Phosphatidylinositol 3-kinase Research Articles

Gas6/AXL Alleviates Hepatic Ischemia/Reperfusion Injury by Inhibiting Ferroptosis via the PI3K/AKT Pathway.

Hepatic ischemia/reperfusion (I/R) injury is a major cause of complications in clinical liver surgery. AXL receptor tyrosine kinase (AXL) is a member of the TAM receptor tyrosine kinase family (TYRO3, AXL, and MERTK). Our previous study has shown that AXL expression was markedly upregulated in liver transplantation patients. However, the underlying mechanism of AXL in hepatic I/R injury remains unclear. A mouse liver warm I/R model and a primary hepatocyte hypoxia/reoxygenation model were established to investigate the role of AXL activation and ferroptosis in hepatic I/R injury by pretreating with recombinant mouse growth arrest-specific protein 6 (AXL activator) or R428 (AXL inhibitor). Moreover, we used LY294002 (phosphatidylinositol 3-kinase [PI3K] inhibitor) to evaluate the relationship between the PI3K/AKT (the Ser and Thr kinase AKT) pathway and ferroptosis in hepatic I/R injury. Hepatic I/R injury decreased phosphorylation AXL expression and enhanced ferroptosis in liver transplantation patients and hepatic I/R-subjected mice. AXL activation attenuated lipid peroxidation and ferroptosis in hepatic I/R injury in vivo and in vitro. Inhibition of AXL activation exacerbated liver pathological damage and liver dysfunction, as well as iron accumulation and lipid peroxidation in hepatic I/R injury. Mechanistically, activated growth arrest-specific protein 6/AXL and its downstream PI3K/AKT signaling pathway inhibited ferroptosis during hepatic I/R injury. AXL activation protects against hepatic I/R injury by preventing ferroptosis through the PI3K/AKT pathway. This study is the first investigation on the AXL receptor and ferroptosis, and activating AXL to mitigate ferroptosis may be an innovative therapeutic strategy to combat hepatic I/R injury.

Beneficial Effects of Echinacoside on Cognitive Impairment and Diabetes in Type 2 Diabetic db/db Mice.

Cognitive dysfunction is an important comorbidity of diabetes. Insulin resistance may play a critical role in diabetes-related cognitive impairment. Echinacoside (ECH), a natural phenylethanoid glycoside, is the active component of anti-diabetes prescriptions in traditional Chinese medicine. Its effect on modulating insulin resistance has been confirmed but modulating neurodegenerative disease remains unclear. Db/db mice, a spontaneous type 2 diabetes mode, were intragastrically administered ECH by 300 mg/kg or an equivalent volume of saline. Weight, blood glucose, and insulin resistance index were measured. Morris water maze test was performed to observe the compound effects on cognition. Hippocampal lesions were observed by histochemical analysis. In db/db mice, ECH alleviated diabetes symptoms, memory loss, and hippocampal neuronal damage. Next, the expression of CD44 and phosphorylated tau was upregulated in diabetic mice. In addition, the insulin receptor substrate-1/phosphatidylinositol 3-kinase /protein kinase B signaling pathway was dysregulated in diabetic mice. All these dysregulations could be reversed by ECH. This study provides theoretical support and experimental evidence for the future application of ECH in diabetic cognition dysfunction treatment, promoting the development of traditional medicines.

Endothelial Cell-Derived Exosomes Inhibit Osteoblast Apoptosis and Steroid-Induced Necrosis of Femoral Head Progression by Activating the PI3K/Akt/Bcl-2 Pathway

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed in vitro, which included cell migration, viability, and apoptosis. In vivo, a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&E) were used to observe femoral trabeculae. Our in vitro results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts in vitro. Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. In vivo, increased bone volume per tissue volume (BV/TV) p=0.031, trabecular thickness (Tb.Th) p=0.020, and decreased separation (Tb.Sp) p=0.040 were observed in SNFH rats treated with EC-exos. H&E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.

ABCG2 Mediates Resistance to the Dual EGFR and PI3K Inhibitor MTX-211 in Cancer Cells

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

Evaluation of copanlisib in combination with eribulin in triple negative breast cancer patient-derived xenograft models.

The phosphoinositide 3-kinase (PI3K) pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the anti-tumor effect in TNBC. A panel of 8 TNBC patient-derived xenograft (PDX) models were tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment induced signaling changes were examined by reverse phase protein array, immunohistochemistry, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). Compared to each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin induced mitotic arrest. The combination enhanced induction of apoptosis compared to each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan of PDX tumor and AKT phosphorylation by immunohistochemistry. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin. These data led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC.

Curcumin Improves Neurogenesis in Alzheimer’s Disease Mice via the Upregulation of Wnt/β-Catenin and BDNF

Adult neurogenesis in the dentate gyrus (DG) is impaired during Alzheimer’s disease (AD) progression. Curcumin has been reported to reduce cell apoptosis and stimulate neurogenesis. This study aimed to investigate the influence of curcumin on adult neurogenesis in AD mice and its potential mechanism. Two-month-old male C57BL/6J mice were injected with soluble β-amyloid (Aβ1–42) using lateral ventricle stereolocalization to establish AD models. An immunofluorescence assay, including bromodeoxyuridine (BrdU), doublecortin (DCX), and neuron-specific nuclear antigen (NeuN), was used to detect hippocampal neurogenesis. Western blot and an enzyme-linked immunosorbent assay (ELISA) were used to test the expression of related proteins and the secretion of brain-derived neurotrophic factor (BDNF). A Morris water maze was used to detect the cognitive function of the mice. Our results showed that curcumin administration (100 mg/kg) rescued the impaired neurogenesis of Aβ1–42 mice, shown as enhanced BrdU+/DCX+ and BrdU+/NeuN+ cells in DG. In addition, curcumin regulated the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) -mediated glycogen synthase kinase-3β (GSK3β) /Wingless/Integrated (Wnt)/β-catenin pathway and cyclic adenosine monophosphate response element-binding protein (CREB)/BDNF in Aβ1–42 mice. Inhibiting Wnt/β-catenin and depriving BDNF could reverse both the upregulated neurogenesis and cognitive function of curcumin-treated Aβ1–42 mice. In conclusion, our study indicates that curcumin, through targeting PI3K/Akt, regulates GSK3β/Wnt/β-catenin and CREB/BDNF pathways, improving the adult neurogenesis of AD mice.

Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases

Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

P,p'-DDT induces apoptosis in human endometrial stromal cells via the PI3K/AKT pathway and oxidative stress.

Bis-[4-chlorophenyl]-1,1,1-trichloroethane (DDT), one of the most widely used synthetic pesticides, is an endocrine-disrupting chemical with the potential to interfere with the human reproductive system. The effects of DDT and one of its metabolites, p,p'-DDT, on human endometrial stromal cells (ESCs) and health outcomes remain unknown. In this study, we investigated whether p,p'-DDT induces an imbalance in cell proliferation and apoptosis in human ESCs via oxidative stress. We assessed apoptosis in ESCs by quantifying the expression of markers associated with both intrinsic and extrinsic pathways. Additionally, we measured levels of reactive oxygen species (ROS), antioxidant enzyme activity, and estrogen receptors (ERs). We also examined changes in signaling involving nuclear factor kappa-light-chain-enhancer of activated B cells. Following treatment with 1,000 pg/mL of p,p'-DDT, we observed an increase in Bax expression, a decrease in Bcl-2 expression, and increases in the expression of caspases 3, 6, and 8. We also noted a rise in the generation of ROS and a reduction in glutathione peroxidase expression after treatment with p,p'-DDT. Additionally, p,p'-DDT treatment led to changes in ER expression and increases in the protein levels of phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (phospho-AKT), and phospho-extracellular signal-regulated kinase (phospho-ERK). p,p'-DDT was found to induce apoptosis in human ESCs through oxidative stress and an ER-mediated pathway. The activation of the PI3K/AKT and ERK pathways could represent potential mechanisms by which p,p'-DDT prompts apoptosis in human ESCs and may be linked to endometrial pathologies.

Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

ObjectiveOver the past decade, heat shock protein 90 (HSP90) inhibitors have emerged as promising anticancer drugs in solid and hematological malignancies. Flavokawain C (FKC) is a naturally occurring chalcone that has been found to exert considerable anti-tumor efficacy by targeting multiple molecular pathways. However, the efficacy of FKC has not been studied in nasopharyngeal carcinoma (NPC). Metabolic abnormalities and uncontrolled angiogenesis are two important features of malignant tumors, and the occurrence of these two events may involve the regulation of HSP90B1. Therefore, this study aimed to explore the effects of FKC on NPC proliferation, glycolysis, and angiogenesis by regulating HSP90B1 and the underlying molecular regulatory mechanisms.MethodsHSP90B1 expression was analyzed in NPC tissues and its relationship with patient’s prognosis was further identified. Afterward, the effects of HSP90B1 on proliferation, apoptosis, glycolysis, and angiogenesis in NPC were studied by loss-of-function assays. Next, the interaction of FKC, HSP90B1, and epidermal growth factor receptor (EGFR) was evaluated. Then, in vitro experiments were designed to analyze the effect of FKC treatment on NPC cells. Finally, in vivo experiments were allowed to investigate whether FKC treatment regulates proliferation, glycolysis, and angiogenesis of NPC cells by HSP90B1/EGFR pathway.ResultsHSP90B1 was highly expressed in NPC tissues and was identified as a poor prognostic factor in NPC. At the same time, knockdown of HSP90B1 can inhibit the proliferation of NPC cells, trigger apoptosis, and reduce glycolysis and angiogenesis. Mechanistically, FKC affects downstream EGFR phosphorylation by regulating HSP90B1, thereby regulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. FKC treatment inhibited the proliferation, glycolysis, and angiogenesis of NPC cells, which was reversed by introducing overexpression of HSP90B1. In addition, FKC can affect NPC tumor growth and metastasis in vivo by regulating the HSP90B1/EGFR pathway.ConclusionCollectively, FKC inhibits glucose metabolism and tumor angiogenesis in NPC by targeting the HSP90B1/EGFR/PI3K/Akt/mTOR signaling axis.

237 Disrupted insulin signaling due to an acute immune response in swine

Abstract Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Altered insulin signaling can occur due to metabolic state, pathogen exposure, and autoimmune disorders. Lipopolysaccharide (LPS), an endotoxin secreted produced by gram-negative bacteria, can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance have not been elucidated. Crossbred nursery pigs [n = 58, initial body weight (BW) = 12.1 kg ± 0.23 kg] were randomly assigned into two experimental subsets, where 10 animals were designated for repeated blood sampling and 48 animals underwent timed euthanasia for tissue collection. For repeated blood sampling, jugular catheters were placed under anesthesia and the animals were given 48 h to recover before being randomly assigned to treatments. These treatments were an intraperitoneal injection of either 15µg/kg BW LPS or an equal volume of 0.9% Saline (n = 5 pigs/treatment). Blood was collected for 48 h followed by euthanasia. The other 48 pigs were randomly assigned to the same treatments, and then randomly assigned to euthanasia timepoints of 0, 3, 6, 12, 24 or 48 h post injection for tissue collection. Among the catheterized pigs, 2-h post LPS challenge there was a decrease in serum insulin concentration (P < 0.05) and mild hyperglycemia (P < 0.05) post challenge. At 12- and 24-h post LPS challenge there was a decrease in insulin (P < 0.05) and a trend hyperglycemia was also apparent at 24 h (P = 0.063). In liver tissue, insulin related genes exhibited alterations post LPS injection. Phosphoinositide 3-kinase (pi3k) and insulin receptor substrate 1 (irs1) expression were decreased at 3 and 6 h (p< 0.05) and protein kinase b (akt) expression were decreased at 3 h (P < 0.05) post challenge. The expression of insulin inhibitor growth factor receptor bound protein 10 (grb10) was decreased at 6-, 12-, and 24-h after challenge (P < 0.05). Fructose-1-6-bisphosphatase (f16bp) expression was reduced at 3-, 6-, and 12-h (P < 0.05) in the liver while phosphoenolpyruvate carboxykinase (pepck) expression was decreased at 3- and 6-h (P < 0.01) post challenge. Other metabolic genes were decreased at 3-, 6-, and 12-h post LPS challenge, including fatty acid synthase (fas; P < 0.05), carbohydrate response element binding protein (chrebp; P < 0.05), and acetyl CoA carboxylase (acc; P < 0.05). Protein abundance of Akt 3 h after LPS injection was decreased in the liver (P < 0.05). In skeletal muscle 6 h post LPS injection, insulin receptor (insr), insulin-like growth factor receptor (igfr) and fas were all increased (P < 0.05) These results indicate disrupted insulin signaling at various levels after an acute immune response.

Therapeutic Effects of Melatonin in the Regulation of Ferroptosis: A Review of Current Evidence.

Ferroptosis is implicated in the pathogenesis of multiple diseases, including neurodegenerative diseases, cardiovascular diseases, kidney pathologies, ischemia-reperfusion injury, and cancer. The current review article highlights the involvement of ferroptosis in traumatic brain injury, acute kidney damage, ethanol-induced liver injury, and PM2.5-induced lung injury. Melatonin, a molecule produced by the pineal gland and many other organs, is well known for its anti- aging, anti-inflammatory, and anticancer properties and is used in the treatment of different diseases. Melatonin's ability to activate anti-ferroptosis pathways including sirtuin (SIRT)6/p- nuclear factor erythroid 2-related factor 2 (Nrf2), Nrf2/ antioxidant responsive element (ARE)/ heme oxygenase (HO-1)/SLC7A11/glutathione peroxidase (GPX4)/ prostaglandin-endoperoxide synthase 2 (PTGS2), extracellular signal-regulated kinase (ERK)/Nrf2, ferroportin (FPN), Hippo/ Yes-associated protein (YAP), Phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) and SIRT6/ nuclear receptor coactivator 4 (NCOA4)/ ferritin heavy chain 1 (FTH1) signaling pathways suggests that it could serve as a valuable therapeutic agent for preventing cell death associated with ferroptosis in various diseases. Further research is needed to fully understand the precise mechanisms by which melatonin regulates ferroptosis and its potential as a therapeutic target.

Capsaicin attenuates Porphyromonas gingivalis-suppressed osteogenesis of periodontal ligament stem cells via regulating mitochondrial function and activating PI3K/AKT/mTOR pathway.

Prevention of periodontal bone resorption triggered by Porphyromonas gingivalis (P. gingivalis) is crucial for dental stability. Capsaicin, known as the pungent ingredient of chili peppers, can activate key signaling molecules involved in osteogenic process. However, the effect of capsaicin on osteogenesis of periodontal ligament stem cells (PDLSCs) under inflammation remains elusive. P. gingivalis culture suspension was added to mimic the inflammatory status after capsaicin pretreatment. The effects of capsaicin on the osteogenesis of PDLSCs, as well as mitochondrial morphology, Ca2+ level, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and osteogenesis-regulated protein expression levels were analyzed. Furthermore, a mouse experimental periodontitis model was established to evaluate the effect of capsaicin on alveolar bone resorption and the expression of osteogenesis-related proteins. Under P. gingivalis stimulation, capsaicin increased osteogenesis of PDLSCs. Not surprisingly, capsaicin rescued the damage to mitochondrial morphology, decreased the concentration of intracellular Ca2+ and ROS, enhanced MMP and activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. The invivo results showed that capsaicin significantly attenuated alveolar bone loss and augmented the expression of bone associated proteins. Capsaicin increases osteogenesis of PDLSCs under inflammation and reduces alveolar bone resorption in mouse experimental periodontitis.

Abstract PO4-26-06: Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers

Abstract Introduction Triple negative breast cancer (TNBC) is an aggressive cancer defined by the absence of estrogen receptors (ER), progesterone receptors (PR), and lack of human epidermal growth factor receptor 2 (HER-2). We previously identified molecular subgroups with distinct mutational, epigenetic, and clinicopathologic signatures. Here, we sought to identify signaling pathways using targeted RNA expression-based pathway analysis. Methods We analyzed 44 TNBC cases diagnosed at NYU Langone Health between March 2011 and April 2018. Samples were compared based on histology, immunohistochemistry, and epigenetics. We assessed mRNA expression levels of pathway-specific target genes with RT-qPCR using the OncoSIGNal pathway profiling assay. Signal transduction pathway analyses were used to measure activity of the estrogen receptor (ER), androgen receptor (AR), phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), Hedgehog (HH), Notch, and transforming growth factor beta (TGF- β) pathways. Results DNA methylation Cluster 1, which is enriched for apocrine histology and PI3K/Akt mutations, showed no increased activity in PI3K pathway by RNA expression, but showed increase of AR and MAPK signaling, with a single case of increased ER signaling in the absence of ESR1 activating mutations. In contrast, both DNA methylation Cluster 2 (no recurrent mutations) and Cluster 3 (DNA instability cluster) showed upregulation in PI3K pathway in 4/7 (57%) and 11/25 (44%) of cases, respectively. PI3K pathway activation was invariably associated with high Ki-67 >15%, however not all high Ki-67 cases showed high PI3K activity. Invasive ductal carcinoma (IDC) morphology was characterized by a nearly even split between MAPK (13/28, 46%) and PI3K (12/28, 44%), with single HH and TGF- β driven cases. In 5/28 (18%) of IDC we did not identify an upregulated pathway. Discussion We show that RNA expression-based pathway activity and mutational and epigenetic analyses provide complimentary information about the molecular landscape of TNBC. PI3K mutations are early drivers and lead to distinct epigenetic signatures, however they are not associated with increased PI3K pathway activity by RNA expression. In contrast, TNBC without PI3K mutations may show activation of the PI3K pathway in the absence of PI3K/Akt mutations. Our observation suggests that distinct biomarkers may be differently suited to predict response to PI3K inhibitors in TNBC. This may include expanding the number of patients for which PI3K/Akt pathway inhibition might be a therapeutic option, as well as explaining the lack of response in PI3K mutated tumors. Citation Format: Chanel Schroff, Lawrence Lin, Dianne van Strijp, Daniel Roses, Sigi Neerken, Freya Schnabel, Farbod Darvishian, Matija Snuderl. Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers [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 PO4-26-06.

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.

Abstract PS17-06: A phase I trial of the PI3K inhibitor (PI3Ki) copanlisib and fulvestrant in combination with continuous or intermittent abemaciclib in patients with estrogen receptor-positive (ER+), HER2-negative (HER2-) metastatic breast cancer

Abstract Background: Activation of the phosphoinositide 3-kinase (PI3K) pathway is a prominent resistance mechanism to endocrine therapy and CDK4/6 inhibition (CDK4/6i) in ER+, HER2- breast cancer. There is reciprocal crosstalk between PI3K and cell cycle regulatory pathways. Triplet therapy that targets ER, CDK4/6, and PI3K prevents or delays disease progression in preclinical models of ER+, HER2- breast cancer. However, the toxicities of triplet therapy hinder its clinical development. Copanlisib (COP) is an intravenous (IV) PI3Ki with potent activity against α and δ isoforms. Intermittent high-dose PI3Ki is expected to be less toxic and more effective than continuous daily dosing. We set out to determine the recommended phase 2 dose (RP2D) for the COP/Fulvestrant (FUL)/Abemaciclib (ABE) combination. Methods: Eligible patients (pts) included men or women with ER+, HER2- metastatic breast cancer (MBC), with no more than 1 prior chemotherapy in the metastatic setting, and no limits on prior endocrine or targeted therapy. Prior FUL, CDK4/6i and PI3K/mTORi were allowed. Pts with HbA1c >8.5% were excluded. Pts received FUL 500 mg IM standard dosing and COP/ABE at assigned doses. Premenopausal women also received a GnRH agonist. Dose-limiting toxicity (DLT) was assessed during the first 28-day cycle. Adverse events (AEs) were graded according to NCI-CTCAE v5.0. Dose escalation/de-escalation was based on continual reassessment (CRM) and the RP2D was defined as the dose with the highest probability of the DLT rate within the acceptable 25~35%. Results: Between June 2020 and June 2023, 24 pts with ER+, HER2- MBC were enrolled. Table 1 shows the dose levels (DLs) and DLTs. The first 10 pts were enrolled to Part A on two consecutive DLs of COP (DL1: 45 mg IV D1 and D15, n=7; DL2: 45 mg IV D1, D8, and D15, n=3), with ABE fixed at 100 mg PO BID continuously. DL2a exceeded the maximum tolerated dose (MTD). DL1a was tolerable based on Cycle 1 AEs. However, during subsequent cycles, only 1 of the 7 pts in DL1a maintained the intended dose of ABE due to neutropenia, others reduced ABE to 50mg bid (n=5) or discontinued (n=1). This triggered a protocol amendment, adding Part B with ABE administered 5 days on and 2 days off (5-on/2-off) intermittent schedule. 14 pts were enrolled to Part B in 2 consecutive DLs of COP (DL1b: n=7; DL2b: n=7), the same as in Part A. ABE was fixed at 100 mg PO Bid 5-on/2-off. DL2b exceeded the MTD. DL1b was tolerable based on Cycle 1 AEs. In addition, in subsequent cycles, only 1 of 7 pts on DL1b required dose reduction of ABE to 50mg bid. RP2D is therefore defined as DL1b. Grade (G) 4 AEs were rare. The most common all cycle G3 AEs included transient hypertension (45%), rash (30%), anemia (25%), neutropenia (25%), and AST elevation (20%). As of 6/14/2023, 22 pts, with a median of 1 prior metastatic regimen (range 0-5), prior CDK4/6i in 18 (82%), liver mets in 8 (36%), and PIK3CA mutation in 8 (50%) of 16 pts with known mutation status, were evaluable for response. There were 5 partial responses and 3 stable diseases lasting for ≥24 weeks. Clinical benefit rate was 36.4% (8/22, 95%CI: 17.19% ~ 59.34%). Responses were observed in pts regardless of prior CDK4/6i, FUL, or PIK3CA mutation. Conclusion: COP/FUL/intermittent ABE can be safely administered with fair overall tolerability. Preliminary anti-tumor activity was observed, which will be further examined in the randomized phase II trial (NCT 03939897). Table 1 Dose-limiting Toxicities by Dose Level Citation Format: Cynthia Ma, Jingqin Luo, Jessica Moss, Maryann Kwa, Ritesh Parajuli, Katia Khoury, Emily Douglas, Ashley Frith, Caron Rigden, Foluso Ademuyiwa, Shana Thomas, Brittney Haas, Gerburg Wulf, Claire Dees, Rabih Said. A phase I trial of the PI3K inhibitor (PI3Ki) copanlisib and fulvestrant in combination with continuous or intermittent abemaciclib in patients with estrogen receptor-positive (ER+), HER2-negative (HER2-) metastatic 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 PS17-06.

Abstract PO1-26-03: Activity of alpelisib in a panel of breast XPDX models harboring hotspot and uncommon PIK3CA mutations

Abstract Background: Therapies targeting mutated phosphoinositide 3-kinase (PI3K) p110α catalytic subunit (PIK3CA) cancers remain an active field of research. Alpelisib was approved in combination with fulvestrant in ER+/HER2- breast cancer patients harboring PIK3CA mutations on hotspot amino acids E542, E545 Q546 or H1047. While mutation at these sites have been widely studied, they only comprise ~25% of identified variants in this protein. Recent studies have reported other mutations, including those at the c-terminus, which can lead to constitutive activation of PIK3CA; however, whether these variants are sensitive to agents like alpelisib is unclear. To better understand these variants and their role in PIK3CA-mediated breast cancer, we established a panel of PIK3CA-mutated ER+ and ER- breast XPDX models and evaluated each in vivo with single agent alpelisib. Methods: Ninety previously developed XPDX models representing PIK3CA-mutated ER+ and ER- breast cancer were evaluated in this study. Models were grown subcutaneously in female athymic nude mice supplemented with estradiol in drinking water when necessary and ER expression confirmed at multiple passes; PIK3A variants were determined by WES and RNAseq. For in vivo studies, alpelisib was administered once daily by oral gavage at 35 mg/kg through study completion. Endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C< 0%) versus Day 0 tumor volume was also reported. Results: 60% of PIK3CA-mutated models were found ER+ with 40% also HER2+ while 20% of ER- PIK3CA-mutated models were also HER2+. H1047X mutations accounted for 40% of all PIK3CA variants, 75% of which were single or dual PIK3CAH1047R. Several hotspot-mutated models were sensitive to alpelisib some with regressions including ST986 (E542K), ST1097 (E454K) and STF040 (H1047R), as well as ST1245C (R88Q/N1044H), ST2076 (102-103ins/R357Q) and ST4176 (E726K/H1047R). HER2+ models were less sensitive to alpelisib although STM148D (H1047L) reported tumor regressions. Interestingly, ST1799/PBR (E542K/H1065L), a palbociclib resistant clone of the ER+ parent ST1799 model was resistant to alpelisib while the parent was found sensitive. Conclusion: We have benchmarked a panel of PIK3CA-mutated breast XPDX models with alpelisib and compared activity of models harboring hotspot versus uncommon PIK3CA mutations with ER and HER2 expression. This data is a valuable tool in further developing PIK3CA inhibitors and identifying new variant targets in breast cancer. Citation Format: Mercedes Castaneda, Alyssa Simonson, Johnnie Flores, Maci DeBoer, Jim Lund, Tahmi Rouzbahan, Kyriakos P. Papadopoulos, Amy Lang, Gladys Rodriguez, Maryam Elmi, Arthur Rosenthal, Drew Rasco, Victor Moreno, Amita Patnaik, Tatiana Hernandez, Emiliano Calvo, Lon Smith, Manish Sharma, Muralidhar Beeram, Michael Wick. Activity of alpelisib in a panel of breast XPDX models harboring hotspot and uncommon PIK3CA mutations [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 PO1-26-03.

Abstract PO2-04-10: Effect of Alpelisib Dose Modification for AE Management on Progression-Free Survival and Treatment Duration in SOLAR-1 and BYLieve Clinical Trials

Abstract Background: Alpelisib (ALP) is an α-selective phosphatidylinositol 3-kinase (PI3K) inhibitor and degrader approved with fulvestrant (FUL) for patients (pts) with phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated, hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2−) advanced breast cancer (ABC) following progression on/after endocrine therapy-based treatment (tx). Toxicity limited ALP exposure in some pts in the SOLAR-1 and BYLieve studies. On-target effects of PI3K inhibitors, including hyperglycemia, rash, and diarrhea, are among the most common adverse events (AEs) associated with ALP. During SOLAR-1, median onset times were 15 and 13 days for grade ≥3 hyperglycemia and rash and 46 days for grade 2/3 diarrhea. ALP dose modification can help manage AEs. We report efficacy of ALP + FUL as second-line (2L) tx in pts who underwent ALP dose reduction (DR). Methods: Pts with PIK3CA mutations from SOLAR-1 and BYLieve, treated with ALP + FUL in 2L, were included. Landmark (LM) analyses were performed at 1, 2, 3, 4, 5, and 6 mo after tx initiation to evaluate the association between DR status (yes, no) with progression-free survival (PFS) and duration of tx (DoT), respectively. Pts with PFS or exposure time less than each LM time point were excluded from each LM analysis; pts were then grouped by DR (yes, no) before each LM time point. Median PFS and DoT were obtained using a modified Kaplan-Meier method. Alternatively, Cox regression analysis was used to assess the effect of DR on PFS and DoT, where 2 time-dependent variables were included separately in the model as a covariate: 1) DR status, and 2) DR groups (300 to 250 mg [1 DR] vs no DR, 300 to 250 to 200 mg [2 DR] vs no DR). Association between AEs and DR was evaluated descriptively. A matched analysis of AEs will be presented. Results: In all, the analysis included 212 pts in 2L: 77 from SOLAR-1, and 95 and 40 from BYLieve Cohorts A and C, respectively. Of these pts, 92 (43.4%) had no DR and 120 (56.6%) had DRs: 70 (58.3%) had 1 DR, 43 (35.8%) had 2, and 7 (5.8%) had other types of DR. 25 of 92 pts (27.2%) with no DR and 40 of 120 pts (33.3%) with DR had ≥12-mo exposure to ALP. Hazard ratios (HRs) for PFS and DoT in DR status for the LM analysis are shown (Table). HRs (95% CI) for PFS from the time-dependent covariate analyses were 1.28 (0.94-1.74) with DR status as the covariate, and 1.24 (0.88-1.76) and 1.21 (0.79-1.84) with DR group (1 DR vs no DR, 2 DR vs no DR) as the covariate, respectively; for DoT, these were 1.34 (1.00-1.79) for DR status, and 1.30 (0.93-1.80) and 1.30 (0.87-1.93) for DR group, respectively. During the treatment period, the incidence rate for grade ≥3 AEs was the highest for pts with 2 DR and lowest for those with no DR. Similarly, incidence of AEs leading to DR or tx discontinuation was higher in pts with 2 DR than 1 DR. The post-DR incidence rate for grade ≥3 AE was similar for pts with 2 DR or 1 DR. A matched analysis will assess the relationship between DR group and AE. Conclusions: In this pooled analysis, regardless of if/when DR occurred, PFS was similar and DoT may be sustained with ALP + FUL. ALP DR can potentially reduce AEs, which may allow pts to remain on tx. Using DR as an AE management strategy may enable pts with PIK3CA-mutated, HR+, HER2− ABC to optimize tx duration and clinical benefit of ALP in the 2L. Landmark Analyses of PFS and Tx Duration by DR Group (Yes vs No; Pooled FAS) Citation Format: Hope Rugo, Alessandra Gennari, Stephen Chia, Dejan Juric, Neil Vasan, Sherko Küemmel, Patrick Neven, Florence Lerebours, Manuel Ruíz - Borrego, Pedram Razavi, Jyotika Singh, Yogesh Chattar, Murat Akdere, Eva Ciruelos. Effect of Alpelisib Dose Modification for AE Management on Progression-Free Survival and Treatment Duration in SOLAR-1 and BYLieve Clinical Trials [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 PO2-04-10.