PI3K Pathway Inhibitors Research Articles

Fetuin-B Interacts With Insulin Receptor-β and Promotes Insulin Resistance in Retina Cells.

The purpose of this study was to investigate the correlation between insulin and Fetuin-B (FETUB) and the influence of FETUB on insulin signaling pathway in diabetic retinopathy (DR). Enzyme-linked immunosorbent assay (ELISA) was used to analyze FETUB and insulin levels in the serum and aqueous fluid of patients with DR and healthy controls. Quantitative PCR (q-PCR), Western blotting, and ELISA were used to examine FETUB expression in ARPE-19, BV2, and Müller cells under insulin stimulation. Co-immunoprecipitation was used to investigate the interaction of FETUB with insulin receptor-β (IRβ). Insulin resistance (IR)-BV2 and IR-Müller cells were treated with FETUB recombinant protein or FETUB short hairpin RNA (shRNA) to explore the influence of FETUB on insulin signaling pathway in DR. LY294002 (a PI3K pathway inhibitor) was used to determine whether FETUB affects glucose metabolism via the PI3K/Akt pathway. In aqueous fluid, FETUB concentrations were positively correlated with insulin levels. FETUB expression increased in Müller and BV2 cells under insulin regulation, and FETUB interacted with IRβ in retinal cells and mice retina. The interaction between IRβ and FETUB increased in BV2 and Müller cells under high-glucose than in controls. Insulin signaling pathway activation was suppressed in FETUB recombinant protein-treated BV2 and Müller cells but increased in FETUB shRNA-transfected cells. FETUB shRNA could not reverse LY294002-mediated inhibition of glucose transporter-4 expression. Retinal cells are the source of insulin-regulated FETUB. The FETUB interacts with IRβ and affects insulin signaling pathway in BV2 and Müller cells. FETUB may aggravate IR in BV2 and Müller cells via the PI3K/Akt pathway.

Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors.

The sudden rise of the SARS-CoV-2 virus and the delay in the development of effective therapeutics to mitigate it made evident a need for ways to screen for compounds that can block infection and prevent further pathogenesis and spread. Yet, identifying effective drugs efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, yet as the SARS-CoV-2 mutated, these antibodies became ineffective. Small molecule antivirals were identified using pseudovirus constructs to recapitulate infection in non-human cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce virus infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e. PIK3i, mTORi, multiple RTKi) that could be effective against SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.

Cancer Therapy with Quinoxaline Derivatives: Dual Inhibition of Pi3k and Mtor Signaling Pathways

Quinoxaline is a heterocyclic compound with a benzene ring fused to a pyrazine ring. It learned much about his ability to cure many diseases, including cancer. Phosphoinositide 3kinase (PI3K) and the mTOR (mammalian target of rapamycin) are the 3 essential pathways which regulate the growth, survival, and development of cells. Dysregulation of such pathways is often seen in cancer, making them attractive targets for cancer therapy. In particular, several quinoxaline derivatives show promise as two inhibitors of the mTOR/PI3K signalling pathway, which is often dysregulated in cancer along with many other diseases. One such example is PX866, which has shown activity against several cancers in previous studies. In previous studies, another quinoxaline derivative, PKI587, also showed strong inhibitory activity against mTOR and PI3K. PKI587 was researched in several clinical trials for treating many cancer types, which include non-small cell lung as well as breast cancer. Dual inhibitors of mTOR and PI3K, including quinoxaline derivatives, inhibit cell growth and cancer by blocking the signalling of these two important factors. In recent years, quinoxaline derivatives have emerged as potent dual inhibitors of PI3K and mTOR, and in this review, we explore the latest developments in this area.

Fluid shear stress-mediated Piezo1 alleviates osteocyte apoptosis by activating the PI3K/Akt pathway

Glucocorticoid-induced osteoporosis serves as a primary cause for secondary osteoporosis and fragility fractures, representing the most prevalent adverse reaction associated with prolonged glucocorticoid use. In this study, to elucidate the impact and underlying mechanisms of fluid shear stress (FSS)-mediated Piezo1 on dexamethasone (Dex)-induced apoptosis, we respectively applied Dex treatment for 6 h, FSS at 9 dyne/cm2 for 30 min, Yoda1 treatment for 2 h, and Piezo1 siRNA transfection to intervene in MLO-Y4 osteocytes. Western blot analysis was used to assess the expression of Cleaved Caspase-3, Bax, Bcl-2, and proteins associated with the PI3K/Akt pathway. Additionally, qRT-PCR was utilized to quantify the mRNA expression levels of these molecules. Hoechst 33258 staining and flow cytometry were utilized to evaluate the apoptosis levels. The results indicate that FSS at 9 dyne/cm2 for 30 min significantly upregulates Piezo1 in osteocytes. Following Dex-induced apoptosis, the phosphorylation levels of PI3K and Akt are markedly suppressed. FSS-mediated Piezo1 exerts a protective effect against Dex-induced apoptosis by activating the PI3K/Akt pathway. Additionally, downregulating the expression of Piezo1 in osteocytes using siRNA exacerbates Dex-induced apoptosis. To further demonstrate the role of the PI3K/Akt signaling pathway, after intervention with the PI3K pathway inhibitor, the activation of the PI3K/Akt pathway by FSS-mediated Piezo1 in osteocytes was significantly inhibited, reversing the anti-apoptotic effect. This study indicates that under FSS, Piezo1 in MLO-Y4 osteocytes is significantly upregulated, providing protection against Dex-induced apoptosis through the activation of the PI3K/Akt pathway.

A kinome drug screen identifies multi-TKI synergies and ERBB2 signaling as a therapeutic vulnerability in MYC/TYR subgroup atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a rare, devastating, and largely incurable pediatric brain tumor. Although recent studies have uncovered 3 molecular subgroups of ATRTs with distinct disease patterns, and signaling features, the therapeutic profiles of ATRT subgroups remain incompletely elucidated. We examined the effect of 465 kinase inhibitors on a panel of ATRT subgroup-specific cell lines. We then applied multiomics analyses to investigate the underlying molecular mechanism of kinase inhibitor efficacy in ATRT subgroups. We observed that ATRT cell lines are broadly sensitive to inhibitors of the PI3K and MAPK signaling pathways, as well as CDKs, AURKA/B kinases, and polo-like kinase 1. We identified 2 classes of multikinase inhibitors predominantly targeting receptor tyrosine kinases including PDGFR and EGFR/ERBB2 in MYC/TYR ATRT cells. The PDGFRB inhibitor, Dasatinib, synergistically affected MYC/TYR ATRT cell growth when combined with broad-acting PI3K and MAPK pathway inhibitors, including Rapamycin and Trametinib. We observed that MYC/TYR ATRT cells were also distinctly sensitive to various inhibitors of ERBB2 signaling. Transcriptional, H3K27Ac ChIPSeq, ATACSeq, and HiChIP analyses of primary MYC/TYR ATRTs revealed ERBB2 expression, which correlated with differential methylation and activation of a distinct enhancer element by DNA looping. Significantly, we show the brain penetrant EGFR/ERBB2 inhibitor, Afatinib, specifically inhibited in vitro and in vivo growth of MYC/TYR ATRT cells. Taken together, our studies suggest combined treatments with PDGFR and ERBB2-directed TKIs with inhibitors of the PI3K and MAPK pathways as an important new therapeutic strategy for the MYC/TYR subgroup of ATRTs.

Combination Therapy Approach to Overcome the Resistance to PI3K Pathway Inhibitors in Gynecological Cancers.

The PI3K signaling pathway plays an essential role in cancer cell proliferation and survival. PI3K pathway inhibitors are now FDA-approved as a single agent treatment or in combination for solid tumors such as renal cell carcinoma or breast cancer. However, despite the high prevalence of PI3K pathway alterations in gynecological cancers and promising preclinical activity in endometrial and ovarian cancer models, PI3K pathway inhibitors showed limited clinical activity in gynecological cancers. In this review, we provide an overview on resistance mechanisms against PI3K pathway inhibitors that limit their use in gynecological malignancies, including genetic alterations that reactivate the PI3K pathway such as PIK3CA mutations and PTEN loss, compensatory signaling pathway activation, and feedback loops causing the reactivation of the PI3K signaling pathway. We also discuss the successes and limitations of recent clinical trials aiming to address such resistance mechanisms through combination therapies.

First-in-human study of RLY-2608, a pan-mutant and isoform selective PI3Kα inhibitor, as a single agent in patients (pts) with advanced solid tumors and in combination with fulvestrant in pts with advanced breast cancer.

TPS1128 Background: Oncogenic mutations in PIK3CA occur in ~35% of HR+, HER2– breast cancer (BC) and are frequent in multiple solid tumors. The approval of alpelisib, a non-mutant selective PI3Kα inhibitor, validates PI3Kα as a therapeutic target. However, inhibition of wild-type PI3Kα leads to significant toxicity, including hyperglycemia and rash. RLY-2608, a novel, oral allosteric PI3Kα inhibitor, is designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition for greater target coverage, improved tolerability, and anti-tumor activity. ReDiscover (NCT05216432) is the ongoing, first-in-human study to investigate the maximum tolerated dose (MTD), recommended phase 2 doses (RP2Ds), safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor activity of RLY-2608 monotherapy in pts with PIK3CA-mutant advanced solid tumors; and of RLY-2608 plus fulvestrant with or without ribociclib in pts with PIK3CA-mutant, HR+, HER2– BC. To date RLY-2608 plus fulvestrant has completed dose escalation; 400 and 600 mg BID is being investigated in the combination regimens, based on safety, PK, PD, and anti-tumor activity. Methods: This global, multi-center, dose escalation/expansion study of RLY-2608 monotherapy includes adult pts with advanced solid tumors who are refractory or intolerant to, or have declined, standard therapy. RLY-2608 is also being investigated in combination with fulvestrant and in combination with fulvestrant and ribociclib, each in previously treated pts with HR+, HER2– advanced/metastatic BC. Eligibility criteria include presence of a PIK3CA mutation (blood or tumor) per local assessment, ECOG performance status 0–1, evaluable disease per RECIST 1.1, and no prior treatment with a PI3K pathway inhibitor. RLY-2608 is administered daily in 4-week cycles. Adverse events (AEs) per CTCAE v5, PK, biomarkers (pAKT, mutant ctDNA and insulin pathway markers) and anti-tumor activity are assessed serially. Dose escalation employs a Bayesian Optimal Interval design to identify the MTD. Following dose escalation, expansion cohorts are enrolling patients with select PIK3CA-mutated solid tumors for treatment with RLY-2608 monotherapy and patients with HR+, HER2– metastatic BC for treatment with RLY-2608 plus fulvestrant with or without ribociclib. Primary endpoints are MTD/RP2D and AE profile; key secondary endpoints are PIK3CA genotype in blood and tumor, PK, biomarkers, and overall response rate. All dose expansion cohorts in ReDiscover are now enrolling in the United States and Europe. Clinical trial information: NCT05216432 .

Genomic characterization reveals distinct mutational landscapes and therapeutic implications between different molecular subtypes of triple-negative breast cancer

Triple-negative breast cancer (TNBC) has high heterogeneity, poor prognosis, and limited treatment success. Recently, an immunohistochemistry-based surrogate classification for the “Fudan University Shanghai Cancer Center (FUSCC) subtyping” has been developed and is considered more suitable for clinical application. Seventy-one paraffin-embedded sections of surgically resected TNBC were classified into four molecular subtypes using the IHC-based surrogate classification. Genomic analysis was performed by targeted next-generation sequencing and the specificity of the subtypes was explored by bioinformatics, including survival analysis, multivariate Cox regression, pathway enrichment, Pyclone analysis, mutational signature analysis and PHIAL analysis. AKT1 and BRCA1 mutations were identified as independent prognostic factors in TNBC. TNBC molecular subtypes encompass distinct genomic landscapes that show specific heterogeneities. The luminal androgen receptor (LAR) subtype was associated with mutations in PIK3CA and PI3K pathways, which are potentially sensitive to PI3K pathway inhibitors. The basal-like immune-suppressed (BLIS) subtype was characterized by high genomic instability and the specific possession of signature 19 while patients in the immunomodulatory (IM) subtype belonged to the PD-L1 ≥ 1% subgroup with enrichment in Notch signaling, suggesting a possible benefit of immune checkpoint inhibitors and Notch inhibitors. Moreover, mesenchymal-like (MES) tumors displayed enrichment in the receptor tyrosine kinase (RTK)-RAS pathway and potential sensitivity to RTK pathway inhibitors. The findings suggest potential treatment targets and prognostic factors, indicating the possibility of TNBC stratified therapy in the future.

Revolutionizing Glioblastoma Treatment: A Comprehensive Overview of Modern Therapeutic Approaches.

Glioblastoma is the most common malignant primary brain tumor in the adult population, with an average survival of 12.1 to 14.6 months. The standard treatment, combining surgery, radiotherapy, and chemotherapy, is not as efficient as we would like. However, the current possibilities are no longer limited to the standard therapies due to rapid advancements in biotechnology. New methods enable a more precise approach by targeting individual cells and antigens to overcome cancer. For the treatment of glioblastoma, these are gamma knife therapy, proton beam therapy, tumor-treating fields, EGFR and VEGF inhibitors, multiple RTKs inhibitors, and PI3K pathway inhibitors. In addition, the increasing understanding of the role of the immune system in tumorigenesis and the ability to identify tumor-specific antigens helped to develop immunotherapies targeting GBM and immune cells, including CAR-T, CAR-NK cells, dendritic cells, and immune checkpoint inhibitors. Each of the described methods has its advantages and disadvantages and faces problems, such as the inefficient crossing of the blood-brain barrier, various neurological and systemic side effects, and the escape mechanism of the tumor. This work aims to present the current modern treatments of glioblastoma.

Abstract PO3-04-11: Unmet clinical need in patients with pre-treated hormone receptor-positive/HER2-negative (HR+/HER2–) metastatic breast cancer in routine care: a targeted literature review

Abstract Background Treatment options remain limited to chemotherapy (CT) for patients with HR+/HER2− metastatic breast cancer (MBC) who have progressed on endocrine therapy (ET) with/without CDK4/6 or PI3K pathway inhibitors (i). We aimed to review the current unmet need in patients who had received ≥ 1 prior systemic therapy in terms of ET resistance as well as CT survival outcomes, tolerability and discontinuation. Methods MEDLINE, Embase and the Cochrane Library were searched via Ovid for observational studies (≥ 50 patients who had received ≥ 1 prior systemic therapy, January 2018 – January 2023). Abstracts from relevant congresses (January 2021 – February 2023) were reviewed. All screening was performed in a single-blind manner by one reviewer and any uncertainties were resolved by a senior reviewer. Results We included 72 full-text publications and 48 congress abstracts (US based 39%, European 28%, Asian 23%). Median treatment duration was 1.9–16.2 months (m) for ET (7 studies) and 2.0–24.5 m for ET-based therapy (5 studies). Exceptional responders (10% of patients treated for the longest time) received ET for ≥ 43.6 m (1 study, N = 4195). The proportion of patients with ET-resistant disease ranged widely (3–100%; 22 studies) and increased by line (L) of therapy: 14–19% of patients treated in 1L had primary resistance, 14–49% in ≥ 2L and 27–68% in ≥ 3L; acquired resistance was reported for 26–38% of patients in 1L, 51–57% in ≥ 2L, 32–60% in ≥ 3L (2 studies). One or more prior lines of CT had a negative impact on median overall survival (mOS; 5 studies) and median progression-free survival (mPFS; 11 studies) in subsequent therapy lines regardless of the chosen treatment; prior CT had no effect on mOS in 3 studies, no effect on mPFS in 4 studies. Consecutive CT cycles (primarily after ET + CDK4/6i) steadily lost clinical benefit (Table), with mPFS with 2 to 5 lines of CT: 7.6 m, 4.8 m, 4.2 m and 3.2 m, and mOS: 13.5 m, 9.1 m, 6.8 m and 7.2 m, respectively (1 study, N = 266). Paclitaxel + bevacizumab as second CT had a mPFS and a mOS of 8.6 m and 19.8 m, respectively (1 study, N = 538). Eribulin use in ≥ 2L had a mPFS of 4.6–10.0 m (2 studies) and a mOS of 10.1–11.2 m (2 studies). Patients who progressed on CDK4/6i as best response and those with a mPFS of < 6 m on ET benefited more from CT than ET in ≥ 2L with a mPFS of 7.1–8.1 m compared with 1.7–3.9 m, respectively (2 studies). Discontinuation due to toxicity was 2–36% (3 studies) for CT in 2L and dose reduction was required in 19–37% of patients (2 studies). Common grade 3/4 adverse events in patients receiving CT were leukopenia (17%), neutropenia (7%), thrombocytopenia (3%) and pneumonitis (1–3%) (3 studies). Conclusions Our review of real-world evidence highlights the limitations of repeated CT for patients with ET-resistant HR+/HER2− MBC, characterized by diminishing clinical benefit and significant toxicity burden. Results are limited by treatment and study population heterogeneity. Clinical outcomes are expected to improve with the use of new therapies, for which real-world evidence has yet to accrue. Table Real-world clinical outcomes with chemotherapy from observational studies CI, confidence interval; CT, chemotherapy; L, line; mOS, median overall survival; mPFS, median progression-free survival; NR, not reported Citation Format: Laura Spring, Simon M Collin, Inderjit K Dhillon, Gráinne Long, Evelina Bertranou, Komal Jhaveri, Aditya Bardia. Unmet clinical need in patients with pre-treated hormone receptor-positive/HER2-negative (HR+/HER2–) metastatic breast cancer in routine care: a targeted literature review [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 PO3-04-11.

Abstract LB174: OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer

Abstract Background: Dysregulation of PI3K signaling is a common driver of solid tumors, with the PI3KαH1047R activating mutation found in 4% of all cancers. Because many cancers, including breast and lung cancers, can metastasize to the CNS, brain penetration should be a key feature of new therapies. The FDA-approved PI3K inhibitor alpelisib lacks CNS activity, and brain-penetrant alternatives such as buparlisib and paxalasib were not approved due to toxicity associated with a lack of mutant selectivity. OKI-219 is a potent, highly selective PI3KαH1047R inhibitor that has shown preclinical anti-tumor activity in multiple cancer models, both as a monotherapy and in combination with HER2- and ER-mediated therapeutics. Here, we demonstrate its brain penetrance across multiple animal species and anti-tumor activity in an intracranial mouse model of PI3KαH1047R mutant breast cancer. Materials and methods: Brain penetrance in mouse, rat, dog, and monkey was quantified by the ratio of unbound drug concentrations in brain to plasma (Kp,uu). Plasma, brain, and cerebrospinal fluid (CSF) concentrations of OKI-219 were measured by LC/MS, and protein binding in plasma, CSF, and brain homogenate were measured by rapid equilibrium dialysis. Additionally, free drug levels in the brain were directly measured by microdialysis in rats. Mice were implanted subcutaneously and intracranially with PI3KαH1047R xxT47D-luc tumors and tumor growth was measured by calipers (subcutaneous tumors) and luminescence (intracranial tumors). Results: At the Cmax of a pharmacologically active 10 mg/kg PO dose, OKI-219 had a Kp,uu of 0.34, 0.61, and 0.58 in rat, dog, and monkey CSF, respectively. Additionally, OKI-219 had an average Kp,uu of 0.67 over 24h post-dose as measured by brain microdialysis in rats at 30 mg/kg, with a dose-proportionate increase in brain free drug levels at 300 mg/kg. After 21 days of treatment, OKI-219 induced a statistically significant dose-dependent anti-tumor effect on both flank and brain xxT47D-luc tumors. Conclusion: Here we show that OKI-219 had consistently good brain penetrance across multiple species by multiple methods of measuring free drug levels in the brain. We further demonstrated that OKI-219 had anti-tumor activity against xxT47D-luc tumors in an intracranial mouse model that is similar to activity in the same tumor when implanted subcutaneously, demonstrating that the brain levels achieved were pharmacologically active. These data indicate the potential for activity of OKI-219 in CNS disease settings, including metastatic brain cancer, which are not currently addressed with existing PI3K pathway inhibitors. OnKure has initiated the PIKture Phase I clinical study (OKI-219-101) to evaluate OKI-219 in patients with PI3KαH1047R-mutated solid tumors. Citation Format: Alexander A. Strait, Molly A. Taylor, Kevin S. Litwiler, Qian Zhao, David A. Mareska, Mark L. Boys, Yevgeniy Izrayelit, Richard Woessner, James D. Winkler. OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB174.

Abstract 3369: Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage

Abstract Background: Endometrial cancer (EC) is the most common gynecologic malignancy with increasing incidence and mortality in the U.S. Among the four subtypes characterized by the Cancer Genome Atlas, the serous-like copy number high (CNH) subtype which occurs in 15-25% of EC is associated with poor prognosis and platinum resistance, requiring alternative treatment options. ~85% CNH EC cases have impaired G1/S checkpoint regulation, making cells rely on ATR-mediated G2/M cell cycle checkpoints for optimal DNA replication and repair. Additionally, over 80% of ECs exhibit PI3K pathway activation (e.g., PIK3CA mutations), linked to enhanced DNA repair and drug resistance. Therefore, we hypothesized that dual ATR and PI3Kα inhibition would enhance DNA damage, increasing cell death in EC. Methods: CNH EC cell lines included platinum-resistant (KLE, MFE280, HEC1A, ARK2) as well as platinum-sensitive (ARK1) lines used to evaluate the activity of ATR inhibitor (ATRi) camonsertib and PI3Kα inhibitor (PI3Kαi) inavolisib. Cell growth was assessed by 3-day XTT and 9-12-day colony-forming assays. The degree of combination synergy, additivity, or antagonism was calculated using SynergyFinder with a reference highest single agent (HSA) model. An HSA synergy score > 10 indicates synergy, -10 to 10 suggests additivity and < -10 represents antagonism. DNA damage endpoints were measured by alkaline comet assay and immunoblotting for γ-H2AX. Data were repeated in triplicate, analyzed using one-way ANOVA test, and shown as mean ± SD. P < 0.05 was statistically significant. Results: All tested EC cell lines showed sensitivity to clinically attainable doses of ATRi monotherapy (IC50 0.09 - 0.98 μM), but varying sensitivity to PI3Kαi monotherapy (IC50 0.03 - 67.01 μM) unrelated to their PIK3CA mutation status. Combination treatment using clinically attainable concentrations (camonsertib < 6 μM; inavolisib < 10 μM) yielded additivity in all cell lines independent of platinum sensitivity (HSA synergy scores -0.8-10.0). Notably, increased cytotoxic effects were observed in both PIK3CA wild-type (KLE) and PIK3CA-mutant (MFE280 and HEC1A) platinum-resistant lines by colony-forming assays (4.8- to 15.4-fold decrease in colony-forming ability relative to ATRi; 1.5- to 3.5-fold decrease relative to PI3Kαi). ATRi and PI3Kαi combination induced greater DNA damage, evidenced by elevated mean comet tail moment relative to ATRi (increased 2.5- to 6.7-fold; P < 0.01) or PI3Kαi (increased 2.1- to 5.6-fold; P < 0.01) regardless of PIK3CA mutation status. Increased γ-H2AX levels were found in HEC1A cells with combination treatment (3.2-fold increase relative to ATRi; 16-fold increase relative to PI3Kαi). Conclusion: Our results suggest that dual inhibition of ATR and PI3K pathways induces greater cell death by increasing DNA damage in platinum-resistant EC cells independent of PIK3CA mutation status. Citation Format: Kristen R. Ibanez, Tzu-Ting Huang, Sotirios Sotiriou, Yvonne G. Lin, Jung-Min Lee. Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3369.

Abstract 1955: Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer

Abstract Phosphoinositide 3-kinase (PI3K) is a key regulator of cell proliferation, survival, and metabolism. Hotspot mutations in the PIK3CA gene constitutively activate the catalytic subunit of the PI3Kα isoform and are found in 10-30% of solid tumors. The development of PI3K pathway inhibitors has been hampered by poor drug tolerance and release of negative feedback loops, leading to rapid reactivation of signaling in response to reversible inhibitors. To remedy these challenges, we developed highly selective covalent PI3Kα inhibitors that irreversibly bind to a non-conserved cysteine (C862) located 11 Å outside the ATP-binding site. Here we present the properties of an optimized drug-like covalent lead, dubbed compound 9, and describe the generation of a biotinylated probe developed to be used in conjunction with compound 9 to monitor PI3Kα target occupancy in relation to downstream PI3K signaling outputs. Biochemical investigations of compound 9 using TR-FRET assays showed high affinity binding (Ki), high reaction rates (kinact) for covalent bond formation with PI3Kα, and negligible off-target reactivity. Cellular NanoBRET assays revealed that compound 9 diffuses three times more rapidly into cells as compared to BYL719 (alpelisib), and leads to rapid and potent on-target engagement. Covalent bond formation to residue C862 of PI3Kα was further confirmed by X-ray crystallography. In PIK3CA mutant cancer cell lines, covalent binding of PI3Kα occurred at low nanomolar concentrations and led to persistent inhibition of Akt/PKB phosphorylation for more than 72 hours after drug removal. Notably, investigation of PI3Kα re-synthesis after labeling with compound 9 showed a negligible reappearance in cancer cell lines, which indicates that persistent suppression of PI3Kα signaling by compound 9 results in a much longer half-life for PI3Kα than previously reported. The irreversible inactivation of PI3Kα by compound 9 provides a marked gain in growth inhibition potency (10-600-fold) in PIK3CA mutant cancer cell lines in comparison to clinical reversible PI3Kα inhibitors and allows for intermittent dosing without compromising efficacy. These data collectively demonstrate that compound 9 enables precise and selective targeting of PI3Kα with high potency and long-lasting efficacy in cancer models. The new class of irreversible PI3Kα inhibitors have a unique pharmacology among PI3K inhibitors characterized by strong decoupling of drug exposure from efficacy which may provide opportunities to lower treatment burden in patients. Citation Format: Theodora A. Constantin, Lukas Bissegger, Erhan Keleş, Luka Raguž, Clara Orbegozo, Thorsten Schaefer, Isobel Barlow-Busch, John E. Burke, Chiara Borsari, Matthias P. Wymann. Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1955.

Abstract 7196: Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines

Abstract Background: Endocrine therapy (ET) is the standard of care for hormone receptor positive (HR+) breast cancer (BC), but ET resistance develops in most patients (pts). One resistance mechanism involves cross talk between the PI3K-AKT-mTOR and estrogen receptor (ER) signaling pathways, resulting in increased ER signaling and tumor proliferation. This is mitigated by mTOR inhibition as demonstrated by improved outcomes with exemestane plus the mTOR inhibitor (mTORi) everolimus in pts with HR+ advanced BC. While PI3K inhibitors (PI3Ki [e.g. alpelisib]) are efficacious in pts with HR+, PIK3CA-mutated BC, resistance can occur due to acquired genomic alterations in the PI3K-AKT-mTOR pathway. Combination therapy that targets a critical downstream node, mTOR, could delay or overcome acquired resistance due to activation of upstream components of the PI3K-AKT-mTOR pathway. nab-Sirolimus is an intravenously administered, albumin-bound nanoparticle form of the mTORi sirolimus, with significantly greater tumor accumulation, mTOR target suppression, and improved antitumor effects than oral mTORis in nonclinical tumor models. We evaluated the antiproliferative and cytotoxic effects of nab-sirolimus in combination with ET or PI3Kis in BC cell lines. Methods: HR+, PI3K-mutated BC cell lines (MCF7 or MDA-MB-361) were incubated for 5 days with increasing concentrations of nab-sirolimus and the selective ER degrader fulvestrant. MDA-MB-361 cells or HR-negative (HR−), PI3K-mutated BC cells, MDA-MD-453, were incubated for 5 days with increasing concentrations of nab-sirolimus and PI3K-AKT-mTOR pathway inhibitors gedatolisib or alpelisib. Antiproliferative/cytotoxic effects of single-agent and combination treatment were assessed via an automated trypan blue exclusion assay. Results: Addition of 20 or 80 nM nab-sirolimus to fulvestrant (12.5-500 nM) markedly decreased cell viability and nearly doubled cell death in MCF7 and MDA-MB-361 cells. Antiproliferative effects of low-dose gedatolisib (2.5-10 nM) were enhanced by 61-71% when nab-sirolimus (20 or 80 nM) was added to MDA-MB-453 cells. While low doses (2.5, 5, and 10 nM) of gedatolisib alone led to limited cytotoxicity (4.2-9.6%), up to 48.9% of cell death occurred when 20 or 80 nM nab-sirolimus was added. Results were similar with alpelisib (1 uM) plus nab-sirolimus (20 or 80 nM). Western blot showed increased p4EBP1 in response to fulvestrant or PI3Ki treatment, which was reversed with the addition of nab-sirolimus. Conclusions: nab-Sirolimus enhanced the cytotoxic effects of fulvestrant in HR+ BC cells and of PI3Kis in HR+ and HR− BC cells. Addition of nab-sirolimus to ET or other PI3K-AKT-mTOR pathway inhibitors may overcome mechanisms of resistance; further exploration is warranted to elucidate clinical relevance. Citation Format: Khine Nyein Myint, Sean Wallace, Shihe Hou, Maria Zalath, Brian McMorran, Andrew Kwon, Igor Vivanco. Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7196.

Somatic Loss-of-Function PIK3R1 and Activating Non-hotspot PIK3CA Mutations Associated with Capillary Malformation with Dilated Veins (CMDV)

Common capillary malformations (CMs) are red vascular skin lesions, most commonly associated with somatic activating GNAQ or GNA11 mutations. We focused on CMs lacking such a mutation to identify novel genetic causes. We used targeted next-generation-sequencing on 82 lesions. Bioinformatic analysis allowed the identification of 9 somatic pathogenic variants in PIK3R1 and PIK3CA, encoding for the regulatory and catalytic subunits of the PI3K kinase, respectively. Re-characterization of these lesions unraveled a common phenotype: a pale Capillary Malformation associated with visible Dilated Veins (CMDV). Primary-endothelial cells from two PIK3R1-mutated lesions were isolated and PI3K-AKT-mTOR and RAS-RAF-MAPK signaling were assessed by western-blot. This unveiled an abnormal increase in AKT phosphorylation, effectively reduced by PI3K pathway inhibitors, such as mTOR, AKT and PIK3CA-inhibitors. The effects of mutant PIK3R1 were further studied using zebrafish embryos. Endothelium-specific expression of PIK3R1 mutants resulted in abnormal development of the posterior capillary-venous plexus. In summary, CMDV emerges as a clinical entity associated with somatic pathogenic variants in PIK3R1 or PIK3CA (non-hotspot). Our findings suggest that the activated AKT signaling can be effectively reversed by PI3K-pathway inhibitors. Additionally, the proposed zebrafish model, holds promise as a valuable tool for future drug screening aimed at developing patient-tailored treatments.

Pharmacological Mechanisms of Kirenol against Ovarian Carcinoma: A Network Pharmacology and Experimental Validation Study In Vitro.

Ovarian carcinoma is an aggressive gynecological malignancy. Kirenol, a diterpene compound, has recently gained attention for its potential anticancer properties. However, its exact anti-tumor mechanism remains largely unexplored. In this study, we explored the inhibitory effects of Kirenol on ovarian cancer using network pharmacology and in vitro experiments and elucidated its underlying mechanisms. Through the utilization of molecular docking, we established a network of proteinprotein interactions (PPI), which unveiled CDK4 as an essential target. Additionally, gene enrichment and pathway analysis highlighted the significance of the PI3K/AKT pathway. The viability of ovarian cancer cells and normal ovarian epithelial cells was evaluated using CCK8 assays to determine the effect of Kirenol. Following in vitro tests, cell colony formation, wound healing, flow cytometry, and Western blotting were conducted to assess its impact on cell proliferation, metastasis, apoptosis, and the cell cycle. Kirenol significantly reduced the viability of ovarian cancer cells (SKOV3 and A2780) compared to normal ovarian epithelial cells (IOSE-80). Moreover, Kirenol efficiently suppressed the growth and movement, caused a cell cycle halt, and stimulated programmed cell death in SKOV3 and A2780 cells. Through molecular analysis, it was observed that Kirenol increased the expression of Bax while decreasing the expression of MMP2, MMP9, and Bcl-2. It also attenuated the phosphorylation of PI3K, AKT, and RB and downregulated CDK4 and CCND1 expression. Notably, co-treatment with the PI3K pathway inhibitor LY294002 enhanced the inhibitory effect of Kirenol on ovarian cancer cells. In summary, the combined results of our network pharmacology analysis and in vitro tests emphasized that Kirenol hinders the growth of ovarian cancer cells, causes cell cycle arrest, enhances apoptosis, and hampers migration, possibly by regulating the PI3K/AKT/CDK4 signaling pathway.

Notch signaling regulates Th17 cells differentiation through PI3K/AKT/mTORC1 pathway and involves in the thyroid injury of autoimmune thyroiditis.

Autoimmune Thyroiditis (AIT) is the most common thyroid disease; however, there were no measures to prevent the progression of the disease. The present study attempts to identify that Notch signaling regulates the differentiation of T helper 17 (Th17) cells by activating downstream Phosphatidylinositol-3 kinase/protein kinase/mechanistic target of rapamycin complex 1 (PI3K/AKT/mTORC1) pathway participating in the thyroid injury of the experimental autoimmune thyroiditis (EAT). In vivo experiments, mice were randomly divided into 4 groups: a control group, an EAT group, and two groups with LY294002 treatment (pTg plus 25mg/kg or 50mg/kg LY294002, respectively). The degrees of thyroiditis were evaluated, and the percentage of Th17 cells, expression of interleukin-17A (IL-17A), and the main components of the Notch-PI3K signaling pathway were detected in different groups. In vitro experiments, two different dosages of LY294002 (25 and 50μM) were used to intervene splenic mononuclear cells (SMCs) from EAT mice to further evaluate the regulatory effect of Notch-PI3K pathway on Th17 cells. Our data demonstrate that the infiltration of Th17 cells and the expressions of IL-17A, Notch, hairy and split 1 (Hes1), p‑AKT (Ser473), p‑AKT (Thr308), p‑mTOR (Ser2448), S6K1, and S6K2 increased remarkably in EAT mice. After PI3K pathway was blocked, the degrees of thyroiditis were significantly alleviated, and the proportion of Th17 cells, the expression of IL-17A, and the above Notch-PI3K pathway-related molecules decreased in a dose-dependent manner. Additionally, the proportion of Th17 cells was positively correlated with the concentration of serum thyroglobulin antibody (TgAb), IL-17A, and Notch-PI3K pathway-related molecules mRNA levels. Notch signal promotes the secretion of IL-17A from Th17 cells by regulating the downstream PI3K/AKT/mTORC1 pathway through Hes-Phosphatase and tensin homolog (PTEN) and participates in thyroid autoimmune damage, and the PI3K pathway inhibitor may play important effects on AIT by affecting Th17 cells differentiation.

A phase 1 study of triple-targeted therapy with BRAF, MEK, and AKT inhibitors for patients with BRAF-mutated cancers.

Aberrant PI3K/AKT signaling in BRAF-mutant cancers contributes to resistance to BRAF inhibitors. The authors examined dual MAPK and PI3K pathway inhibition in patients who had BRAF-mutated solid tumors (ClinicalTrials.gov identifier NCT01902173). Patients with BRAF V600E/V600K-mutant solid tumors received oral dabrafenib at 150mg twice daily with dose escalation of oral uprosertib starting at 50mg daily, or, in the triplet cohorts, with dose escalation of both oral trametinib starting at 1.5mg daily and oral uprosertib starting at 25mg daily. Dose-limiting toxicities (DLTs) were assessed within the first 56days of treatment. Radiographic responses were assessed at 8-week intervals. Twenty-seven patients (22 evaluable) were enrolled in parallel doublet and triplet cohorts. No DLTs were observed in the doublet cohorts (N=7). One patient had a DLT at the maximum administered dose of triplet therapy (dabrafenib 150mg twice daily and trametinib 2mg daily plus uprosertib 75mg daily). Three patients in the doublet cohorts had partial responses (including one who had BRAF inhibitor-resistant melanoma). Two patients in the triplet cohorts had a partial response, and one patient had an unconfirmed partial response. Pharmacokinetic data suggested reduced dabrafenib and dabrafenib metabolite exposure in patients who were also exposed to both trametinib and uprosertib, but not in whose who were exposed to uprosertib without trametinib. Concomitant inhibition of both the MAPK and PI3K-AKT pathways for the treatment of BRAF-mutated cancers was well tolerated, leading to objective responses, but higher level drug-drug interactions affected exposure to dabrafenib and its metabolites.

Inhibition of the PI3K/AKT/mTOR pathway in pancreatic cancer: is it a worthwhile endeavor?

Pancreatic cancer (PC) is an aggressive disease that is challenging to treat and is associated with a high mortality rate. The most common type of PC is pancreatic ductal adenocarcinoma (PDAC), and the existing treatment options are insufficient for PDAC patients. Due to the complexity and heterogeneity of PDAC, personalized medicine is necessary for effectively treating this illness. To achieve this, it is essential to understand the mechanism of PDAC carcinogenesis. Targeted therapies are a promising strategy to improve patient outcomes. Aberrant activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway plays a crucial role in PC pathogenesis, from initiation to progression. This review provides a comprehensive overview of the current state of knowledge regarding the PI3K pathway in PDAC, summarizes clinical data on PI3K pathway inhibition in PDAC, and explores potential effective combinations that are a promising direction requiring further investigation in PDAC.

Novel sulfonyl-substituted tetrandrine derivatives for colon cancer treatment by inducing mitochondrial apoptosis and inhibiting PI3K/AKT/mTOR pathway

Tetrandrine (TET) possesses multiple pharmacological activities and could suppress tumor proliferation via PI3K pathway inhibition. However, inferior antitumor activity and potential toxicity limit its clinical application. In the present study, a series of 14-sulfonamide and sulfonate TET derivatives were designed, synthesized, and evaluated for biological activities. Through structural-activity relationship studies, compound 3c with α, β-unsaturated carbonyl group exhibited the most potent activity against all tested tumor cell lines (including Hela, HCT116, HepG2, MCF-7, and SHSY5Y), as well as negligible toxicity against normal cell lines LO2 and HEK293. Additionally, compound 3c effectively inhibited HCT116 and CT26 cell proliferation in vitro with increased cell proportion in the G2/M phase, activated the mitochondrial apoptosis pathway, and induced colon cancer cell apoptosis by suppressing the PI3K/AKT/mTOR pathway. The further molecular docking results confirmed that compound 3c is potentially bound to multiple residues in PI3K with a stronger binding affinity than TET. Ultimately, compound 3c dramatically suppressed tumor growth in the CT26 xenograft tumor model, without noticeable visceral toxicity detected in the high-dose group. In summary, compound 3c might present new insights for designing new PI3K inhibitors and be a potential candidate for colon cancer treatment.