistance to V. alginolyticus, whereas the activation of AMPK by metformin decreased the shrimp's disease resistance. Among the mTOR downstream targets, the HIF-1α expression in shrimp treated with dsAMPK significantly increased at 48 h but returned to normal levels when shrimp were treated with dsAMPK and either dsRheb or dsTOR. Immune responses such as respiratory burst, lysozyme activity, and phagocytic activity increased, while superoxide dismutase activity decreased following the knockdown of the AMPK gene compared to the control group. However, co-injection with dsAMPK and dsTOR or dsRheb restored immune responses to normal levels. Collectively, these results demonstrate that the inactivation of AMPK may ameliorate shrimp's innate immune response to recognize and defend against pathogens via the AMPK/mTOR1 pathway.

mTOR1c Activation with the Leucine "Trigger" for Prevention of Sarcopenia in Older Adults During Lockdown.

Sarcopenia and muscle wasting have many negative impacts on health and well-being. Evidence suggests that high rates of COVID-19 hospitalizations and lockdown conditions will lead to a marked increase in musculoskeletal disorders associated with sarcopenia in older adults. The molecular etiology of sarcopenia is complex, but physical inactivity, poor diet, and age diminished ability to stimulate muscle protein synthesis (MPS) remain important drivers. A body of evidence shows that, acting through the highly conserved nutrient sensor pathway mTORc1, the branch chain amino acid leucine can trigger and enhance MPS in older adults, and thus has a role in the medical management of sarcopenia. Whey protein-enriched enteral supplements are a low cost, easily accessible source of highly bioavailable leucine used clinically in older adults for preservation of lean body mass in long-term care setting. Therefore, given the evidence of leucine's ability to stimulate MPS in older adults, we argue that meal supplementation with whey-enriched enteral products, which can provide the 3-5 g of leucine necessary to trigger MPS in older adults, should be given serious consideration by medical and nutrition professionals to potentially mitigate muscle wasting and sarcopenia risk associated with prolonged COVID-19 lockdown measures.

Hypo-Expression of Tuberin Promotes Adenomyosis via the mTOR1-Autophagy Axis.

Adenomyosis (AM) is a disease in which endometrial tissue invades the myometrium and has a 10-60% prevalence in reproductive-aged women. TSC2 regulates autophagy via mTOR1 signalling in colorectal cancer and endometrial carcinoma. Dysregulation of autophagy is implicated in adenomyosis pathogenesis. However, whether TSC2 participates in adenomyosis via autophagy remains obscure. Here, we found that the expression of TSC2 in adenomyosis was significantly decreased than that in normal endometrium during the secretory phase. Moreover, TSC2 and autophagy marker expression was significantly lower in ectopic lesions than in eutopic samples. TSC2 downregulation inhibited autophagy through mTOR1 signalling pathway activation in endometrial cells, leading to excessive proliferation, migration, and EMT; TSC2 overexpression induced the opposite effects. Rapamycin treatment suppressed cell proliferation, migration and EMT in the absence of TSC2. In parallel, an autophagy-specific inhibitor (SAR-405) restored migration and EMT under rapamycin treatment in TSC2-knockdown Ishikawa cells. Finally, SAR-405 treatment promoted EMT and migration of overexpressing cells. Collectively, our results suggest that TSC2 controls endometrial epithelial cell migration and EMT by regulating mTOR1-autophagy axis activation and that hypo-expression of TSC2 in the endometrium might promote adenomyosis.

PPP1CA/YAP/GS/Gln/mTORC1 pathway activates retinal Müller cells during diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-loss complication caused by diabetes with high prevalence. During DR, the retinal microvascular injury and neurodegeneration derived from chronic hyperglycemia have attracted global attention to retinal Müller cells (RMCs), the major macroglia in the retina contributes to neuroprotection. Protein Phosphatase 1 Catalytic Subunit Alpha (PPP1CA) dephosphorylates the transcriptional coactivator Yes-associated protein (YAP) to promote the transcription of glutamine synthetase (GS). GS catalyzes the transformation of neurotoxic glutamate (Glu) into nontoxic glutamine (Gln) to activate the mammalian target of rapamycin complex 1 (mTORC1), which promotes the activation of RMCs. In this study, in vitro MIO-M1 cell and in vivo mouse high-fat diet and streptozotocin (STZ)-induced diabetic model to explore the role of the PPP1CA/YAP/GS/Gln/mTORC1 pathway on the activation of MRCs during DR. Results showed that PPP1CA promoted the dephosphorylation and nuclear translocation of YAP in high glucose (HG)-exposed MIO-M1 cells. YAP transcribed GS in HG-exposed MIO-M1 cells in a TEAD1-dependent and PPP1CA-dependent way. GS promoted the biosynthesis of Gln in HG-exposed MIO-M1 cells. Gln activated mTORC1 instead of mTORC2 in HG-exposed MIO-M1 cells. The proliferation and activation of HG-exposed MIO-M1 cells were PPP1CA/YAP/GS/Gln/mTORC1-dependent. Finally, RMC proliferation and activation during DR were inhibited by the PPP1CA/YAP/GS/Gln/mTORC1 blockade. The findings supplied a potential idea to protect RMCs and alleviate the development of DR.

Therapeutic potential of the new TRIB3-mediated cell autophagy anticancer drug ABTL0812 in endometrial cancer.

OBJECTIVES: The PI3K/AKT/mTOR pathway is frequently overactivated in endometrial cancer (EC). We assessed the efficacy of ABTL0812, a novel first-in-class molecule presenting a unique mechanism of action inhibiting this pathway. METHODS: We investigated the effects of ABTL0812 on proliferation, cell death and modulation of intracellular signaling pathways in a wide panel of endometrioid and non-endometrioid cell lines, an inducible PTEN knock-out murine model, and two patient-derived xenograft murine models of EC. Then, TRIB3 expression was evaluated as potential ABTL0812 pharmacodynamic biomarker in a Phase 1b/2a clinical trial. RESULTS: ABTL0812 induced an upregulation of TRIB3 expression, resulting in the PI3K/AKT/mTOR axis inhibition and autophagy cell death induction on EC cells but not in healthy endometrial cells. ABTL0812 treatment also impaired PTEN knock-out cells to progress from hyperplasia to cancer. The therapeutic effects of ABTL0812 were demonstrated in vivo. ABTL0812 increased TRIB3 mRNA levels in whole blood samples of eight EC patients, demonstrating that TRIB3 mRNA could be used as a pharmacodynamic biomarker to monitor the ABTL0812 treatment. CONCLUSIONS: ABTL0812 may represent a novel and highly effective therapeutic agent by inducing TRIB3 expression and autophagy in EC patients, including those with poorer prognosis.

Dietary starch types affect liver nutrient metabolism of finishing pigs.

This study aimed to evaluate the effect of different starch types on liver nutrient metabolism of finishing pigs. In all ninety barrows were randomly allocated to three diets with five replicates of six pigs, containing purified waxy maize starch (WMS), non-waxy maize starch (NMS) and pea starch (PS) (the amylose to amylopectin ratios were 0·07, 0·19 and 0·28, respectively). After 28 d of treatments, two per pen (close to the average body weight of the pen) were weighed individually, slaughtered and liver samples were collected. Compared with the WMS diet, the PS diet decreased the activities of glycogen phosphorylase, phosphoenolpyruvate carboxykinase and the expression of phosphoenolpyruvate carboxykinase 1 in liver (P0·05). Compared with the WMS diet, the PS diet reduced the expressions of glutamate dehydrogenase and carbamoyl phosphate synthetase 1 in liver (P<0·05). PS diet decreased the expression of the insulin receptor, and increased the expressions of mammalian target of rapamycin complex 1 and ribosomal protein S6 kinase ß-1 in liver compared with the WMS diet (P<0·05). These findings indicated that the diet with higher amylose content could down-regulate gluconeogenesis, and cause less fat deposition and more protein deposition by affecting the insulin/PI3K/protein kinase B signalling pathway in liver of finishing pigs.

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002.

The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC50 on ATP concentration that allows prediction of the IC50 at different ATP concentrations in enzyme and cellular assays. Comparison of drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K/PTEN/AKT/mTOR1 network in order to understand mechanisms of drug sensitivity and resistance in different cancer cell lines. We suggest that using these models in a systems biology investigation of the PI3K/AKT/mTOR1 signalling in cancer cells can bridge the gap between direct drug target action and the therapeutic response to these drugs and their combinations.

Metabolic adaptation of skeletal muscle to hyperammonemia drives the beneficial effects of l-leucine in cirrhosis.

BACKGROUND & AIMS: Increased skeletal muscle ammonia uptake with loss of muscle mass adversely affects clinical outcomes in cirrhosis. Hyperammonemia causes reduced protein synthesis and sarcopenia but the cellular responses to impaired proteostasis and molecular mechanism of l-leucine induced adaptation to ammonia induced stress were determined. METHODS: Response to activation of amino acid deficiency sensor, GCN2, in the skeletal muscle from cirrhotic patients and the portacaval anastomosis (PCA) rat were quantified. During hyperammonemia and l-leucine supplementation, protein synthesis, phosphorylation of eIF2α, mTORC1 signaling, l-leucine transport and response to l-leucine supplementation were quantified. Adaptation to cellular stress via ATF4 and its target GADD34 were also determined. RESULTS: Activation of the eIF2α kinase GCN2 and impaired mTORC1 signaling were observed in skeletal muscle from cirrhotic patients and PCA rats. Ammonia activated GCN2 mediated eIF2α phosphorylation (eIF2α-P) and impaired mTORC1 signaling that inhibit protein synthesis in myotubes and MEFs. Adaptation to ammonia induced stress did not involve translational reprogramming by activation transcription factor 4 (ATF4) dependent induction of the eIF2α-P phosphatase subunit GADD34. Instead, ammonia increased expression of the leucine/glutamine exchanger SLC7A5, l-leucine uptake and intracellular l-leucine levels, the latter not being sufficient to rescue the inhibition of protein synthesis, due to potentially enhanced mitochondrial sequestration of l-leucine. l-leucine supplementation rescued protein synthesis inhibition caused by hyperammonemia. CONCLUSIONS: Response to hyperammonemia is reminiscent of the cellular response to amino acid starvation, but lacks the adaptive ATF4 dependent integrated stress response (ISR). Instead, hyperammonemia-induced l-leucine uptake was an adaptive response to the GCN2-mediated decreased protein synthesis. LAY SUMMARY: Sarcopenia or skeletal muscle loss is the most frequent complication in cirrhosis but there are no treatments because the cause(s) of muscle loss in liver disease are not known. Results from laboratory experiments in animals and muscle cells were validated in human patients with cirrhosis to show that ammonia plays a key role in causing muscle loss in patients with cirrhosis. We identified a novel stress response to ammonia in the muscle that decreases muscle protein content that can be reversed by supplementation with the amino acid l-leucine.