Mechanistic Target Of Rapamycin Inhibitors Research Articles

COVID-19 in Lymphangioleiomyomatosis: An International Study of Outcomes and Impact of Mechanistic Target of Rapamycin Inhibition

Lymphangioleiomyomatosis is a rare low-grade neoplastic lung disease that occurs sporadically in women or in people with tuberous sclerosis complex. Mechanistic target of rapamycin (mTOR) inhibitors are used for patients with progressive or severe pulmonary impairment or extrapulmonary manifestations.1

Research Highlights

C3 Complement Inhibition Prevents Antibody-mediated Rejection and Prolongs Renal Allograft Survival in Sensitized Nonhuman Primates Schmitz R, Fitch ZW, Schroder PM, et al. Nat Commun. 2021;12:5456. Current therapeutic strategies for desensitization in renal transplantation have shown limited efficacy.1 Because a major downstream effector arm of donor-specific antibody (DSA) is the complement cascade,2 complement inhibition strategies are gaining interest.3 Following C1 and C5 inhibition strategies, the C3 inhibitor Cp40 has recently been added to this field of promising candidates with a successful first-in-human study (NCT03316521) of its derivate AMY-101. In the study from Schmitz et al,4 Cp40 treatment was assessed in a nonhuman primate sensitized renal transplantation model, in which animals received donor skin transplants before the kidney transplant. Induction therapy with rhesus-specific antithymocyte globulin and maintenance therapy with tacrolimus, mycophenolate mofetil, and methylprednisolone were administered with (n = 6) or without (n = 5) peritransplant Cp40 treatment (day –2 to day 14). Although all animals showed a significant DSA increase after transplant, Cp40 treatment reduced acute antibody-mediated rejection and prolonged graft survival clearly (15.5 versus 4 d). Although initial lymphocyte depletion was not affected by Cp40, activation and proliferation proved to be markedly disrupted. Interestingly, virtually no intragraft C3d deposition could be detected during the treatment period with Cp40, which contrasted with controls and treated animals after discontinuation of Cp40. Within the treatment group, there were early (ie, during Cp40 treatment) and late rejectors. Although overall DSA levels were similar, late rejectors had less antibody-mediated rejection and less graft infiltration with CD68+ macrophages in parallel to downregulated complement-related genes. Late rejectors also had higher numbers of CD4+ CD25+ FoxP3+ regulatory T cells 1 wk after transplant, reduced T- and B-cell proliferation, and lower immunoglobulin (Ig)M DSAs with lower complement activation. The authors concluded that because of heterogeneity of IgM DSA levels, Cp40 doses could have been insufficient in animals bearing the highest immunologic burden. These data provide crucial evidence for C3 inhibition in a relevant preclinical model. Notably, overall DSA levels were not predictive of rejection in this study, yet treatment success correlated with IgM DSA load. This study sets the stage for early phase clinical trials of Cp40 in transplantation in which identification and stratification of potential benefit and dosage requirements in patient populations could be incorporated according to IgM DSA levels. An Aged Immune System Drives Senescence and Aging of Solid Organs Yousefzadeh MJ, Flores RR, Zhu Y, et al. Nature. 2021;594:100–105. The decline of immune responses with aging, termed immunosenescence, is linked to increased morbidity and mortality, likely related to reduced immunosurveillance, poor antipathogen immunity, and reduced vaccination responses.1,2 Given the broad role of the immune system in homeostasis, targeting immune cells to influence systemic aging may be a useful strategy for the reversal of age-related tissue pathology. In the landmark study from Yousefzadeh et al in Nature,3 the authors knocked out Ercc1 (which encodes the crucial DNA repair protein endonuclease ERCC1-XPF) in mouse hematopoietic cells, thereby selectively altering senescence in lymphoid organs. The impact of this genetic modification was evaluated in the context of aging. Compared with normal aging in wild-type mice, mutant mice developed peripheral leukopenia in adulthood and overall immune cells decreased in all compartments, as did thymic and splenic weights. Lymphoid tissues were less active and delayed-type hypersensitivity reactions demonstrated decreased innate and adaptive immune functions in older mutant mice, whereas younger ones had similar responses to wild-type littermates. Mutant mice displayed increased oxidative stress in lymphoid tissues and senescence onset in several immune cell subsets when compared with aged wild-type mice. Notably, older mutant mice had systemic changes associated with normal aging measured by DNA damage, oxidative stress, and senescence markers in many solid organ tissues, with peripheral senescence chronologically following immune senescence. Tissue damage appeared in nondeleted organs such as the liver, pancreas, intervertebral discs, brain, and muscles. Accordingly, the lifespan of mutant mice was markedly reduced. Interestingly, some of these effects could be transferred by simple transplantation of aged donor cells into separate recipient mice. Here, transplantation of aged or senescent splenocytes into reporter mice appeared to result in an acceleration of systemic aging and a decreased lifespan. Conversely, adoptive transfer of young wild-type splenocytes was able to attenuate peripheral organ senescence and tissue damage in older recipients. Finally, mechanistic target of rapamycin inhibition with rapamycin mitigated immune senescence characteristics connected with systemic aging. These findings underscore the driving role of immunosenescence in systemic aging, related tissue damage, and fatal outcomes. Additionally, the data highlight some of the molecular mechanisms linking aging and immunosenescence, which may be amenable to therapeutic targeting.

Epithelial X-Box Binding Protein 1 Coordinates Tumor Protein p53-Driven DNA Damage Responses and Suppression of Intestinal Carcinogenesis

Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested invitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. Invivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein1. Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.

Epilepsy in the mTORopathies: opportunities for precision medicine.

The mechanistic target of rapamycin signalling pathway serves as a ubiquitous regulator of cell metabolism, growth, proliferation and survival. The main cellular activity of the mechanistic target of rapamycin cascade funnels through mechanistic target of rapamycin complex 1, which is inhibited by rapamycin, a macrolide compound produced by the bacterium Streptomyces hygroscopicus. Pathogenic variants in genes encoding upstream regulators of mechanistic target of rapamycin complex 1 cause epilepsies and neurodevelopmental disorders. Tuberous sclerosis complex is a multisystem disorder caused by mutations in mechanistic target of rapamycin regulators TSC1 or TSC2, with prominent neurological manifestations including epilepsy, focal cortical dysplasia and neuropsychiatric disorders. Focal cortical dysplasia type II results from somatic brain mutations in mechanistic target of rapamycin pathway activators MTOR, AKT3, PIK3CA and RHEB and is a major cause of drug-resistant epilepsy. DEPDC5, NPRL2 and NPRL3 code for subunits of the GTPase-activating protein (GAP) activity towards Rags 1 complex (GATOR1), the principal amino acid-sensing regulator of mechanistic target of rapamycin complex 1. Germline pathogenic variants in GATOR1 genes cause non-lesional focal epilepsies and epilepsies associated with malformations of cortical development. Collectively, the mTORopathies are characterized by excessive mechanistic target of rapamycin pathway activation and drug-resistant epilepsy. In the first large-scale precision medicine trial in a genetically mediated epilepsy, everolimus (a synthetic analogue of rapamycin) was effective at reducing seizure frequency in people with tuberous sclerosis complex. Rapamycin reduced seizures in rodent models of DEPDC5-related epilepsy and focal cortical dysplasia type II. This review outlines a personalized medicine approach to the management of epilepsies in the mTORopathies. We advocate for early diagnostic sequencing of mechanistic target of rapamycin pathway genes in drug-resistant epilepsy, as identification of a pathogenic variant may point to an occult dysplasia in apparently non-lesional epilepsy or may uncover important prognostic information including, an increased risk of sudden unexpected death in epilepsy in the GATORopathies or favourable epilepsy surgery outcomes in focal cortical dysplasia type II due to somatic brain mutations. Lastly, we discuss the potential therapeutic application of mechanistic target of rapamycin inhibitors for drug-resistant seizures in GATOR1-related epilepsies and focal cortical dysplasia type II.

Propionate and Dietary Fermentable Fibers Upregulate Intestinal Heat Shock protein70 in Intestinal Caco-2 Cells and Mouse Colon.

Short-chain fatty acids (SCFAs), including propionate, are major metabolites of intestinal microorganisms and play an essential role in regulating intestinal epithelial integrity. Heat shock proteins (HSPs) promote cellular homeostasis under physiological and stressed conditions. This study aimed to investigate the regulation of intestinal HSP70 by propionate in human intestinal Caco-2 cells and the colon of fermentable dietary fiber (DF)-fed mice and germ-free mice. The results showed that propionate increased Hspa1a (HSP70 mRNA) level in Caco-2 cells, upregulated HSP70 protein, and phosphorylation of heat shock factor 1; however, the latter two were reduced by mitogen-activated protein kinases and the mechanistic target of rapamycin inhibitors. Feeding fermentable DFs, such as guar gum (GG) and partially hydrolyzed GG, increased both cecal SCFAs and colonic HSP70 expression, both of which were reduced in germ-free mice than in specific-pathogen-free mice. Collectively, the propionate-induced HSP70 expression was shown to be possibly involved in intestinal homeostasis.

Efficacy, Retention and Tolerability of Everolimus in Patients with Tuberous Sclerosis Complex: A Survey-Based Study on Patients' Perspectives.

BackgroundThe approval of everolimus (EVE) for the treatment of angiomyolipoma (2013), subependymal giant cell astrocytoma (2013) and drug-refractory epilepsy (2017) in patients with tuberous sclerosis complex (TSC) represents the first disease-modifying treatment option available for this rare and complex genetic disorder.ObjectiveThe objective of this study was to analyse the use, efficacy, tolerability and treatment retention of EVE in patients with TSC in Germany from the patient’s perspective.MethodsA structured cross-age survey was conducted at 26 specialised TSC centres in Germany and by the German TSC patient advocacy group between February and July 2019, enrolling children, adolescents and adult patients with TSC.ResultsOf 365 participants, 36.7% (n = 134) reported the current or past intake of EVE, including 31.5% (n = 115) who were taking EVE at study entry. The mean EVE dosage was 6.1 ± 2.9 mg/m2 (median: 5.6 mg/m2, range 2.0–15.1 mg/m2) in children and adolescents and 4 ± 2.1 mg/m2 (median: 3.7 mg/m2, range 0.8–10.1 mg/m2) in adult patients. An early diagnosis of TSC, the presence of angiomyolipoma, drug-refractory epilepsy, neuropsychiatric manifestations, subependymal giant cell astrocytoma, cardiac rhabdomyoma and overall multi-organ involvement were associated with the use of EVE as a disease-modifying treatment. The reported efficacy was 64.0% for angiomyolipoma (75% in adult patients), 66.2% for drug-refractory epilepsy, and 54.4% for subependymal giant cell astrocytoma. The overall retention rate for EVE was 85.8%. The retention rates after 12 months of EVE therapy were higher among adults (93.7%) than among children and adolescents (88.7%; 90.5% vs 77.4% after 24 months; 87.3% vs 77.4% after 36 months). Tolerability was acceptable, with 70.9% of patients overall reporting adverse events, including stomatitis (47.0%), acne-like rash (7.7%), increased susceptibility to common infections and lymphoedema (each 6.0%), which were the most frequently reported symptoms. With a total score of 41.7 compared with 36.8 among patients not taking EVE, patients currently being treated with EVE showed an increased Liverpool Adverse Event Profile. Noticeable deviations in the sub-items ‘tiredness’, ‘skin problems’ and ‘mouth/gum problems’, which are likely related to EVE-typical adverse effects, were more frequently reported among patients taking EVE.ConclusionsFrom the patients’ perspective, EVE is an effective and relatively well-tolerated disease-modifying treatment option for children, adolescents and adults with TSC, associated with a high long-term retention rate that can be individually considered for each patient. Everolimus therapy should ideally be supervised by a centre experienced in the use of mechanistic target of rapamycin inhibitors, and adverse effects should be monitored on a regular basis.

Comparative metabolomics and lipidomics study to evaluate the metabolic differences between first- and second-generation mammalian or mechanistic target of rapamycin inhibitors.

Mammalian or mechanistic target of rapamycin (mTOR) drives its fundamental cellular functions through two distinct catalytic subunits, mTORC1 and mTORC2, and is frequently dysregulated in most cancers. To treat cancers, developed mTOR inhibitors have been classified into first and second generations based on their ability to inhibit single (first-generation) and dual (second-generation) mTOR subunits. However, the underlying metabolic differences due to the effects of first- and second-generation mTOR inhibitors have not been clearly evaluated. In this study, rapamycin (sirolimus) and AZD8055 and PP242 were selected as first- and second-generation mTOR inhibitors, respectively, to evaluate the metabolic differences due to these two generations of mTOR inhibitors after a single oral dose using untargeted metabolomics and lipidomics approaches. The metabolic differences at each time point were compared using multivariate analysis. The multivariate and data analyses showed that metabolic disparity was more prominent within 8h after drug administration and a broad class of metabolites were affected by the administration of both generations of mTOR inhibitors. Among the metabolite classes, changes in the pattern of fatty acids and glycerophospholipids were opposite, specifically at 4 and 8h between the two generations of mTOR inhibitors. We speculate that the inhibition of the mTORC2 subunit by the second-generation mTOR inhibitor may have resulted in a distinct metabolic pattern between the first- and second-generation inhibitors. Finally, the findings of this study could assist in a more detailed understanding of the key metabolic differences caused by first- and second-generation mTOR inhibitors.

Gynecological sarcomas: literature review of 2020.

This article, focus on recently published data of the last 18 months on the management of gynecologic sarcomas. Different tools have been studied to identify the differences between benign from malignant uterine conjonctive tumor.Molecular biology impact more and more on the diagnosis of uterine sarcoma with new definitions of very specific groups. This will make it possible to better define the last group of endometrial sarcoma which has been defined as undifferentiated.In several articles, surgical approaches and fertility-sparing surgery were described including the role of surgery for recurrences.Some other articles have evaluated the potential benefice of adjuvant therapy for uterine sarcoma with early stages.Several new targeted therapies are in development. Notably deoxyribonucleic acid repair machinery in uterine leiomyosarcoma and also immune therapies, transforming growth factor beta pathway, mechanistic target of rapamycin inhibitor, anti angiogenics, etc. This last year the potential interest for uterine sarcoma increased, demonstrated by the increasing number of publications in the literature compared to previous years. Despite this greater interest over time, the standard of care for uterine sarcoma does not change and we are always waiting for new innovative therapies able to change routine practice and survival of patients. Currently, the result of different clinical trials, which include new options as targeted molecular approach or immune checkpoint inhibitors are closed to be reported.

Alpelisib in combination with everolimus ± exemestane in solid tumours: Phase Ib randomised, open-label, multicentre study

Background and purposeCombined mTORC1 inhibition with everolimus (EVE) and phosphatidylinositol 3-kinase catalytic subunit p110α blockade with alpelisib (ALP) has demonstrated synergistic efficacy in preclinical models and supports testing the combination of ALP and EVE in the clinical setting. The primary objective was to determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE) of ALP in combination with EVE and in combination with EVE and exemestane (EXE) and subsequently assess safety, preliminary efficacy and effect of ALP on the pharmacokinetics of EVE and determine the magnitude of the drug-drug interaction. Patients and methodsDose escalation phases were conducted in patients with advanced solid tumours and in postmenopausal women with hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2–) advanced breast cancer (ABC). The dose expansion phase was conducted in patients with pancreatic neuroendocrine tumour and renal cell carcinoma (RCC) (both mechanistic target of rapamycin inhibitor [mTORi]–naive), in patients with mTORi-pretreated solid tumours and in postmenopausal women with HR+, HER2– ABC. ResultsDuring the doublet escalation phase, dose-limiting toxicities (DLTs) were reported in 5 of 10 (50%) patients: one patient had grade (Gr) 2 hyperglycemia and one patient had Gr 3 diarrhoea in the 300 mg dose group, one patient had Gr 2 hyperglycemia and one patient had Gr 4 hypocalcaemia in the 250 mg dose group, and one patient in the 200 mg dose group had Gr 3 diarrhoea and Gr 3 stomatitis. The combination of ALP 250 mg + EVE 2.5 mg was declared as the MTD/RDE in subjects with advanced solid tumours. In the triplet escalation phase, one patient who received ALP 200 mg + EVE 2.5 mg + EXE 25 mg had a DLT of Gr 3 acute kidney injury. This dose combination was declared as the MTD and RDE in subjects with advanced HR-positive HER2-negative BC. The common adverse events (≥30% patients), occurring across all phases, were hyperglycaemia, stomatitis, diarrhoea, nausea, asthenia, decreased appetite and fatigue. The sixteen-week progression-free survival rate was 52.4% (90% confidence interval [CI]: 32.8, 71.4) in the RCC cohort, 35.3% (90% CI: 16.6, 58.0) in the prior pNET cohort and 30.0% (90% CI: 8.7, 60.7) in the prior mTORi cohort. The pharmacokinetics of 2.5 mg of EVE was largely unchanged in the presence of ALP, independent of the dose (250 mg or 300 mg). There were no clinically relevant drug-drug interactions observed between ALP and EVE. ConclusionThe overall safety profile of ALP with EVE and EXE is manageable and reversible; no unexpected safety signals were noted compared with the individual safety profiles. Pharmacokinetics of ALP, EVE and EXE was largely unchanged in combination with each other.

Mechanistic Target of Rapamycin Inhibitors in Renal Cell Carcinoma: Potential, Limitations, and Perspectives.

Several elements highlight the importance of the mechanistic target of rapamycin (mTOR) in the biology of renal cell carcinoma (RCC). mTOR signaling pathway is indeed frequently activated in RCC, inducing cancer cell proliferation and survival. In addition, mTOR promotes tumor angiogenesis and regulates the expression of hypoxia-inducible factors that play an important role in a subset of RCC. Despite mTOR protumorigenic effects, mTOR inhibitors have failed to provide long-lasting anticancer benefits in RCC patients, highlighting the need to readdress their role in the treatment of RCC. This review aims to present the rationale and limitations of targeting mTOR in RCC. Future roles of mTOR inhibitors in the treatment of RCC are also discussed, in particular in the context of immunotherapies.

Inhibition of MTOR signaling impairs rat embryo organogenesis by affecting folate availability.

Mechanistic target of rapamycin (MTOR) is essential for embryo development by acting as a nutrient sensor to regulate cell growth, proliferation and metabolism. Folate is required for normal embryonic development and it was recently reported that MTOR functions as a folate sensor. In this work, we tested the hypothesis that MTOR functions as a folate sensor in the embryo and its inhibition result in embryonic developmental delay affecting neural tube closure and that these effects can be rescued by folate supplementation. Administration of rapamycin (0.5 mg/kg) to rats during early organogenesis inhibited embryonic ribosomal protein S6, a downstream target of MTOR Complex1, markedly reduced embryonic folate incorporation (-84%, P < 0.01) and induced embryo developmental impairments, as shown by an increased resorption rate, reduced embryo somite number and delayed neural tube closure. These alterations were prevented by folic acid administered to the dams. Differently, although an increased rate of embryonic rotation defects was observed in the rapamycin-treated dams, this alteration was not prevented by maternal folic acid supplementation. In conclusion, MTOR inhibition during organogenesis in the rat resulted in decreased folate levels in the embryo, increased embryo resorption rate and impaired embryo development. These data suggest that MTOR signaling influences embryo folate availability, possibly by regulating the transfer of folate across the maternal-embryonic interface.

Trehalose Inhibits Human Immunodeficiency Virus Type 1 Infection in Primary Human Macrophages and CD4+ T Lymphocytes through Two Distinct Mechanisms.

Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a nontoxic, nonreducing disaccharide that induces autophagy through an MTOR-independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose-dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy, including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2, and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5 Additionally, trehalose downregulated the expression of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages, which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar trehalose at doses safely achieved in humans inhibits HIV through two mechanisms: (i) decreased entry through the downregulation of CCR5 in T cells and decreased CD4 expression in both T cells and macrophages and (ii) degradation of intracellular HIV through the induction of MTOR-independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, nontoxic sugar.IMPORTANCE Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of the mechanistic target of rapamycin (MTOR) has cellular effects that may alter HIV infection through other mechanisms. Here, we examined the HIV-inhibitory effects of the MTOR-independent inducer of autophagy, trehalose. Of note, we identified that in addition to the inhibition of the intracellular replication of HIV by autophagy, trehalose decreased viral entry in human primary macrophages and CD4+ T cells through the downregulation of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages. Thus, we showed that trehalose uniquely inhibits HIV replication through inhibition of viral entry and intracellular degradation in the two most important target cells for HIV infection.

Immunosuppression Withdrawal in Liver Transplant Recipients on Sirolimus.

As conversion from calcineurin inhibitor to sirolimus (SRL), a mechanistic target of rapamycin inhibitor (mTOR-I), has been shown to enhance immunoregulatory profiles in liver transplant (LT) recipients (LTRs), mTOR-I therapy might allow for increased success of immunosuppression (IS) withdrawal. Our aim was to determine if operational tolerance could be observed in LTRs withdrawn from SRL and if blood/graft tolerance biomarkers were predictive of successful withdrawal. We performed a prospective trial of SRL monotherapy withdrawal in nonimmune, nonviremic LTRs > 3 years post-LT. SRL was weaned over ~6 months, and biopsies were performed 12 months postweaning or at concern for acute rejection. Twenty-one LTRs consented; 6 were excluded due to subclinical acute rejection on baseline biopsy or other reasons, and 15 underwent weaning (age 61.3±8.8years; LT to SRL weaning 6.7±3years). Eight (53%) achieved operational tolerance (TOL). Of the 7 who were nontolerant (non-TOL), 6 had mild acute rejection on biopsy near the end of weaning or at study end; 1 was removed from the trial due to liver cancer recurrence. At baseline preweaning, there were statistically increased blood tolerogenic dendritic cells and cell phenotypes correlating with chronic antigen presentation in the TOL versus non-TOL groups. A previously identified biopsy gene signature accurately predicted TOL versus non-TOL in 12/14 LTRs before weaning. At study end, biopsy staining revealed statistically significant increases in antigen-presenting cell:leukocyte pairings, FOXP3+ /CD4+ T cells, Tbet+ /CD8+ T cells, and lobular dendritic cells in the non-TOL group. This study evaluated IS withdrawal directly from mTOR-I therapy in LTRs and achieved > 50% operational tolerance. Preweaning gene expression and peripheral blood mononuclear cell profiling may be useful as predictors of successful mTOR-I therapy withdrawal. NCT02062944.

Lymphangioleiomyomatosis: a clinical review.

Lymphangioleiomyomatosis (LAM) is a diffuse cystic lung disease. There are two main types of LAM: sporadic, and LAM associated with the tuberous sclerosis complex (TSC), which is caused by mutations in the TSC1 and TSC2 genes. LAM is characterised by cystic lung disease resulting in progressive dyspnoea, renal angiomyolipomas and lymphatic complications. Pneumothorax occurs frequently (70%) and definitive management with pleurodesis is recommended as the risk of recurrence is high. Characteristic thin-walled cysts are seen on computed tomography and the presence of elevated serum levels of a vascular endothelial growth factor-D has good diagnostic specificity. Currently, no single clinical or serological factor has been shown to predict prognosis. However, over the past decade, significant advances in our understanding of the pathophysiology of LAM has led to improved recognition of this rare disease and identification of treatment options. Mechanistic target of rapamycin inhibitors slow the rate of lung function decline and can resolve chylous effusion and regress angiomyolipomas. Life expectancy in patients with LAM is favourable, with a mean transplant-free survival >20 years from the time of diagnosis. Continued advances in understanding the molecular basis of LAM will lead to improved therapeutic targets and the development of more robust prognostic indicators.Educational aimsTo illustrate the clinical features, common presentations and radiological features of LAMTo outline the diagnostic approach to LAM, including the role of VEGF-DTo review the current prognostic indicators in LAM, and outline the impact of lung function, hormonal status, VEGF-D and clinical presentation on outcomeTo inform clinicians on the management options for LAM both pharmacological and nonpharmacological

IL-2 enhances ex vivo–expanded regulatory T-cell persistence after adoptive transfer

AbstractAs regulatory T cell (Treg) adoptive therapy continues to develop clinically, there is a need to determine which immunomodulatory agents pair most compatibly with Tregs to enable persistence and stabilize suppressor function. Prior work has shown that mechanistic target of rapamycin inhibition can increase the stability of thymic Tregs. In this study, we investigated the transcriptomic signatures of ex vivo–expanded Tregs after adoptive transfer in the setting of clinically relevant immunosuppression using a nonhuman primate (NHP) model as a prelude to future transplant studies. Here, we found that adding interleukin-2 (IL-2) to rapamycin in vivo supported a logarithmic increase in the half-life of adoptively transferred carboxyfluorescein diacetate succinimidyl ester–labeled, autologous NHP Tregs, effectively doubling the number of cells in the peripheral blood Treg compartment compared with Treg infusion when rapamycin was given alone. Using single-cell transcriptomics, we found that transferred ex vivo–expanded Tregs initially exhibit a gene expression signature consistent with an activated state. Moreover, those cells with the highest levels of activation also expressed genes associated with p53-mediated apoptosis. In contrast, transferred Tregs interrogated at day +20 posttransfer demonstrated a gene signature more similar to published profiles of resting Tregs. Together, these preclinical data further support combining IL-2 and rapamycin in vivo as adjunctive therapy for ex vivo–expanded adoptively transferred Tregs and suggest that the activation status of ex vivo–expanded Tregs is critical to their persistence.

Long-Term Redistribution of Peripheral Lymphocyte Subpopulations after Switching from Calcineurin to mTOR Inhibitors in Kidney Transplant Recipients.

Classical immunosuppression based on steroids, calcineurin inhibitors, and mycophenolate results in several unwanted effects and unsatisfactory long-term outcomes in kidney transplantation (KT). New immunosuppressors search for fewer adverse events and increased graft survival but may have a distinct impact on graft function and immunological biomarkers according to their mechanism of action. This prospective study evaluates the immunological effect of tacrolimus to serine/threonine protein kinase mechanistic target of rapamycin inhibitors (mTORi) conversion in 29 KT recipients compared with 16 controls maintained on tacrolimus. We evaluated renal function, human leukocyte antigen (HLA) antibodies and peripheral blood lymphocyte subsets at inclusion and at 3, 12, and 24 months later. Twenty immunophenotyped healthy subjects served as reference. Renal function remained stable in both groups with no significant change in proteinuria. Two patients in the mTORi group developed HLA donor-specific antibodies and none in the control group (7% vs. 0%, p = 0.53). Both groups showed a progressive increase in regulatory T cells, more prominent in patients converted to mTORi within the first 18 months post-KT (p < 0.001). All patients showed a decrease in naïve B cells (p < 0.001), excepting those converted to mTORi without receiving steroids (p = 0.31). Transitional B cells significantly decreased in mTORi patients (p < 0.001), independently of concomitant steroid treatment. Finally, CD56bright and CD94/NK group 2 member A receptor positive (NKG2A+) Natural Killer (NK) cell subsets increased in mTORi- compared to tacrolimus-treated patients (both p < 0.001). Patients switched to mTORi displayed a significant redistribution of peripheral blood lymphocyte subpopulations proposed to be associated with graft outcomes. The administration of steroids modified some of these changes.

Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy

ABSTRACT Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.

Differential regulation of autophagy during metabolic stress in astrocytes and neurons

ABSTRACT Macroautophagy/autophagy is a key homeostatic process that targets cytoplasmic components to the lysosome for breakdown and recycling. Autophagy plays critical roles in glia and neurons that affect development, functionality, and viability of the nervous system. The mechanisms that regulate autophagy in glia and neurons, however, are poorly understood. Here, we define the molecular underpinnings of autophagy in primary cortical astrocytes in response to metabolic stress, and perform a comparative study in primary hippocampal neurons. We find that inducing metabolic stress by nutrient deprivation or pharmacological inhibition of MTOR (mechanistic target of rapamycin kinase) robustly activates autophagy in astrocytes. While both paradigms of metabolic stress dampen MTOR signaling, they affect the autophagy pathway differently. Further, we find that starvation-induced autophagic flux is dependent on the buffering system of the starvation solution. Lastly, starvation conditions that strongly activate autophagy in astrocytes have less pronounced effects on autophagy in neurons. Combined, our study reveals the complexity of regulating autophagy in different paradigms of metabolic stress, as well as in different cell types of the brain. Our findings raise important implications for how neurons and glia may collaborate to maintain homeostasis in the brain. Abbreviations ACSF: artificial cerebrospinal fluid; baf A1: bafilomycin A1; EBSS: earle’s balanced salt solution; GFAP: glial fibrillary acidic protein; Glc: glucose; GM: glial media; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; p-RPS6: phospho-RPS6; p-ULK1: phospho-ULK1; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; ULK1: unc-51-like kinase 1.

Cannabidiol Elevates Mechanistic Target of Rapamycin Inhibitor Levels in Patients With Tuberous Sclerosis Complex

BackgroundThe mechanistic target of rapamycin inhibitors everolimus and sirolimus have activity against multiple manifestations of tuberous sclerosis complex and are approved to treat astrocytomas, angiomyolipomas, lymphangioleiomyomatosis, and epilepsy. Cannabidiol is a novel antiepileptic medication. There is lack of information regarding drug-drug interactions between mechanistic target of rapamycin inhibitors and cannabidiol in clinical practice. MethodsWe reviewed patients with tuberous sclerosis complex who were treated with a mechanistic target of rapamycin inhibitor (everolimus, sirolimus) and cannabidiol. Clinical information, mechanistic target of rapamycin inhibitor and cannabidiol dosing, concomitant antiepileptic drugs, as well as laboratory and adverse events were reviewed before and after initiation of cannabidiol. ResultsA total of 25 patients were treated with cannabidiol and a mechanistic target of rapamycin inhibitor (18 everolimus, seven sirolimus). All mechanistic target of rapamycin inhibitor levels were drawn as troughs. Levels were significantly higher in 76% patients after cannabidiol treatment (P = 0.0003). Median change from baseline was +9.8 ng/mL for everolimus and +5.1 ng/mL for sirolimus. Adverse events occurred in 40%, with diarrhea being the most frequent adverse event occurring in three patients. No severe adverse events occurred during the treatment period. ConclusionsCannabidiol resulted in increased serum levels of everolimus and/or sirolimus. Some patients experienced doubling or tripling of their mechanistic target of rapamycin inhibitor trough following the addition of cannabidiol. In some cases, this resulted in clinical toxicity, as well as laboratory abnormalities. Awareness of this interaction can lead clinicians to evaluate serum levels and other safety laboratory studies more closely, and thereby avoid potentially significant adverse effects. In patients known to be prone to mechanistic target of rapamycin inhibitor toxicity, preemptive reduction in dose may be warranted upon initiation of cannabidiol.

Rare patients in routine care: Treatment and outcome in advanced papillary renal cell carcinoma in the prospective German clinical RCC-Registry.

Non‐clear cell renal cell carcinoma is a very rare malignancy that includes several histological subtypes. Each subtype may need to be addressed separately regarding prognosis and treatment; however, no Phase III clinical trial data exist. Thus, treatment recommendations for patients with non‐clear cell metastatic RCC (mRCC) remain unclear. We present first prospective data on choice of first‐ and second‐line treatment in routine practice and outcome of patients with papillary mRCC. From the prospective German clinical cohort study (RCC‐Registry), 99 patients with papillary mRCC treated with systemic first‐line therapy between December 2007 and May 2017 were included. Prospectively enrolled patients who had started first‐line treatment until May 15, 2016, were included into the outcome analyses (n = 82). Treatment was similar to therapies used for clear cell mRCC and consisted of tyrosine kinase inhibitors, mechanistic target of rapamycin inhibitors and recently checkpoint inhibitors. Median progression‐free survival from start of first‐line treatment was 5.4 months (95% confidence interval [CI], 4.1–9.2) and median overall survival was 12.0 months (95% CI, 8.1–20.0). At data cutoff, 73% of the patients died, 6% were still observed, 12% were lost to follow‐up, and 9% were alive at the end of the individual 3‐year observation period. Despite the lack of prospective Phase III evidence in patients with papillary mRCC, our real‐world data reveal effectiveness of systemic clear cell mRCC therapy in papillary mRCC. The prognosis seems to be inferior for papillary compared to clear cell mRCC. Further studies are needed to identify drivers of effectiveness of systemic therapy for papillary mRCC.