mTOR Inhibitor Research Articles

Oroxylin A-induced Trained Immunity Promotes LC3-associated Phagocytosis in Macrophage in Protecting Mice Against Sepsis.

Sepsis is defined as a dysregulated host response to infection that leads to multiorgan failure. Innate immune memory, i.e., "trained immunity", can result in stronger immune responses and provide protection against various infections. Many biological agents, including β-glucan, can induce trained immunity, but these stimuli may cause uncontrolled inflammation. Oroxylin A (OA) is an active flavonoid compound that is derived from Scutellaria baicalensis.OA is an agonist for inducing trained immunity in vivo and in vitro, and β-glucan was used as a positive control. The protective effects of OA-induced trained immunity were evaluated in mouse models that were established by either lipopolysaccharide (LPS) administration or caecal ligation and puncture (CLP). The expression of inflammatory factors and signaling pathway components involved in trained immunity was evaluated in vitro using qRT‒PCR, western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). Flow cytometry and confocal microscopy were used to examine reactive oxygen species (ROS) levels and phagocytosis in trained macrophages. A PCR array was used to screen genes that were differentially expressed in trained macrophages.Here, we revealed that OA alleviated sepsis via trained immunity. OA-treated macrophages displayed increased glycolysis and mTOR phosphorylation, and mTOR inhibitors suppressed OA-induced trained immunity by effectively reprogramming macrophages. The PCR array revealed key genes in the mTOR signaling pathway in OA-treated macrophages. Furthermore, OA targeted the Dectin-1-syk axis to promote LC3-associated phagocytosis (LAP) by trained macrophages, thereby enhancing the ability of these macrophages to protect against infection. This ability could be transferred to a new host via the adoptive transfer of peritoneal macrophages.This study is the first to provide new insights into the potential of OA-induced trained immunity to be used as a strategy to protect mice against sepsis by promoting LAP by macrophages.

MTOR promotes an inflammatory response through the HIF1 signaling pathway in ulcerative colitis

The imbalance between T helper cell 17 （Th17）and regulatory T cells (Treg) cells leading to inflammation has an important role in the pathogenesis of ulcerative colitis (UC). Mammalian target of rapamycin (mTOR) can regulate the differentiation of T cells, but the specific pathway leading mTOR to regulate Th17/Treg cells in UC remains unclear. Our aim with this study was to investigate the effects of mTOR overexpression and silencing on the hypoxia inducible factor-1α (HIF-1α) − Th17/Treg signaling pathway. To mimic a human study, we established a colon cancer epithelial cell line (HT-29) co-culture system with human CD4+ T cells, and we treated the cells with TNF-α. We observed the effects of mTOR on the HIF-Th17/Treg signaling pathway to determine whether mTOR is involved in the regulatory mechanism.Under the stimulation of TNF-α, the levels of HIF-1α in CD4+T cells were increased in the HT-29 co-culture with CD4+ T cells, promoting glycolysis, increasing the Th17 proportion, decreasing the Treg proportion, increasing the pro-inflammatory factors levels, and decreasing the anti-inflammatory factors levels. Moreover, after mTOR silencing, the HIF-1α level and cell glycolysis levels decreased, Th17 cell differentiation decreased, the pro-inflammatory factor levels decreased, and the anti-inflammatory factor levels increased. In contrast, mTOR overexpression lead to the opposite results.mTOR promotes inflammation by regulating the HIF signaling pathway during UC, and silencing mTOR may alleviate inflammation. An mTOR inhibitor is a potential therapeutic target for UC treatment.

Cannabidiol (Epidiolex)

CADTH recommends that Epidiolex should be reimbursed by public drug plans as adjunctive therapy for the treatment of seizures associated with tuberous sclerosis complex (TSC) in patients aged 2 years and older if certain conditions are met. Epidiolex should only be covered for the treatment of patients who are aged 2 years and older with seizures caused by TSC despite receiving other antiseizure medications. Epidiolex should only be reimbursed in patients who have at least 8 seizures over a 28-day period that are not controlled despite receiving 2 or more antiseizure medications if it is prescribed by physicians with experience in the diagnosis and management of patients living with TSC and if the cost of Epidiolex is reduced. Epidiolex should not be reimbursed for use in combination with mTOR inhibitors or recreational or medicinal cannabis.

NRASQ61R mutation drives elevated angiopoietin-2 expression in human endothelial cells and a genetic mouse model.

Angiopoietin-2 (Ang-2) is increased in the blood of patients with kaposiform lymphangiomatosis (KLA) and kaposiform hemangioendothelioma (KHE). While the genetic causes of KHE are not clear, a somatic activating NRASQ61R mutation has been found in the lesions of KLA patients. Our study tested the hypothesis that the NRASQ61R mutation drives elevated Ang-2 expression in endothelial cells. Ang-2 was measured in human endothelial progenitor cells (EPC) expressing NRASQ61R and a genetic mouse model with endothelial targeted NRASQ61R. To determine the signaling pathways driving Ang-2, NRASQ61R EPC were treated with signaling pathway inhibitors. Ang-2 levels were increased in EPC expressing NRASQ61R compared to NRASWT by Western blot analysis of cell lysates and ELISA of the cell culture media. Ang-2 levels were elevated in the blood of NRASQ61R mutant mice. NRASQ61R mutant mice also had reduced platelet counts and splenomegaly with hypervascular lesions, like some KLA patients. mTOR inhibitor rapamycin attenuated Ang-2 expression by NRASQ61R EPC. However, MEK1/2 inhibitor trametinib was more effective blocking increases in Ang-2. Our studies show that the NRASQ61R mutation in endothelial cells induces Ang-2 expression in vitro and in vivo. In cultured human endothelial cells, NRASQ61R drives elevated Ang-2 through MAP kinase and mTOR-dependent signaling pathways.

Chordoma Genetic Aberrations and Targeted Therapies Panorama: A Systematic Literature Review.

Background: Chordomas pose a challenge in treatment due to their local invasiveness, high recurrence, and potential lethality. Despite being slow-growing and rarely metastasizing, these tumors often resist conventional chemotherapies (CTs) and radiotherapies (RTs), making surgical resection a crucial intervention. However, achieving radical resection for chordomas is seldom possible, presenting therapeutic challenges. The accurate diagnosis of these tumors is vital for their distinct prognoses, yet differentiation is hindered by overlapping radiological and histopathological features. Fortunately, recent molecular and genetic studies, including extracranial location analysis, offer valuable insights for precise diagnosis. This literature review delves into the genetic aberrations and molecular biology of chordomas, aiming to provide an overview of more successful therapeutic strategies. Methods: A systematic search was conducted across major medical databases (PubMed, Embase, and Cochrane Library) up to 28 January 2023. The search strategy utilized relevant Medical Subject Heading (MeSH) terms and keywords related to "chordomas", "molecular biology", "gene aberrations", and "target therapies". The studies included in this review consist of preclinical cell studies, case reports, case series, randomized controlled trials, non-randomized controlled trials, and cohort studies reporting on genetic and biological aberrations in chordomas. Results: Of the initial 297 articles identified, 40 articles were included in the article. Two tables highlighted clinical studies and ongoing clinical trials, encompassing 18 and 22 studies, respectively. The clinical studies involved 185 patients diagnosed with chordomas. The tumor sites were predominantly sacral (n = 8, 44.4%), followed by clivus (n = 7, 38.9%) and lumbar spine (n = 3, 16.7%). Primary treatments preceding targeted therapies included surgery (n = 10, 55.6%), RT (n = 9, 50.0%), and systemic treatments (n = 7, 38.9%). Various agents targeting specific molecular pathways were analyzed in the studies, such as imatinib (a tyrosine kinase inhibitor), erlotinib, and bevacizumab, which target EGFR/VEGFR. Common adverse events included fatigue (47.1%), skin reactions (32.4%), hypertension (23.5%), diarrhea (17.6%), and thyroid abnormalities (5.9%). Clinical outcomes were systematically assessed based on progression-free survival (PFS), overall survival (OS), and tumor response evaluated using RECIST or CHOI criteria. Notably, stable disease (SD) occurred in 58.1% of cases, and partial responses (PRs) were observed in 28.2% of patients, while 13.7% experienced disease progression (PD) despite targeted therapy. Among the 22 clinical trials included in the analysis, Phase II trials were the most prevalent (40.9%), followed by I-II trials (31.8%) and Phase I trials (27.3%). PD-1 inhibitors were the most frequently utilized, appearing in 50% of the trials, followed by PD-L1 inhibitors (36.4%), CTLA-4 inhibitors (22.7%), and mTOR inhibitors (13.6%). Conclusions: This systematic review provides an extensive overview of the state of targeted therapy for chordomas, highlighting their potential to stabilize the illness and enhance clinical outcomes.

Disparities in PI3K/mTOR inhibitor use, toxicities, and outcomes among patients with metastatic breast cancer.

Novel agents such as PI3K and mTOR inhibitors (PI3K/mTORi) have expanded treatment options in metastatic breast cancer (MBC). Nevertheless, mortality rates remain disproportionately high for Black patients and patients with lower socioeconomic status. Furthermore, clinical trials for these novel agents lacked diversity, so their toxicity profile in minority populations is uncertain. We conducted a retrospective analysis of EHR-derived data from the Flatiron Health Database for patients with HR+, HER2- MBC. Multivariable logistic regression was used to evaluate factors associated with PI3K/mTORi use and toxicity outcomes. A total of 9169 patients with MBC were included in our analysis, of which 1780 (19.4%) received a PI3K/mTORi. We estimated the conditional total effect of insurance through Medicaid, and found lower odds of use of PI3K/mTORi among patients on Medicaid compared to those with commercial insurance (OR 0.73, 95% CI 0.54-0.99, p = 0.049). Odds of PI3K/mTORi use were higher for patients treated at an academic center (OR 1.28, CI 1.06-1.55, p = 0.01). Modeled as a controlled direct effect, Black/African American (Black/AA) race had no impact on odds of PI3K/mTOR use. Black/AA patients had twice the odds of developing hyperglycemia on PI3K/mTORi compared to White patients (OR 2.02, CI 1.24-3.39, p < 0.01). This analysis of real-world data suggests that the use of PI3K/mTORi is influenced by socioeconomic factors. We also found racial disparities in toxicity outcomes, with Black/AA patients having twice the risk of hyperglycemia. Our findings call for greater efforts to ensure access to novel treatments and improve their tolerability in diverse populations.

Comparative effects of progestin-based combination therapy for endometrial cancer or atypical endometrial hyperplasia: a systematic review and network meta-analysis

ObjectivesThe objective of this network meta-analysis is to systematically compare the efficacy of diverse progestin-based combination regimens in treating patients diagnosed with endometrial cancer or atypical endometrial hyperplasia. The primary goal is to discern the optimal combination treatment regimen through a comprehensive examination of their respective effectiveness.MethodsWe systematically searched four prominent databases: PubMed, Web of Science, Embase, and Cochrane Central Register of Controlled Trials, for randomized controlled trials addressing the efficacy of progestins or progestin combinations in the treatment of patients with endometrial cancer or atypical endometrial hyperplasia. The search spanned from the inception of these databases to December 2023. Key outcome indicators encompassed survival indices, criteria for assessing efficacy, as well as pregnancy and relapse rate. This study was registered in PROSPERO (CRD42024496311).ResultsFrom the 1,558 articles initially retrieved, we included 27 studies involving a total of 5,323 subjects in our analysis. The results of the network meta-analysis revealed that the mTOR inhibitor+megestrol acetate (MA)+tamoxifen regimen secured the top rank in maintaining stable disease (SD) (SUCRA=73.4%) and extending progression-free survival (PFS) (SUCRA=72.4%). Additionally, the progestin combined with tamoxifen regimen claimed the leading position in enhancing the partial response (PR) (SUCRA=75.2%) and prolonging overall survival (OS) (SUCRA=80%). The LNG-IUS-based dual progestin regimen emerged as the frontrunner in improving the complete response (CR) (SUCRA=98.7%), objective response rate (ORR) (SUCRA=99.1%), pregnancy rate (SUCRA=83.7%), and mitigating progression (SUCRA=8.0%) and relapse rate (SUCRA=47.4%). In terms of safety, The LNG-IUS-based dual progestin regimen had the lowest likelihood of adverse events (SUCRA=4.2%), while the mTOR inhibitor regimen (SUCRA=89.2%) and mTOR inbitor+MA+tamoxifen regimen (SUCRA=88.4%) had the highest likelihood of adverse events.ConclusionsPatients diagnosed with endometrial cancer or atypical endometrial hyperplasia exhibited the most favorable prognosis when undergoing progestin combination therapy that included tamoxifen, mTOR inhibitor, or LNG-IUS. Notably, among these options, the LNG-IUS-based dual progestin regimen emerged as particularly promising for potential application.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO, identifier CRD42024496311.

The activity of pyrazolo[4,3-e][1,2,4]triazine and pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide derivatives in monolayer and spheroid breast cancer cell cultures

In the last decade, an increasing interest in compounds containing pyrazolo[4,3-e][1,2,4]triazine moiety is observed. Therefore, the aim of the research was to synthesise a novel sulphonyl pyrazolo[4,3-e][1,2,4]triazines (2a, 2b) and pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide derivatives (3a, 3b) to assess their anticancer activity. The MTT assay showed that 2a, 2b, 3a, 3b have stronger cytotoxic activity than cisplatin in both breast cancer cells (MCF-7 and MDA-MB-231) and exhibited weaker effect on normal breast cells (MCF-10A). The obtained results showed that the most active compound 3b increased apoptosis via caspase 9, caspase 8, and caspase 3/7. It is worth to note that compound 3b suppressed NF-κB expression and promoted p53, Bax, and ROS which play important role in activation of apoptosis. Moreover, our results confirmed that compound 3b triggers autophagy through increased formation of autophagosomes, expression of beclin-1 and mTOR inhibition. Thus, our study defines a possible mechanism underlying 3b-induced anti-cancer activity against breast cancer cell lines.

Abstract PO1-24-04: Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is frequently activated in breast cancer (BC). Current standard-of-care therapy options for patients with advanced BC (ABC) include PAM inhibitors (PAMi), such as everolimus and alpelisib, and hormonal therapy, such as letrozole and fulvestrant. Most PAMi selectively inhibit one or only a few PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the multiple PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, can be more effective in BC cells than a PAMi targeting single PAM pathway components. Methods: A panel of BC cell lines with mutated or non-mutated PAM pathway genes (PIK3CA, PTEN, AKT) were evaluated for their sensitivity to gedatolisib and other PAMi (PI3K-α: alpelisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, growth rate inhibition, and cell death were evaluated by luciferase- and fluorescence-based assays, both in monolayer cultures and three-dimensional (3D) cultures on reconstituted basement membrane. Flow cytometry analytical assays were conducted to assess DNA synthesis (EdU incorporation), protein synthesis (OPP incorporation), and PAM pathway activity (RPS6 and 4EBP1 phosphorylation). BC xenograft studies evaluating gedatolisib in vivo were also performed. Results: Gedatolisib strongly inhibited PAM pathway activity and reduced cell viability and growth rate in the cell lines tested. Compared to the other PAMi, gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects, regardless of the cell lines’ PI3K pathway mutational status. Gedatolisib effects were confirmed in 3D culture on reconstituted basement membrane, where gedatolisib inhibited tumor cell spheroid growth and induced regression. Mechanistically, gedatolisib decreased both DNA and protein synthesis more effectively than the other PAMi tested. Gedatolisib also exerted superior anti-proliferative and cytotoxic effects relative to the other PAMi tested in estrogen receptor-positive BC cell lines concomitantly treated with fulvestrant. BC xenografts confirmed growth inhibitory effects of gedatolisib in vivo. Conclusions: The pan-PI3K/mTOR inhibitor, gedatolisib, may more effectively address potential drug resistance mechanisms associated with narrowly targeted PAM inhibitors. Gedatolisib has previously demonstrated promising preliminary clinical efficacy and safety data in ABC in combination with hormonal therapy. A Phase 3 study (VIKTORIA-1) evaluating gedatolisib plus fulvestrant with and without palbociclib is underway in patients with ABC. Citation Format: Stefano Rossetti, Aaron Broege, Adrish Sen, Salmaan Khan, Ian MacNeil, Jhomary Molden, Igor Gorbatchevsky, Brian F. Sullivan, Lance Laing. Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-04.

Abstract PO5-04-01: Inetetamab combined with sirolimus and chemotherapy in HER2 positive metastatic breast cancer patients with abnormal activation of Pi3k/Akt/mTOR pathway after trastuzumab treatment

Abstract Objective: To explore the efficacy and safety of Inetetamab Combined with Sirolimus and Chemotherapy in the treatment of human epidermal factor receptor 2 (HER2) positive metastatic breast cancer patients with abnormal activation of Pi3k/Akt/mTOR (PAM) pathway after Trastuzumab treatment. Methods: This prospective multicenter clinical study enrolled HER2 positive metastatic breast cancer patients at the National Cancer Center in China from July 2021 to September 2022 with PAM pathway mutation confirmed by histology or peripheral blood genetic testing. The inclusion criteria included: 1) Recurrent and metastatic breast cancer patients with HER2 positive confirmed histologically; 2) ECOG PS score ≤ 2; 3) Patients who have progressed after previous treatment with Trastuzumab; 4) Patients with PAM pathway mutation confirmed by Genetic testing; 5) Patients' cardiac, pulmonary, liver and bone marrow functions are basically normal; 6) Patients voluntarily sign informed consent forms. The exclusion criteria included: 1) Patients previously treated with mTOR inhibitors or pyrotinib; 2) Patients who have used chronic Corticosteroid for more than 3 months or used Immunosuppressive drug within 4 weeks; 3) Patients with symptomatic central nervous system metastasis; 4) Patients with left ventricular Ejection fraction&amp;lt; 50% or clinical manifestations of obvious arrhythmia, myocardial ischemia, severe atrioventricular block, cardiac insufficiency, and severe valvular disease; 5)Diagnosed with other malignant tumors within five years. Patients were randomly divided into trial group and control group. The patients in the trial group received inetetamab combined with sirolimus and chemotherapy, and control group received pyrotinib and chemotherapy. RECISTv1.1 standard was used to evaluate the efficacy of patients. Descriptive statistics are used to summarize clinical pathological features. Kaplan Meier method was used to draw survival curves. Log-rank test was used to compare progression free survival (PFS) differences between groups. P&amp;lt; 0.05 was considered statistically significant. Results:A total of 59 HER2 positive metastatic breast cancer patients with abnormal activation of PAM pathway were included, of which 37 patients received the treatment of inetetamab group and 22 patients received the treatment of pyrotinib group. The median follow-up time was 5.33 months. In the intent-to-treat set, the median PFS of patients in the inetetamab group was 4.64 months, which was shorter than 5.69 months in the pyrotinib group. There was no statistically significant difference between the two groups (P=0.51; HR=0.41; 95% CI, 0.12-1.45). The objective response rate (ORR) of the inetetamab group and the pyrotinib group were 28.1% and 29.4%, respectively. In terms of safety, the incidence of adverse events (AE) in the experimental group was (33/37) 89.1%, while (16/21) 76.2% in the control group. The incidence of AE above level 3 in the inetetamab group and pyrotinib group were (13/37) 35.1% and (3/21) 14.3%, respectively. Conclusion:For the metastatic HER2 positive breast cancer patients with abnormal activation of PAM pathway treated with trastuzumab previously, the combination of inetetamab with sirolimus and chemotherapy regimen are equivalent to the combination of pyrotinib and chemotherapy regimen. It demonstrated that combination of inetetamab with sirolimus and chemotherapy could be one of the treatment options for the metastatic HER2 positive breast cancer patients. Citation Format: Qiao Li, Dan Lv, Xiaoying Sun, Mengyuan Wang, Li Cai, Feng Liu, Chenghui Li, Jiuda Zhao, Jing Sun, Yehui Shi, Fei Ma. Inetetamab combined with sirolimus and chemotherapy in HER2 positive metastatic breast cancer patients with abnormal activation of Pi3k/Akt/mTOR pathway after trastuzumab treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-01.

Abstract PO2-20-02: A Phase 3 study of gedatolisib plus fulvestrant with and without palbociclib in patients with HR+/HER2- advanced breast cancer previously treated with a CDK4/6 inhibitor plus a non-steroidal aromatase inhibitor (VIKTORIA-1)

Abstract Patients who receive frontline CDK4/6 inhibitor (CDK4/6i) therapy eventually experience disease progression. Resistance to CDK4/6i is likely a transient adaptive mechanism that may be reversed by inhibition of the PI3K/mTOR pathway. Thus, combination of CDK4/6i and PI3K/mTORi after disease progression on CDK4/6i could restore sensitivity to CDK4/6i and prevent activation of the PI3K/mTOR pathway. This hypothesis was evaluated in a Phase 1b study (Layman SABCS 2021) of gedatolisib (geda), a potent inhibitor of PI3K and mTOR. Subjects with HR+/HER2- ABC treated with prior CDK4/6i received geda (180 mg IV weekly for 3 weeks, then one week off) with palbociclib (P) and fulvestrant (F). Median PFS was 12.9 months with 63% overall response rate. Efficacy was observed regardless of PIK3CA mutation status (Wesolowski SABCS 2022). Geda was well tolerated, with few discontinuations due to treatment-related adverse events (4%). The most common AE was stomatitis; hyperglycemia of any grade occurred in 26% of patients. This preliminary data, dosing schedule, and study population characteristics form the basis for the Phase 3 trial, VIKTORIA-1. This Phase 3 multinational clinical trial will evaluate geda and F with or without P in patients with HR+/HER2- ABC previously treated with any CDK4/6i in combination with a non-steroidal aromatase inhibitor (AI) therapy. Those without tumor PIK3CA mutations will be assigned to Study 1 (n=351) and randomized to Arm A (geda, P, and F), Arm B (geda and F), or Arm C (F). Those with PIK3CA mutations will be assigned to Study 2 (n=350) and randomized to Arm D (geda, P, and F), Arm E (alpelisib and F), or Arm F (geda and F). Key eligibility criteria include adults with confirmed metastatic or locally advanced breast cancer, any menopausal status, radiologically evaluable disease, and prior CDK4/6i treatment in combination with a non-steroidal AI. Prior hormonal therapy, including SERDs, is allowed. Key exclusion criteria include prior treatment with a PI3K, Akt, or mTOR inhibitor, prior treatment with chemotherapy for advanced disease, more than two lines of prior endocrine therapy, bone only disease with no soft tissue components, active CNS metastases, and type 1 diabetes or uncontrolled type 2 diabetes. The primary endpoint is PFS assessed by blinded independent central review per RECIST v1.1. Secondary endpoints included overall survival (OS), safety and tolerability, ORR, duration of response, time to response, CBR, quality of life, and pharmacokinetics. Enrollment is ongoing. This trial abstract was previously presented at the 2022 San Antonio Breast Cancer Symposium, December 6-10, 2022. Citation Format: Sara Hurvitz, Fabrice André, Massimo Cristofanilli, Giuseppe Curigliano, Antonio Giordano, Hyo Han, Miguel Martín, Barbara Pistilli, Hope Rugo, Robert Wesolowski, Igor Gorbatchevsky, Sibylle Loibl. A Phase 3 study of gedatolisib plus fulvestrant with and without palbociclib in patients with HR+/HER2- advanced breast cancer previously treated with a CDK4/6 inhibitor plus a non-steroidal aromatase inhibitor (VIKTORIA-1) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-20-02.

Abstract PO4-23-09: Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells

Abstract In HER2-enriched (HER2E) breast cancer (BC), the dual HER2/EGFR inhibitor Lapatinib is a standard of care for advanced staged disease. Anti-HER2 therapies disrupt tumor progression through inhibition of the PI3K/AKT pathway. Some treated tumors will override inhibition by shifting to MAPK/ERK signaling to fuel cell growth, thus fostering resistance to anti-HER2 therapy. The tyrosine receptor kinase FGFR4 is included in the profile for a HER2E subtype. FGFR4 is overexpressed in ~30% of BC and can signal through PI3K/AKT and MAPK/ERK. Inhibitors for FGFR4 such as BLU554 are being investigated in phase I/II clinical trials for hepatocellular carcinoma, showing success in tumors that are positive for FGFR4’s preferred ligand FGF19. According to cBioPortal patient data, FGF19 is amplified in ~20% of BC, and has been shown to trigger an autocrine loop in triple negative BC through FGFR4. We examined BLU554 as a dual therapeutic with Lapatinib and dissected intracellular signaling stemming from the combination in HER2E BC. MDA-MB-453 BC cells (HER2+FGFR4+) were used for a dual strategy with BLU554 and Lapatinib. Cell viability assays were performed at matched doses, then as a dose response matrix, ranging from 0.1-20 µM. Findings were used for synergy analysis using synergyfinder.fimm.fi. Western blots determined protein activity in response to treatments. Ridaforolimus (mTOR inhibitor) was used in combination with either BLU554 or Lapatinib to assess MAPK/ERK and PI3K/AKT pathway crosstalk in MB-453 cells. Additionally, FGF19 was added to cells after a 2-hour pre-treatment to challenge BLU554 + Lapatinib and compare changes in signaling with FGF19 present. MB-453 cells treated with BLU554 decreased cell viability similarly to Lapatinib at 10 µM (65% [SD ± 3.3] and 63% [SD ± 2.1], respectively). Dual treatment decreased viability compared to single agents at all concentrations but the greatest difference was observed at 20 µM (38.5% in dual compared to Lapatinib [p-value = 0.00006]; 45.1% in dual compared to BLU554 [p-value = 0.00016]). Dose-response matrix of dual treatment yielded a ZIP synergy score of 16.852 ± 2.97 (&amp;gt;10 is synergistic), with the greatest synergy and inhibition at 10 µM and 20 µM of both drugs. BLU554 caused a therapeutic potency shift of Lapatinib, such that lower doses of Lapatinib (0.1 µM, 1 µM) are effective at higher doses of BLU554 (10 µM, 20 µM). Westerns of MB-453 with one dose of BLU554 increased phospho-ERK1/2 (pERK1/2) at 16- or 48-hr time points versus control, but when BLU554 was re-dosed then pERK1/2 was successfully inhibited. Both pAKT and pS6RP were increased by re-dosing BLU554. Cells treated for 2 hrs with Lapatinib decreased pAKT and pS6RP and did not change pERK, whereas a 2-hr treatment of BLU554 increased pAKT and decreased pERK. Dual treatment exhibited inhibition of ERK, AKT, 4EBP1, and S6RP phosphorylation all to a greater extent than single agents in combination with Ridaforolimus. Challenge by ectopic FGF19 of BLU554 + Lapatinib was unable to restore downstream signaling of AKT and ERK. BLU554 synergistically interacts with Lapatinib to decrease cell viability while increasing its therapeutic potency. Lapatinib primarily inhibits AKT signaling, whereas BLU554 targets ERK. Dual treatment simultaneously blocks MAPK/ERK and PI3K/AKT signaling and yields decreases of mTOR downstream effectors pS6RP and p4EBP1. These effects are maintained even when challenged with FGF19, suggesting that dual inhibition of FGFR4 and HER2 could prevent pathway activation from external stimuli. Citation Format: Jordan Beardsley, Joseph Goode, Deborah Altomare. Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-09.

Abstract PO3-14-01: Heterogeneity of tumor immune microenvironment to predict everolimus efficacy in premenopausal women with hormone receptor-positive/human epidermal growth factor receptor 2-negative adavanced breast cancer

Abstract Objective Everolimus (EVE), a mammalian target of rapamycin (mTOR) inhibitor, has been shown to prolong survival of patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-), advanced breast cancer (ABC) with endocrine resistance. Preclinical studies have suggested that mTOR is closely associated with the tumor immune microenvironment (TME). We aimed to explore the tumor heterogeneity of TME between patients with different response to everolimus (EVE), and identify potential markers to predict efficacy of EVE in premenopausal patients with HR+/HER2−, advanced breast cancer (ABC) suffering endocrine resistance. Methods Patients were divided into two groups according to the best response to EVE: the responsive subgroup (RS) and the non-responsive subgroup (NRS). Postoperative tumor tissue from patients who received EVE was collected for whole exome sequencing. Transcript abundance of 126 distinct tumor regions of interest (ROIs) were quantitated using digital spatial profiling, including 42 tumor cell zones (TCZs), 42 immune cell zones (ICZs), and 42 stroma cell zones (SCZs). Based on investigation in multiple discrete areas, differences in cell components, function and pathway, gene expression and immune cell infiltration between RS and NRS were investigated. Results Totally, 93 differentially expressed genes (DEGs) were identified in the ICZs, 174 DEGs in the TCZs, and 197 DEGs in the SCZs, using p&amp;lt; 0.05 and |log2FC &amp;gt;1| as the screening criterion. Among all the subgroup analyses on different ROIs, the statistical significance of PIP gene was the highest (ICZs: p=2.6E-12, TCZs: p=1.25E-20, SCZs: p=2.0E-18) in the comparison of RS and NRS. Moreover, we found that PIP was highly expressed in NRS and patients with a progression-free survival &amp;lt; 1 year. The receptor-legend analysis showed that, the intra-and inter-cellular communication in RS was mainly mediated by interaction between CCL-CCR family, while NRS mainly by COL family complex. Comparing all cell components extracted from each ROI, no statistical differences were found between RS and NRS, only a statistical trend in the SCZs (p=0.098). Further cell classification analysis showed fibroblasts highest in TCZs and SCZs, macrophages in ICZs. Compared to NRS, in TCZs, fibroblasts were significantly increased (p=0.01), and plasma cells (p=0.02) and neutrophils (p=0.03) were decreased in RS. In SCZs, the downregulated fibroblasts (p=0.04) and enhanced plasma cells (p=0.01) were observed in NRS. In ICZs, pDCs (p=0.048) and neutrophils (p=0.006) were significantly elevated in NRS compared with RS. Conclusion The utility of digital spatial gene expression profiling might provide some references to predict the efficacy of EVE in premenopausal patients with HR+/HER2- ABC with endocrine resistance. Citation Format: Yujing Tan, Fei Ma, Jiayu Wang, Pin Zhang, Binghe Xu, Liyan Xue, Ying Fan. Heterogeneity of tumor immune microenvironment to predict everolimus efficacy in premenopausal women with hormone receptor-positive/human epidermal growth factor receptor 2-negative adavanced breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-01.

Abstract PO2-06-07: A Randomized Phase II Comparison of Single-Agent Carboplatin versus the Combination of Carboplatin and Everolimus for the Treatment of Advanced Triple Negative Breast Cancer

Abstract Background: Both TNBC and BRCA-1 associated breast cancers are sensitive to DNA cross-linking agents such as platinum compounds due to defective DNA repair by homologous recombination. TNBCs are also associated with a high frequency of PTEN loss, which can lead to activation of the mTOR pathway resulting in tumor cell growth and proliferation. mTOR activation can confer resistance to platinum agents, and this phenomenon may be reversible by the addition of an mTOR inhibitor, such as everolimus. A prior phase II single arm trial of carboplatin and everolimus in patients (pts) with advanced TNBC demonstrated good tolerability and preliminary efficacy. Methods: We conducted a phase II, multicenter, randomized trial in pts with advanced TNBC, who had received 0-3 prior lines of therapy. Pts were randomly assigned (in a 2:1 ratio) to carboplatin and everolimus or carboplatin alone. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), overall response rate (ORR), clinical benefit rate (CBR), and safety. We planned to enroll 72 pts which would provide &amp;gt; 80% power for a one-sided log-rank test at the 5% level of significance, to detect a 2.5 month improvement in median PFS between treatment groups, assuming a median PFS of 2.5 months in the control group. An interim analysis for futility was planned after 36 events with a stopping boundary of P &amp;lt;0.375 for the comparison of PFS, using an Obrien-Fleming spending function. Of note, the trial was stopped earlier than planned based on a sensitivity/tipping point analysis which indicated that terminating the trial 3 months ahead of schedule would have no substantial impact on the primary endpoint. Results: A total of 56 pts were randomized between 2015 and 2022, of whom 36 received carboplatin/everolimus and 20 carboplatin alone. The median age of the population was 62.8 years (range: 33-87) and about 20% of pts had BRCA-1 or BRCA-2 mutations. In the overall population, pts had received a median of 1 (range 1-3) prior line of therapy in the metastatic setting, with 75% of pts receiving prior chemotherapy and 47% prior carboplatin. The median PFS was significantly improved in pts who received carboplatin and everolimus (4.7 months) versus those who received carboplatin alone (2.1 months; HR: 0.37; 95% CI: 0.19-0.72; p=0.0042). Overall survival was 22.1 months in pts on the combination versus 14.4 months on carboplatin alone (HR: 0.73; 95% CI: 0.30-1.72; p=0.3106). CBR was 61% with the combination vs 50% with carboplatin monotherapy, as shown in Table 1. Most common adverse events (AEs) on carboplatin and everolimus included thrombocytopenia (81%), anemia (69%), leukopenia (67%), fluid retention (64%), and neutropenia (61%). Overall, Grade 3/4 events occurred in 75% of pts (83% of pts on combination vs 60% on carboplatin alone) and were primarily anemia (40% vs 17%), thrombocytopenia (47% vs 0%), and neutropenia (20% vs 0%). Of note, there was an increase in thrombocytopenia with the addition of everolimus (All Grades: 81% vs 35%, Grade 3/4: 39% vs 0%). Conclusions: Treatment options for TNBC are limited due to a lack of targeted therapies. The combination of carboplatin and everolimus in this study was associated with a 63% reduction in risk of progression or death in pts with metastatic TNBC. The regimen was well tolerated and provides a promising treatment option for pts with advanced TNBC. Table 1. Results in Patients on Carboplatin and Everolimus versus Carboplatin Alone NE= Not Estimable; 1Unevaluable patients treated as non-responders in CBR and ORR calculations. Citation Format: Rima Patel, Jami Fukui, Paula Klein, Erin Moshier, Charles Shapiro, Anupama Goel, Julie Fasano, Theresa Shao, Aarti Bhardwaj, Rita Vaccaro, Gargi Atul Joshi, Joseph Sparano, Amy Tiersten. A Randomized Phase II Comparison of Single-Agent Carboplatin versus the Combination of Carboplatin and Everolimus for the Treatment of Advanced Triple Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-07.

3-Epi-betulinic acid 3-O-β-D-glucopyranoside (eBAG) induces autophagy by activation of AMP-activated protein kinase in hepatocellular carcinoma

3-Epi-betulinic acid 3-O-β-D-glucopyranoside (eBAG) is a pentacyclic triterpene mainly distributed in food and medicinal plants, which exhibits various pharmacological properties. However, whether these functions are attributed to eBAG or additional components in these plants remain unknown. Herein, we report that eBAG exerted an inhibitory activity against hepatocellular carcinoma and esophageal cancer cells. EBAG induced non-apoptotic cell death in hepatocellular carcinoma cells. The eBAG-induced cell death was inhibited by knock-down of autophagy related gene (ATG) 5 and ATG7, by administration of 3-methyladenine, a selective autophagy inhibitor that suppresses phosphoinositide 3-kinase (PI3K), and by chloroquine, a classic autophagy flux inhibitor. We demonstrated that eBAG induced an autophagy-mediated cell death. Application of eBAG mimicked cellular bioenergetics depletion leading to the reduction of intracellular ATP, activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR. Co-treatment with compound C, an AMPK inhibitor, abrogated cell death induced by eBAG. We further validated the anti-tumor effect of eBAG in the murine xenograft model of hepatocellular carcinoma and found that eBAG treatment promoted the induction of autophagy and reduction of tumor growth in mice. As a functional food ingredient, eBAG is a potential therapeutic agent for the treatment of hepatocellular carcinoma and esophageal cancer.

Chemically engineered mTOR-nanoparticle blockers enhance antitumour efficacy

Hepatocellular carcinoma (HCC) is a highly prevalent and deadly type of cancer, and although pharmacotherapy remains the cornerstone of treatment, therapeutic outcomes are often unsatisfactory. Pharmacological inhibition of mammalian target of rapamycin (mTOR) has been closely associated with HCC regression. Herein, we covalently conjugated AZD8055, a potent mTORC1/2 blocker, with a small panel of unsaturated fatty acids via a dynamically activating linkage to enable aqueous self-assembly of prodrug conjugates to form mTOR nanoblockers. Cell-based experiments were carried out to evaluate the effects of the nanoblocker against hepatocellular carcinoma (HCC) cells. The orthotopic and subcutaneous HCC mouse models were established to examine its antitumour activity. Among several fatty acids as promoieties, linoleic acid-conjugated self-assembling nanoblocker exhibited optimal size distribution and superior physiochemical properties. Compared with free agents, PEGylated AZD8055 nanoblocker (termed AZD NB) was pharmacokinetically optimized after intravenous administration. Invivo investigations confirmed that AZD NB significantly suppressed tumour outgrowth in subcutaneous HCCLM3 xenograft, Hepatoma-22, and orthotopic Hepa1-6 liver tumour models. Strikingly, treatment with AZD NB, but not free agent, increased intratumour infiltration of IFN-γ+CD8+ T cells and CD8+ memory T cells, suggesting a potential role of the mTOR nanoblocker to remodel the tumour microenvironment. Overall, a single conjugation with fatty acid transformed a hydrophobic mTOR blocker into a systemically injectable nanomedicine, representing a facile and generalizable strategy for improving the therapeutic index of mTOR inhibition-based cancer therapy. The mTOR inhibition by chemically engineered nanoblocker presented here had enhanced efficacy against tumours compared with the pristine drug and thus has the potential to improve the survival outcomes of patients with HCC. Additionally, this new nanosystem derived from co-assembling of small-molecule prodrug entities can serve as a delivery platform for the synergistic co-administration of distinct pharmaceutical agents. This work was supported by the National Natural Science Foundation of China (32171368,81721091), the Zhejiang Provincial Natural Science Foundation of China (LZ21H180001), the Jinan Provincial Laboratory Research Project of Microecological Biomedicine (JNL-2022039c and JNL-2022010B), State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (zz202310), and Natural Science Foundation of Shandong Province (ZR2023ZD59).

Everolimus treatment enhances inhibitory immune checkpoint molecules’ expression in monocyte-derived dendritic cells

BackgroundAntigen-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. MethodsTo 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. ResultsThe 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. ConclusionThe 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.

Salutary Effects of mTOR Inhibition on Deep Veins in Vivo: Results from the Sirolimus Trial with Drug-Eluting Balloon (STRIDE)

Salutary Effects of mTOR Inhibition on Deep Veins in Vivo: Results from the Sirolimus Trial with Drug-Eluting Balloon (STRIDE)

Therapeutic effect of Topical Sirolimus on Facial Angiofibromas in Patients of Tuberous Sclerosis Complex

Abstract Introduction: Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder. Facial angiofibromas are the most common cutaneous findings of TSC. Treatment modalities such as laser, surgery, and/or cryotherapy are employed. Topical therapy with Sirolimus, an mTOR inhibitor, showed beneficial effects. Objective: To study the effects of topical sirolimus (0.1%) on Facial Angiofibromas in patients of TSC. Methodology : Four patients with facial angiofibromas were included. They applied Sirolimus preparation twice daily, for 3 months. The Facial Angiofibroma Severity Index (FASI) was recorded pre-intervention, at 3 months and after 6 months. Results: All the patients showed a reduction in the FASI score at the end of three months of therapy. In three patients, on discontinuing therapy, there was no change in the FASI score at the end of six months, i.e., FASI 3 and FASI 6 were the same. Conclusion: Topical sirolimus is an effective treatment for facial angiofibroma in patients with TSC.

Ziprasidone triggers inflammasome signaling via PI3K-Akt-mTOR pathway to promote atrial fibrillation

BackgroundSecond-generation antipsychotics increase the risk of atrial fibrillation. This study explores whether the atypical antipsychotic ziprasidone triggers inflammasome signaling, leading to atrial arrhythmia. MethodsElectromechanical and pharmacological assessments were conducted on the rabbit left atria (LA). The patch-clamp technique was used to measure ionic channel currents in single cardiomyocytes. Detection of cytosolic reactive oxygen species production was performed in atrial cardiomyocytes. ResultsThe duration of action potentials at 50 % and 90 % repolarization was dose-dependently shortened in ziprasidone-treated LA. Diastolic tension in LA increased after ziprasidone treatment. Ziprasidone-treated LA showed rapid atrial pacing (RAP) triggered activity. PI3K inhibitor, Akt inhibitor and mTOR inhibitor abolished the triggered activity elicited by ziprasidone in LA. The NLRP3 inhibitor MCC950 suppressed the ziprasidone-induced post-RAP-triggered activity. MCC950 treatment reduced the reverse-mode Na+/Ca2+ exchanger current in ziprasidone-treated myocytes. Cytosolic reactive oxygen species production decreased in ziprasidone-treated atrial myocytes after MCC950 treatment. Protein levels of inflammasomes and proinflammatory cytokines, including NLRP3, caspase-1, IL-1β, IL-18, and IL-6 were observed to be upregulated in myocytes treated with ziprasidone. ConclusionsOur findings suggest ziprasidone induces atrial arrhythmia, potentially through upregulation of the NLRP3 inflammasome and enhancement of reactive oxygen species production via the PI3K/Akt/mTOR pathway.