Phosphatidylinositol 3-kinase Signaling Research Articles

Idebenone Attenuates Diabetic Retinopathy by Modulating Autophagy Via Targeting Akt Signaling.

Diabetic Retinopathy (DR) is a common microvascular issue caused by diabetes. Idebenone (IDE) is a coenzyme Q10 analog and antioxidant that has been utilized in the treatment of neurodegenerative diseases. Our goal was to investigate how IDE might treat diabetic retinopathy. An in vivo DR model was established by injecting a single dose of streptozotocin (STZ). Rats were treated with IDE, and their vascular function was measured by ultrasound. The retina structure was checked by haematoxylin and eosin (HE) staining. The expression of biomarkers of autophagy and apoptosis was measured by western blotting assay. The retina endothelial cell line RF/6A was stimulated with high glucose (HG) and treated with IDE. Cell proliferation and apoptosis were assessed using the Edu assay, TUNEL assay, and flow cytometry, respectively. Reduced peak systolic velocity (PSV), mean velocity (MV), end-diastolic velocity (EDV), and increased pulsatility index (PI) and resistance index (RI) were observed in diabetic rats; however, these traits were reversed by IDE therapy. IDE alleviated the STZ-induced disordered retina structure. The IDE administration suppressed DR-induced apoptosis and autophagy both in vivo and in vitro. IDE suppressed the activation of Phosphatidylinositol 3 kinase (PI3K) signaling. Activation of PI3K abolished the IDE-alleviated retina damage and cell death. IDE regulated the autophagy of retina cells to alleviate diabetic retinopathy via regulating the PI3K signaling pathway.

Small molecule modulation of insulin receptor-insulin like growth factor-1 receptor heterodimers in human endothelial cells

ObjectivesThe insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of two extracellular α-subunits and two transmembrane β -subunits. Insulin αβ and insulin like growth factor-1 αβ hemi-receptors can heterodimerize to form hybrids composed of one IR αβ and one IGF-1R αβ. The function of hybrids in the endothelium is unclear. We sought insight by developing a small molecule capable of reducing hybrid formation in endothelial cells. MethodsWe performed a high-throughput small molecule screening, based on a homology model of the apo hybrid structure. Endothelial cells were studied using western blotting and qPCR to determine the effects of small molecules that reduced hybrid formation. ResultsOur studies unveil a first-in-class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effects on IR or IGF-1R. This small molecule reduced expression of the negative regulatory p85α subunit of phosphatidylinositol 3-kinase, increased basal phosphorylation of the downstream target Akt and enhanced insulin/insulin-like growth factor-1 and shear stress-induced serine phosphorylation of Akt. In primary saphenous vein endothelial cells (SVEC) from patients with type 2 diabetes mellitus undergoing coronary artery bypass (CABG) surgery, hybrid receptor expression was greater than in patients without type 2 diabetes mellitus. The small molecule significantly reduced hybrid expression in SVEC from patients with type 2 diabetes mellitus. ConclusionsWe identified a small molecule that decreases the formation of IR: IGF-1R hybrid receptors in human endothelial cells, without significant impact on the overall expression of IR or IGF-1R. In HUVECs, reduction of IR: IGF-1R hybrid receptors leads to an increase in insulin-induced serine phosphorylation of the critical downstream signalling kinase, Akt. The underpinning mechanism appears, at least in part to involve the attenuation of the inhibitory effect of IR: IGF-1R hybrid receptors on PI3-kinase signalling.

Regulation of PI3K signaling in cancer metabolism and PI3K-targeting therapy.

The phosphatidylinositol-3-kinase (PI3K) signaling plays a key role in various cellular functions and is frequently activated in cancer, making it an attractive therapeutic target. The PI3K signaling pathway influencing glucose metabolism, lipid synthesis, nucleotide production, and protein synthesis, all of which contribute to cancer cell proliferation and survival. It enhances glucose uptake through the activation of glucose transporters and glycolysis, while also promoting lipid synthesis via downstream factors like mTORC1. This pathway boosts nucleotide synthesis by regulating transcription factors like MYC, activating key enzymes for purine and pyrimidine production. Additionally, due to its essential role in cancer cell growth, the PI3K pathway is a key target for anticancer therapies. However, treatment using PI3K inhibitors alone has limitations, including drug resistance and significant side effects such as hyperglycemia, fatigue, and liver dysfunction. Clinical trials have led to the development of isoform-specific PI3K inhibitors to reduce toxicity. Combining PI3K inhibitors with other treatments, such as hormone therapy or surgery, may improve efficacy and minimize side effects. Further research is needed to fully understand the mechanisms of PI3K inhibitors and improve individualized treatment approaches. In this review, we introduce the characteristic of three classes of PI3Ks, discuss the regulation of cancer metabolism including the control of glucose uptake, glycolysis, de novo lipid synthesis, nucleotide synthesis and protein synthesis, and review the current statuses of different PI3K inhibitors therapy.

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking.

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFRα signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFRα signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

Pericyte-to-Endothelial Cell Communication via Tunneling Nanotubes Is Disrupted by a Diol of Docosahexaenoic Acid.

The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte-endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte-endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy.

Extracellular Matrix Sulfation in the Tumor Microenvironment Stimulates Cancer Stemness and Invasiveness.

Tumor extracellular matrices (ECM) exhibit aberrant changes in composition and mechanics compared to normal tissues. Proteoglycans (PG) are vital regulators of cellular signaling in the ECM with the ability to modulate receptor tyrosine kinase (RTK) activation via their sulfated glycosaminoglycan (sGAG) side chains. However, their role on tumor cell behavior is controversial. Here, it is demonstrated that PGs are heavily expressed in lung adenocarcinoma (LUAD) patients in correlation with invasive phenotype and poor prognosis. A bioengineered human lung tumor model that recapitulates the increase of sGAGs in tumors in an organotypic matrix with independent control of stiffness, viscoelasticity, ligand density, and porosity, is developed. This model reveals that increased sulfation stimulates extensive proliferation, epithelial-mesenchymal transition (EMT), and stemness in cancer cells. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K) signaling axis is identified as a mediator of sulfation-induced molecular changes in cells upon activation of a distinct set of RTKs within tumor-mimetic hydrogels. The study shows that the transcriptomic landscape of tumor cells in response to increased sulfation resembles native PG-rich patient tumors by employing integrative omics and network modeling approaches.

Differential gene expression and pathway analysis in growth hormone-secreting pituitary tumors according to granulation pattern.

This study investigated differential gene expression between granulation patterns in growth hormone (GH)-secreting pituitary tumors, aiming to elucidate novel transcriptomes that explain clinical variances in patients with acromegaly. Transcriptome analysis was conducted on 6 normal pituitary tissues and 15 GH-secreting pituitary tumors, including 9 densely granulated somatotroph tumors (DGSTs) and 6 sparsely granulated somatotroph tumors (SGSTs). We identified 3111 differentially expressed genes (DEGs) in tumors compared to normal pituitaries, with 1117 DEGs unique to a specific granulation within tumors. SGST showed enrichment of neuronal development and acute inflammatory response pathways, along with a significant enhancement of JAK-STAT, phosphatidylinositol 3-kinase, and MAPK signaling. The results suggest that granulation-specific gene expression may underpin diverse clinical presentations in acromegaly, highlighting the potential for further investigation into these transcriptomic variations and their roles in disease pathology, particularly the involvement of genes linked to neuronal development, inflammatory response, and JAK-STAT signaling in SGST.

Dual Inhibition of PI3 Kinase and MAP Kinase Signaling Pathways in Intrahepatic Cholangiocellular Carcinoma Cell Lines Leads to Proliferation Arrest but Not Apoptosis.

Cholangiocellular carcinoma (CCA) is the second most common primary liver cancer, with increasing incidence worldwide and inadequate therapeutic options. Intra- and extrahepatic bile ducts have distinctly different embryonic origins and developmental behavior, and accordingly, intra- and extrahepatic CCAs (ICC vs. ECC) are molecularly different. A promising strategy in oncotherapy is targeted therapy, targeting proteins that regulate cell survival and proliferation, such as the MAPK/ERK and PI3K/AKT/mTOR signaling pathways. Inhibitors of these pathways have been tested previously in CCA cell lines. However, these cell lines could not be clearly assigned to ICC or ECC, and the results indicated apoptosis induction by targeted therapeutics. We tested targeted therapeutics (selumetinib, MK2206) in three defined ICC cell lines (HuH28, RBE, SSP25). We observed additive effects of the dual inhibition of the two pathways, in accordance with the inhibition of phospho-AKT and phospho-ERK1/2 expression. Proliferation was blocked more effectively with dual inhibition than with each single inhibition, but cell numbers did not drop below baseline. Accordingly, we observed G1 phase arrest but not apoptosis or cell death (measured by cleaved caspase-3, AIFM1 regulation, sub-G0/G1 phase). We conclude that the dual inhibition of the MAPK/ERK and PI3K/AKT/mTOR pathways is highly effective to block the proliferation of ICC cell lines in vitro; however, potential clinical applications must be critically examined, as a proliferation block could also induce resistance to standard therapies.

Large-scale control over collective cell migration using light-controlled epidermal growth factor receptors.

Receptor tyrosine kinases (RTKs) are thought to play key roles in coordinating cell movement at single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggested these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled EGF receptor (OptoEGFR) can be deployed in epithelial cell lines for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by PI 3-kinase signaling, rather than diffusible signals, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications including wound healing and tissue morphogenesis.

Exploring active ingredients and mechanisms of Coptidis Rhizoma-ginger against colon cancer using network pharmacology and molecular docking.

Colon cancer is the most prevalent and rapidly increasing malignancy globally. It has been suggested that some of the ingredients in the herb pair of Coptidis Rhizoma and ginger (Zingiber officinale), a traditional Chinese medicine, have potential anti-colon cancer properties. This study aimed to investigate the molecular mechanisms underlying the effects of the Coptidis Rhizoma-ginger herb pair in treating colon cancer, using an integrated approach combining network pharmacology and molecular docking. The ingredients of the herb pair Coptidis Rhizoma-ginger, along with their corresponding protein targets, were obtained from the Traditional Chinese Medicine System Pharmacology and Swiss Target Prediction databases. Target genes associated with colon cancer were retrieved from the GeneCards and OMIM databases. Then, the protein targets of the active ingredients in the herb pair were identified, and the disease-related overlapping targets were determined using the Venn online tool. The protein-protein interaction (PPI) network was constructed using STRING database and analyzed using Cytoscape 3.9.1 to identify key targets. Then, a compound-target-disease-pathway network map was constructed. The intersecting target genes were subjected to Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for colon cancer treatment. Molecular docking was performed using the Molecular Operating Environment (MOE) software to predict the binding affinity between the key targets and active compounds. Besides 1922 disease-related targets, 630 targets associated with 20 potential active compounds of the herb pair Coptidis Rhizoma-ginger were collected. Of these, 229 intersection targets were obtained. Forty key targets, including STAT3, Akt1, SRC, and HSP90AA1, were further analyzed using the ClueGO plugin in Cytoscape. These targets are involved in biological processes such as miRNA-mediated gene silencing, phosphatidylinositol 3-kinase (PI3K) signaling, and telomerase activity. KEGG enrichment analysis showed that PI3K-Akt and hypoxia-inducible factor 1 (HIF-1) signaling pathways were closely related to colon cancer prevention by the herb pair Coptidis Rhizoma-ginger. Ten genes (Akt1, TP53, STAT3, SRC, HSP90AA1, JAK2, CASP3, PTGS2, BCl2, and ESR1) were identified as key genes for validation through molecular docking simulation. This study demonstrated that the herb pair Coptidis Rhizoma-ginger exerted preventive effects against colon cancer by targeting multiple genes, utilizing various active compounds, and modulating multiple pathways. These findings might provide the basis for further investigations into the molecular mechanisms underlying the therapeutic effects of Coptidis Rhizoma-ginger in colon cancer treatment, potentially leading to the development of novel drugs for combating this disease.

Integration of Multicomplex‐Based Pharmacophore Modeling and Molecular Docking in Machine Learning‐Based Virtual Screening: Toward the Discovery of Novel PI3K Inhibitors

AbstractThe phosphatidylinositol‐3 kinase (PI3K) pathway is a crucial intracellular signaling pathway within living cells. The hyperactivation of PI3K signaling cascades is a common occurrence in human cancers, rendering PI3K a promising therapeutic target. Although several PI3K inhibitors are already available on the market, the adverse side effects of current therapies continue to highlight the necessity for the development of novel PI3K inhibitors. In this study, a virtual screening strategy employing naïve Bayesian classification (NBC) models, based on multicomplex‐based molecular docking and pharmacophore modeling, is developed. First, the docking accuracy and scoring reliability of four docking software are assessed, and Glide demonstrated higher predictability for PI3K inhibitors. Second, pharmacophore models are generated based on the current reported PI3K‐inhibitor interactions, and five pharmacophore hypotheses displayed significant capability in discriminating active PI3K molecules from inactive ones. Subsequently, three NBC models are constructed based on molecular docking and/or pharmacophore models, and the validation results showed that the NBC model, combining multicomplex‐based molecular docking and pharmacophore, significantly improved the hit rate of virtual screening against PI3K. Finally, the optimal NBC model is employed for virtual screening against the ChEMBL database, leading to the identification of multiple molecules with high potential as active PI3K inhibitors.

EGFR and PI3K Signalling Pathways as Promising Targets on Circulating Tumour Cells from Patients with Metastatic Gastric Adenocarcinoma.

The prognosis for metastatic gastric adenocarcinoma (mGAC) remains poor. Gene alterations in receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and their downstream effectors including catalytic subunit alpha of the phosphatidylinositol 3-kinase (PIK3CA) are common in mGAC. Targeted RTK and phosphatidylinositol-3-kinase (PI3K) treatments have demonstrated clinical benefits in other solid tumours and are key potential targets for clinical development against mGAC given the presence of recurrent alterations in these pathways. Furthermore, combination RTK/PI3K treatments may overcome compensatory mechanisms that arise using monotherapies, leading to improved patient outcomes. Herein, we investigated RTK/PI3K single and combination drug responses against our unique human mGAC-derived PIK3CA gain-of-function mutant, human epidermal growth factor receptor 2 (HER2)-negative, EGFR-expressing circulating tumour cell line, UWG02CTC, under two- and three-dimensional culture conditions to model different stages of metastasis. UWG02CTCs were highly responsive to the PI3K p110α-subunit targeted drugs PIK-75 (IC50 = 37.0 ± 11.1 nM) or alpelisib (7.05 ± 3.7 µM). Drug sensitivities were significantly increased in 3D conditions. Compensatory MAPK/ERK pathway upregulation by PI3K/Akt suppression was overcome by combination treatment with the EGFR inhibitor gefitinib, which was strongly synergistic. PIK-75 plus gefitinib significantly impaired UWG02CTC invasion in an organotypic assay. In conclusion, UWG02CTCs are a powerful ex vivo mGAC drug responsiveness model revealing EGFR/PI3K-targeted drugs as a promising combination treatment option for HER2-negative, RAS wild-type mGAC patients.

Abstract PO3-02-03: Ketogenic diet and letrozole to inhibit PI3K signaling in ER+ breast cancer

Abstract Obesity is associated with an increased risk of breast cancer recurrence, similar in magnitude to the reduction in risk seen with the use of adjuvant chemotherapy. However, whether and how obesity causes that increased risk of recurrence remains unknown. Plausible mechanisms include obesity-mediated increases in estrogen signaling, inflammatory conditions resulting from obesity, or effects on insulin or other growth factor signaling pathways resulting from the insulin-resistant state that often accompanies obesity. While phosphatidyl inositol 3-kinase (PI3K) signaling is the normal, biological effector of insulin signaling, abnormal activation of PI3K is of particular concern in breast cancer, where it is associated with resistance to endocrine therapies and HER2-targeted therapies. We hypothesize that at least part of the adverse outcome associated with obesity results from aberrant activation of PI3K signaling in tumors. This may confer resistance to established therapies, or directly stimulate tumor growth (particularly in tumors with existing PI3K pathway mutations), or both. We tested a dietary intervention designed to alter insulin resistance pathophysiology coupled with endocrine therapy in the well-established neoadjuvant treatment paradigm in breast cancer. This will allow us to study the effects of the dietary intervention directly on tumor biology. The primary objective of this neoadjuvant study was to assess feasibility and tolerability of 2 weeks of a very low-carbohydrate ketogenic diet in combination with letrozole for patients with early stage operable ER+ disease. Up to 36 patients will be enrolled for this pilot and feasibility study (24 in the treatment group and 12 controls). Baseline metabolic parameters will be measured and the treatment group will begin a dietitian-supervised 2-week diet to induce a ketogenic state, along with letrozole 2.5 mg daily. The control group will receive only letrozole. At the end of 2 weeks, patients will proceed with surgical treatment of their breast cancer. We will report the primary outcome measure of feasibility, assessed by determining 1) the proportion of patients in the diet group achieving ketosis, 2) adherence to the diet and 3) participant-reported measures of stress, fatigue, and emotional health. For both groups, cell proliferation will be measured in a tumor biopsy obtained from the surgical specimen and compared with the pre-treatment diagnostic biopsy. Correlative studies will evaluate tumor markers of insulin/PI3K signaling before and after the intervention and between diet and control. Citation Format: Brent Rexer, Vandana Abramson, Sara Nunnery, Sonya Reid, Laura Kennedy, Melinda Sanders, Jessica Bennett, Paula Gonzalez-Ericsson, Violeta Sanchez. Ketogenic diet and letrozole to inhibit PI3K signaling in ER+ 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 PO3-02-03.

Abstract ED10-02: Identifying Collateral Vulnerabilities in the AKT pathway for Therapeutic Benefit

Abstract The PI 3-kinase (PI3K) and AKT signaling pathway plays a critical role in regulating all aspects of normal cellular physiology, and is also frequently deregulated in human pathophysiologies, most evidently in cancer and diabetes. Three AKT isoforms exist in humans encoded by distinct genes (AKT1, AKT2, AKT3), and although originally thought to function redundantly, many studies have shown that AKT isoforms have non-overlapping and unique roles in both normal physiology and disease. Similarly, genetic lesions in the PIK3CA and AKT oncogenes have been described, and many of the genes that contribute to PI3K/AKT pathway activation and also signal termination have been found to be altered in human cancers. Numerous drugs that inhibit PI3K as well AKT have been developed for therapeutic use in patients, and many of these are being evaluated in late-stage clinical trials. During the seminar, I will review the major mechanisms that contribute to AKT hyperactivation in cancer. Genetic lesions in the PI3K/AKT pathway in human cancers will also be discussed, as well as efforts to target this pathway therapeutically, including our recent efforts aimed at developing new AKT degrader compounds. I will further present new studies using CRISPR-based genome wide synthetic lethal screens to identify genes that synergize with PI3K and AKT pathway inhibition to induce a cytotoxic response in TNBC addicted to pathway hyperactivation. I will present studies on genes and mechanisms that represent collateral vulnerabilities to pathway inhibition and our efforts at designing combination therapy strategies for cancer patients with genetic aberrations in the PI3K and AKT pathway. Citation Format: A. Toker. Identifying Collateral Vulnerabilities in the AKT pathway for Therapeutic Benefit [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 ED10-02.

Rational drug design from phosphatidylinositol 3-kinase-α inhibitors through molecular docking and 3D-QSAR methodologies for cancer immunotherapy

Dysregulation or aberrant activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is commonly observed in various cancers and is associated with tumor growth, metastasis, and resistance to therapy. Targeting PI3K-α with appropriate inhibitors can disrupt this pathway, hindering cancer progression, and potentially enhancing the immune system’s ability to recognize and eliminate cancer cells. In this study, we aimed to design a novel and potent inhibitor of PI3K-α for cancer immunotherapy using rational drug design techniques, including virtual screening, molecular docking, and 3D-QSAR. We obtained the human PI3K-α protein (6PYS) complexed with (3S)-3-benzyl-3-methyl-5-[5-(2-methylpyrimidin-5-yl)pyrazolo[1,5-a]pyrimidin-3-yl]-1,3-dihydro-2H-indol-2-one (PJ5) from the RCSB Protein Data Bank. Virtual screening of ligands, integrated with predictive computational molecular docking and 3D-field-based-QSAR, was implemented using appropriate Schrödinger Maestro modules. Rational drug design was also carried out, and its clinical relevance was validated across several ADMET descriptors. Docking results suggested that a hybrid of sulfonamide and pyridine-based heterocyclic compounds, functionalized with potent moieties derived from alkaloids, exhibited adequate synergistic biological effects capable of enhancing sufficient biological activity against PI3K-α. A field-based 3D-QSAR model was built on four partial least squares factors, and five statistical metrics were employed to validate the model. The newly designed ligand from this approach, named 6’-amino-5’-(2-fluoro-1,3-oxazol-5-yl)-N-{[3-(hydroxymethyl)oxetan-3-yl]methyl}-3-methyl-[2,3’-bipyridine]-6-sulfonamide or T85, exhibited a predicted bioactivity (pIC50) of 8.25. The predicted ADMET properties of T85 fell reasonably within the range of recommended standards, especially adhering to Lipinski’s rule of five and Jorgensen’s rule of three. In conclusion, the results of this study offer significant insights into in silico drug design using a rational approach, which could expedite the discovery and development of new drug molecules.

The PI3K/AKT/mTOR signaling pathway in breast cancer: Review of clinical trials and latest advances.

Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer mortality in women. As the phosphatidylinositol 3-kinase (PI3K) signaling pathway is involved in a wide range of physiological functions of cells including growth, proliferation, motility, and angiogenesis, any alteration in this axis could induce oncogenic features; therefore, numerous preclinical and clinical studies assessed agents able to inhibit the components of this pathway in BC patients. To the best of our knowledge, this is the first study that analyzed all the registered clinical trials investigating safety and efficacy of the PI3K/AKT/mTOR axis inhibitors in BC. Of note, we found that the trends of PI3K inhibitors in recent years were superior as compared with the inhibitors of either AKT or mTOR. However, most of the trials entering phase III and IV used mTOR inhibitors (majorly Everolimus) followed by PI3K inhibitors (majorly Alpelisib) leading to the FDA approval of these drugs in the BC context. Despite favorable efficacies, our analysis shows that the majority of trials are utilizing PI3K pathway inhibitors in combination with hormone therapy and chemotherapy; implying monotherapy cannot yield huge clinical benefits, at least partly, due to the activation of compensatory mechanisms. To emphasize the beneficial effects of these inhibitors in combined-modal strategies, we also reviewed recent studies which investigated the conjugation of nanocarriers with PI3K inhibitors to reduce harmful toxicities, increase the local concentration, and improve their efficacies in the context of BC therapy.

Cytotoxic and Antiproliferative Activity of LASSBio-2208 and the Attempts to Determine Its Drug Metabolism and Pharmacokinetics In Vitro Profile.

Inappropriate expression of histone deacetylase (HDAC-6) and deregulation of the phosphatidylinositol 3-kinase (PI3K) signalling pathway are common aberrations observed in cancers. LASSBio-2208, has been previously described as a dual inhibitor in the nanomolar range of HDAC-6 and PI3Kα and is three times more potent in inhibiting HDAC-6. In this paper we described the cytotoxic and antiproliferative potency of LASSBio-2208 on different tumour cell lines, its possible synergism effect in association with PI3K and HDAC-6 inhibitors, and its drug metabolism and pharmacokinetics (DMPK) in vitro profile. Our studies have demonstrated that LASSBio-2208 has moderate cytotoxic potency on breast cancer cell line MCF-7 (IC50 = 23 µM), human leukaemia cell line CCRF-CEM (IC50 = 8.54 µM) and T lymphoblast cell line MOLT-4 (IC50 = 7.15 µM), with no cytotoxic effect on human peripheral blood mononuclear cells (hPBMC). In addition, it has a good antiproliferative effect on MCF-7 cells (IC50 = 5.44 µM), low absorption by parallel artificial membrane permeability-gastrointestinal tract (PAMPA-GIT) and low permeation by parallel artificial membrane permeability-blood-brain barrier (BBB) (PAMPA-BBB), exhibiting high metabolic stability in rat plasma. Moreover, LASSBio-2208 exhibited synergism when combined with getadolisib and tubastatin A, using the concentrations corresponding to their CC50 values on MOLT-4 and CCRF-CEM cells.

Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion.

Plasmodium falciparum is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after P. falciparum invasion, while β-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by P. falciparum phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in P. falciparum-infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.

M6A/m1A/m5C-Associated Methylation Alterations and Immune Profile in MDD

Major depressive disorder (MDD) is a prevalent psychiatric condition often accompanied by severe impairments in cognitive and functional capacities. This research was conducted to identify RNA modification-related gene signatures and associated functional pathways in MDD. Differentially expressed RNA modification-related genes in MDD were first identified. And a random forest model was developed and distinct RNA modification patterns were discerned based on signature genes. Then, comprehensive analyses of RNA modification-associated genes in MDD were performed, including functional analyses and immune cell infiltration. The study identified 29 differentially expressed RNA modification-related genes in MDD and two distinct RNA modification patterns. TRMT112, MBD3, NUDT21, and IGF2BP1 of the risk signature were detected. Functional analyses confirmed the involvement of RNA modification in pathways like phosphatidylinositol 3-kinase signaling and nucleotide oligomerization domain (NOD)-like receptor signaling in MDD. NUDT21 displayed a strong positive correlation with type 2 T helper cells, while IGF2BP1 negatively correlated with activated CD8 T cells, central memory CD4 T cells, and natural killer T cells. In summary, further research into the roles of NUDT21 and IGF2BP1 would be valuable for understanding MDD prognosis. The identified RNA modification-related gene signatures and pathways provide insights into MDD molecular etiology and potential diagnostic biomarkers.

Abstract B028: Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia

Abstract Somatic mutations in the phosphatidylinositol 3-kinase (PI3K) pathway occur in 84% of endometrial carcinomas (EC) and up to 68% of endometrial hyperplasia (EH). Regulation of PI3K/Akt/mTOR signaling is integral for normal endometrial function and is governed by tumor suppressor genes PTEN and PIK3R1 and proto-oncogene PIK3CA. While the actions of PTEN and PIK3CA are in direct opposition as a phosphatase and kinase, respectively, PIK3R1 regulates PI3K/Akt/mTOR signaling in two distinct ways. As the regulatory subunit of the PI3K enzyme, PIK3R1 controls the catalytic activity of PIK3CA and augments PTEN activity. Mutations or alterations in PIK3R1 are present in 33% of EC and 9% of EH, the majority of which lead to loss of PIK3R1 protein (p85a). Both loss of PTEN and PIK3R1 lead to increased proliferation through PI3K/Akt/mTOR signaling, but the structural, functional, and mechanistic implications of PIK3R1 loss in the endometrial epithelium (EE) in vivo are largely unexplored. To further interrogate the changes in the endometrium resulting from PIK3R1 loss, we have created a murine model of endometrial epithelial-specific PIK3R1 loss driven by Pax8-Cre. Mice were estrus cycle staged and sacrificed in the diestrus phase to reduce confounding variables when assessing phenotypes between genotypes. PIK3R1 loss was confirmed by immunohistochemistry (IHC). Histology and immunostaining of control and PIK3R1 knockout (KO) mice were assessed by IHC and immunofluorescence. We found that PIK3R1 loss in the EE leads to EH distinct from what is observed with PTEN loss. In contrast to the EH with atypia seen In PTEN KO mice, the EH resulting from PIKR1 loss more closely resembles EH without atypia. The endometrium of PIK3R1 KO mice is characterized by thickened luminal epithelia with jagged edges and irregularities, mild glandular crowding, and occasional nuclear atypia. PIK3R1 KO mice also showed an increase in proliferation (Ki-67) and downstream PI3K signaling (phospho-S6) in the EE. Gene expression changes occurring within the EE from PIK3R1 KO and Cre-negative control mice were also assessed by RNA-seq. In vivo genomic analyses were compared to in vitro knockdown of PIK3R1 and publicly available patient data. We found that PIK3R1 loss leads to an increase in signaling through the PI3K/Akt/mTOR and MAPK pathways. Our data show that the effect of PIK3R1 loss is sufficient to cause EH and may have shared and distinct actions from PTEN. Citation Format: Shannon K. Harkins, Hilary J. Skalski, Amelia Arendt, Laura Pavliscak, Mike R. Wilson, Ronald L. Chandler. Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B028.