mTOR In Patients Research Articles

The relation of mTOR with diabetic complications and insulin resistance in patients with type 2 diabetes mellitus

BackgroundDysregulation of the mechanistic target of rapamycin (mTOR) has been related to several metabolic conditions, notably obesity and type 2 diabetes (T2DM). This study aimed to evaluate the role of mTOR in patients with T2DM, and its relationship with insulin resistance and microvascular complications.MethodsThis case-control study was conducted on 90 subjects attending the Outpatient Internal Medicine Clinic in Damanhur Teaching Hospital. Subjects were divided into 3 groups, Group I: 20 healthy controls, Group II: 20 subjects with T2DM without complications, and Group III: 50 subjects with T2DM with microvascular complications. An Enzyme-linked immunosorbent assay was used to measure serum mTOR levels. T2DM and diabetic complications were defined according to the diagnostic criteria of the American Diabetes Association.ResultsThe results revealed significant positive correlations to HbA1c (r = 0.530, P < 0.001), fasting glucose (r = 0.508, P < 0.001), and HOMA- IR (r = 0.559, P < 0.001), and a significant negative correlation to eGFR (r=-0.370, P = 0.002). Multivariate analysis revealed an independent association of mTOR and HbA1c values with the presence of microvascular complications. The prediction of microvascular complications was present at a cutoff value of 8 ng/ml mTOR with a sensitivity of 100% and specificity of 95% with an AUC of 0.983 and a p-value < 0.001.ConclusionmTOR is a prognostic marker of diabetic microvascular and is associated with insulin resistance in patients with T2DM.Trial RegistrationThe study was conducted following the Declaration of Helsinki, and approved by the Ethics Committee of Alexandria University (0201127, 19/7/2018).

Pro-oxidation status of rotator cuff tissue and expression of Beclin-1 and mTOR in patients with rotator cuff injury

To explore pro-oxidative state of rotator cuff tissue and expression levels of Beclin-1 and mam-malian target of rapamycin(mTOR) in patients with acute and chronic rotator cuff injury, and then analyzed relationship between rotator cuff injury and oxidative stress and autophagy. Forty patients with rotator cuff injury were seleceted from July 2019 to December 2020, and divided into male chronic injury group, male acute injury group, female chronic injury group, and female acute injury group, 10 patients in each group. All patients were performed rotator cuff repair under arthroscopy. The sample of tendon at the rotator cuff injury site of the patient was taken during operation, and total reactive oxygen species (ROS) and superoxide dismutase(SOD) were detected by detection kit;expression of Beclin-1 and mTOR mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and Western-blot was applied to detect protein expression of Beclin-1 and p-mTOR/mTOR. There were no significant difference in expression of ROS, SOD, Beclin-1mRNA and mTOR mRNA between male and female chronic injury groups, and between male and female acute injury groups (P>0.05); ROS, SOD and Beclin-1mRNA in male chronic injury group were higher than those in male chronic injury group, while mTOR mRNAand protein decreased (P<0.05);ROS, SOD and Beclin-1 mRNA in female chronic injury group were up-regulated compared with female acute injury group, while mTOR mRNA was down-regulated (P<0.05). Chronic rotator cuff injury is more likely to stimulate the pro-oxidation state of rotator cuff tissue than acute rotator cuff injury, which could up-regulating expression of autophagy factor Beclin-1 and down-regulating expression of mTOR. Therefore, patients with chronic rotator cuff injury may have higher levels of oxidative stress and autophagy.

New prospectives on treatment opportunities in RASopathies.

The RASopathies are a group of clinically defined developmental syndromes caused by germline variants of the RAS/mitogen-activated protein (MAPK) cascade. The prototypic RASopathy is Noonan syndrome, which has phenotypic overlap with related disorders such as cardiofaciocutaneous syndrome, Costello syndrome, Noonan syndrome with multiple lentigines, and others. In this state-of-the-art review, we summarize current knowledge on unmet therapeutic needs in these diseases and novel treatment approaches informed by insights from RAS/MAPK-associated cancer therapies, in particular through inhibition of MEK1/2 and mTOR in patients with severe disease manifestations. We explore the possibilities of integrating a larger arsenal of molecules currently under development into future care plans. Lastly, we describe both medical and ethical challenges and opportunities for future clinical trials in the field.

Functional and structural analyses of novel Smith-Kingsmore Syndrome-Associated MTOR variants reveal potential new mechanisms and predictors of pathogenicity.

Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder characterized by macrocephaly/megalencephaly, developmental delay, intellectual disability, hypotonia, and seizures. It is caused by dominant missense mutations in MTOR. The pathogenicity of novel variants in MTOR in patients with neurodevelopmental disorders can be difficult to determine and the mechanism by which variants cause disease remains poorly understood. We report 7 patients with SKS with 4 novel MTOR variants and describe their phenotypes. We perform in vitro functional analyses to confirm MTOR activation and interrogate disease mechanisms. We complete structural analyses to understand the 3D properties of pathogenic variants. We examine the accuracy of relative accessible surface area, a quantitative measure of amino acid side-chain accessibility, as a predictor of MTOR variant pathogenicity. We describe novel clinical features of patients with SKS. We confirm MTOR Complex 1 activation and identify MTOR Complex 2 activation as a new potential mechanism of disease in SKS. We find that pathogenic MTOR variants disproportionately cluster in hotspots in the core of the protein, where they disrupt alpha helix packing due to the insertion of bulky amino acid side chains. We find that relative accessible surface area is significantly lower for SKS-associated variants compared to benign variants. We expand the phenotype of SKS and demonstrate that additional pathways of activation may contribute to disease. Incorporating 3D properties of MTOR variants may help in pathogenicity classification. We hope these findings may contribute to improving the precision of care and therapeutic development for individuals with SKS.

Altered serum levels of autophagy proteins Beclin-1 and mTOR in patients with exudative age-related macular degeneration.

Autophagy is a key process in the maintenance of cellular survival and homeostasis. Inhibition of autophagy results in degenerative changes resembling ageing. We wondered if autophagy can contribute to the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate the serum concentrations of two key autophagy regulators, Beclin-1 and mechanistic target of rapamycin (mTOR), in patients with exudative AMD. This retrospective case-control study included 38 patients with exudative AMD and 36 sex- and age-matched controls selected among senile cataract patients. Circulating Beclin-1 and mTOR were assessed using an enzyme-linked immunosorbent assay. The proteins levels were correlated with age, sex, duration of ocular symptoms, as well as angiographic and optical coherence tomography findings. Serum Beclin-1 levels were much lower in patients with AMD than in controls (median, 0.100 ng/ml versus 1.123 ng/ml; p = 0.0033), while mTOR levels did not differ (median, 4.377 ng/ml versus 3.608 ng/ml; p = 0.4522). Participants of the study older than 70 years had lower Beclin-1 levels than younger ones (p = 0.0444). However, this difference was the most evident in patients with AMD (p = 0.0024). Serum mTOR levels increased with age. In patients with AMD, lower mTOR levels were associated with drusen, while higher levels were observed in those with a fibrous scar in the contralateral eye (p = 0.0212). Our findings suggest that circulating Beclin-1 decreases with age and that is downregulated in patients with AMD.

A systematic review and meta-analysis protocol of clinical characteristics and prognostic significance of mammalian target of rapamycin for gastric cancer patients.

Background:The high morbidity and mortality of Gastric cancer (GC) is seriously endangered human health. Owing to the low rate of early diagnosis and human body can resistant to the anti-tumor drugs, so an early diagnostic biology marker is essential. However, recently studies indicated that Mammalian target of rapamycin (mTOR) is usually frequently deregulated in many cancers, especially in GC. And the efficacy of mTOR inhibitor was promising in a phase II clinical trial which could inhibited the proliferation of GC cells and delayed tumor progression. Therefore, mTOR were identified as a potential prognosis biomarker for GC, and its inhibitor will be promising in anti-GC therapy. The main aim of this systematic review and meta-analysis is to investigate the relationships between the expression level and prognostic value of mTOR in patients with GC.Methods:Four electronic databases were systematically searched as follow: the PubMed, EMbase, Web of Science databases, and the Cochrane Library. All the data will be extracted by independent researchers from the eligible studies with the inclusion and exclusion criteria. And the data will be analyzed through STATA 12.0 software.Results and conclusion:This meta-analysis indicated that overexpressed mTOR was significantly in predicting a poorer prognosis for GC patients. The expression level of mTOR should be considered as a potential independent prognostic predictor for GC patients.Protocol registration number:CRD42020159690.

Constitutive activation of mTORC1 signaling induced by biallelic loss-of-function mutations in SZT2 underlies a discernible neurodevelopmental disease.

There have been increasing number of reports of SZT2-related neurological diseases, the main symptoms of which are epilepsy, developmental delay, macrocephaly and a dysmorphic corpus callosum. SZT2 functions as a regulator of mechanistic target of rapamycin complex 1 (mTORC1) signaling in cultured human cell lines and mouse tissues. However, it remains to be determined whether mutations in SZT2 in human patients alter mTORC1 signaling. In this study, we aimed to investigate the functional consequence of biallelic SZT2 variants in Epstein-Barr virus-induced lymphoblastoid cell lines (LCLs) established from two patients with a typical SZT2-related neurodevelopmental disease. Increased phosphorylation of S6 kinase and S6 was identified in patient-derived cell lines under amino acid-starved condition, suggestive of constitutive hyperactivation of mTORC1 signaling. This result was validated by constitutive lysosomal localization of mTOR in patients’ LCLs. Furthermore, patients’ LCLs display an excessive response to slight amino acid stimulation. Our data suggest the loss-of-function nature of SZT2 mutations in the patients, and consequent hyperactivation of mTORC1 signaling in response to both amino acid starvation and stimulation in their LCLs. By these functional analyses, the pathogenicity of newly identified SZT2 variants can be determined, allowing for more detailed characterization of genotype-phenotype correlations.

RNA-seq reveals aurora kinase-driven mTOR pathway activation in patients with sarcomatoid metastatic renal cell carcinoma.

Sarcomatoid metastatic renal cell carcinoma (mRCC) is associated with a poor prognosis, and the biology of the disease has been inadequately characterized. RNA sequencing (RNA-seq) was performed on adjacent benign, clear cell, and sarcomatoid components from clinical specimens with sarcomatoid mRCC. M phase and cell-cycle pathways were enriched in sarcomatoid versus adjacent clear cell components, suggesting greater cell proliferation. The expression of aurora kinase A (AURKA) was increased as part of these pathways, and its increased expression was validated by quantitative PCR (qPCR). Immunohistochemical (IHC) analysis revealed that AURKA levels were increased in sarcomatoid tissue compared with their benign or clear cell parts. The increase in AURKA correlated with increased mTOR pathway activity, as evidenced by increased expression of phosphorylated mTOR (S2448) and ribosomal protein S6K (T389). When AURKA was stably expressed in a RCC cell line (Renca), it resulted in increased expression and activity of mTOR, suggesting that overexpression of AURKA can activate the mTOR pathway. These results warrant the analysis of a larger clinical cohort and suggest that targeting AURKA and/or mTOR in patients with sarcomatoid mRCC should be explored. Comparative RNA-seq of adjacent sarcomatoid and clear cell histology of RCC indicates a proliferative phenotype and increased AURKA-dependent activation of mTOR signaling in sarcomatoid RCC, which could be targeted by available agents.

Highlights of This Issue

An analysis by Sowalsky and colleagues of metastatic castration-resistant prostate cancer (mCRPC) specimens using paired-end RNA-seq identified a series of likely driver mutations, genomic fusions, and lncRNAs. The authors also found an unexpectedly high level of sequence reads mapping to introns, the result of inefficient splicing at canonical splice junctions, which were more abundant in the mCRPC samples than in normal prostate or primary prostate cancer. Inefficient splicing in advanced prostate cancer may provide a selective advantage through effects on microRNA networks, but it may also render tumors vulnerable to agents that target steps in RNA splicing.Lukin and colleagues show that in response to transient treatment with DNA damaging agents, wild-type p53 cells reversibly arrest and repair the damage. In contrast, p53-null cells undergo cell death, suggesting that acute treatments can exploit differences between wild-type and p53-null cells. Although p53 status has been implicated as a clinical predictor of chemotherapeutic efficacy, studies have not always aligned. These data support the use of transient treatment paradigms to exploit the reversibility of the p53-dependent checkpoint and justify the use of p53 status in clinical decision making. The molecular basis for this appears to involve the temporal regulation of p53-dependent gene expression.ATM loss-of-function has been described in hematologic malignancies such as mantle cell lymphoma (MCL). Given the importance of ATM to genome integrity, small-molecule ATR inhibitors may have therapeutic utility in MCL. Here, a focused siRNA screen by Menezes and colleagues not only confirmed ATM but also identified additional replication checkpoint proteins that are synthetically lethal with ATR inhibition. In both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function compared with wild-type MCL cells. These data indicate that ATR inhibitors, as a monotherapy, have therapeutic potential and that ATR utilizes a complex set of proteins to maintain genomic stability that could be further exploited.Sarcomatoid tumors are a rare and aggressive histologic subset of renal cell carcinoma (RCC), and one that is generally refractory to current treatments. Little is known about the molecular biology of this disease; therefore, Pal and colleagues performed RNA-seq on metastatic RCC (mRCC) patient samples. Importantly, follow-up analysis of a large cohort revealed changes in cell proliferation pathways, including altered expression of Aurora kinase (AURKA) and mTOR signaling. These results not only warrant further preclinical workup and analysis of a larger clinical cohort but also suggest that combined or individual therapeutic targeting of AURKA and mTOR in patients with sarcomatoid mRCC should be explored.

Mechanisms of activity of the TORC1 inhibitor everolimus in Waldenstrom macroglobulinemia.

The TORC1 inhibitor everolimus has previously shown significant activity as a single agent in hematologic malignancies, with reported responses of 30% to 70% in Waldenstrom macroglobulinemia. However, the specific mechanisms by which this class of mTOR inhibitors exerts anti-Waldenstrom macroglobulinemia activity have not been fully investigated. We therefore sought to dissect the mechanisms of everolimus-dependent modulation of Waldenstrom macroglobulinemia cell survival. We confirmed that everolimus targets mTOR in patients treated with everolimus and responding to therapy. We evaluated the effect of everolimus on proliferation and survival of primary Waldenstrom macroglobulinemia cells, as well as of other IgM-secreting lymphoma cell lines. Everolimus-dependent mechanisms of induced apoptosis and its effect on Waldenstrom macroglobulinemia cells in the context of bone marrow microenvironment have been also evaluated. miRNA-155 loss-of-function studies were conducted. Moreover, the combinatory effect of bortezomib and rituximab has been tested. We showed that everolimus targeted mTOR downstream signaling pathways, ex vivo, in patients responding to everolimus treatment. Everolimus induced toxicity in primary Waldenstrom macroglobulinemia cells, as well as in other IgM-secreting lymphoma cells, supported by cell-cycle arrest and caspase-dependent and -independent induction of apoptosis. Importantly, everolimus targeted Waldenstrom macroglobulinemia cells even in the context of bone marrow milieu, where it affected migration, adhesion, and angiogenesis. Everolimus-dependent anti-Waldenstrom macroglobulinemia activity was partially driven by miRNA-155. Moreover, everolimus synergized with bortezomib and rituximab in targeting Waldenstrom macroglobulinemia cells, as shown by synergistic inhibition of p65/ and p50/NF-κB activities. These findings provide a better understanding of the mechanisms that are responsible for everolimus-induced anti-Waldenstrom macroglobulinemia activity.

Abstract A167: First-in-patient study of PF-05212384, a small molecule intravenous dual inhibitor of PI3K and mTOR in patients with advanced cancer: Update on safety, efficacy, and pharmacology.

Abstract Background: Activation of the PI3K pathway, by PIK3CA mutation or amplification or PTEN deletion, has been implicated in a wide variety of human cancers. PF-05212384 is a dual-specificity inhibitor of PI3K and mTOR that is formulated for intravenous (IV) administration. PF-05213384 is the first IV PI3K/mTOR inhibitor to be tested in a clinical trial. Methods: In this ongoing phase 1 trial, PF-05212384 was administered once weekly, using a modified continual reassessment method (CRM) for estimation of the maximum tolerated dose (MTD). Assessments included safety (NCI CTC AE v4.0), pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity (RECIST v1.1). PD assessments included blood glucose and immunohistochemistry analysis of phosphoprotein biomarkers in tumor and surrogate tissue (hair follicles). The population was actively enriched for patients with dysregulation in the PI3K pathway. Results: As of 15 March 2011, 47 patients have been enrolled at doses ranging from 10 mg to 319 mg. Median age was 58. Represented tumor types included CRC (8), NSCLC (4), ovarian/endometrial/cervical (8), salivary (3) and gastric/esophageal (3). Analysis of archived tumor biopsies revealed PI3K pathway alterations in 12/26 evaluable patients; alterations included PTEN loss, PIK3CA mutation or amplification. PF-05212384 was well tolerated with the most common AEs being mucositis (44%), nausea (42%), fatigue (28%), rash (28%), and hyperglycemia (26%). DLTs included mucositis, rash, transaminase elevation, and hyperglycemia. An MTD of 154 mg weekly has been estimated. PF-05212384 is eliminated with a half-life of approximately 16 hours, with low clearance (5–9 L/h) and relatively high volume of distribution (54–144 L). Changes in blood glucose have been observed in 13 patients, including 2 patients with Grade 3 hyperglycemia, a known effect of PI3K/Akt pathway suppression. No objective tumor response has been observed; 12 patients have achieved stable disease (SD) in excess of 16 weeks, with 9 patients on study for ≥24 weeks and one patient (salivary) on study for &amp;gt;46 weeks. Enrollment to MTD expansion cohorts is ongoing. Conclusions: Weekly administration of PF-05212384 is safe and tolerable across dose levels with multiple patients deriving clinical benefit with prolonged SD. The occurrence of hyperglycemia is suggestive of target modulation. Updated data for safety, PK, PD and antitumor activity will be presented Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A167.

Abstract B163: First-in-patient study of PF-04691502, a small molecule intravenous dual inhibitor of PI3K and mTOR in patients with advanced cancer: Update on safety, efficacy, and pharmacology.

Abstract Background: Activation of the PI3K pathway has been implicated in the growth and survival of a wide variety of human cancers. Mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, are very common in human cancers, having been observed in prostate, breast and colorectal tumors, among others. Phosphatase and tensin homolog (PTEN) deletion, leading to the activation of Akt signaling has been observed in many types of malignancies including prostate cancer, breast cancer, and glioma. PF-04691502 is an orally available dual-specificity inhibitor of PI3K and mTOR which is being evaluated for the treatment of human cancers. Methods: PF-04691502 is evaluated in a Phase 1 dose escalation study at doses ranging from 2 to 11 mg once daily (QD) using the standard 3+3 dose escalation method. Assessments include safety (NCI CTC AE v4.0), pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity. A maximum tolerated dose (MTD) of 8 mg QD has been determined. PD assessments include blood glucose and immunohistochemistry analysis of phosphoproteins in tumor and surrogate tissue (hair follicles). Antitumor activity is assessed per RECIST version 1.1. Results: As of 15 August 2011, a total of 30 patients have been dosed at 2, 4, 8 and 11 mg QD. The median age of patients was 62; median baseline ECOG PS was 0. Represented tumor types have included CRC (8), NSCLC (4), and breast (2), among others. Analysis of archived tumor biopsies revealed PI3K pathway alterations in 7/13 evaluable patients; alterations included PTEN loss, PIK3CA mutation or amplification. PF-04691502 has been well tolerated with the most common treatment-related AEs being fatigue, nausea, vomiting, decreased appetite and rash. One patient has experienced DLT (Fatigue) at the 8 mg QD dose level, and two patients experienced DLT at 11 mg (Fatigue, Rash). Preliminary PK data indicate that PF-04691502 is eliminated with a half life of approximately 11–15 hours, with low clearance and a relatively high volume of distribution. Eleven patients have experienced hyperglycemia, a known effect of PI3K/Akt pathway suppression. No objective tumor response has been observed; 5 patients have experienced stable disease (SD) in excess of 16 weeks, with one patient (CRC) remaining on study for 39 weeks and one patient (HER2+ BC) for 27 weeks. Enrollment to MTD expansion cohorts is ongoing. Conclusions: Daily oral administration of PF-04691502 appears safe and tolerable across multiple dose levels, with multiple patients deriving clinical benefit with prolonged SD. Multiple observations of hyperglycemia are suggestive of target modulation. Updated data for safety, PK, PD and antitumor activity will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B163.

387 First-in-human study of PF-05212384, a small molecule intravenous dual inhibitor of PI3K and mTOR in patients with advanced cancer: preliminary report on safety and pharmacokinetics

First-in-human (FIH) research is a translational process to move a new potential therapy from bench to bedside. Major ethical challenges of an FIH trial arise because of the indeterminate nature of the risks involved and the controversial risk-benefit justification. Severe adverse events and death of subjects who participated in FIH research in the past have led to an increased attention on ethical considerations in the design and conduct of such research. Furthermore, novel therapies in the current decade, such as molecular-targeted, gene transfer, and pluripotent stem cells therapies, have led to numerous emerging ethical challenges or different ethical assessment and justification frameworks for FIH research. This article presents, discusses, and interlinks ethical considerations and challenges in FIH research through a review of related ethical principles and their application to each ethical issue with given examples. Possible solutions to address each ethical challenge are presented. The scope of this article focuses on 4 major ethical issues in FIH research: risk-benefit assessment and justification for the conduct of research, selection of a suitable target population, informed consent, and conflict of interest.

383 First-in-human study of PF-04691502, a small molecule oral dual inhibitor of PI3K and mTOR in patients with advanced cancer: Preliminary report on safety and pharmacokinetics

383 First-in-human study of PF-04691502, a small molecule oral dual inhibitor of PI3K and mTOR in patients with advanced cancer: Preliminary report on safety and pharmacokinetics

PI3K/Akt/mTOR pathway as a target for cancer therapy

The PI3K/Akt/mTOR pathway regulates several normal cellular functions that are also critical for tumorigenesis, including cellular proliferation, growth, survival and mobility. Components of this pathway are frequently abnormal in a variety of tumors, making them an attractive target for anti-cancer therapy. Inhibition of mTOR in patients with cancer became more feasible after the development of rapamycin analogs with improved pharmacologic properties. The promising activity of these agents in early clinical trials has led to the development of ongoing phase III trials in renal cell carcinoma and breast cancer. Future studies are needed to identify the patients most likely to benefit from this form of therapy, and to define its role in combination with chemotherapy, hormones and growth factor inhibitors.