mTOR Inhibitor Research Articles

Fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans.

Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse models. Previous work supports that C. elegans fmo-2 promotes longevity, stress resistance, and healthspan by rewiring endogenous metabolism. However, there are five C. elegans FMOs and five mammalian FMOs, and it is not known whether promoting longevity and health benefits is a conserved role of this gene family. Here, we report that expression of C. elegans fmo-4 promotes lifespan extension and paraquat stress resistance downstream of both dietary restriction and inhibition of mTOR. We find that overexpression of fmo-4 in just the hypodermis is sufficient for these benefits, and that this expression significantly modifies the transcriptome. By analyzing changes in gene expression, we find that genes related to calcium signaling are significantly altered downstream of fmo-4 expression. Highlighting the importance of calcium homeostasis in this pathway, fmo-4 overexpressing animals are sensitive to thapsigargin, an ER stressor that inhibits calcium flux from the cytosol to the ER lumen. This calcium/ fmo-4 interaction is solidified by data showing that modulating intracellular calcium with either small molecules or genetics can change expression of fmo-4 and/or interact with fmo-4 to affect lifespan and stress resistance. Further analysis supports a pathway where fmo-4 modulates calcium homeostasis downstream of activating transcription factor-6 ( atf-6 ), whose knockdown induces and requires fmo-4 expression. Together, our data identify fmo-4 as a longevity- promoting gene whose actions interact with known longevity pathways and calcium homeostasis.

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

BackgroundRenal 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 designWe 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.ResultsEY-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.ConclusionTaken 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.

Gedatolisib shows superior potency and efficacy versus single-node PI3K/AKT/mTOR inhibitors in breast cancer models

The PI3K, AKT, and mTOR (PAM) pathway is frequently dysregulated in breast cancer (BC) to accommodate high catabolic and anabolic activities driving tumor growth. Current therapeutic options for patients with hormone receptor (HR) + / HER2- advanced BC (ABC) include PAM inhibitors that selectively inhibit only one PAM pathway node, which can lead to drug resistance as cells rapidly adapt to maintain viability. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, may be more effective in BC cells than single-node PAM inhibitors by limiting adaptive resistances. By using multiple functional assays, a panel of BC cell lines was evaluated for their sensitivity to four different PAM inhibitors: gedatolisib (pan-PI3K/mTOR inhibitor), alpelisib (PI3Kα inhibitor), capivasertib (AKT inhibitor), and everolimus (mTORC1 inhibitor). Gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects regardless of the PAM pathway mutational status of the cell lines compared to the single-node PAM inhibitors. The higher efficacy of gedatolisib was confirmed in three-dimensional culture and in BC PDX models. Mechanistically, gedatolisib decreased cell survival, DNA replication, cell migration and invasion, protein synthesis, glucose consumption, lactate production, and oxygen consumption more effectively than the other PAM inhibitors tested. These results indicate that inhibition of multiple PAM pathway nodes by a pan-PI3K/mTOR inhibitor like gedatolisib may be more effective at inducing anti-tumor activity than single-node PAM inhibitors. A global Phase 3 study is currently evaluating gedatolisib plus fulvestrant with and without palbociclib in patients with HR+/HER2− ABC.

ISKNV Triggers AMPK/mTOR-Mediated Autophagy Signaling through Oxidative Stress, Inducing Antioxidant Enzyme Expression and Enhancing Viral Replication in GF-1 Cells.

Infectious spleen and kidney necrosis virus (ISKNV) infections can induce the process of host cellular autophagy but have rarely been identified within the molecular autophagy signaling pathway. In the present study, we demonstrated that ISKNV induces ROS-mediated oxidative stress signals for the induction of 5'AMP-activated protein kinase/mechanistic target of rapamycin kinase (AMPK/mTOR)-mediated autophagy and upregulation of host antioxidant enzymes in fish GF-1 cells. We also examined ISKNV-induced oxidative stress, finding that reactive oxidative species (ROS) increased by 1.5-fold and 2.5-fold from day 2 to day 3, respectively, as assessed by the H2DCFDA assay for tracing hydrogen peroxide (H2O2), which was blocked by NAC treatment in fish GF-1 cells. Furthermore, ISKNV infection was shown to trigger oxidative stress/Nrf2 signaling from day 1 to day 3; this event was then correlated with the upregulation of antioxidant enzymes such as Cu/ZnSOD and MnSOD and was blocked by the antioxidant NAC. Using an MDC assay, TEM analysis and autophagy marker LC3-II/I ratio, we found that ROS stress can regulate autophagosome formation within the induction of autophagy, which was inhibited by NAC treatment in GF-1 cells. Through signal analysis, we found that AMPK/mTOR flux was modulated through inhibition of mTOR and activation of AMPK, indicating phosphorylation levels of mTOR Ser 2448 and AMPK Thr 172 from day 1 to day 3; however, this process was reversed by NAC treatment, which also caused a reduction in virus titer (TCID50%) of up to 1000 times by day 3 in GF-1 cells. Thus, ISKNV-induced oxidative stress signaling is blocked by antioxidant NAC, which can also either suppress mTOR/AMPK autophagic signals or reduce viral replication. These findings may provide the basis for the creation of DNA control and treatment strategies.

Phase III Randomized, Placebo-Controlled Trial of Endocrine Therapy ± 1 Year of Everolimus in Patients With High-Risk, Hormone Receptor-Positive, Early-Stage Breast Cancer.

Phosphatidylinositol 3-kinase/AKT-serine threonine kinase/mammalian target of rapamycin (mTOR) pathway abnormalities contribute to endocrine resistance. Everolimus, an mTOR inhibitor, improved progression-free survival in hormone receptor-positive metastatic breast cancer (BC) when combined with endocrine therapy (ET). In this phase III randomized, placebo-controlled trial, we assessed the efficacy of everolimus + ET as adjuvant therapy in high-risk, hormone receptor-positive, human epidermal growth factor receptor 2-negative BC after adjuvant/neoadjuvant chemotherapy. Patients were randomly assigned 1:1 to physician's choice ET and 1 year of everolimus (10 mg orally once daily) or placebo stratified by risk group. The primary end point was invasive disease-free survival (IDFS) evaluated by a stratified log-rank test with the hazard ratio (HR) estimated by Cox regression. Subset analyses included preplanned evaluation by risk group and exploratory analyses by menopausal status and age. Secondary end points included overall survival (OS) and safety. Everolimus did not improve IDFS/OS when added to ET in patients with early-stage high-risk, hormone receptor-positive BC. One thousand and nine hundred thirty-nine patients were randomly assigned with 1,792 eligible for analysis. Overall, no benefit of everolimus was seen for IDFS (HR, 0.94 [95% CI, 0.77 to 1.14]) or OS (HR, 0.97 [95% CI, 0.75 to 1.26]). The assumption of proportional hazards was not met suggesting significant variability in the HR over time since the start of treatment. In an unplanned subgroup analysis among postmenopausal patients (N = 1,221), no difference in IDFS (HR, 1.08 [95% CI, 0.86 to 1.36]) or OS (HR, 1.19 [95% CI, 0.89 to 1.60]) was seen. In premenopausal patients (N = 571), everolimus improved both IDFS (HR, 0.64 [95% CI, 0.44 to 0.94]) and OS (HR, 0.49 [95% CI, 0.28 to 0.86]). Treatment completion rates were lower in the everolimus arm compared with placebo (48% v 73%) with higher grade 3 and 4 adverse events (35% v 7%). One year of adjuvant everolimus + ET did not improve overall outcomes. Subset analysis suggests mTOR inhibition as a possible target for patients who remain premenopausal after chemotherapy.

A comparative analysis of clinical outcomes between conversion to mTOR inhibitor and calcineurin inhibitor reduction in managing BK viremia among kidney transplant patients

BK virus-associated nephropathy (BKVAN) exerts a substantial impact on allograft survival, however, the absence of robust clinical evidence regarding treatment protocols adds to the complexity of managing this condition. This study aimed to compare the two treatment approaches. The study population consisted of patients who underwent kidney transplantation between January 2016 and June 2020 at two tertiary hospitals in Korea. Patients diagnosed with BK viremia were evaluated based on their initial viral load and the treatment methods. The ‘Reduction group’ involved dose reduction of tacrolimus while the ‘Conversion group’ included tacrolimus discontinuation and conversion to sirolimus. A total of 175 patients with an initial viral load (iVL) ≥ 3 on the log10 scale were evaluated within two iVL intervals (3–4 and 4–5). In the iVL 4–5 interval, the Reduction group showed potential effectiveness in terms of viral clearance without statistically significant differences. However, within the iVL 3–4 interval, the Reduction group demonstrated superior viral clearance and a lower incidence of biopsy-proven acute rejection (BPAR) than the Conversion group. The renal function over 12 months after BKV diagnosis showed no statistically significant difference. Reducing tacrolimus compared to converting to mTORi would be a more appropriate treatment approach for BK viral clearance in kidney transplantation. Further research is warranted in a large cohort of patients.

Arginine Regulates Skeletal Muscle Fiber Type Formation via mTOR Signaling Pathway.

The composition of skeletal muscle fiber types affects the quality of livestock meat and human athletic performance and health. L-arginine (Arg), a semi-essential amino acid, has been observed to promote the formation of slow-twitch muscle fibers in animal models. However, the precise molecular mechanisms are still unclear. This study investigates the role of Arg in skeletal muscle fiber composition and mitochondrial function through the mTOR signaling pathway. In vivo, 4-week C56BL/6J male mice were divided into three treatment groups and fed a basal diet supplemented with different concentrations of Arg in their drinking water. The trial lasted 7 weeks. The results show that Arg supplementation significantly improved endurance exercise performance, along with increased SDH enzyme activity and upregulated expression of the MyHC I, MyHC IIA, PGC-1α, and NRF1 genes in the gastrocnemius (GAS) and quadriceps (QUA) muscles compared to the control group. In addition, Arg activated the mTOR signaling pathway in the skeletal muscle of mice. In vitro experiments using cultured C2C12 myotubes demonstrated that Arg elevated the expression of slow-fiber genes (MyHC I and Tnnt1) as well as mitochondrial genes (PGC-1α, TFAM, MEF2C, and NRF1), whereas the effects of Arg were inhibited by the mTOR inhibitor rapamycin. In conclusion, these findings suggest that Arg modulates skeletal muscle fiber type towards slow-twitch fibers and enhances mitochondrial functions by upregulating gene expression through the mTOR signaling pathway.

Focal cortical dysplasia II caused by brain somatic mutation of IRS-1 is associated with ERK signaling pathway activation.

Somatic mutations have been identified in 10% to 63% of focal cortical dysplasia type II samples, primarily linked to the mTOR pathway. When the causative genetic mutations are not identified, this opens the possibility of discovering new pathogenic genes or pathways that could be contributing to the condition. In our previous study, we identified a novel candidate pathogenic somatic variant of IRS-1 c.1791dupG in the brain tissue of a child with focal cortical dysplasia type II. This study further explored the variant's role in causing type II focal cortical dysplasia through in vitro overexpression in 293T and SH-SY5Y cells and in vivo evaluation via in utero electroporation in fetal brains, assessing effects on neuronal migration, morphology, and network integrity. It was found that the mutant IRS-1 variant led to hyperactivity of p-ERK, increased cell volume, and was predominantly associated with the MAPK signaling pathway. In vivo, the IRS-1 c.1791dupG variant induced abnormal neuron migration, cytomegaly, and network hyperexcitability. Notably, the ERK inhibitor GDC-0994, rather than the mTOR inhibitor rapamycin, effectively rescued the neuronal defects. This study directly highlighted the ERK signaling pathway's role in the pathogenesis of focal cortical dysplasia II and provided a new therapeutic target for cases of focal cortical dysplasia II that are not treatable by rapamycin analogs.

Mitochondrial Dysfunction in Systemic Lupus Erythematosus with a Focus on Lupus Nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting mostly women of child-bearing age. Immune dysfunction in SLE results from disrupted apoptosis which lead to an unregulated interferon (IFN) stimulation and the production of autoantibodies, leading to immune complex formation, complement activation, and organ damage. Lupus nephritis (LN) is a common and severe complication of SLE, impacting approximately 30% to 40% of SLE patients. Recent studies have demonstrated an alteration in mitochondrial homeostasis in SLE patients. Mitochondrial dysfunction contributes significantly to SLE pathogenesis by enhancing type 1 IFN production through various pathways involving neutrophils, platelets, and T cells. Defective mitophagy, the process of clearing damaged mitochondria, exacerbates this cycle, leading to increased immune dysregulation. In this review, we aim to detail the physiopathological link between mitochondrial dysfunction and disease activity in SLE. Additionally, we will explore the potential role of mitochondria as biomarkers and therapeutic targets in SLE, with a specific focus on LN. In LN, mitochondrial abnormalities are observed in renal cells, correlating with disease progression and renal fibrosis. Studies exploring cell-free mitochondrial DNA as a biomarker in SLE and LN have shown promising but preliminary results, necessitating further validation and standardization. Therapeutically targeting mitochondrial dysfunction in SLE, using drugs like metformin or mTOR inhibitors, shows potential in modulating immune responses and improving clinical outcomes. The interplay between mitochondria, immune dysregulation, and renal involvement in SLE and LN underscores the need for comprehensive research and innovative therapeutic strategies. Understanding mitochondrial dynamics and their impact on immune responses offers promising avenues for developing personalized treatments and non-invasive biomarkers, ultimately improving outcomes for LN patients.

MTOR: Its Critical Role in Metabolic Diseases, Cancer, and the Aging Process.

The mammalian target of rapamycin (mTOR) is a pivotal regulator, integrating diverse environmental signals to control fundamental cellular functions, such as protein synthesis, cell growth, survival, and apoptosis. Embedded in a complex network of signaling pathways, mTOR dysregulation is implicated in the onset and progression of a range of human diseases, including metabolic disorders such as diabetes and cardiovascular diseases, as well as various cancers. mTOR also has a notable role in aging. Given its extensive biological impact, mTOR signaling is a prime therapeutic target for addressing these complex conditions. The development of mTOR inhibitors has proven advantageous in numerous research domains. This review delves into the significance of mTOR signaling, highlighting the critical components of this intricate network that contribute to disease. Additionally, it addresses the latest findings on mTOR inhibitors and their clinical implications. The review also emphasizes the importance of developing more effective next-generation mTOR inhibitors with dual functions to efficiently target the mTOR pathways. A comprehensive understanding of mTOR signaling will enable the development of effective therapeutic strategies for managing diseases associated with mTOR dysregulation.

Racial differences in genomic profiles and targeted treatment use in ER+ HER2- metastatic breast cancer.

1017 Background: Despite well-documented disparities in survival between patients (pts) with Black and White racial backgrounds, there are limited data on racial equity of targeted treatment use for pts with estrogen receptor positive, HER2 negative (ER+/HER2-) metastatic breast cancer (MBC). We evaluated differences in circulating tumor DNA (ctDNA) genomic alterations in Black and White pts with ER+/HER2- MBC. Methods: This retrospective cohort study analyzed a database of 1327 pts with MBC who were treated at Washington University in St. Louis, Massachusetts General Hospital, and Northwestern University and had ctDNA testing through the commercially available Guardant360 assay. Progesterone receptor (PR) status of the ER+/HER2- pts was identified to categorize pts as ER+/PR+/HER2- (N=60 Black, N=435 White) and ER+/PR-/HER2- (N=24 Black, N=237 White). Pts were evaluated by self-reported race and use of PIK3CA, CDK4/6, and mTOR inhibitors, either through clinical trial enrollment or after FDA approval. A multivariate logistic analysis was performed to determine genomic and prognostic differences by race. Overall survival (OS) from time of first ctDNA testing was determined by subgroup. Results: Black pts with PIK3CA single nucleotide variants (SNVs) were significantly less likely to receive targeted therapy than White pts (5.9%, 1/17 pts vs. 28.8%, 45/156 pts, Fisher’s exact test p=0.045), while there was no difference in use of CDK4/6 or mTOR inhibitors. None of the Black pts with PIK3CA mutations were enrolled in clinical trials vs. 11.5% of White pts. Black pts with ER+/PR+/HER2- tumors were more likely to have CCND1 copy number variation (cnv) (odds ratio (OR) 2.4, 95% confidence interval (CI) 1.1-5.4, p=0.030) when compared with White pts. Black pts with PR- tumors were more likely to have de novo metastatic disease (OR 3.4, 95% CI 1.1-10.8, p=0.034) and GATA3SNV (OR 3.5, 95% CI 1.04-11.6, p=0.044). KRAS cnv was more common in White PR- pts (HR 2.9, CI 1.02-8.3, p=0.045). There were significant differences in OS between Black and White pts with ER+/PR+/HER2- tumors (median OS of 25 vs. 32 months, respectively) and for Black and White pts with ER+/PR-/HER2- MBC, (median OS of 9.1 vs. 21 months, respectively, log-rank test p=0.00093). Conclusions: To our knowledge, this is the largest clinicogenomic dataset comparing genomic alterations and targeted treatment use by race in MBC. We identified significant disparities in the use of PI3K inhibitors for Black pts while other drugs that do not require genomic profiling were similarly utilized. OS and clinical trial enrollment disparities are consistent with prior findings and must continue to be addressed. Further research is needed to validate these findings, elucidate the factors contributing to these disparities, and explore interventions to improve racial disparities in use of precision medicine therapies and enrollment in clinical trials.

PD-L1 regulates tumor proliferation and T-cell function in NF2-associated meningiomas.

Programmed death-ligand 1 (PD-L1) expression is an immune evasion mechanism that has been demonstrated in many tumors and is commonly associated with a poor prognosis. Over the years, anti-PD-L1 agents have gained attention as novel anticancer therapeutics that induce durable tumor regression in numerous malignancies. They may be a new treatment choice for neurofibromatosis type 2 (NF2) patients. The aims of this study were to detect the expression of PD-L1 in NF2-associated meningiomas, explore the effect of PD-L1 downregulation on tumor cell characteristics and T-cell functions, and investigate the possible pathways that regulate PD-L1 expression to further dissect the possible mechanism of immune suppression in NF2 tumors and to provide new treatment options for NF2 patients. PD-L1 is heterogeneously expressed in NF2-associated meningiomas. After PD-L1 knockdown in NF2-associated meningioma cells, tumor cell proliferation was significantly inhibited, and the apoptosis rate was elevated. When T cells were cocultured with siPD-L1-transfected NF2-associated meningioma cells, the expression of CD69 on both CD4+ and CD8+ T cells was partly reversed, and the capacity of CD8+ T cells to kill siPD-L1-transfected tumor cells was partly restored. Results also showed that the PI3K-AKT-mTOR pathway regulates PD-L1 expression, and the mTOR inhibitor rapamycin rapidly and persistently suppresses PD-L1 expression. Invivo experimental results suggested that anti-PD-L1 antibody may have a synergetic effect with the mTOR inhibitor in reducing tumor cell proliferation and that reduced PD-L1 expression could contribute to antitumor efficacy. Targeting PD-L1 could be helpful for restoring the function of tumor-infiltrating lymphocytes and inducing apoptosis to inhibit tumor proliferation in NF2-associated meningiomas. Dissecting the mechanisms of the PD-L1-driven tumorigenesis of NF2-associated meningioma will help to improve our understanding of the mechanisms underlying tumor progression and could facilitate further refinement of current therapies to improve the treatment of NF2 patients.

Antitumor activity of nab-sirolimus versus mTOR inhibitors temsirolimus, sirolimus, and everolimus in A549 NSCLC xenografts.

e15096 Background: The mTORC1 pathway, often activated by mutations in genes like TSC1, TSC2, PIK3CA, PTEN, STK11, and KEAP1, plays a pivotal role in cancer progression; in some settings, alterations in STK11 and KEAP1 may be associated with treatment resistance and poor prognosis. Despite the broad importance of the mTORC1 pathway in cancer cell growth and survival, mTOR inhibitors (mTORis) temsirolimus (TEM), sirolimus (SIRO), and everolimus (EVE) have limited clinical application in the cancer setting. nab-Sirolimus, an injectable form of albumin-bound SIRO that leverages unique transport properties of albumin known to increase tumor drug accumulation, is approved for treatment of malignant PEComa. Here, we report the antitumor activity of nab-sirolimus in comparison to other mTORis in A549 NSCLC ( KRAS G12S, STK11 Q37*, and KEAP1 G333C) xenografts, and the correlation of antitumor activity, tumor PK profile, and mTOR pathway suppression. Methods: Athymic mice bearing subcutaneous A549 NSCLC xenografts were treated with either saline or equal weekly doses of nab-sirolimus (administered intravenously [IV]; 5 or 15 mg/kg/week), TEM (IV; 5 mg/kg/week), or SIRO or EVE (both oral; 15 mg/kg/week). Tumors were harvested at different time points after mTORi treatment and analyzed for tumor drug levels (LC-MS/MS) and mTOR pathway biomarkers (pS6 and p4EBP1) via western blot (WB). Results: In A549 xenografts, nab-sirolimus (IV) treatment resulted in significantly greater suppression of tumor growth compared with TEM (IV), SIRO (oral), and EVE (oral) (Table). Average intratumoral drug concentrations 24 hours after IV mTORi treatment were significantly higher with nab-sirolimus (420-539 ng/g) compared with TEM (TEM [parent] 34.9 ng/g; SIRO [active metabolite measured from TEM] 13.2 ng/g) and compared with 7-day steady-state concentrations for oral SIRO (17 ng/g) and EVE (10 ng/g). WB confirmed that nab-sirolimus consistently inhibited mTOR targets pS6 and p4EBP1, whereas TEM, SIRO, and EVE were less effective. Conclusions: nab-Sirolimus resulted in significantly higher intratumoral drug concentration, stronger inhibition of mTOR targets, and greater antitumor activity compared to other IV and oral mTORis in a KRAS/STK11/KEAP1 mutated NSCLC xenograft model. These results support further clinical evaluation of nab-sirolimus as a single agent or in combination with other therapeutic agents in oncology. [Table: see text]

Clinical efficacy and safety of mTOR inhibitor in patients with advanced unresectable epithelioid hemangioendothelioma (EHE).

e23540 Background: Epithelioidhemangioendothelioma is a rare vascular soft tissue sarcoma, 90% of which contains WWTR1-CAMTA1 fusion gene. Studies have shown that mTOR inhibitors may have a certain effect on EHE patients. Methods: A retrospective analysis of the efficacy and safety of mTOR inhibitor everolimus in the treatment of advanced unresectable epithelioidhemangioendothelioma in our hospital in the past one year. Patients with advanced epithelioidhemangioendothelioma (EHE) confirmed by consultation in the pathological center received this regimen, everolimus 10mg, once a day orally for 28 days, every 4 weeks as a treatment cycle until the disease progressed or showed intolerable toxicity. Results: There were 3 females and 5 males, and their average age was 41.8 years old. All of them were unresectable with liver, lung, and shoulder primary lesions, and lungs, liver, pleura and bone metastasis. Ki67 is from 2% to 10%. 3 patients who received molecular pathological examination were all identified the WWTR1-CAMTA1 fusion gene, and one of them also had EWSR1 chromosome translocation. Previous antineoplastic treatments included gemcitabine, cisplatin, albumin paclitaxel, epirubicin, dacarbazine chemotherapy, anlotinib targeting, and toripalimab immune therapy. 5 patients were treated with first-line drugs, 2 cases with second-line and 1 case with third-line. In terms of overall efficacy, 1 case was not evaluated, 2 cases lost follow-up due to poor tolerance and economic factors, 2 cases were included in the clinical trails after reaching tumor control for 3 months, and the other 3 cases achieved tumor remission with a median PFS of 11.0 months, and the best curative effect was SDa. Severe oral mucositis can greatly affect the medication experience of patients, which can be relieved after symptomatic medication, or when interstitial pneumonia and hemogram decrease significantly, we can consider the oral administration of everolimus 5mg. Conclusions: At present, there is no standard treatment for EHE, we can roughly see the efficacy of mTOR inhibitor everolimus in some EHE patients, mainly disease stability. Emphasis on side effect management, improve tolerance, and systematic treatment of EHE needs to be further explored.

Sequential use of PI3K/AKT/mTOR pathway inhibitors alpelisib and everolimus in patients with hormone receptor-positive (HR+) metastatic breast cancer.

1057 Background: Alpelisib (A), a PI3K inhibitor, is FDA-approved with fulvestrant for patients with PIK3CA-mutated HR+/HER2- MBC. Everolimus (E), an mTOR inhibitor, is FDA-approved with endocrine therapy (ET) regardless of PI3K/AKT/mTOR pathway mutation status. The efficacy and safety of sequential treatment with these pathway inhibitors is not well described. Methods: This single-center retrospective cohort study identified patients with HR+/HER2- MBC treated with both A and E sequentially, each in combination with ET, in either order from 2012-2023. We collected information on patient demographics, clinical and pathologic characteristics, treatment history, treatment adverse effects, and dates of disease progression (defined as radiographic or clinical progression leading to treatment change) from medical records. Patients treated first with E then A were categorized as Group 1 and those first with A then E as Group 2. Rates of prior CDK4/6i use were compared between groups with the chi-square test; differences in prior lines of therapy were assessed with the Wilcoxon rank sum test. Median PFS1 (PFS on 1st pathway inhibitor) and PFS2 (PFS on 2nd pathway inhibitor) were computed using the Kaplan-Meier method and the impact of prior treatment on PFS2 between groups was compared using Cox proportional hazards analysis. Results: 115 patients with HR+/HER2- MBC were included in this study. There were 62 (54%) patients in Group 1 and 53 (46%) patients in Group 2. Median prior lines of therapy in the metastatic setting for Group 1 was 4 (range 1-13) and for Group 2 was 3 (range 1-8; p=0.08). Median number of intervening therapies between E and A in Group 1 was 2 (range 0-8) and between A and E in Group 2 was 0 (range 0-4; p<0.001). 34/62 (55%) patients in Group 1 and 45/53 (85%) in Group 2 received prior CDK4/6i ( p<0.001). In Group 1, 49/62 (79%) of patients discontinued E for progression and 13 (21%) discontinued for toxicity. Median PFS1 was 9.2 months (95%CI 5.5-13.0) and PFS2 was 5.0 months (95%CI 4.6-7.9). In Group 2, 34/53 (64%) of patients discontinued A for progression and 19 (36%) discontinued for toxicity. Median PFS1 was 9.3 months (95%CI 7.0-14.0) and PFS2 was 3.5 months (95%CI 3.0-4.1). After adjusting for prior CDK4/6i use and prior lines of therapy (total and intervening), PFS2 was not significantly different between the groups. Genomic information will be included at the time of presentation. Conclusions: This is the largest retrospective study to-date of patients treated sequentially with PI3K/AKT/mTOR pathway inhibitors. PFS was longer with the 1st pathway inhibitor than with the 2nd regardless of sequence. This has implications for optimal integration of capivasertib, an AKT inhibitor, into HR+ MBC treatment. Prospective studies testing optimal sequencing are needed.

Regulation of the ubiquitin-proteasome system and proteostasis with mTOR inhibition in cytochrome c oxidase deficiency

Regulation of the ubiquitin-proteasome system and proteostasis with mTOR inhibition in cytochrome c oxidase deficiency

MTOR Inhibition by Drug-Coated Balloon

mTOR Inhibition by Drug-Coated Balloon

Fulvestrant with or without the cyclin-dependent kinase 7 (CDK7) inhibitor samuraciclib in advanced hormone receptor positive (HR+) breast cancer after CDK4/6 inhibition: Phase II SUMIT-BC study.

TPS1126 Background: CDK7 enhances oncogene transcription and upregulates anti-apoptotic genes, accelerates the cell cycle via CDK phosphorylation, and activates estrogen receptors (ERs) that can drive resistance to hormonal therapy in cancer. CDK7 inhibition therefore represents a potential anticancer strategy in cancer. Samuraciclib (CT7001) is a small molecule, ATP competitive, selective oral inhibitor of CDK7 that inhibits these key effects of CDK7. Samuraciclib had a favorable safety profile and showed clinical activity when combined with fulvestrant in a phase I study in patients with HR+/HER2− advanced breast cancer (BC) who had previously been treated with a CDK4/6 inhibitor. Patients with no detectable TP53 mutation in ctDNA at baseline and those without baseline liver metastases appeared to have better outcomes. The instant release capsule formulation of samuraciclib used in this study released large amounts of samuraciclib high in the gastrointestinal (GI) tract, which may have contributed to the observed low grade GI adverse events, which were managed with prophylaxis and counselling (1). Methods: A phase 2 open-label randomized study has been designed to evaluate the efficacy, safety, pharmacokinetics (PK), and quality of Life (QoL) of samuraciclib combined with fulvestrant compared to fulvestrant monotherapy (SUMIT-BC; NCT05963984). Eligible patients (n=60) are ≥18 years, have histologically or cytologically confirmed ER+/HER2− advanced or metastatic BC not amenable to resection or radiotherapy of curative intent, have received an aromatase inhibitor combined with a CDK4/6 inhibitor in the adjuvant or advanced setting, are receiving a luteinizing hormone-releasing hormone agonist if pre/perimenopausal, and have RECIST v1.1 evaluable disease. Prior SERD, mTOR inhibition, or chemotherapy for advanced BC are not permitted. All patients undergo baseline Guardant360 ctDNA analysis to establish TP53 and other mutation status. Patients are randomized 1:1:1 to fulvestrant alone (500 mg IM administered on days 1, 15 and 29 and then monthly), fulvestrant and samuraciclib 240 mg QD, or fulvestrant and samuraciclib 360 mg QD. In preparation for phase 3 development, a novel single tablet formulation of samuraciclib is used, which may enhance GI tolerability. Patients undergo RECIST v1.1 evaluation at baseline, every 8 weeks until week 48, and every 12 weeks thereafter. The primary endpoint is clinical benefit response at 24 weeks. Secondary endpoints are tolerability, progression free survival, overall response rate, duration of response, and PK of fulvestrant and samuraciclib. QoL (Functional Assessment of Cancer Therapy - Breast questionnaire [FACT-B]) and correlations between TP53 mutation and efficacy/safety will be assessed. 1. Coombes et al. Nature Comms 2023. Clinical trial information: NCT05963984 .

N2M2/NOA-20: Phase I/IIa umbrella trial of molecularly matched targeted therapies plus radiotherapy in patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation.

2000 Background: Patients with glioblastoma without MGMT promoter hypermethylation are unlikely to benefit from temozolomide (TMZ). Trials aiming at replacing TMZ with targeted agents in not molecularly selected patient populations have failed. Methods: This phase I/IIa umbrella trial aimed at showing safety, feasibility, and preliminary efficacy of targeted compounds in addition to standard radiotherapy initiated within 42 days postoperatively. Molecular diagnostics and bioinformatic evaluation are performed within 28 days after surgery. Stratification for treatment takes place in five subtrials, including alectinib, idasanutlin, palbociclib, vismogedib and temsirolimus, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asunercept and TMZ as standard of care. Primary objective of the phase I parts of the trial was dose finding or dose validation. In the phase IIa trials, centrally determined progression-free survival at six months (PFS-6) is used as endpoint for efficacy with interim analyses for futility ( H0: p=0.231). Results: From May 2018 through July 2022, 301 patients were enrolled and 228 treated in 13 German NOA sites. The alectinib and vismodegib subtrials were closed since no molecularly matching patients were accrued; the idasanutlin subtrial was closed prior to the optimal dose at nine patients at discretion of the company providing the drug. The TMZ subtrial showed a PFS-6 of 18.52% (10/54 patients) (p=0.831) and a median overall survival (OS) of 12.1 months. Asunercept: PFS-6 of 15.4% (4/26) (p=0.8825) and OS of 12.8 months. Atezolizumab: PFS-6 of 21.4% (9/42) (p=0.660) and OS of 11.7 months. Palbociclib with patients demonstrating CDK4 amplification or CDKN2A/B codeletion: PFS-6 of 24.4% (10/41) (p=0.4823) and OS of 12.6 months. Temsirolimus with patients demonstrating mTOR activation: PFS-6 of 39.1% (18/46) (p=0.0109) and OS of 15.4 months. The regimen-limiting toxicity (RLT)-rate is 34.8%, which is insignificantly above the predefined unacceptable rate for RLTs of 30%. Most RLTs had severity grade 3, one RLT had severity grade 4. No RLTs resulted in death. Conclusions: N2M2 allows for elaborate molecular testing being integrated into the treatment decision and efficient determination of treatment activity for patients with newly diagnosed glioblastoma. There is clinical activity of temsirolimus in patients with tumors harboring an activated mTOR pathway although this is not positively prognostic without mTOR inhibition; there is no clinical activity for asunercept and atezolizumab in not molecularly selected patients and also palbociclib in molecularly selected patients. Clinical trial information: NCT03158389 .

Revealing ANXA6 as a Novel Autophagy-related Target for Pre-eclampsia Based on the Machine Learning

Background: Preeclampsia (PE) is a severe pregnancy complication associated with autophagy. Objective: This research sought to uncover autophagy-related genes in pre-eclampsia through bioinformatics and machine learning. Methods: GSE75010 from the GEO series was subjected to WGCNA to identify key modular genes in PE. Autophagy genes retrieved from the THANATOS overlapped with the modular genes to yield PErelated autophagy genes. Furthermore, the crucial step involved the utilization of two machine learning algorithms (LASSO and SVM-RFE) for dimensionality reduction. The candidate gene was further verified by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. Preliminary experiments were conducted on HTR-8/SVneo cell lines to explore the role of candidate genes in autophagy regulation. Results: WGCNA identified 291 genes from 5 hubs, and after overlapping with 1087 autophagy-related genes obtained from THANATOS, 42 PE-related ARGs were identified. ANXA6 was recognized as a potential target through SVM-RFE and LASSO analyses. The mRNA and protein expression of ANXA6 were verified in placenta samples. In HTR8/SVneo cells, modulating ANXA6 expression altered autophagy levels. Knocking down ANXA6 resulted in an anti-autophagy effect, which was reversed by treatment with CAL101, an inhibitor of PI3K, Akt, and mTOR. Conclusion: We observed that ANXA6 may serve as a possible PE action target and that autophagy may be crucial to the pathogenesis of PE.