Subunit Of Phosphoinositide 3-kinase Research Articles

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12wk of age. Both sexes had smaller marrow areas from 6wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Regulatory roles of Osteopontin in lung epithelial inflammation and epithelial-telocyte interaction.

Lung epithelial cells play important roles in lung inflammation and injury, although mechanisms remain unclear. Osteopontin (OPN) has essential roles in epithelial damage and repair and in lung cancer biological behaviours. Telocyte (TC) is a type of interstitial cell that interacts with epithelial cells to alleviate acute inflammation and lung injury. The present studies aim at exploring potential mechanisms by which OPN regulates the epithelial origin lung inflammation and the interaction of epithelial cells with TCs in acute and chronic lung injury. The lung disease specificity of OPN and epithelial inflammation were defined by bioinformatics. We evaluated the regulatory roles of OPN in OPN-knockdown or over-expressed bronchial epithelia (HBEs) challenged with cigarette smoke extracts (CSE) or in animals with genome OPN knockout (gKO) or lung conditional OPN knockout (cKO). Acute lung injury and chronic obstructive pulmonary disease (COPD) were induced by smoking or lipopolysaccharide (LPS). Effects of OPN on PI3K subunits and ERK were assessed using the inhibitors. Spatialization and distribution of OPN, OPN-positive epithelial subtypes, and TCs were defined by spatial transcriptomics. The interaction between HBEs and TCs was assayed by the co-culture system. Levels of OPN expression increased in smokers, smokers with COPD, and smokers with COPD and lung cancer, as compared with healthy nonsmokers. LPS and/or CSE induced over-production of cytokines from HBEs, dependent upon the dysfunction of OPN. The severity of lung inflammation and injury was significantly lower in OPN-gKO or OPN-cKO mice. HBEs transferred with OPN enhanced the expression of phosphoinositide 3-kinase (PI3K)CA/p110α, PIK3CB/p110β, PIK3CD/p110δ, PIK3CG/p110γ, PIK3R1, PIK3R2 or PIK3R3. Spatial locations of OPN and OPN-positive epithelial subtypes showed the tight contact of airway epithelia and TCs. Epithelial OPN regulated the epithelial communication with TCs, and the down-regulation of OPN induced more alterations in transcriptomic profiles than the up-regulation. Our data evidenced that OPN regulated lung epithelial inflammation, injury, and cell communication between epithelium and TCs in acute and chronic lung injury. The conditional control of lung epithelial OPN may be an alternative for preventing and treating epithelial-origin lung inflammation and injury.

PIK3CB as a potential target to regulate chemosensitivity in glioblastoma.

e14051 Background: Glioblastoma (GBM) is the most common primary central nervous system malignancy but exhibits universally poor outcomes in part due to chemoresistance. Temozolomide (TMZ) can significantly improve overall survival, but resistance mechanisms limit its consistent efficacy. The phosphoinositide 3 kinase (PI3K) pathway is involved in multiple TMZ resistance mechanisms and is highly druggable; however, pan-inhibition of its four grossly homologous but functionally distinct catalytic subunits (PIK3CA/B/D/G encode p110α/β/δ/γ, respectively) was unsuccessful clinically. Therefore, by understanding how the individual PI3K subunits regulate TMZ sensitivity, a more selective treatment strategy can be developed to combat chemoresistance. Methods: Differential expression of PI3K subunit genes was assessed using RNA sequencing (RNAseq) results obtained from public databases (e.g., DepMap, The Cancer Tissue Genome Atlas) for GBM cell lines and patient tumors expressing high or low O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair enzyme highly implicated in TMZ resistance. PI3K protein levels were assessed using immunohistochemistry of gliomas available at The Human Protein Atlas (THPA). PI3K gene expression was correlated with PI3K pathway activation, quantified as protein kinase B phosphorylation (pAKT). PI3K mRNAs and pAKT were then correlated with the half maximal inhibitory concentrations (IC50s) of TMZ in GBM cell lines to evaluate for a relationship between PI3K gene expression, PI3K pathway activity, and TMZ sensitivity. Finally, hazard ratios were used to compare PI3K gene expression in patient tumors with survival and response to chemotherapy to determine if expression was predictive of prognosis and/or chemosensitivity. Results: PIK3CB was the only PI3K gene enriched in both GBM cell lines and tumors, demonstrating significantly higher expression than all other PI3K genes in GBM (P < 0.0001), independent of MGMT levels. PIK3CB expression remained the highest even after controlling for GBM risk factors such as tumor recurrence, isocitrate dehydrogenase (IDH) mutations, molecular subtypes, or patient gender/age differences. Levels of the p110β protein were also significantly higher than those of other PI3K catalytic subunits (P < 0.001). PIK3CB expression was positively correlated with pAKT levels (P < 0.05), but only in cell lines and tumors with low MGMT activity. Both PIK3CB expression and pAKT levels correlated positively with TMZ IC50s (P < 0.05) in MGMT-deficient GBMs, suggesting an inverse relationship between PIK3CB and TMZ sensitivity. Finally, better prognosis and significant survival benefit with chemotherapy were both associated with lower levels of PIK3CB in MGMT-deficient patients. Conclusions: PIK3CB may selectively regulate chemoresistance in GBM, suggesting a role for p110β selective inhibition to improve chemosensitivity.

Overcoming temozolomide resistance in glioblastoma through selective inhibition of phosphoinositide 3-kinase catalytic subunit.

e14045 Background: Glioblastoma is one of medicine's most difficult-to-treat cancers due to its inevitable resistance to chemotherapies, such as temozolomide (TMZ). Tumor resistance to TMZ often comes from overactivation of phosphoinositide 3-kinase (PI3K), making this enzyme an ideal anti-cancer drug target; however, in practice, non-selective targeting of PI3K has resulted in undesirable clinical outcomes. Therefore, it is necessary to explore each subunit of PI3K to determine their effect on chemoresistance in order to develop therapeutics to overcome said resistance. PI3K has four homologous yet functionally distinct catalytic subunits: p110α, p110β, p110δ, and p110γ. Preliminary in silico analysis of each subunit has demonstrated that p110β is more highly expressed in glioblastoma than the other subunits. Here we report that TMZ resistance in glioblastoma is driven primarily by activation of p110β. Methods: Glioblastoma cell lines with high p110β levels (SF295 and U87MG) and low p110β levels (A172 and LN229) were cultured. CRISPR-Cas9 was used to knockout p110α, p110β, or p110δ in cultured cell lines. The viability of cultured cells was measured using an MTS viability assay. Newly cultured wild-type cells were treated with IC50 doses of selective p110β inhibitor TGX-221 and TMZ. Mice bearing SF295 xenograft tumors were treated with DMSO, TGX-221, and/or TMZ. A Bliss model of independence was used to assess the association between TGX-221 and TMZ with synergistic and antagonistic patterns measured using excess over Bliss (EOB) scores. Results: There was a significant reduction in viability among SF295 p110β-knockout cells (p < 0.001) and an additive cytotoxic effect following treatment with TMZ. The LN229 p110β-knockout cells treated with TMZ did not reduce cell viability. Similarly, no substantial declines in viability were observed in the p110α- or p110δ-knockout cells before or after TMZ treatment in either cell line. In newly cultured wild-type p110β-high and p110β-low glioblastoma cell lines, TGX-221 or TMZ alone were insufficient to inhibit cell viability; however, when combined, TGX-221 and TMZ synergistically decreased cell survival with an EOB of 45.5% in U87MG cells and 26.3% in SF295 cells. No additive effects were detected in the p110β-low cell lines following TGX-221 and TMZ treatment. Additionally, TGX-221 combined with TMZ resulted in a drastic reduction in xenograft tumor growth, which was significantly greater than TGX-221 (p < 0.02) or TMZ (p < 0.003) treatment alone. Conclusions: Inhibition of p110β via CRISPR-Cas9 knockout or pharmaceutical blockade restores TMZ sensitivity in p110β-high glioblastoma cells and xenograft glioblastoma tumors. These results demonstrate the varying importance of PI3K catalytic subunits and necessitate further exploration into p110β as a potential drug target to overcome chemoresistance in glioblastoma.

The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.

IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitrobinding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.

CSIG-21. EXPLORING THE PHENOTYPIC EFFECTS OF TOLL SIGNALING PATHWAYS IN TUMOR PROGRESSION IN EGFR-DRIVEN GLIOBLASTOMA MODELS

Abstract The treatment options for Glioblastoma (GBM) are currently limited to resection and chemotherapy approaches, but resistance to chemotherapy is common in patients. There is an urgent need for further understanding of the underlying biology of tumorigenesis in order to develop new treatment options. Previous genomic work on GBMs has shown that the epidermal growth factor receptor (EGFR) is frequently amplified and constitutively active in GBM tumors. To discover new factors that contribute to progression of EGFR-driven GBMs, we used our Drosophila GBM model in which GBM-like glial neoplasia can be induced by glial-specific overexpression of constitutively active forms of EGFR and dp110, a subunit of Phosphoinositide 3-kinase (PI3K), using the UAS-GAL4 gene expression system. Using our Drosophila GBM model system, we performed a modifier screen in which we searched for genes and pathways that worsened or ‘enhanced’ glial neoplasia when specifically overexpressed or activated in neoplastic glia. From our screen, we identified the Toll pathway, which in Drosophila activates an NF-κB signaling pathway. Furthermore, the Toll pathway normally functions in innate immune and injury responses as well as in development and cell proliferation. Using our Drosophila GBM model, we are studying how the Toll signaling pathway and its effectors, including NF-κB, contribute to enhanced growth of neoplastic glial cells. Furthermore, using well-defined mouse genetic models and human patient-derived GBM models, we are examining whether Toll pathway regulatory and signaling mechanisms that we have uncovered in Drosophila function in EGFR-mutant GBM tumor stem cells to promote GBM progression and chemotherapy resistance.

Lack of Functional P110δ Affects Expression of Activation Marker CD80 but Does Not Influence Functions of Neutrophils.

Neutrophils are specialized immune cells that are essential constituents of the innate immune response. They defend the organism against pathogens through various mechanisms. It was reported that phosphatidylinositols are key players in neutrophil functions, especially in the activity of class-I phosphoinositide 3-kinases (PI3Ks). P110δ, one of the PI3K subunits, is mostly expressed in immune cells, and its activity plays an important role in inflammatory responses. The aim of this study was to investigate the role of p110δ in neutrophil antimicrobial functions, activation status and cytokine production. To this end, we used bone marrow and splenic neutrophils isolated from a murine model expressing catalytically inactive p110δD910A/D910A. The level of phagocytosis and degranulation, the expressions of activation markers and cytokine production were determined by flow cytometry. ROS generation and NET release were assessed by fluorometry and fluorescent microscopy. We observed a significantly higher percentage of CD80-positive cells among the splenic granulocytes and found granulocytes subpopulations of differing phenotypes between WT and p110δD910A/D910A mice by multiparametric tSNE analysis. Moreover, we detected some differences in the expressions of activation markers, intracellular production of cytokines and bacterial killing. However, we did not observe any alterations in the selected neutrophil functions in p110δ mutant mice. Altogether, our data suggest that the catalytic p110 subunit(s), other than p110δ, is a key player in most neutrophil functions in mice. A follow-up study to correlate these in vitro results with in vivo observations is highly recommended.

Precision medicine: Novel PI3K inhibitor as cancer treatment.

e15093 Background: Phosphoinositide 3-kinase (PI3K) is an attractive anti-cancer drug target due to its promotion of cell growth and proliferation; however, available pan-PI3K inhibitors currently lack specificity, leading to excessive drug toxicity and adverse effects. Selectide-18 is a novel PI3K inhibitor that targets PI3K’s catalytic subunit p110β, resulting in reduced viability of cancer cells. The p110β subunit is the most prominent PI3K subunit in many chemoresistant tumors. Previous research has revealed a unique 18-residue long motif within the C2 domain of p110β. This motif is not found in other catalytic subunits (p110α, p110δ, or p110γ), making it a promising drug target. Selectide-18 is a memetic peptide drug modeled after this motif and has been shown to effectively inhibit p110β binding without affecting other isoforms, separating it from currently available pan-PI3K inhibitors. While Selectide-18 has been successful in inhibiting PI3K in previous studies, it has poor cell permeability due to its large size. Previous findings have revealed functional redundancies among Selectide-18’s residues. This study aims to remove these redundancies and determine the optimal formulation of Selectide-18 through in silico analysis. Methods: An in silico protocol was developed to determine the optimal formulation of Selectide-18. By utilizing the PEP-FOLD server, 455 peptide models were created using 91 unique fragments of the Selectide-18 amino acid sequence ranging in size from 6 to 17 residues. Physical conformation of each model was compared to wild-type Selectide-18 using root-mean-square deviation data collected using PyMOL and UCSF Chimera software. Computational docking through AutoDock Vina was performed to test the binding affinity of each model to the active site of regulatory subunit p85α. The distance between each drug and the active site was measured with UCSF Chimera’s distance function. Finally, ClusPro docking of drug-bound p85α/p110β was compared to wild-type p85α/p110β, as well as non-truncated Selectide-18-bound p85α/p110β. Statistical significance was determined using a Student's t-test. Results: Select truncated models demonstrated physical confirmation (RMSD = 0.024), binding affinity (-8.74 kcal/mol), and simulated inhibitory capabilities comparable to Selectide-18. A specific group of variants comprised of 9 amino acids (9 residue_6K-14Q) had the shortest formulation without significant declines in binding affinity. Conclusions: Selectide-18 is a selective PI3K inhibitor that can be optimized by removing functionally redundant amino acids. A 9-residue variant ranging from amino acids 6Lys to 14Gln showed the most promise and will be used in future studies to reformulate Selectide-18. Being half the size of Selectide-18, this optimized drug is anticipated to have improved cell permeability without sacrificing inhibitory function.

Analysing the cellular mechanisms underlying activated PI3K δ Syndrome (APDS) reveals differences between APDS1 and APDS2

Abstract APDS is a primary immunodeficiency condition caused by heterozygous gain-of-function mutations in PIK3CD (APDS1), which encodes the leukocyte-restricted p110-δ catalytic subunit of phosphoinositide 3-kinase (PI3K) or heterozygous loss-of-function mutations in PIK3R1 (APDS2), which encodes the regulatory subunit of PI3K. PI3K is activated downstream of many receptors expressed by lymphocytes and plays important roles in their activation and differentiation. The increased PI3K activity in cells from APDS patients leads to multiple immune manifestations including lymphoproliferation, respiratory tract infections, Th2-related pathologies, impaired Ab responses and autoimmunity. We previously dissected the cellular defects that lead to disease in APDS1 by studying patient cells and our novel mouse model. This revealed changes in lymphocytes including increased memory T cells, defective Tfh function, decreased isotype switching and a break in B cell tolerance and production of autoantibodies. We have now generated a mouse model of APDS2 (Pik3r1E11SpD) and use this together with studies of patients’ cells to determine whether increased PI3K signalling due to loss of regulatory function causes the same cellular dysregulation. While we observed many of the same changes previously observed in APDS1, including altered cytokine production and decreased isotype switching, we also identified multiple differences in the cellular phenotype in APDS2 compared to APDS1. Together, these studies reveal that although APDS1 and 2 both cause increased PI3K signalling and result in similar clinical phenotypes, there are distinctions between how these two types of mutations affect cellular function. Supported by grants from the NHMRC and an AAI Careers in Immunology Fellowship

Inhibition of the PI 3-kinase pathway disrupts the unfolded protein response and reduces sensitivity to ER stress-dependent apoptosis.

Class Ia phosphoinositide 3-kinases (PI3K) are critical mediators of insulin and growth factor action. We have demonstrated that the p85α regulatory subunit of PI3K modulates the unfolded protein response (UPR) by interacting with and regulating the nuclear translocation of XBP-1s, a transcription factor essential for the UPR. We now show that PI3K activity is required for full activation of the UPR. Pharmacological inhibition of PI3K in cells blunts the ER stress-dependent phosphorylation of IRE1α and PERK, decreases induction of ATF4, CHOP, and XBP-1 and upregulates UPR target genes. Cells expressing a human p85α mutant (R649W) previously shown to inhibit PI3K, exhibit decreased activation of IRE1α and PERK and reduced induction of CHOP and ATF4. Pharmacological inhibition of PI3K, overexpression of a mutant of p85α that lacks the ability to interact with the p110α catalytic subunit (∆p85α) or expression of mutant p85α (R649W) in vivo, decreased UPR-dependent induction of ER stress response genes. Acute tunicamycin treatment of R649W+/- mice revealed reduced induction of UPR target genes in adipose tissue, whereas chronic tunicamycin exposure caused sustained increases in UPR target genes in adipose tissue. Finally, R649W+/- cells exhibited a dramatic resistance to ER stress-dependent apoptosis. These data suggest that PI3K pathway dysfunction causes ER stress that may drive the pathogenesis of several diseases including Type 2 diabetes and various cancers.

PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3‐kinase (PI3K) and its product phosphatidylinositol (3,4,5)‐trisphosphate (PIP 3). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP 3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP 3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP 3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK.

The 1918 influenza A virus (IAV) caused the worst flu pandemic in human history. Non-structural protein 1 (NS1) is an important virulence factor of the 1918 IAV and antagonizes host antiviral immune responses. NS1 increases virulence by activating phosphoinositide 3-kinase (PI3K) via binding to the p85β subunit of PI3K. Intriguingly, unlike the NS1 of other human IAV strains, 1918 NS1 hijacks another host protein, CRK, to form a ternary complex with p85β, resulting in hyperactivation of PI3K. However, the molecular basis of the ternary interaction between 1918 NS1, CRK, and PI3K remains elusive. Here, we report the structural and thermodynamic bases of the ternary interaction. We find that the C-terminal tail (CTT) of 1918 NS1 remains highly flexible in the complex with p85β. Thus, the CTT of 1918 NS1 in the complex with PI3K can efficiently hijack CRK. Notably, our study indicates that 1918 NS1 enhances its affinity to p85β in the presence of CRK, which might result in enhanced activation of PI3K. Our results provide structural insight into how 1918 NS1 hijacks two host proteins simultaneously.

Abstract P4-06-07: PIK3CA-mutant breast phyllodes tumors show a uniformly aggressive histology and significant mutual exclusivity with MED12 mutation

Abstract Introduction: De-regulation of the class I phosphoinositide 3’-kinase (PI3K) pathway has long been known to contribute to the development and progression of many tumors. However, the FDA only recently approved the first selective inhibitor of PIK3CA, the p110-alpha catalytic subunit of PI3K, specifically for use in treatment of a subset of PIK3CA-mutant breast carcinomas. Given the emergence of this therapeutic class of agents, we sought to identify other subsets of breast tumors that may be driven largely by PIK3CA mutations and which therefore could be candidates for these therapies. In breast fibroepithelial neoplasms, mutations in PIK3CA have been occasionally reported in borderline and malignant phyllodes tumors. In contrast, mutations in MED12 are common and recurrent across the entire spectrum of benign and malignant breast fibroepithelial tumors, and are enriched in borderline/malignant phyllodes cases that still have benign fibroadenoma-like areas. In the current study, we sought to define the histologic and molecular features of PIK3CA-mutant phyllodes tumors. Methods: From 2014 to 2019, we analyzed clinical tumor samples using comprehensive genomic profiling by a hybrid capture-based DNA sequencing platform. We searched our case archive to find breast phyllodes tumors with known or likely pathogenic alterations in PIK3CA and other known tumor-related genes. All cases were clinically advanced. We reviewed pathology reports, histopathology, and patient clinical data. Results: We identified 12 (16%) of 76 breast phyllodes tumors in our case archive as PIK3CA-mutant. Median patient age for PIK3CA-mutant phyllodes tumors was 56 years. Cases consisted of 6 primary tumors, 2 local recurrences, and 4 lung metastases. Primary tumor size measured from 38 to 220 mm (median 100 mm; mean 114 mm). Digital slides were available for histology review in 9 cases. Cases showed uniformly malignant histology, with no benign or fibroadenoma-like regions. 3 cases showed malignant heterologous elements. Compared to the rest of our breast phyllodes tumor cohort, PIK3CA-mutant cases showed significantly fewer pathogenic genomic alterations in MED12 (8% vs. 55%, p=0.0037) and TP53 (17% vs. 56%, p=0.0245), as well as a trend to fewer mutations in RB1 (0% vs. 22%, p=0.11). The other most frequently mutated genes in the PIK3CA-mutant group were TERTp (70%), CDKN2A (67%), and NF1 (50%). Conclusions: PIK3CA-mutant phyllodes tumors define a unique subset of tumors characterized by aggressive histologic features and largely lacking the MED12 mutations seen in many fibroepithelial neoplasms. These findings provide compelling rationale for comprehensive genomic profiling of advanced cases of this disease in an effort to inform therapeutic options including clinical trials of PI3K-targeted agents in this setting. Citation Format: Erik A Williams, Ethan S Sokol, Dean C Pavlick, Nikunj Shah, Julia A Elvin, Jo-Anne Vergilio, Jonathan K Killian, Nhu Ngo, Douglas Lin, Vincent A Miller, Jeffrey S Ross. PIK3CA-mutant breast phyllodes tumors show a uniformly aggressive histology and significant mutual exclusivity with MED12 mutation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-07.

Abstract P4-10-04: Clinical outcomes of alpelisib in hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer by next-generation sequencing-detected PIK3CA alteration status and phosphatase and tensin homolog loss: Biomarker analysis from the SOLAR-1 study

Introduction: Hyperactivation of the phosphoinositide-3-kinase (PI3K) pathway is a frequent event in human cancers and can be a result of phosphatase and tensin homolog (PTEN) loss, activation of receptor tyrosine kinases, or alterations in PI3K isoforms. About 40% of hormone receptor-positive (HR+), human epidermal growth factor receptor-2-negative (HER2-) breast cancers harbor alterations in the PIK3CA gene, which encodes the alpha subunit of PI3K (p110α). Mutations in PIK3CA can cause PI3K pathway hyperactivation, which may contribute to endocrine resistance. Alpelisib (ALP) is an α-selective PI3K inhibitor that, in combination with fulvestrant (FUL), demonstrated improved median progression-free survival (PFS) vs placebo (PBO) + FUL (11.0 vs 5.7 mo, respectively; hazard ratio [HR] 0.65; 95% confidence interval [CI], 0.50-0.85; P Methods: SOLAR-1 is a phase 3, randomized, double-blind study of ALP 300 mg (or PBO) once daily + FUL 500 mg every 28 days + Cycle 1 Day 15 in men and postmenopausal women with HR+, HER2- ABC. Retrospectively, PIK3CA alterations (mutations and amplifications) were assessed by NGS and PTEN levels were assessed by immunohistochemistry. PTEN loss was defined based on an H-score Results: Of 572 pts in SOLAR-1, 341 (60%) had a PIK3CA mutation in tumor tissue by PCR, valid NGS results were available for 404 (71%), and PTEN levels were available for 401 (70%). Of the pts with available PTEN results, 34 (8%) had PTEN loss. PFS results and HRs, by PIK3CA alteration status as detected by NGS and PTEN status, are shown in the Table. In pts whose tumors had any PIK3CA alteration (n=239), which included a small group of pts with alterations not detectable by the PCR-based test used for screening in SOLAR-1 (n=31), mPFS was improved in the ALP + FUL vs PBO + FUL arms. In the small group of pts (n=19) whose tumors had PTEN loss but no PIK3CA alteration, ALP showed an improved mPFS and favorable HR vs the placebo arm. In pts whose tumors had multiple alterations (n=44), HR and mPFS benefits of ALP were observed, albeit with wide 95% CIs. Conclusions: These data demonstrate the efficacy of ALP in pts whose tumors harbor PIK3CA alterations as detected by NGS, including alterations that were not detectable by the PCR-based test used in SOLAR-1. Additional analyses suggest that ALP is also effective in pts with single and multiple PIK3CA mutations and in those whose tumors do not harbor a PIK3CA alteration but do have PTEN loss; however, low patient numbers in the PTEN loss subgroups may limit conclusions. Citation Format: Dejan Juric, Fabrice Andre, Christian F. Singer, Joohyuk Sohn, Mario Campone, Sibylle Loibl, Pierfranco Conte, Hiroji Iwata, Eva Ciruelos, Ingrid A. Mayer, Albert Reising, Chong Ma, Michelle Miller, Naveen Babbar, Hope S. Rugo. Clinical outcomes of alpelisib in hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer by next-generation sequencing-detected PIK3CA alteration status and phosphatase and tensin homolog loss: Biomarker analysis from the SOLAR-1 study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-04.

Von Hippel–Lindau tumor suppressor (VHL) stimulates TOR signaling by interacting with phosphoinositide 3-kinase (PI3K)

Cell growth is positively controlled by the phosphoinositide 3-kinase (PI3K)-target of rapamycin (TOR) signaling pathway under conditions of abundant growth factors and nutrients. To discover additional mechanisms that regulate cell growth, here we performed RNAi-based mosaic analyses in the Drosophila fat body, the primary metabolic organ in the fly. Unexpectedly, the knockdown of the Drosophila von Hippel-Lindau (VHL) gene markedly decreased cell size and body size. These cell growth phenotypes induced by VHL loss of function were recovered by activation of TOR signaling in Drosophila Consistent with the genetic interactions between VHL and the signaling components of PI3K-TOR pathway in Drosophila, we observed that VHL loss of function in mammalian cells causes decreased phosphorylation of ribosomal protein S6 kinase and Akt, which represent the main activities of this pathway. We further demonstrate that VHL activates TOR signaling by directly interacting with the p110 catalytic subunit of PI3K. On the basis of the evolutionarily conserved regulation of PI3K-TOR signaling by VHL observed here, we propose that VHL plays an important role in the regulation and maintenance of proper cell growth in metazoans.

Seeing double can be a good thing

Cancer Many human breast cancers harbor activating mutations in PIK3CA , the gene coding for the catalytic subunit of phosphoinositide 3-kinase (PI3K). Clinical trials are underway to evaluate the efficacy of PI3K inhibitors in cancer patients. Vasan et al. found unexpectedly that a subset of breast cancers harbor not one—but two— PIK3CA mutations, and the mutations occur on the same allele (see the Perspective by Toker). In model systems, the double mutations hyperactivate PI3K signaling and enhance tumor growth. Preliminary analysis of clinical trial data suggests that breast cancers with double mutations are more responsive to PI3K inhibitors than those with a single mutation. PIK3CA mutational status could help identify the breast cancer patients most likely to benefit from these drugs. Science , this issue p. [714][1]; see also p. [685][2] [1]: /lookup/doi/10.1126/science.aaw9032 [2]: /lookup/doi/10.1126/science.aaz4016

The Ciliary Muscle and Zonules of Zinn Modulate Lens Intracellular Hydrostatic Pressure Through Transient Receptor Potential Vanilloid Channels.

PurposeLenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn. Here, we have examined if ciliary muscle tension influenced the lens intracellular hydrostatic pressure gradient.MethodsWe measured the ciliary body position and intracellular hydrostatic pressures in mouse lenses while pharmacologically causing relaxation or contraction of the ciliary muscle. We also used inhibitors of TRPV1 and TRPV4, in addition to phosphoinositide 3-kinase (PI3K) p110α knockout mice and immunostaining of phosphorylated protein kinase B (Akt), to determine how changes in ciliary muscle tension resulted in altered hydrostatic pressure.ResultsCiliary muscle relaxation increased the distance between the ciliary body and the lens and caused a decrease in intracellular hydrostatic pressure that was dependent on intact zonules and could be blocked by inhibition of TRPV4. Ciliary contraction moved the ciliary body toward the lens and caused an increase in intracellular hydrostatic pressure and Akt phosphorylation that required intact zonules and was blocked by either inhibition of TRPV1 or genetic deletion of the p110α catalytic subunit of PI3K.ConclusionsThese results show that the hydrostatic pressure gradient within the lens was influenced by the tension exerted on the lens by the ciliary muscle through the zonules of Zinn. Modulation of the gradient of intracellular hydrostatic pressure in the lens could alter the water content, and the gradient of refractive index.

TIM-1 As a Signal Receptor Triggers Dengue Virus-Induced Autophagy.

Dengue virus (DENV) infection triggers the activation of autophagy to facilitate the viral replication cycle from various aspects. Although a number of stimulators are proposed to activate autophagy, none of them appears prior to the uncoating process. Given that T-cell immunoglobulin and mucin domain 1 (TIM-1) receptor is a putative DENV receptor and promotes apoptotic body clearance by autophagy induction, it raises the possibility that TIM-1 may participate in the activation of DENV-induced autophagy. In this study, confocal images first revealed the co-localization of TIM-1 with autophagosomes in DENV-induced autophagy rather than rapamycin-induced autophagy, suggesting the co-transportation of TIM-1 with DENV during infection. The treatment of siRNA to knockdown TIM-1 expression in DENV-infected GFP-microtubule-associated protein light chain 3 (LC3)-Huh7.5 cells revealed that TIM-1 is required not only for DENV cellular internalization but also for autophagy activation. Furthermore, knockdown p85, a subunit of phosphoinositide 3-kinases (PI3Ks), which is co-localized with TIM-1 at rab5-positive endosomes caused the reduction of autophagy, indicating that TIM-1-mediated DENV-induced autophagy requires p85. Taken together, the current study uncovered TIM-1 as a novel factor for triggering autophagy in DENV infection through TIM-1-p85 axis, in addition to serving as a DENV receptor.

C-SH2 point mutation converts p85\u03b2 regulatory subunit of phosphoinositide 3-kinase to an anti-aging gene

Insulin interacts with the insulin receptor, and the activated receptor promotes activity of the phosphoinositide-3 kinase (PI3K) enzyme. A decrease in insulin or insulin-like growth factor 1 (IGF-1) signaling increases the lifespan in mammalian species. We found that a point mutation in the C-SH2 domain of the p85β regulatory subunit of PI3K results in a prolonged lifespan. In p85β mutant cells, nerve growth factor (NGF) activates the longevity protein FOXO, and the mutant p85β gene produces strong resistance to oxidative stress, which contributes to aging. The p85β gene mutation causes increased serum insulin and low blood glucose in p85β mutant transgenic mice. Our results indicate that the p85β mutant allele alters the activity of downstream targets of PI3K by NGF and platelet-derived growth factor (PDGF) but not by insulin. We report that a point mutation in the C-SH2 domain of p85β transforms p85β into a novel anti-aging gene by abnormally regulating PI3K.

Cellular and molecular mechanisms of PIK3CA-related vascular anomalies.

The phosphoinositide 3-kinase (PI3K) pathway is a major mediator of growth factor signaling, cell proliferation and metabolism. Somatic gain-of-function mutations in PIK3CA, the catalytic subunit of PI3K, have recently been discovered in a number of vascular anomalies. The timing and origin of these mutations remain unclear although they are believed to occur during embryogenesis. The cellular origin of these lesions likely involves endothelial cells or an early endothelial cell lineage. This review will cover the diseases and syndromes associated with PIK3CA mutations and discuss the cellular origin, pathways and mechanisms. Activating PIK3CA ‘hot spot’ mutations have long been associated with a multitude of cancers allowing the development of targeted pharmacological inhibitors that are FDA-approved or in clinical trials. Current and future therapeutic approaches for PIK3CA-related vascular anomalies are discussed.