Target Of Rapamycin Inhibitors Research Articles

In Vitro Interactions of Antifungal Agents and Everolimus Against Aspergillus Species.

Multiple cellular activities, including protein and lipid synthesis, ribosome biogenesis, and metabolic processes, are regulated by the target of rapamycin (TOR) pathway. Recent research suggests that the TOR might play an important role in various physiological functions of pathogenic fungi, such as nutrient sensing, stress response, and cell cycle progression. Given their robust immunosuppressant and antitumor activities, TOR inhibitors are widely used in clinical settings. In the present study, a microdilution checkerboard-based approach was employed to assess the interactions between the oral mammalian target of rapamycin (mTOR) inhibitor everolimus (EVL) and antifungal agents in the treatment of Aspergillus species derived from 35 clinical isolates in vitro. The results revealed that EVL exhibited promising inhibitory synergy with itraconazole (ITC), posaconazole (POS), and amphotericin B (AMB) for 85.7%, 74.2%, and 71.4%, respectively. In contrast, EVL exhibited minimal synergistic inhibitory activity (14.3%) when applied in combination with voriconazole (VRC). Antagonistic interactions were not observed. In vivo experiments conducted in Galleria mellonella revealed that EVL in combination with antifungal agents improved the larva survival rates in the ITC, VRC, POS, and AMB groups by 18.3%, 13.3%, 26.7%, and 13.3%, respectively. These data suggest that the combination treatment with antifungal agents and antifungal agents holds promise as a means of alleviating clinical aspergillosis.

ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase.

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses such as drought, partly by modulating growth through poorly understood mechanisms. Here, we show that ABA-triggered repression of cell proliferation in the Arabidopsis thaliana root meristem relies on the swift subcellular relocalization of SNF1-RELATED KINASE 1 (SnRK1). Under favorable conditions, the SnRK1 catalytic subunit, SnRK1α1, is enriched in the nuclei of root cells, and this is accompanied by normal cell proliferation and meristem size. Depletion of two key drivers of ABA signaling, SnRK2.2 and SnRK2.3, causes constitutive cytoplasmic localization of SnRK1α1 and reduced meristem size, suggesting that, under nonstress conditions, SnRK2s promote growth by retaining SnRK1α1 in the nucleus. In response to ABA, SnRK1α1 translocates to the cytoplasm, and this is accompanied by inhibition of target of rapamycin (TOR), decreased cell proliferation, and reduced meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1α1 relocalization to the cytoplasm and ABA-elicited inhibition of TOR. Furthermore, fusing SnRK1α1 to an SV40 nuclear localization signal leads to defective ABA-dependent TOR repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK1α1 subcellular localization are crucial for inhibiting TOR and root growth in response to ABA. Rapid relocalization of central regulators such as SnRK1 may represent a general strategy of eukaryotic organisms to respond to environmental changes.

Investigating a novel role of LARP along the algal TOR pathway

Over the last few decades, global warming has become an increasingly desperate rallying cry for the need to find and optimize efficient renewable energy sources.1 Microalgae are considered apromising source of energy because they contain a high concentration of lipids, but due to inefficiencies in their lipid production, microalgae cannot produce enough biofuel to replace current fuel needs.2Chlamydomonas reinhardtiiis a unicellular green alga that is a great candidate for manipulation and an excellent model organism used to better understand algal metabolic pathways and improve biofuels. Our previous research has shown that inhibiting the target of rapamycin (TOR) kinase of C. reinhardtii with AZD8055 causes the growth of the cell cultures to stagnate, the photosynthetic efficiency to decrease, as well as a direct link between oxidative signaling and the upregulation of TAGs.3 Additionally, our work has indicated that La‐related protein 1 (LARP1) is a TOR target and involved in propagating the intracellular response following inhibition. In mammalian systems, LARP1 interacts directly with mTOR and regulates protein synthesis by binding and stabilizing mRNA before recruiting polysomes to initiate translation. LARP1 is essential in mammalian systems, and deficiencies lead to inhibited growth, defects, and increased apoptosis. However, LARP1 deficiency in C. reinhardtii does not cause the same phenotype, indicating a distinct uncharacterized role in phototrophic organisms.This study seeks to understand this role by using a LARP1 knockout mutant to compare the physiological changes following inhibition with the parent strain. Both strains were assessed for biochemical composition, cell size, and photosynthetic electron fluxes. Together, these data support LARP1 as a regulator of protein degradation along the TOR pathway. The differential delineation of metabolic processes in LARP1 knockouts provides a framework for further TOR pathway characterization in phototrophic eukaryotes.Literature References1 IPCC, 2014: Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (eds.)]. IPCC, Geneva, Switzerland, 151 pp.2 Sharma, Kalpesh K., Holger Schuhmann, and Peer M. Schenk.. High Lipid Induction in Microalgae for Biodiesel Production. 2012. Energies. 5:1532‐1553.3 Ford, M. M., Smythers, A. L., McConnell, E. W., Lowery, S. C., Kolling, D. R. J., & Hicks, L. M. (2019). Inhibition of TOR in Chlamydomonas reinhardtii leads to rapid cysteine oxidation reflecting sustained physiological changes. Cells, 8(10), 1171.

TOR inhibition primes immunity and pathogen resistance in tomato in a salicylic acid‐dependent manner

All organisms need to sense and process information about the availability of nutrients, energy status, and environmental cues to determine the best time for growth and development. The conserved target of rapamycin (TOR) protein kinase has a central role in sensing and perceiving nutritional information. TOR connects environmental information about nutrient availability to developmental and metabolic processes to maintain cellular homeostasis. Under favourable energy conditions, TOR is activated and promotes anabolic processes such as cell division, while suppressing catabolic processes. Conversely, when nutrients are limited or environmental stresses are present, TOR is inactivated, and catabolic processes are promoted. Given the central role of TOR in regulating metabolism, several previous works have examined whether TOR is wired to plant defence. To date, the mechanisms by which TOR influences plant defence are not entirely clear. Here, we addressed this question by testing the effect of inhibiting TOR on immunity and pathogen resistance in tomato. Examining which hormonal defence pathways are influenced by TOR, we show that tomato immune responses and disease resistance to several pathogens increase on TOR inhibition, and that TOR inhibition‐mediated resistance probably requires a functional salicylic acid, but not jasmonic acid, pathway. Our results support the notion that TOR is a master regulator of the development–defence switch in plants.

Cardiovascular Protection Associated With Cilostazol, Colchicine, and Target of Rapamycin Inhibitors.

An alteration in extracellular matrix (ECM) production by vascular smooth muscle cells is a crucial event in the pathogenesis of vascular diseases such as aging-related, atherosclerosis and allograft vasculopathy. The human target of rapamycin (TOR) is involved in the synthesis of ECM by vascular smooth muscle cells. TOR inhibitors reduce arterial stiffness, blood pressure, and left ventricle hypertrophy and decrease cardiovascular risk in kidney graft recipients and patients with coronary artery disease and heart allograft vasculopathy. Other drugs that modulate ECM production such as cilostazol and colchicine have also demonstrated a beneficial cardiovascular effect. Clinical studies have consistently shown that cilostazol confers cardiovascular protection in peripheral vascular disease, coronary artery disease, and cerebrovascular disease. In patients with type 2 diabetes, cilostazol prevents the progression of subclinical coronary atherosclerosis. Colchicine reduces arterial stiffness in patients with familial Mediterranean fever and patients with coronary artery disease. Pathophysiological mechanisms underlying the cardioprotective effect of these drugs may be related to interactions between the cytoskeleton, TOR signaling, and cyclic adenosine monophosphate (cAMP) synthesis that remain to be fully elucidated. Adult vascular smooth muscle cells exhibit a contractile phenotype and produce little ECM. Conditions that upregulate ECM synthesis induce a phenotypic switch toward a synthetic phenotype. TOR inhibition with rapamycin reduces ECM production by promoting the change to the contractile phenotype. Cilostazol increases the cytosolic level of cAMP, which in turn leads to a reduction in ECM synthesis. Colchicine is a microtubule-destabilizing agent that may enhance the synthesis of cAMP.

Immunosuppression, Compliance, and Tolerance After Orthotopic Liver Transplantation: State of the Art.

Orthotopic liver transplantation is the treatment of choice for several otherwise irreversible forms of acute and chronic liver diseases. Early implemented immunosuppressant regimens have had disappointing results with high rejection rates. However, new drugs have reduced the daily immunosuppression requirements, thereby improving graft and patient survival as well as kidney function. Liver rejection is a T-cell-driven immune response and is the active target of immunosuppressive agents. Immunosuppressants can be divided into pharmacological or biological drugs: the gold standard is the calcineurin inhibitors, steroids, mycophenolate mofetil, and mechanistic target of rapamycin inhibitors. Compliance with these agents is essential, although they can increase the risk of infections and neoplastic diseases. In some patients, graft tolerance can be achieved. Graft tolerance is defined as the absence of acute and chronic rejection in a graft, with normal function and histology in an immunosuppression-free, fully immunocompetent host, usually as the final result of a successful attempt at immunosuppression withdrawal. The occurrence of immunosuppressive-related complications has led to new protocols aimed at protecting renal function and preventing de novo cancer and dysmetabolic syndrome. The backbone of immunosuppression remains calcineurin inhibitors in association with other drugs, mainly over the short-term period. To avoid rejection and the side effects on renal dysfunction, de novo cancer, and cardiovascular syndrome, optimal long-term immunosuppressive therapy should be tailored in liver transplant recipients.

Effectiveness of first-line therapy in patients with advanced non-clear renal cell carcinoma (nccRCC).

304 Background: Current treatment principles for advanced nccRCC have been largely extrapolated from guidelines for clear cell RCC. Given the emerging randomized data for select nccRCC subtypes, real-world outcomes for these patients are informative particularly in the contemporary checkpoint inhibitor era. Methods: We performed an analysis using the Canadian Kidney Cancer information system (CKCis), a prospective database involving 14 academic centers, on nccRCC patients undergoing first-line systemic therapy between January 2011 – December 2019. Treatment groups were defined as receipt of: vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGF-TKI), mammalian target of rapamycin inhibitors (mTORi), and PD-1/PD-L1 immune checkpoint inhibitors (ICI, mono- or combination therapy). Primary outcome was 1-yr overall survival (OS) rate. Secondary outcomes were median time to treatment failure ((TTF, months), defined as treatment discontinuation, change or death) and objective response rate (ORR, %). Results: We identified 265 nccRCC patients: 204 (77.0%) received VEGF-TKI, 19 (7.2%) received mTORi and 42 (15.8%) received ICI-based first-line therapy (Table). Overall, median age was 64 years, 75% were male, 84% were classified as IMDC intermediate/poor risk, and 16% underwent prior nephrectomy. Twenty-three percent of patients were enrolled in clinical trials. Patients received primarily sunitinib (81%) or pazopanib (15%) in the VEGF-TKI group (other: 4%), while mTORi-treated patients received temsirolimus (74%) or everolimus (26%). For the ICI-based treatment group, most patients received combination therapy as ipilimumab-nivolumab (71%) or pembrolizumab-axitinib (26%), with 3% receiving ICI monotherapy. 1-yr OS was 65.2% for VEGF-TKI, 57.9% for mTORi and 69.0% for ICI-treated patients. Median TTF was 3.3 for VEGF-TKI, 3.5 for mTORi and 7.1 mos for ICI-treated patients. ORR was 17%, 5%, and 37% respectively for the VEGF-TKI, mTORi and ICI-treated groups. Conclusions: We describe the effectiveness of first-line therapy for patients with nccRCC from a national database. This real-world data suggests an association between first-line ICI-based therapies and improved outcomes, albeit with cabozantinib not available for the indication during this time. Our data supports consensus recommendations for preferred use of ICI-based or VEGF-TKI over mTORi as first-line therapy in nccRCC.[Table: see text]

Target of rapamycin (TOR) regulates the response to low nitrogen stress via autophagy and hormone pathways in Malus hupehensis.

Target of rapamycin (TOR) is a highly conserved master regulator in eukaryotes; it regulates cell proliferation and growth by integrating different signals. However, little is known about the function of TOR in perennial woody plants. Different concentrations of AZD8055 (an inhibitor of TOR) were used in this study to investigate the role of TOR in the response to low nitrogen (N) stress in the wild apple species Malus hupehensis. Low N stress inhibited the growth of M. hupehensis plants, and 1 μM AZD alleviated this effect. Plants supplied with 1 μM AZD had higher photosynthetic capacity, which promoted the accumulation of biomass, as well as higher contents of N and anthocyanins and lower content of starch. Exogenous application of 1 μM AZD also promoted the development of the root system. Plants supplied with at least 5 μM AZD displayed early leaf senescence. RNA-seq analysis indicated that TOR altered the expression of genes related to the low N stress response, such as genes involved in photosystem, starch metabolism, autophagy, and hormone metabolism. Further analysis revealed altered autophagy in plants supplied with AZD under low N stress; the metabolism of plant hormones also changed following AZD supplementation. In sum, our findings revealed that appropriate inhibition of TOR activated autophagy and jasmonic acid signaling in M. hupehensis, which allowed plants to cope with low N stress. Severe TOR inhibition resulted in the excessive accumulation of salicylic acid, which probably led to programmed cell death in M. hupehensis.

Autophagy inducers lead to transient accumulation of autophagosomes in Arabidopsis roots.

This study reveals that plant roots show a rapid termination of autophagy induction, offering a plant model for studying how excessive autophagy is deterred. In eukaryotes, autophagy is an intracellular mechanism that is important for recycling nutrients by degrading various macromolecules and organelles in vacuoles and lysosomes. Autophagy is induced when the nutrient supply to plant cells is limited. The protein kinase target of rapamycin (TOR) complex negatively regulates autophagy when nutrients are present in adequate amounts. The TOR inhibitor AZD8055 is an autophagy inducer that is useful for studying starvation-induced autophagy in plant cells. The mechanism by which AZD8055 increases the autophagic flux in plant cells has not been studied in detail. Here, we show that AZD8055-induced autophagy requires phosphatidylinositol 3-kinase activity and canonical AUTOPHAGY-RELATED (ATG) genes in Arabidopsis thaliana. Autophagic flux rapidly increased in seedlings treated with AZD8055. Unexpectedly, autophagy induction was transient in root cells and terminated earlier than in cotyledon cells. Transient induction is partly caused by a temporary effect of AZD8055 on phagophore initiation. These findings indicate a TOR-independent mechanism for terminating autophagy induction, thereby paving the way for elucidating how excess autophagy is prevented in plant roots.

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

Adverse events of targeted therapies approved for women's cancers.

Breast cancer and gynecologic cancers affect >3 million women worldwide each year. With advances in precision medicine, a growing number of targeted therapies have been approved recently, and new therapeutic classes have emerged, including cell cycle inhibitors for hormone receptor positive breast cancer, antibody drug conjugate for human epidermal growth factor receptor 2 positive and triple negative breast cancer, and poly-ADP-ribose polymerase inhibitors for ovarian cancer. This article focuses first on the challenges for health care systems to address the specificities of each emerging targeted therapy and new issues raised by oral antitumor treatments, including individualization of prescriptions, drug–drug interaction assessment, pharmaceutical counseling, patient education, and outpatient management. Then, we provide an overview of the main adverse effects of targeted therapies approved for breast and gynecologic cancers, such as hematologic toxicity of cyclin-dependent kinase 4/6 inhibitors and poly-ADP-ribose polymerase inhibitors, metabolic disorders of phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin inhibitors, and cardiovascular toxicity of agents targeting human epidermal growth factor receptor 2.

Consequences of Mammalian Target of Rapamycin Inhibition on Adeno-Associated Virus Hepatic Transduction Efficacy.

The efficiency of recombinant adeno-associated virus (AAV) vectors transducing host cells is very low, limiting their therapeutic potential in patients. There are several cellular pathways interacting and interfering with the journey of the AAV from the cell surface to the nucleus, opening the possibility to enhance AAV transduction by modifying these interactions. In this study, we explored the results of AAV hepatic transduction when different mammalian target of rapamycin (mTOR) inhibitors, rapamycin, MLN0128, RapaLink-1, were used in preconditioned juvenile and adult mice. We confirmed rapamycin as an AAV hepatic transduction enhancer in juvenile and adult mice; however, RapaLink-1, a stronger mTOR inhibitor and a clear hepatic autophagy inducer, had no positive effect. Moreover, MLN0128 reduced AAV hepatic transduction. Therefore, our results show a complex interaction between the mTOR pathway and AAV-mediated hepatic transduction and indicate that mTOR inhibition is not a straightforward strategy for improving AAV transduction. More studies are necessary to elucidate the molecular mechanisms involved in the positive and negative effects of mTOR inhibitors on AAV transduction efficiency.

Huge Renal Angiomyolipoma in a Child with Tuberous Sclerosis Complex: A Diagnostic and Therapeutic Dilemma

Tuberous Sclerosis Complex (TSC) is a rare neuro-cutaneous disorder that is associated with the development of benign hamartomas including renal angiomyolipoma (RAML). TSC associated RAML are usually asymptomatic, but it carries a life-threatening bleeding risk. We are sharing a case of a 5-year-old girl who was diagnosed to have TSC with associated subependymal giant cell astrocytoma, cardiac rhabdomyoma and autism. She presented with a history of worsening abdominal distension over 3 weeks duration and clinically noted to be pale with a ballotable left flank mass. Ultrasound and CT scan found to have multiple RAML in both kidneys with a huge mass on the left side. The mass represented a huge RAML (8cm) with aneurysmal formation with suspicion of intratumoral bleeding. The option of conservative management with mammalian target of rapamycin inhibitor followed with partial nephrectomy has been questioned with its life-threatening risk of bleeding and inability to do biopsy to rule out the possibility of renal cell carcinoma. Decision for nephrectomy was then made clearer following a MAG-3 scan which revealed only 11% differential function of the left kidney. She underwent a total left nephrectomy uneventfully and intraoperatively noted to have an enlarging lesion as compared to the previous imaging; 15cm in largest diameter. Histopathological finding was consistent with multifocal angiomyolipoma with intratumoral haematoma. Decision for nephrectomy in TSC-associated RAML need to be justified carefully in view of its risk of losing the contralateral kidney following the disease progression which may end up with life-long renal replacement therapy.International Journal of Human and Health Sciences Supplementary Issue-2: 2021 Page: S26

Pharmacotherapy for Seizures in Tuberous Sclerosis Complex.

Epilepsy is one of the main symptoms affecting the lives of individuals with tuberous sclerosiscomplex (TSC), causing a high rate of morbidity. Individuals with TSC can present with various types of seizures, epilepsies, and epilepsy syndromes that can coexist or appear in relation to age. Focal epilepsy is the most frequent epilepsy typewith two developmental and epileptic encephalopathies: infantile spasms syndrome and Lennox-Gastaut syndrome. Active screening and early management of epilepsy is recommended in individuals with TSC to limit its consequences and its impact on quality of life,cognitive outcome and the economic burden of the disease. The progress in the knowledge of the mechanisms underlying epilepsy in TSC has paved the way for new concepts in the management of epilepsy related to TSC. In addition, we are moving from traditional "reactive" and therapeutic choices with current antiseizure medications used after the onset of seizures, to a proactive approach, aimed at predicting and preventing epileptogenesis and the onset of epilepsy with vigabatrin, and to personalized treatments with mechanistic therapies, namely mechanistic/mammalian target of rapamycin inhibitors. Indeed, epilepsy linked to TSC is one of the only epilepsies for which a predictive and preventive approach can delay seizure onset and improve seizure response. However, the efficacy of such interventions on long-term cognitive and psychiatric outcomes is still under investigation.

Transition to Targeted Therapies Improved the Prognosis and Increased the Utilization of Medical Treatments among Patients with Synchronous Metastatic Renal Cell Cancer

Since the introduction of targeted therapies (TTs) for metastatic renal cell cancer (mRCC) in 2005, a limited amount of epidemiological data on efficacy of modern drug therapies for synchronous mRCC has been published. We present a comprehensive nationwide cohort including all cases of primarily metastasized renal cell cancer among adults diagnosed between 2005 and 2010, based on data from the Finnish Cancer Registry and patient records from treating hospitals. Applied treatment protocols and survival outcomes were analyzed. A total of 977 patients were included in the analysis; 499 patients were diagnosed between 2005 and 2007 and 478 patients were diagnosed between 2008 and 2010. The median overall survival (OS) was 8.80 months (95% confidence interval (CI): 7.60–10.02). The median OS of the patients diagnosed at the latter era was significantly better (11.1; 95% CI: 8.8–13.4 vs. 7.0; 95% CI: 5.7–8.3 months, p ≤ 0.001). A total number of 524 (53.8%) patients received drug therapy. Altogether, TTs including tyrosine kinase inhibitors, mammalian target of rapamycin inhibitors (mTORi), and vascular endothelial growth factor inhibitor covered 331 (63.2%) of first-line treatments, whereas interferon and its combinations with chemotherapy were used for 186 (35.5%) patients. The median OS rates for TT and interferon as first-line therapy groups were 19.9 (16.9–22.8) and 14.9 (12.3–17.4) months, respectively. The OS for patients who did not receive drug therapy after cytoreductive nephrectomy was dismal. We found that the OS estimate of mRCC patients in Finland has improved since the introduction of tyrosine kinase inhibitors. However, the prognosis remains poor for frail, elderly patients with an impaired performance status.

MO088CLINICOPATHOLOGICAL FEATURES OF THROMBOTIC MICROANGIOPATHY

Abstract Background and Aims TMA is a rare and severe endothelial lesion with thrombus formation and vascular occlusion with organ damage. It can be a complication of several clinical conditions. The aim of this study was to characterize clinical and pathological findings of patients with confirmed kidney TMA. Method We performed a retrospective observational descriptive study that analyzed all patients with TMA diagnosed by kidney biopsy, in two hospital centers, from January 2015 until December 2020. Clinical, laboratorial, pathologic characteristics were analyzed as also therapeutic performed and kidney and patient outcomes. Results Our sample had 14 patients, the majority female (n=11, 78%) with a mean age of 45±10 years-old and 12 (80%) were Caucasian. Six (40%) had past medical history of HTA, 2 (13%) had Multiple Myeloma, 4 (26%) transplant (3 kidney, 1 lung) and none had positive viral serology’s. Prodromal symptoms included diarrhea in 2 (13%) patients, neurological in 3 (20%) and previous infection in 3 (20%). Mean laboratorial values were: Hemoglobin 7.8 g/dL, platelets 161.000/uL; LDH 586 UL; bilirubin 1.38 mg/dL, haptoglobin 1,55 g/L. Only 5 had schistocytes. ADAMTS13 activity was >10% in all performed and in all, Coombs test were negative. C3 was consumed in 3 (20%) patients and C4 in 2 (13%) but complement phenotypic study was normal in all performed patients. Auto-immune study revealed: 1 positive ANA, dsDNA negative in all but one with positive c1q antibody. One had positive antiphospholipid syndrome (AFS) antibodies and one scleroderma antibodies. Main kidney presentation was acute kidney injury in 8 (53%) patients, with mean eGFR 14ml/min/1,73m2. Mean proteinuria was 2,5 g/24h and half (50%) had hematuria. Kidney biopsies had a mean of 11,85 glomerulus, of which 14% sclerotic, mean interstitial fibrosis and tubular atrophy score 1,7. Acute tubular necrosis was present in 7 (46%) and inflammatory infiltrate in all, endotheliosis in 13 (86%), mesangiolysis in 3 (20%), arteriolar thrombus in 10 (66%), glomerular thrombus in 6 (40%), ischemic glomerulus in 12 (80%) and double-contour appearance in 7 (46%). TMA etiologies were the following: 2 malignant hypertension, 2 proteasome inhibitor (PI), 1 scleroderma, 1 lupus, 1 AFS, 2 calcineurin and mammalian target of rapamycin inhibitors, 2 antibody mediated rejection (AMR), 1 methylmalonic acidemia and 2 undetermined. Treatment performed encompassed: hypertension management, withdrawal od PI, changing immunosuppression, AMR treatment, cyanocobalamin, lupus induction treatment and eculizumab. Kidney recovery was observed in 8 patients (53%). Patient survival at 6 months was 73%. Conclusion These study results highlight the vast context of conditions that TMA can appear. Most of them had only with mild or even absent clinical and laboratory features. Therefore, a high index of suspicion is required to diagnose TMA, allowing targeted treatment to preserve of kidney function.

MO573CNI AND MTORI-BASED REGIMENS ON FIVE-YEAR COURSE OF BONE MINERAL DENSITY SCORES AFTER RENAL TRANSPLANTATION

Abstract Background and Aims Since glucocorticoids are used in low maintenance doses today, the relationship between calcineurin inhibitors (CNI) and osteoporosis after solid organ transplantation has gained clinical significance. In contrast, (mammalian target of rapamycin inhibitors) mTORi agents are shown to have an antiresorptive effect on bone by reducing osteoclastic activity in vitro and in vivo. We investigated the bone mineral density (BMD) changes on an extremely selective group of incident renal transplant recipients under CNI or mTORi-based maintenance treatment regimens during the first five-year posttransplant course. Method This study consists of renal allograft recipients with a dialysis history of less than one year. Patients older than 50 years old, using CNI - mTORi combinations, patients with a history of a prior renal transplant, acute rejection, CNI – mTORi switch after posttransplantation first three months, bisphosphonate treatment, parathyroidectomy, diuretic use, and malignancy excluded to eliminate all confounding factors for bone mineral loss. First and fifth-year BMD scores and simultaneous laboratory parameters were evaluated. Results CNI (n=21) and mTORi (n=17) groups had similar demographics, dialysis vintages, cumulative steroid doses, first and fifth-year parathormone, calcium, phosphate, magnesium, alkaline phosphatase, and 25-OH-vitamin D levels. The femur neck scores of the CNI group decreased from -0.82(±0.96) to -1.52(±0.92) (p=0.020). We observed a significant decrease in the CNI group compared to the mTORi group [-0.70(±0.68) and 0.30(±0.36), respectively; p<0.01] when BMD score changes evaluated among years. The mean femur neck score of the mTORi group insignificantly increased from -1.13(±0.65) to -0.82(±0.56) at the fifth-year DXA scan (p=0.230). Although statistically insignificant, similar trends were also observed in L1-4 scores. Conclusion Our study suggests that CNI-based treatment is associated with decreased femur neck and L1-4 BMD scores, and mTORi-based treatment tends to be beneficial presumably due to their antiresorptive effects in the posttransplant five-year follow-up. Our findings may shed light on long-term maintenance therapy management in renal transplant patients with bone disease if supported by future studies.

TOR Inhibitors Synergistically Suppress the Growth and Development of Phytophthora infestans, a Highly Destructive Pathogenic Oomycete.

Phytophthora infestans, one of most famous pathogenic oomycetes, triggered the Great Irish Famine from 1845 to 1852. The target of rapamycin (TOR) is well known as a key gene in eukaryotes that controls cell growth, survival and development. However, it is unclear about its function in controlling the mycelial growth, sporulation capacity, spore germination and virulence of Phytophthora infestans. In this study, key components of the TOR signaling pathway are analyzed in detail. TOR inhibitors, including rapamycin (RAP), AZD8055 (AZD), KU-0063794 (KU), and Torin1, inhibit the mycelial growth, sporulation capacity, spore germination, and virulence of Phytophthora infestans with AZD showing the best inhibitory effects on Phytophthora infestans. Importantly, compared with a combination of RAP + KU or RAP + Torin1, the co-application of RAP and AZD show the best synergistic inhibitory effects on P. infestans, resulting in the reduced dosage and increased efficacy of drugs. Transcriptome analysis supports the synergistic effects of the combination of RAP and AZD on gene expression, functions and pathways related to the TOR signaling pathway. Thus, TOR is an important target for controlling Phytophthora infestans, and synergism based on the application of TOR inhibitors exhibit the potential for controlling the growth of Phytophthora infestans.

Lupeol induces autophagy and apoptosis with reduced cancer stem-like properties in retinoblastoma via phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin inhibition.

To evaluate the anticancer effects of lupeol in retinoblastoma cells. WERI-Rb-1 and Y-79 cell lines were used to evaluate the anticancer effect of lupeol. After lupeol treatment, the viability, proliferation, apoptosis, cancer stem-like properties, autophagy and in vivo tumour xenograft formation were detected. In this study, lupeol decreased cell viability in both WERI-Rb-1 and Y-79 cell lines. Lupeol could also inhibit proliferation and induce apoptosis of RB cells, with increased Bax level and decreased Ki67, survivin and Bcl-2 levels. Furthermore, lupeol could suppress the spheroid formation and stem-like properties of RB cells. Moreover, LC3 II/LC3 I ratio and the levels of Beclin1 and ATG7 were increased after lupeol treatment, indicating that lupeol could induce autophagy in RB cells. Next, the inhibitory effect of lupeol on the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway was observed. In tumour-bearing mice, lupeol suppressed tumour growth, and this might relate to its role in cell apoptosis, autophagy and stem-like properties. Lupeol suppressed proliferation and cancer stem-like properties, and promoted autophagy and apoptosis of RB cells by restraining the PI3K/AKT/mTOR pathway.

Experience using mTOR inhibitors for subependymal giant cell astrocytoma in tuberous sclerosis complex at a single facility

BackgroundSubependymal giant cell astrocytoma (SEGA) is occasionally seen in tuberous sclerosis complex (TSC). Two main options are currently available for treating SEGA: surgical resection or pharmacotherapy using mammalian target of rapamycin inhibitors (mTORi). We hypothesized that opportunities for surgical resection of SEGA would have reduced with the advent of mTORi.MethodsWe retrospectively reviewed the charts of patients treated between August 1979 and July 2020, divided into a pre-mTORi era group (Pre-group) of patients treated before November 2012, and a post-mTORi era group (Post-group) comprising patients treated from November 2012, when mTORi became available in Japan for SEGA. We compared groups in terms of treatment with surgery or mTORi. We also reviewed SEGA size, rate of acute hydrocephalus, recurrence of SEGA, malignant transformation and adverse effects of mTORi.ResultsIn total, 120 patients with TSC visited our facility, including 24 patients with SEGA. Surgical resection was significantly more frequent in the Pre-group (6 of 7 patients, 86 %) than in the Post-group (2 of 17 patients, 12 %; p = 0.001). Acute hydrocephalus was seen in 1 patient (4 %), and no patients showed malignant transformation of SEGA. The group treated using mTORi showed significantly smaller SEGA compared with the group treated under a wait-and-see policy (p = 0.012). Adverse effects of pharmacotherapy were identified in seven (64 %; 6 oral ulcers, 1 irregular menstruation) of the 11 patients receiving mTORi.ConclusionsThe Post-group underwent surgery significantly less often than the Pre-group. Since the treatment option to use mTORi in the treatment of SEGA in TSC became available, opportunities for surgical resection have decreased in our facility.