Targeting the EGFR pathway: An alternative strategy for the treatment of tuberous sclerosis complex?

INTRODUCTION: Tuberous sclerosis complex (TSC) is caused by variants in TSC1/TSC2, leading to constitutive activation of the mammalian target of rapamycin (mTOR) complex 1. Therapy with everolimus has been approved for TSC, but variations in success are frequent. Recently, caudal late interneuron progenitor (CLIP) cells were identified as a common origin of the TSC brain pathologies such as subependymal giant cell astrocytomas (SEGA) and cortical tubers (CT). Further, targeting the epidermal growth factor receptor (EGFR) with afatinib, which is expressed in CLIP cells, reduces cell growth in cerebral TSC organoids. However, investigation of clinical patient-derived data is lacking. AIMS: Observation of EGFR expression in SEGA, CT and focal cortical dysplasia (FCD) 2B human brain specimen and investigation of whether its inhibition could be a potential therapeutic intervention for these patients. METHODS: Brain specimens of 23 SEGAs, 6 CTs, 20 FCD2Bs and 17 controls were analysed via immunohistochemistry to characterise EGFR expression, cell proliferation (via Mib1) and mTOR signalling. In a cell-based assay using primary patient-derived cells (CT n = 1, FCD2B n = 1 and SEGA n = 4), the effects of afatinib and everolimus on cell proliferation and cell viability were observed. RESULTS: EGFR overexpression was observed in histological sections of SEGA, CT and FCD2B patients. Both everolimus and afatinib decreased the proliferation and viability in primary SEGA, tuber and FCD2B cells. CONCLUSION: Our study demonstrates that EGFR suppression might be an effective alternative treatment option for SEGAs and tubers, as well as other mTOR-associated malformations of cortical development, including FCD2B.

mTOR Inhibition Prolongs Survival and Has Beneficial Effects on Heart Function After Onset of Lamin A/C Gene Mutation Cardiomyopathy in Mice.

BACKGROUND: Mutations in LMNA encoding nuclear envelope proteins lamin A/C cause dilated cardiomyopathy. Activation of the AKT/mTOR (RAC-α serine/threonine-protein kinase/mammalian target of rapamycin) pathway is implicated as a potential pathophysiologic mechanism. The aim of this study was to assess whether pharmacological inhibition of mTOR signaling has beneficial effects on heart function and prolongs survival in a mouse model of the disease, after onset of heart failure. METHODS: We treated male LmnaH222P/H222P mice, after the onset of heart failure, with placebo or either of 2 orally bioavailable mTOR inhibitors: everolimus or NV-20494, a rapamycin analog highly selective against mTORC1. We examined left ventricular remodeling, and the cell biological, biochemical, and histopathologic features of cardiomyopathy, potential drug toxicity, and survival. RESULTS: Everolimus treatment (n=17) significantly reduced left ventricular dilatation and increased contractility on echocardiography, with a 7% (P=0.018) reduction in left ventricular end-diastolic diameter and a 39% (P=0.0159) increase fractional shortening compared with placebo (n=17) after 6 weeks of treatment. NV-20494 treatment (n=15) yielded similar but more modest and nonsignificant changes. Neither drug prevented the development of cardiac fibrosis. Drug treatment reactivated suppressed autophagy and inhibited mTORC1 signaling in the heart, although everolimus was more potent. With regards to drug toxicity, everolimus alone led to a modest degree of glucose intolerance during glucose challenge. Everolimus (n=20) and NV-20494 (n=20) significantly prolonged median survival in LmnaH222P/H222P mice, by 9% (P=0.0348) and 11% (P=0.0206), respectively, compared with placebo (n=20). CONCLUSIONS: These results suggest that mTOR inhibitors may be beneficial in patients with cardiomyopathy caused by LMNA mutations and that further study is warranted.

Everolimus decreases [U-13C]glucose utilization by pyruvate carboxylase in breast cancer cells in vitro and in vivo.

Reprogrammed metabolism is a hallmark of cancer, but notoriously difficult to target due to metabolic plasticity, especially in response to single metabolic interventions. Combining mTOR inhibitor everolimus and mitochondrial complex 1 inhibitor metformin results in metabolic synergy in in vitro models of triple-negative breast cancer. Here, we investigated whether the effect of this drug combination on tumor size is reflected in changes in tumor metabolism using [U-13C]glucose labeling in an MDA-MB-231 triple negative breast cancer xenograft model. The in vitro effects of everolimus and metformin treatment on oxidative phosphorylation and glycolysis reflected changes in 13C-labeling of metabolites in MDA-MB-231 cells. Treatment of MDA-MB-231 xenografts in SCID/Beige mice with everolimus resulted in slower tumor growth and reduced tumor size and tumor viability by 35%. Metformin treatment moderately inhibited tumor growth but did not enhance everolimus-induced effects. High serum levels of everolimus were reached, whereas levels of metformin were relatively low. Everolimus decreased TCA cycle metabolite labeling and inhibited pyruvate carboxylase activity. Metformin only caused a mild reduction in glycolytic metabolite labeling and did not affect pyruvate carboxylase activity or TCA cycle metabolite labeling. In conclusion, treatment with everolimus, but not metformin, decreased tumor size and viability. Furthermore, the efficacy of everolimus was reflected in reduced 13C-labeling of TCA cycle intermediates and reduced pyruvate carboxylase activity. By using in-depth analysis of drug-induced changes in glucose metabolism in combination with measurement of drug levels in tumor and plasma, effects of metabolically targeted drugs can be explained, and novel targets can be identified.

Long-Term Percutaneous Coronary Intervention Outcomes in Chronic Versus Acute Coronary Syndromes (TARGET All Comers Trial).

In the Targeted therapy with a localised abluminal coated, low-dose sirolimus-eluting, biodegreadable polymer coronary stent (TARGET; NCT02520180) All Comers trial the biodegradable polymer (BP) sirolimus-eluting FIREHAWK stent was noninferior to the durable polymer (DP) everolimus-eluting XIENCE stent with respect to target lesion failure (TLF) at 1 and 5 years; however, the long-term safety and efficacy in the setting of acute coronary syndromes (ACS) are not known. We sought to assess the long-term outcomes in ACS versus chronic coronary syndromes (CCS) with BP sirolimus-eluting stent (SES) versus DP everolimus-eluting stent (EES). The TARGET AC study was a multicenter, open-label, noninferiority trial of all comer patients randomly allocated 1:1 to BP SES or DP EES (stratified by ST-elevation myocardial infarction and study site). In this predefined substudy, the outcomes were compared based on clinical presentation (ACS vs CCS) and treatment allocation. A total of 1,653 patients were enrolled (728 with ACS and 922 with CCS), with 94% completing the 5-year follow-up. The baseline characteristics were well-matched between the 2 stent types; however, co-morbidities were more prevalent in the CCS than in the ACS population. TLF (15.5% vs 17.7%, p = 0.24), patient-oriented outcomes (32.0% vs 34.4%, p = 0.31), and stent thrombosis (4.1% vs 3.3%, p = 0.40) were similar between patients with ACS and patients with CCS. In the ACS cohort, the outcomes at 5 years for BP SES versus DP EES were similar for TLF (16.0% vs 14.9%, p = 0.70), ischemia-driven target lesion revascularization (5.6% vs 8.3%, p = 0.17), and definite/probable stent thrombosis (2.7% vs 4.6%, p = 0.18). The same was true for the CCS cohort, with 5-year outcomes for BP SES versus DP EES for TLF (18.0% vs 17.4%, p = 0.82), ischemia-driven target lesion revascularization (6.4% vs 5.0%, p = 0.37), and definite/probable stent thrombosis (3.0% vs 1.8%, p = 0.26). In conclusion, in the TARGET AC trial, 1 in 3 patients had a major adverse event at 5 years, irrespective of CCS or ACS presentation. Long-term, the BP sirolimus-eluting FIREHAWK stent was as safe and effective as the DP everolimus-eluting XIENCE stent across the spectrum of clinical presentations.

Everolimus inhibits hepatoblastoma by inducing autophagy-dependent ferroptosis.

Everolimus, a known inhibitor of the mammalian target of rapamycin (mTOR), has shown uncertain efficacy in treating hepatoblastoma. This study delves into the potential anti-hepatoblastoma properties of everolimus and its intricate relationship with autophagy and ferroptosis, both in vitro and in vivo. In vivo, tumor tissue from hepatoblastoma patient and human hepatoblastoma cell line HuH-6 were xenografted into nude mice to establish xenograft models for observing the effect of everolimus on tumor growth. In vitro, HuH-6 cells were cultured to evaluate the anti-hepatoblastoma activity of everolimus. Transmission electron microscopy and microtubule-associated proteins 1 light chain 3 (LC3), beclin 1, and p62 protein expressions were employed to investigate autophagy. Additionally, indicators of cell apoptosis, reactive oxygen species (ROS) and proteins associated with ferroptosis were measured to evaluate ferroptosis. The results demonstrate that everolimus treatment effectively induced the formation of autophagosomes in hepatoblastoma cells, upregulated the LC3II/I ratio and beclin 1 expression, and downregulated p62 expression, indicating an enhanced autophagy level both in vitro and in vivo. Furthermore, everolimus treatment induced cell apoptosis, increased ROS level, elevated concentrations of malondialdehyde, 4-hydroxynonenal, and iron content, while reducing the ratio of glutathione/oxidized glutathione, and downregulating the protein expression of glutathione peroxidase 4 and solute carrier family 7 member 11, suggesting its ability to induce ferroptosis in hepatoblastoma cells. Importantly, the induction of ferroptosis by everolimus was significantly reversed in the presence of autophinib, an autophagy inhibitor, indicating the autophagy-dependent of everolimus-induced ferroptosis. Taken together, these findings suggest that everolimus holds promise as an effective anti-hepatoblastoma drug, with its mechanism of action potentially involving the induction of autophagy-dependent ferroptosis in hepatoblastoma cells.

Everolimus exerts anticancer effects through inhibiting the interaction of matrix metalloproteinase-7 with syndecan-2 in colon cancer cells.

Previous work showed that matrix metalloproteinase-7 (MMP-7) regulates colon cancer activities through an interaction with syndecan-2 (SDC-2) and SDC-2-derived peptide that disrupts this interaction and exhibits anticancer activity in colon cancer. Here, to identify potential anticancer agents, a library of 1,379 Food and Drug Administration (FDA)-approved drugs that interact with the MMP-7 prodomain were virtually screened by protein-ligand docking score analysis using the GalaxyDock3 program. Among five candidates selected based on their structures and total energy values for interacting with the MMP-7 prodomain, the known mechanistic target of rapamycin kinase (mTOR) inhibitor, everolimus, showed the highest binding affinity and the strongest ability to disrupt the interaction of the MMP-7 prodomain with the SDC-2 extracellular domain in vitro. Everolimus treatment of the HCT116 human colon cancer cell line did not affect the mRNA expression levels of MMP-7 and SDC-2 but reduced the adhesion of cells to MMP-7 prodomain-coated plates and the cell-surface localization of MMP-7. Thus, everolimus appears to inhibit the interaction between MMP-7 and SDC-2. Everolimus treatment of HCT116 cells also reduced their gelatin-degradation activity and anticancer activities, including colony formation. Interestingly, cells treated with sirolimus, another mTOR inhibitor, triggered less gelatin-degradation activity, suggesting that this inhibitory effect of everolimus was not due to inhibition of the mTOR pathway. Consistently, everolimus inhibited the colony-forming ability of mTOR-resistant HT29 cells. Together, these data suggest that, in addition to inhibiting mTOR signaling, everolimus exerts anticancer activity by interfering with the interaction of MMP-7 and SDC-2, and could be a useful therapeutic anticancer drug for colon cancer.NEW & NOTEWORTHY The utility of cancer therapeutics targeting the proteolytic activities of MMPs is limited because MMPs are widely distributed throughout the body and involved in many different aspects of cell functions. This work specifically targets the activation of MMP-7 through its interaction with syndecan-2. Notably, everolimus, a known mTOR inhibitor, blocked this interaction, demonstrating a novel role for everolimus in inhibiting mTOR signaling and impairing the interaction of MMP-7 with syndecan-2 in colon cancer.

Gelatin microgel-coated balloon catheter with enhanced delivery of everolimus for long-term vascular patency.

In-stent restenosis (ISR) after percutaneous coronary intervention is a major reason for limited long-term patency due to complex neointimal proliferation caused by vascular injury. Drug-coated balloon (DCB) has been developed to treat various cardiovascular diseases including ISR by providing anti-proliferative drugs into blood vessel tissues. However, a significant proportion of the drug is lost during balloon tracking, resulting in ineffective drug delivery to the target region. In this study, we report an everolimus-coated balloon (ECB) using everolimus-loaded gelatin-hydroxyphenyl propionic acid microgel (GM) with enhanced everolimus delivery to vascular walls for long-term patency. GM with high drug loading (> 97%) was simply prepared by homogenizing enzyme-mediated crosslinked hydrogels. The optimal condition to prepare GM-coated ECB (GM-ECB) was established by changing homogenization time and ethanol solvent concentration (30 ∼ 80%). In vitro sustained everolimus release for 30 d, and cellular efficacy using smooth muscle cells and vascular endothelial cells were evaluated. Additionally, an in vivo drug transfer levels of GM-ECB using rabbit femoral arteries were assessed with reduced drug loss and efficient drug delivery capability. Finally, using ISR-induced porcine models, effective in vivo vascular patency 4 weeks after treatment of ECBs was also confirmed. Thus, this study strongly demonstrates that GM can be used as a potential drug delivery platform for DCB application. STATEMENT OF SIGNIFICANCE: We report an ECB using everolimus-loaded GM prepared by homogenization of enzymatic cross-linked hydrogel. GM showed efficient drug loading (> 97 %) and controllable size. GM-ECB exhibited potential to deliver everolimus in a sustained manner to target area with drug efficacy and viability against SMC and EC. Although GM-ECB had much lower drug content compared to controls, animal study demonstrated enhanced drug transfer and reduced drug loss of GM-ECB due to the protection of encapsulated drugs by GM, and the possible interaction between GM and endothelium. Finally, vascular patency and safety were assessed using ISR-induced porcine models. We suggest an advanced DCB strategy to alleviate rapid drug clearance by bloodstream while improving drug delivery for a long-term vascular patency.

Combining MTI-31 with RAD001 inhibits tumor growth and invasion of kidney cancer by activating autophagy.

At most of the times, patients who are diagnosed with kidney cancer should be provided with systemic treatment as drug resistance is a challenging issue in the treatment of this disease. The progression of the cancer can be inhibited with the help of mTOR inhibitors namely RAD001 (everolimus) and MTI-31. In literature, it has been revealed that these mTOR inhibitors have the potential to stimulate autophagy. This degradation pathway boosts the survival rate of the cancerous cells that are subjected to anti-cancer therapy. In this study, CCK8, colony formation assays, and ethynyl deoxyuridine (EdU) analysis were conducted to detect cell proliferation. Furthermore, Transwell assays were also conducted for cell migration analysis. In addition to these, the researchers also performed the flow cytometry process to identify the cells that are undergoing apoptosis. In vivo, experiments were conducted to measure the growth of tumors and metastasis. In this study, the treatment provided through a combination of MTI-31 and RAD001 significantly inhibited the kidney cancer cells' proliferation and tumor growth. Furthermore, there was a notable reduction in the migration and invasion of kidney cancer cells upon the neighboring cells. The outcomes from the mechanistic studies infer that the combination of MTI-31 and RAD001 increases the LC3 levels, which in turn translates into the activation of autophagy. To conclude, the combination of MTI-31 and RAD001 improves the anti-cancerous impact produced by RAD001 in vivo through the promotion of autophagy.

Preliminary Investigation on Efficacy and Safety of Substance P-Coated Stent for Promoting Re-Endothelialization: A Porcine Coronary Artery Restenosis Model.

BACKGROUND: Current polymer-based drug-eluting stents (DESs) have fundamental issues about inflammation and delayed re-endothelializaton of the vessel wall. Substance-P (SP), which plays an important role in inflammation and endothelial cells, has not yet been applied to coronary stents. Therefore, this study compares poly lactic-co-glycolic acid (PLGA)-based everolimus-eluting stents (PLGA-EESs) versus 2-methacryloyloxyethyl phosphorylcholine (MPC)-based SP-eluting stents (MPC-SPs) in in-vitro and in-vivo models. METHODS: The morphology of the stent surface and peptide/drug release kinetics from stents were evaluated. The in-vitro proliferative effect of SP released from MPC-SP is evaluated using human umbilical vein endothelial cell. Finally, the safety and efficacy of the stent are evaluated after inserting it into a pig's coronary artery. RESULTS: Similar to PLGA-EES, MPC-SP had a uniform surface morphology with very thin coating layer thickness (2.074 µm). MPC-SP showed sustained drug release of SP for over 2 weeks. Endothelial cell proliferation was significantly increased in groups treated with SP (n = 3) compared with the control (n = 3) and those with everolimus (n = 3) (SP: 118.9 ± 7.61% vs. everolimus: 64.3 ± 12.37% vs. the control: 100 ± 6.64%, p < 0.05). In the animal study, the percent stenosis was higher in MPC-SP group (n = 7) compared to PLGA-EES group (n = 7) (MPC-SP: 28.6 ± 10.7% vs. PLGA-EES: 16.7 ± 6.3%, p < 0.05). MPC-SP group showed, however, lower inflammation (MPC-SP: 0.3 ± 0.26 vs. PLGA-EES: 1.2 ± 0.48, p < 0.05) and fibrin deposition (MPC-SP: 1.0 ± 0.73 vs. PLGA-EES: 1.5 ± 0.59, p < 0.05) around the stent strut. MPC-SP showed more increased expression of cluster of differentiation 31, suggesting enhanced re-endothelialization. CONCLUSION: Compared to PLGA-EES, MPC-SP demonstrated more decreased inflammation of the vascular wall and enhanced re-endothelialization and stent coverage. Hence, MPC-SP has the potential therapeutic benefits for the treatment of coronary artery disease by solving limitations of currently available DESs.

The mitochondriogenic but not the immunosuppressant effects of mTOR inhibitors prompt neuroprotection and delay disease evolution in a mouse model of progressive multiple sclerosis.

INTRODUCTION: Purportedly, the progression of multiple sclerosis (MS) occurs when neurodegenerative processes due to derangement of axonal bioenergetics take over the autoimmune response. However, a clear picture of the causative interrelationship between autoimmunity and axonal mitochondrial dysfunction in progressive MS (PMS) pathogenesis waits to be provided. METHODS: In the present study, by adopting the NOD mouse model of PMS, we compared the pharmacological effects of the immunosuppressants dexamethasone and fingolimod with those of mTOR inhibitors rapamycin and everolimus that, in addition to immunosuppression, also regulate mitochondrial functioning. Female Non-Obese Diabetic (NOD) mice were immunized with MOG35-55 and treated with drugs to evaluate functional, immune and mitochondrial parameters during disease evolution. RESULTS: We found that dexamethasone and fingolimod did not affect the pattern of progression as well as survival. Conversely, mTOR inhibitors rapamycin and everolimus delayed disease progression and robustly extended survival of immunized mice. The same effects were obtained when treatment was delayed by 30 days after immunization. Remarkably, dexamethasone and fingolimod prompted the same degree of immunosuppression of rapamycin within both spleen and spinal cord of mice. However, only rapamycin prompted mitochondriogenesis by increasing mitochondrial content, and expression of several mitochondrial respiratory complex subunits, thereby preventing mtDNA reduction in the spinal cords of immunized mice. These pharmacodynamic effects were not reproduced in healthy NOD mice, suggesting a disease context-dependent pharmacodynamic effect. DISCUSSION: Data corroborate the key role of mitochondriogenesis to treatment of MS progression, and for the first time disclose the translational potential of mTOR inhibitors in PMS therapy.

Cleverballoon: An integrated approach for developing a drug-coated balloon with everolimus.

Coronary artery disease (CAD) is a chronic disease associated with high mortality and morbidity. Although treatment with drug-eluting stents is the most frequent interventional approach for coronary artery disease, drug-coated balloons (DCBs) constitute an innovative alternative, especially in the presence of certain anatomical conditions in the local coronary vasculature. DCBs allow the fast and homogenous transfer of drugs into the arterial wall, during the balloon inflation. Their use has been established for treating in-stent restenosis caused by stent implantation, while recent clinical trials have shown a satisfactory efficacy in de novo small-vessel disease. Several factors affect DCBs performance including the catheter design, the drug dose and formulation. Cleverballoon focuses on the design and development of an innovative DCB with everolimus. For the realization of the development of this new DCB, an integrated approach, including in- vivo, in-vitro studies and in-silico modelling towards the DCB optimization, is presented.Clinical Relevance-The proposed study introduces the integration of in- vivo, in-vitro and in silico approaches in the design and development process of a new DCB, following the principles of 3R's for the replacement, reduction, and refinement of animal and clinical studies.

Everolimus-induced hyperpermeability of endothelial cells causes lung injury.

The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca2+-dependent manner and can lead to lung injury through mTOR-independent mechanisms.

A Stable Micellar Formulation of RAD001 for Intracerebroventricular Delivery and the Treatment of Alzheimer's Disease and Other Neurological Disorders.

A large body of evidence, replicated in many mouse models of Alzheimer's disease (AD), supports the therapeutic efficacy of the oral mammalian target of rapamycin inhibitors (mTOR-Is). Our preliminary data show that intracerebroventricular (ICV) administration of everolimus (RAD001) soon after clinical onset greatly diminished cognitive impairment and the intracellular beta amyloid and neurofibrillary tangle load. However, RAD001 shows >90% degradation after 7 days in solution at body temperature, thus hampering the development of proper therapeutic regimens for patients. To overcome such a drawback, we developed a stable, liquid formulation of mTOR-Is by loading RAD001 into distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) micelles using the thin layer evaporation method. The formulation showed efficient encapsulation of RAD001 and a homogeneous colloidal size and stabilised RAD001, with over 95% of activity preserved after 14 days at 37 °C with a total decay only occurring after 98 days. RAD001-loaded DSPE-PEG2000 micelles were unchanged when stored at 4 and 25 °C over the time period investigated. The obtained formulation may represent a suitable platform for expedited clinical translation and effective therapeutic regimens in AD and other neurological diseases.

Ultrathin, biodegradable polymer-coated everolimus-eluting stents for patients undergoing percutaneous coronary intervention: Final three-year results of the PERFORM-EVER registry.

The registry reports 3-year safety and clinical performance of the ultrathin strut (60 µm) biodegradable polymer-coated Tetrilimus, an everolimus-eluting stent (EES) (Sahajanand Medical Technologies Limited, India), in 'real-world' patients with coronary artery disease. A total of 815 Tetrilimus EES were implanted in 735 lesions in 594 patients. At 3-year follow-up, primary endpoint (target lesion failure, TLF) was reported in 8.6 % patients, including 2.6 % cardiac deaths, 3.5 % myocardial infarction and 2.6 % target lesion revascularization. At three-year, no cases of definite stent thrombosis were reported. The final three-year results of PERFORM-EVER registry endorse the continuous safety and effectiveness Tetrilimus EES.

Effects of Everolimus in Modulating the Host Immune Responses against Mycobacterium tuberculosis Infection.

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (P13K/AKT/mTOR) pathway plays a key role in tuberculosis (TB) pathogenesis and infection. While the activity levels of this pathway during active infection are still debated, manipulating this pathway shows potential benefit for host-directed therapies. Some studies indicate that pathway inhibitors may have potential for TB treatment through upregulation of autophagy, while other studies do not encourage the use of these inhibitors due to possible host tissue destruction by Mycobacterium tuberculosis (M. tb) and increased infection risk. Investigating further clinical trials and their use of pathway inhibitors is necessary in order to ascertain their potential for TB treatment. This paper is particularly focused on the drug everolimus, an mTOR inhibitor. One of the first clinical trials sponsored by the Aurum Institute showed potential benefit in using everolimus as an adjunctive therapy for tuberculosis. Infection with tuberculosis is associated with a metabolic shift from oxidative phosphorylation towards glycolysis. The everolimus arm in the clinical trial showed further reduction than the control for both maximal and peak glycolytic activity. Compared with control, those receiving everolimus demonstrated increased lung function through forced expiratory volume in 1 s (FEV1) measurements, suggesting that everolimus may mitigate inflammation contributing to lung damage.

Signaling Pathways mTOR and ERK as Therapeutic Targets in Sinonasal Intestinal-Type Adenocarcinoma.

Despite advances in surgery and radiotherapy, the overall prognosis of sinonasal intestinal-type adenocarcinoma (ITAC) is poor, and new treatment options are needed. Recent studies have indicated alterations in cellular signaling pathways that may serve as targets for modern inhibitors. Our aim was to evaluate the frequency of mTOR and ERK pathway upregulation in a retrospective series of 139 ITAC and to test the efficacy and mechanism of action of candidate targeted inhibitors in cell line ITAC-3. An immunohistochemical analysis on p-AKT, p-mTOR, p-S6, p-4E-BP1, and p-ERK indicated, respectively, a 68% and 57% mTOR and ERK pathway activation. In vitro studies using low doses of mTOR inhibitor everolimus and ERK inhibitor selumetinib showed significant growth inhibition as monotherapy and especially as combined therapy. This effect was accompanied by the downregulation of mTOR and ERK protein expression. Our data open a new and promising possibility for personalized treatment of ITAC patients.

Mammalian target of rapamycin inhibition impacts energy homeostasis and induces sex-specific body weight loss in humans.

BACKGROUND: Previous data from a 2-year randomized controlled trial (CRAD001ADE12) indicated that mammalian target of rapamycin (mTOR) inhibition by everolimus slowed cyst growth in patients with autosomal-dominant polycystic kidney disease (ADPKD). During the trial, we noted body weight loss in some patients, particularly in women. We hypothesized that everolimus causes body weight reduction by reduced food intake and/or metabolic changes, which could lead to cachexia. METHODS: Within a sub-analysis of the CRAD001ADE12 trial, body weight course was investigated regarding sex-specific differences in 433 adult ADPKD patients (everolimus, n = 215; placebo, n = 218). One hundred four out of 111 patients who participated in the clinical trial centre in Berlin were evaluated under everolimus/placebo therapy (on drug: everolimus, n = 48; placebo, n = 56) and after therapy (off drug: everolimus, n = 15; placebo, n = 18). Eating habits and nutrient/caloric intake were evaluated by validated questionnaires. Systemic and local metabolism was evaluated in four patients after an oral glucose load (OGL) by using calorimetry and adipose/muscle tissue microdialysis. RESULTS: Within the 2-year CRAD001ADE12 trial, a significant body weight loss was observed in female patients on everolimus versus placebo (P = 0.0029). Data of the Berlin Cohort revealed that weight loss was greater in women on everolimus versus men (P < 0.01). After 9 months, women and men had lost 2.6 ± 3.8 and 0.8 ± 1.5 kg (P < 0.05) in body weight, respectively, and after 21 months, they had lost 4.1 ± 6.6 and 1.0 ± 3.3 kg (P < 0.05), respectively. On everolimus, caloric intake was significantly lower in women versus men (1510 ± 128 vs. 2264 ± 216 kcal/day, P < 0.05), caused mainly by a lower fat and protein intake in women versus men. Cognitive restraints, disinhibition and hunger remained unchanged. In a subgroup of patients resting metabolic rate was unchanged whereas OGL-induced thermogenesis was reduced (7 ± 2 vs. 11 ± 2 kcal, P < 0.05). Fasting and OGL-induced fat oxidation was increased (P < 0.05) on versus off everolimus. In adipose tissue, fasting lipolytic activity was increased, but lipolytic activity was inhibited similarly after the OGL on versus off everolimus, respectively. In skeletal muscle, postprandial glucose uptake and aerobic glycolysis was reduced in patients on everolimus. CONCLUSIONS: mTOR inhibition by everolimus induces body weight reduction, specifically in female patients. This effect is possibly caused by a centrally mediated reduced food (fat and protein) intake and by centrally/peripherally mediated increased fat oxidation (systemic) and mobilization (adipose tissue). Glucose uptake and oxidation might be reduced in skeletal muscle. This could lead to cachexia and, possibly, muscle wasting. Therefore, our results have important implications for patients recieving immune-suppressive mTOR inhibition therapy.

Molecular Characteristics of Everolimus-resistant Renal Cell Carcinoma Cells Generated by Continuous Exposure to Everolimus.

BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) inhibitors represent the standard of care for metastatic renal cell carcinoma (RCC). However, treatment outcomes are relatively poor, suggesting a potential problem with tolerating mTOR inhibitors. The aim of this study was to establish everolimus-resistant sublines and to compare their molecular characteristics with those of their counterparts. MATERIALS AND METHODS: Human-derived RCC, Caki-2, and 786-O cells were continuously exposed to everolimus at 1 µM, and the established resistant sublines were designated as Caki/EV and 786/EV, respectively. Cellular characteristics were compared between both cells. RESULTS: Caki/EV and 786/EV cells showed a decrease in sensitivity to everolimus as well as other mTOR inhibitors. Expression of mTOR and its effectors exhibited no alteration in resistant sublines and their counterparts. However, phosphorylation of S6K, an index of mTOR activity, decreased in resistant sublines. PCR array analysis of mTOR signaling pathway-related factors indicated that the expression of INSR, TP53, and IGFBP3 increased in Caki/EV cells, whereas that of TELO2, HRAS, and SGK1 was up-regulated in 786/EV cells. The levels of DDIT4, DEPTOR, HIF1A, and PLD1 mRNAs decreased in both cell lines. CONCLUSION: The novel everolimus-resistant Caki/EV and 786/EV cells exhibited cross-resistance to other mTOR inhibitors and decreased mTOR activity. Furthermore, down-regulation of DDIT4, DEPTOR, HIF1A, and PLD1 may contribute to everolimus resistance.

ONC201/TIC10 enhances durability of mTOR inhibitor everolimus in metastatic ER+ breast cancer.

The mTOR inhibitor, everolimus, is an important clinical management component of metastatic ER+ breast cancer (BC). However, most patients develop resistance and progress on therapy, highlighting the need to discover strategies that increase mTOR inhibitor effectiveness. We developed ER+ BC cell lines, sensitive or resistant to everolimus, and discovered that combination treatment of ONC201/TIC10 with everolimus inhibited cell growth in 2D/3D in vitro studies. We confirmed increased therapeutic response in primary patient cells progressing on everolimus, supporting clinical relevance. We show that ONC201/TIC10 mechanism in metastatic ER+ BC cells involves oxidative phosphorylation inhibition and stress response activation. Transcriptomic analysis in everolimus resistant breast patient tumors and mitochondrial functional assays in resistant cell lines demonstrated increased mitochondrial respiration dependency, contributing to ONC201/TIC10 sensitivity. We propose that ONC201/TIC10 and modulation of mitochondrial function may provide an effective add-on therapy strategy for patients with metastatic ER+ BCs resistant to mTOR inhibitors.

Breast cancer is one of the most frequently diagnosed cancers globally, particularly among women. The most common type of breast cancer expresses a receptor for the hormone estrogen. Many treatments block the activity of estrogen and therefore slow or block the development and spread of this type of breast cancer. For patients with advanced breast cancer, hormone-blocking treatments work best in combination with other drugs, including one called everolimus. However, in many patients the cancer cells become resistant to these therapies, leading to disease progression and decreased survival. To explore treatment strategies that could enhance the effectiveness of existing therapies for breast cancer, Farmaki et al. studied how cancer cells which had become resistant to everolimus responded when treated with an experimental drug called ONC201/TIC10. A combination of everolimus and ONC201/TIC10 inhibited growth of resistant cancer cells that had been grown in a three-dimensional arrangement to mimic human tumors. Moreover, the drug combination effectively targeted breast cancer cells collected from patients whose cancer had progressed while being treated with everolimus, suggesting that ONC201/TIC10 could be relevant in a clinical setting. Finally, molecular and biochemical experiments revealed that the drug ONC201/TIC10 works by disrupting the pathways that everolimus-resistant cancer cells use to generate the energy required to grow and proliferate. Taken together these findings suggest that ONC201/TIC10 may provide an effective add-on therapy for patients with certain types of advanced breast cancer that are no longer responding to everolimus. Before this becomes a reality for patients, however, there will have to be more experimental testing of ONC201/TIC10 to determine optimal dosing and timing strategy for future clinical trials.

One-Month Dual Antiplatelet Therapy in Patients With Chronic and Acute Coronary Syndromes Treated With Bioresorbable Polymer Everolimus-Eluting Stents.

There is a paucity of data regarding the safety of a 1-month dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI) in patients at high bleeding risk (HBR) presenting with acute coronary syndromes (ACS). We aimed to compare the clinical outcomes of patients at HBR with chronic coronary syndrome (CCS) or ACS treated with PCI using bioresorbable polymer everolimus-eluting stent (BP-EES) followed by 1-month DAPT. Patients at HBR who underwent PCI with BP-EES were prospectively enrolled in 10 Italian centers. All patients were treated with 1-month DAPT. In case of need for anticoagulation, patients received an oral anticoagulant in addition to a P2Y12 inhibitor for 1 month, followed by oral anticoagulation only after that. The primary end point was a composite of cardiac death, myocardial infarction, or definite/probable stent thrombosis at 12 months. Overall, 263 patients (59.4%) with CCS and 180 patients (40.6%) with ACS were enrolled. No significant difference was evident between patients with CCS and ACS for the primary end point (4.3% vs 5.6%, respectively, p = 0.497) and for each isolated component. The risk for Bleeding Academic Research Consortium (BARC) type 1 to 5 or type 3 to 5 bleedings was also similar between patients with CCS and ACS (4.3% vs 5.2%, p = 0.677, and 1.6% vs 2.9%, p = 0.351, respectively). In conclusion, among HBR patients with ACS who underwent PCI with BP-EES, a 1-month DAPT strategy is associated with a similar risk of ischemic and bleeding events compared with those with CCS.