Proteomic Changes Induced by the Immunosuppressant Everolimus in Human Podocytes.

mTOR inhibitors (mTOR-Is) may induce proteinuria in kidney transplant recipients through podocyte damage. However, the mechanism has only been partially defined. Total cell lysates and supernatants of immortalized human podocytes treated with different doses of everolimus (EVE) (10, 100, 200, and 500 nM) for 24 h were subjected to mass spectrometry-based proteomics. Support vector machine and partial least squares discriminant analysis were used for data analysis. The results were validated in urine samples from 28 kidney transplant recipients receiving EVE as part of their immunosuppressive therapy. We identified more than 7000 differentially expressed proteins involved in several pathways, including kinases, cell cycle regulation, epithelial-mesenchymal transition, and protein synthesis, according to gene ontology. Among these, after statistical analysis, 65 showed an expression level significantly and directly correlated with EVE dosage. Polo-Like Kinase 1 (PLK1) content was increased, whereas osteopontin (SPP1) content was reduced in podocytes and supernatants in a dose-dependent manner and significantly correlated with EVE dose (p < 0.0001, FDR < 5%). Similar results were obtained in the urine of kidney transplant patients. This study analyzed the impact of different doses of mTOR-Is on podocytes, helping to understand not only the biological basis of their therapeutic effects but also the possible mechanisms underlying proteinuria.

Influence of Tyrosine Kinase Inhibitors and Everolimus on TGFß1 and TGF-ß Receptor 2 in Squamous Cell Carcinoma Cells.

BACKGROUND/AIM: Transforming growth factor-ß (TGF-ß) plays a significant role in the formation of different cancer subtypes. There is evidence that TGF-ß pathways promote cancerogenic cell characteristics but also have tumor-suppressor capabilities. The tyrosine kinase inhibitors nilotinib, dasatinib, erlotinib, gefitinib, and everolimus are approved as targeted therapies for several tumor entities, including head and neck squamous cell carcinoma (HNSCC). This study aimed to investigate the effects of these substances on the expression levels of TGFß1 and TGF-ß receptor type 2 (TGFßR2) in HPV-negative and HPV-positive SCC cell cultures. MATERIALS AND METHODS: Expression patterns of TGFß1 and TGFßR2 were determined using enzyme-linked immunosorbent assay (ELISA) in three HNSCC cell lines (i.e., HNSCC-11A, HNSCC-14C, and CERV196). These cells were incubated with nilotinib, dasatinib, erlotinib, gefitinib, and everolimus (20 µmol/l) and compared to a chemonaive control. An assessment of concentration levels was conducted after 24, 48, 72, and 96 h of treatment. RESULTS: Statistically significant changes in the expression levels of TGFß1 and TGFßR2 were found in all tested cell cultures (p<0.05) compared to the negative control. An increase in TGFß-R2 expression was detected after treatment with most of the tested tyrosine kinase inhibitors, whereas a reduction in TGFß1 was observed. The addition of everolimus had the opposite effect on both TGFßR2 and TGF-B1- expression. CONCLUSION: Expression of TGFß1 and TGFßR2 was detected in all cultured HNSCC cell lines. Nilotinib, dasatinib, erlotinib, gefitinib, and everolimus had an impact on the expression levels of TGFß1 and TGFßR2 in vitro.

Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe.

BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Down-regulation of ABCB1 in Everolimus-resistant Renal Cell Carcinoma Cells.

BACKGROUND/AIM: Everolimus-resistant Caki/EV and 786/EV cells have been established from human derived renal cell carcinoma cells, Caki-2 and 786-O, respectively. These cells exhibit resistance to everolimus and to other mTOR inhibitors and erlotinib. However, the sensitivity of these resistant cells to classical and cytotoxic anticancer drugs remain unclear. The aim of the study was to examine sensitivity of Caki/EV and 786/EV cells to classical and cytotoxic anticancer drugs. MATERIALS AND METHODS: Sensitivity to classical and cytotoxic anticancer drugs in Caki/EV and 786/EV cells was evaluated using the WST-1 (tetrazolium salts) colorimetric assay and was compared to those of the corresponding parental cells. The mRNA expression levels were measured using SYBR® green based quantitative reverse transcription-polymerase chain reaction. RESULTS: Sensitivity to vinblastine, vincristine, paclitaxel, doxorubicin, etoposide, SN-38 (active metabolite of irinotecan), 5-fluorouracil, cisplatin, and carboplatin varied in the resistant cells. Sensitivity to carboplatin and SN-38 was comparable between resistant cells and their parental cells, whereas sensitivity to vinca alkaloids, etoposide, 5-fluorouracil, and cisplatin decreased in the resistant cells. However, sensitivity to paclitaxel and doxorubicin was remarkably enhanced in both resistant cells compared to that of parental cells, this could be partially explained by down-regulation of ABCB1 mRNA expression. CONCLUSION: The everolimus-resistant Caki/EV and 786/EV cells showed cross-resistance to classical and cytotoxic anticancer drugs. However, Caki/EV and 786/EV cells exhibited a remarkable increase in sensitivity to paclitaxel and doxorubicin, and ABCB1 mRNA was down-regulated in response to long-term exposure to everolimus.

Temporal Trends in the Outcomes of Percutaneous Coronary Intervention With Zotarolimus Eluting Stents Versus Everolimus Eluting Stents: A Meta-Analysis of Randomized Controlled Trials.

INTRODUCTION: Long-term follow-up results of various trials comparing Zotarolimus eluting stents (ZES) with Everolimus eluting stents (EES) have been published recently. Additionally, over the last decade, there have been new trials comparing the ZES with various commercially available EES. We aim to conduct an updated meta-analysis in light of new evidence from randomized controlled trials (RCTs) to provide comprehensive evidence regarding the temporal trends in the clinical outcomes. METHODS: A comprehensive literature search was conducted across PubMed, Cochrane, and Embase. RCTs comparing ZES with EES for short (<2 years), intermediate (2-3 years), and long-term follow-ups (3-5 years) were included. Relative risk was used to pool the dichotomous outcomes using the random effects model employing the inverse variance method. All statistical analysis was conducted using Revman 5.4. RESULTS: A total of 18 studies reporting data at different follow-ups for nine trials (n = 14319) were included. At short-term follow-up (<2 years), there were no significant differences between the two types of stents (all-cause death, cardiac death, Major adverse cardiovascular events (MACE), target vessel myocardial infarction, definite or probable stent thrombosis or safety outcomes (target vessel revascularization, target lesion revascularization, target vessel failure, target lesion failure). At intermediate follow-up (2-3 years), EES was superior to ZES for reducing target lesion revascularization (RR = 1.28, 95% CI = 1.05-1.58, p < 0.05). At long-term follow-up (3-5 years), there were no significant differences between the two groups for any of the pooled outcomes (p > 0.05). CONCLUSION: ZES and EES have similar safety and efficacy at short, intermediate, and long-term follow-ups.

Comparison of thrombogenicity in different types of drug-eluting stents during transition from DAPT to SAPT.

BACKGROUND: During the transition from dual antiplatelet therapy (DAPT) to single antiplatelet therapy (SAPT), previous studies have raised concerns about a rebound effect. We compared platelet and inflammatory cell adhesion on different types of stents in the setting of clopidogrel presence and withdrawal. METHODS: In Experiment 1, three pigs were administered with DAPT, that is, clopidogrel and acetylsalicylic acid (ASA), for 7 days. Each animal underwent an extracorporeal carotid arteriovenous shunt model implanted with fluoropolymer-coated everolimus-eluting stent (FP-EES), biodegradable-polymer sirolimus-eluting stent (BP-SES), and biodegradable-polymer everolimus-eluting stents (BP-EES). In Experiment 2, two pigs were administered DAPT, clopidogrel was then withdrawn at day 7, and SAPT with ASA was continued for next 21 days. Then flow-loop experiments with the drawn blood from each time point were performed for FP-EES, BioLinx-polymer zotarolimus-eluting stents (BL-ZES), and BP-EES. The rebound effect was defined as the statistical increase of inflammation and platelet adhesion assessed with immunohistochemistry on the stent-strut level basis from baseline to day-14 or 28. RESULTS: Both experiments showed platelet adhesion value was highest in BP-EES, while the least in FP-EES during DAPT therapy. There was no increase in platelet or inflammatory cell adhesion above baseline values (i.e., no therapy) due to the cessation of clopidogrel on the stent-strut level. Monocyte adhesion was the least for FP-EES with the same trend observed for neutrophil adhesion. CONCLUSIONS: No evidence of rebound effect was seen after the transition from DAPT to SAPT. FP-EES demonstrated the most favorable antithrombotic and anti-inflammatory profile regardless of the different experimental designs.

Prognostic value of EndoPredict test in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative primary breast cancer screened for the randomized, double-blind, phase III UNIRAD trial.

BACKGROUND: The purpose of this study was to evaluate the prognostic value of the multigene EndoPredict test in prospectively collected data of patients screened for the randomized, double-blind, phase III UNIRAD trial, which evaluated the addition of everolimus to adjuvant endocrine therapy in high-risk, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative early breast cancer. PATIENTS AND METHODS: Patients were classified into low or high risk according to the EPclin score, consisting of a 12-gene molecular score combined with tumor size and nodal status. Association of the EPclin score with disease-free survival (DFS) and distant metastasis-free survival (DMFS) was evaluated using Kaplan-Meier estimates. The independent prognostic added value of EPclin score was tested in a multivariate Cox model after adjusting on tumor characteristics. RESULTS: EndoPredict test results were available for 768 patients: 663 patients classified as EPclin high risk (EPCH) and 105 patients as EPclin low risk (EPCL). Median follow-up was 70 months (range 1-172 months). For the 429 EPCH randomized patients, there was no significant difference in DFS between treatment arms. The 60-month relapse rate for patients in the EPCL and EPCH groups was 0% and 7%, respectively. Hazard ratio (HR) supposing continuous EPclin score was 1.87 [95% confidence interval (CI) 1.4-2.5, P < 0.0001]. This prognostic effect remained significant when assessed in a Cox model adjusting on tumor size, number of positive nodes and tumor grade (HR 1.52, 95% CI 1.09-2.13, P = 0.0141). The 60-month DMFS for patients in the EPCL and EPCH groups was 100% and 94%, respectively (adjusted HR 8.10, 95% CI 1.1-59.1, P < 0.0001). CONCLUSIONS: The results confirm the value of EPclin score as an independent prognostic parameter in node-positive, hormone receptor-positive, HER2-negative early breast cancer patients receiving standard adjuvant treatment. EPclin score can be used to identify patients at higher risk of recurrence who may warrant additional systemic treatments.

Primary prophylaxis with mTOR inhibitor enhances T cell effector function and prevents heart transplant rejection during talimogene laherparepvec therapy of squamous cell carcinoma.

The application of mammalian target of rapamycin inhibition (mTORi) as primary prophylactic therapy to optimize T cell effector function while preserving allograft tolerance remains challenging. Here, we present a comprehensive two-step therapeutic approach in a male patient with metastatic cutaneous squamous cell carcinoma and heart transplantation followed with concomitant longitudinal analysis of systemic immunologic changes. In the first step, calcineurin inhibitor/ mycophenolic acid is replaced by the mTORi everolimus to achieve an improved effector T cell status with increased cytotoxic activity (perforin, granzyme), enhanced proliferation (Ki67) and upregulated activation markers (CD38, CD69). In the second step, talimogene laherparepvec (T-VEC) injection further enhances effector function by switching CD4 and CD8 cells from central memory to effector memory profiles, enhancing Th1 responses, and boosting cytotoxic and proliferative activities. In addition, cytokine release (IL-6, IL-18, sCD25, CCL-2, CCL-4) is enhanced and the frequency of circulating regulatory T cells is increased. Notably, no histologic signs of allograft rejection are observed in consecutive end-myocardial biopsies. These findings provide valuable insights into the dynamics of T cell activation and differentiation and suggest that timely initiation of mTORi-based primary prophylaxis may provide a dual benefit of revitalizing T cell function while maintaining allograft tolerance.

Comparison of the effect of Everolimus, Prednisolone, and a combination of both on experimentally induced peritoneal adhesions in rats.

Postoperative intra-abdominal adhesions represent a significant post-surgical problem. Its complications can cause a considerable clinical and cost burden. Herein, our study aimed to investigate the effect of Everolimus on peritoneal adhesion formation after inducing adhesions in rats. In this experimental study, adhesion bands were induced by intraperitoneal injection of 3 ml of 10% sterile talc solution in 64 male albino rats. The first group served as the control group. The second one received oral Prednisolone (1 mg/kg/day), the third received Everolimus (0.1 mg/kg/day), and group four received both drugs with similar dosages for four consecutive weeks. The formation of adhesion bands was qualitatively graded according to the Nair classification. The rats in the control group had extensive adhesions between the abdominal wall and the organs. Regarding substantial adhesion formation, 50% (8/16) of animals in the control group had substantial adhesions, while this rate in the groups receiving Prednisolone, Everolimus, and combination treatment was 31%, 31%, and 31%, respectively. Also, 68.75% (5/11) of the Prednisolone recipients had insubstantial adhesions, the same as Everolimus recipients, while in the combination group, 66.66% (10/15) rats had insubstantial adhesions. Everolimus demonstrated satisfactory results in reducing the rates of induced peritoneal adhesion in an experimental model, similar to Prednisolone and superior to a combination regime.

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 treatment enhances inhibitory immune checkpoint molecules' expression in monocyte-derived dendritic cells.

BACKGROUND: Antigen-specific T-cell immunity is provided by dendritic cells (DCs), which are specialized antigen-presenting cells. Furthermore, they establish a link between innate and adaptive immune responses. Currently, DC modification is a new approach for the therapy of several disorders. During solid organ transplantation, Everolimus, which is a mammalian target of rapamycin (mTOR) inhibitor, was initially utilized to suppress the immune system's functionality. Due to the intervention of Everolimus in various signaling pathways in cells and its modulatory properties on the immune system, this study aims to investigate the effect of treatment with Everolimus on the maturation and expression of immune checkpoint genes in monocyte-derived DCs. METHODS: To isolate monocytes from PBMCs, the CD14 marker was used via the MACS method. Monocytes were cultured and induced to differentiate into monocyte-derived DCs by utilizing GM-CSF and IL-4 cytokines. On the fifth day, immature DCs were treated with Everolimus and incubated for 24 h. On the sixth day, the flow cytometry technique was used to investigate the effect of Everolimus on the phenotypic characteristics of DCs. In the end, the expression of immune checkpoint genes in both the Everolimus-treated and untreated DCs groups was assessed using the real-time PCR method. RESULTS: The findings of this research demonstrated that the administration of Everolimus to DCs led to a notable rise in human leukocyte antigen (HLA)-DR expression and a decrease in CD11c expression. Furthermore, there was a significant increase in the expression of immune checkpoint molecules, namely CTLA-4, VISTA, PD-L1, and BTLA, in DCs treated with Everolimus. CONCLUSION: The findings of this study show that Everolimus can target DCs and affect their phenotype and function in order to shift them toward a partially tolerogenic state. However, additional research is required to gain a comprehensive understanding of the precise impact of Everolimus on the activation status of DCs.

Discovery of a Long Half-Life AURKA Inhibitor to Treat MYC-Amplified Solid Tumors as a Monotherapy and in Combination with Everolimus.

Aurora kinase inhibitors, such as alisertib, can destabilize MYC-family oncoproteins and have demonstrated compelling antitumor efficacy. In this study, we report 6K465, a novel pyrimidine-based Aurora A inhibitor, that reduces levels of c-MYC and N-MYC oncoproteins more potently than alisertib. In an analysis of the antiproliferative effect of 6K465, the sensitivities of small cell lung cancer (SCLC) and breast cancer cell lines to 6K465 were strongly associated with the protein levels of c-MYC and/or N-MYC. We also report DBPR728, an acyl-based prodrug of 6K465 bearing fewer hydrogen-bond donors, that exhibited 10-fold improved oral bioavailability. DBPR728 induced durable tumor regression of c-MYC- and/or N-MYC-overexpressing xenografts including SCLC, triple-negative breast cancer, hepatocellular carcinoma, and medulloblastoma using a 5-on-2-off or once-a-week dosing regimen on a 21-day cycle. A single oral dose of DBPR728 at 300 mg/kg induced c-MYC reduction and cell apoptosis in the tumor xenografts for more than 7 days. The inhibitory effect of DBPR728 at a reduced dosing frequency was attributed to its uniquely high tumor/plasma ratio (3.6-fold within 7 days) and the long tumor half-life of active moiety 6K465. Furthermore, DBPR728 was found to synergize with the mTOR inhibitor everolimus to suppress c-MYC- or N-MYC-driven SCLC. Collectively, these results suggest DBPR728 has the potential to treat cancers overexpressing c-MYC and/or N-MYC.

SYNERGY-Everolimus-Eluting Stent With a Bioabsorbable Polymer in ST-Elevation Myocardial Infarction: CLEAR SYNERGY OASIS-9 Registry.

Our objective was to evaluate the clinical effectiveness of the SYNERGY stent (Boston Scientific Corporation, Marlborough, Massachusetts) in patients with ST-elevation myocardial infarction (STEMI). The only drug-eluting stent approved for treatment of STEMI by the Food and Drug Administration is the Taxus stent (Boston Scientific) which is no longer commercially available, so further data are needed. The CLEAR (Colchicine and spironolactone in patients with myocardial infarction) SYNERGY stent registry was embedded into a larger randomized trial of patients with STEMI (n = 7,000), comparing colchicine versus placebo and spironolactone versus placebo. The primary outcome for the SYNERGY stent registry is major adverse cardiac events (MACE) as defined by cardiovascular death, recurrent MI, or unplanned ischemia-driven target vessel revascularization within 12 months. We estimated a MACE rate of 6.3% at 12 months after primary percutaneous coronary intervention for STEMI based on the Thrombectomy vs percutaneous coronary intervention alone in STEMI (TOTAL) trial. Success was defined as upper bound of confidence interval (CI) to be less than the performance goal of 9.45%. Overall, 733 patients were enrolled from 8 countries with a mean age 60 years, 19.4% diabetes mellitus, 41.3% anterior MI, and median door-to-balloon time of 72 minutes. The MACE rate was 4.8% (95% CI 3.2 to 6.3%) at 12 months which met the success criteria against performance goal of 9.45%. The rates of cardiovascular death, recurrent MI, or target vessel revascularization were 2.7%, 1.9%, 1.0%, respectively. The rates of acute definite stent thrombosis were 0.3%, subacute 0.4%, late 0.4%, and cumulative stent thrombosis of 1.1% at 12 months. In conclusion, the SYNERGY stent in STEMI performed well and was successful compared with the performance goal based on previous trials.

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.

MEK Inhibition for RASopathy-Associated Hypertrophic Cardiomyopathy: Clinical Application of a Basic Concept.

The term "RASopathies" designates a group of developmental syndromes that are caused by activating variants of the rat sarcoma virus protein (RAS)/mitogen-activated protein kinase (MAPK) cascade. The most prevalent clinical diagnosis is Noonan syndrome, and other, less prevalent conditions include Noonan syndrome with multiple lentigines, Costello syndrome, cardiofaciocutaneous syndrome, and others. Hypertrophic cardiomyopathy occurs in 10% of these patients and can be severe and life-threating. Recently, repurposing of medications inhibiting the RAS/MAPK on a compassionate use basis has emerged as a promising concept to improve the outcome of these patients. Herein, we specifically review the role of the RAS/MAPK pathway in RASopathy-associated cardiomyopathy, and discuss the role of small-molecule inhibition in the treatment of this condition. We describe how drug repurposing of trametinib (mitogen-activated protein/extracellular signal-regulated kinase inhibition) and sirolimus/everolimus (mammalian target of rapamycin inhibition) was performed, how genotype-specific therapies are chosen and followed, as well as initial outcomes from early case series. Finally, we lay out the challenges and opportunities for trials that aim to quantify the benefits of this approach.

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 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.

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.

Second-generation everolimus-eluting intracoronary stents: a comprehensive review of the clinical evidence.

Percutaneous coronary intervention with implantation of second-generation drug-eluting stents (DES) has emerged as a mainstay for the treatment of obstructive coronary artery disease given its beneficial impact on clinical outcomes in these patients. Everolimus-eluting stents (EES) are one of the most frequently implanted second-generation DES; their use for the treatment of a wide range of patients including those with complex coronary lesions is supported by compelling evidence. Although newer stent platforms such as biodegradable polymer DES may lower local vessel inflammation, their efficacy and safety have not yet surpassed that of Xience stents. This article summarizes the properties of the Xience family of EES and the evidence supporting their use across diverse patient demographics and coronary lesion morphologies.

Patients with coronary artery disease (CAD) often require treatment for symptoms caused by blockages in coronary arteries. In addition to medical therapy, available procedure options include either coronary artery bypass grafting, a major heart surgery or percutaneous coronary intervention (PCI) with stenting. PCI is a minimally invasive procedure where a metallic stent (a mesh made up of fine metallic network in a tube shape used to keep vessels open) is advanced over a wire through an artery to open the coronary artery blockage. Over the past few decades, improvements in procedure technique and stent material have made PCI a highly safe and efficacious procedure. A newer generation of stents, known as drug-eluting stents (DES), have been developed in which metallic struts are covered with a highly biocompatible polymer (a thin material coating over the metallic mesh) that releases drugs at the blockage site to prevent local cell growth in the vessel wall. Among the second-generation DES, Xience everolimus-eluting stents (EES) have shown better outcomes compared with earlier generations of stents. Another version of DES with biodegradable polymer coating is emerging but their advantage over EES remains uncertain. Currently, Xience EES are one of the most commonly used stents to treat CAD. This manuscript covers an in-depth review of clinical evidence on the performance of Xience stents in a diverse range patient populations.