PI3K couples long-term synaptic potentiation with cofilin recruitment and actin polymerization in dendritic spines via its regulatory subunit p85α.

Long-term synaptic plasticity is typically associated with morphological changes in synaptic connections. However, the molecular mechanisms coupling functional and structural aspects of synaptic plasticity are still poorly defined. The catalytic activity of type I phosphoinositide-3-kinase (PI3K) is required for specific forms of synaptic plasticity, such as NMDA receptor-dependent long-term potentiation (LTP) and mGluR-dependent long-term depression (LTD). On the other hand, PI3K signaling has been linked to neuronal growth and synapse formation. Consequently, PI3Ks are promising candidates to coordinate changes in synaptic strength with structural remodeling of synapses. To investigate this issue, we targeted individual regulatory subunits of type I PI3Ks in hippocampal neurons and employed a combination of electrophysiological, biochemical and imaging techniques to assess their role in synaptic plasticity. We found that a particular regulatory isoform, p85α, is selectively required for LTP. This specificity is based on its BH domain, which engages the small GTPases Rac1 and Cdc42, critical regulators of the actin cytoskeleton. Moreover, cofilin, a key regulator of actin dynamics that accumulates in dendritic spines after LTP induction, failed to do so in the absence of p85α or when its BH domain was overexpressed as a dominant negative construct. Finally, in agreement with this convergence on actin regulatory mechanisms, the presence of p85α in the PI3K complex determined the extent of actin polymerization in dendritic spines during LTP. Therefore, this study reveals a molecular mechanism linking structural and functional synaptic plasticity through the coordinate action of PI3K catalytic activity and a specific isoform of the regulatory subunits.

Overview of the regulation of the class IA PI3K/AKT pathway by SUMO.

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is a major regulator of metabolism, migration, survival, proliferation, and antiviral immunity. Both an overactivation and an inhibition of the PI3K/AKT pathway are related to different pathologies. Activation of this signaling pathway is tightly controlled through a multistep process and its deregulation can be associated with aberrant post-translational modifications including SUMOylation. Here, we review the complex modulation of the PI3K/AKT pathway by SUMOylation and we discuss its putative incvolvement in human disease.

Enhanced STAT3/PIK3R1/mTOR signaling triggers tubular cell inflammation and apoptosis in septic-induced acute kidney injury: implications for therapeutic intervention.

Septic acute kidney injury (AKI) is a severe form of renal dysfunction associated with high morbidity and mortality rates. However, the pathophysiological mechanisms underlying septic AKI remain incompletely understood. Herein, we investigated the signaling pathways involved in septic AKI using the mouse models of lipopolysaccharide (LPS) treatment and cecal ligation and puncture (CLP). In these models, renal inflammation and tubular cell apoptosis were accompanied by the aberrant activation of the mechanistic target of rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3) signaling pathways. Pharmacological inhibition of either mTOR or STAT3 significantly improved renal function and reduced apoptosis and inflammation. Interestingly, inhibition of STAT3 with pharmacological inhibitors or small interfering RNA blocked LPS-induced mTOR activation in renal tubular cells, indicating a role of STAT3 in mTOR activation. Moreover, knockdown of STAT3 reduced the expression of the phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1/p85α), a key subunit of the phosphatidylinositol 3-kinase for AKT and mTOR activation. Chromatin immunoprecipitation assay also proved the binding of STAT3 to PIK3R1 gene promoter in LPS-treated kidney tubular cells. In addition, knockdown of PIK3R1 suppressed mTOR activation during LPS treatment. These findings highlight the dysregulation of mTOR and STAT3 pathways as critical mechanisms underlying the inflammatory and apoptotic phenotypes observed in renal tubular cells during septic AKI, suggesting the STAT3/ PIK3R1/mTOR pathway as a therapeutic target of septic AKI.

Cancer-associated mutations in the p85α N-terminal SH2 domain activate a spectrum of receptor tyrosine kinases.

The phosphoinositide 3-kinase regulatory subunit p85α is a key regulator of kinase signaling and is frequently mutated in cancers. In the present study, we showed that in addition to weakening the inhibitory interaction between p85α and p110α, a group of driver mutations in the p85α N-terminal SH2 domain activated EGFR, HER2, HER3, c-Met, and IGF-1R in a p110α-independent manner. Cancer cells expressing these mutations exhibited the activation of p110α and the AKT pathway. Interestingly, the activation of EGFR, HER2, and c-Met was attributed to the ability of driver mutations to inhibit HER3 ubiquitination and degradation. The resulting increase in HER3 protein levels promoted its heterodimerization with EGFR, HER2, and c-Met, as well as the allosteric activation of these dimerized partners; however, HER3 silencing abolished this transactivation. Accordingly, inhibitors of either AKT or the HER family reduced the oncogenicity of driver mutations. The combination of these inhibitors resulted in marked synergy. Taken together, our findings provide mechanistic insights and suggest therapeutic strategies targeting a class of recurrent p85α mutations.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1.

Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase-binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

Tissue-specific disruption of Kbtbd2 uncovers adipocyte-intrinsic and -extrinsic features of the teeny lipodystrophy syndrome.

Loss of KBTBD2 in all tissues causes the teeny phenotype, characterized by insulin resistance with late failure of insulin production, severe hyperglycemia/diabetes, lipodystrophy, hepatosteatosis, and growth retardation. KBTBD2 maintains insulin sensitivity in adipocytes by restricting the abundance of p85α. However, the possible physiological contribution or contributions of KBTBD2 have not yet been examined in other tissues. Here we show that mice with an adipocyte-specific knockout of Kbtbd2 accumulate p85α in white and brown adipose tissues, causing insulin resistance, moderate rather than severe hyperglycemia, sustained hyperinsulinemia without late failure of insulin production, and lipodystrophy leading to ectopic lipid accumulation in the liver. Adipocyte-extrinsic insulin resistance was observed in liver and muscle. None of these abnormalities were observed in liver- or muscle-specific Kbtbd2 knockout mice. Mice with Kbtbd2 knockout in adipocytes, liver, and muscle all showed normal growth, suggesting that KBTBD2 may be necessary to ensure IGF1 signaling in other tissues, notably bone. While much of the teeny phenotype results from loss of KBTBD2 in adipocytes, some features are adipocyte-extrinsic.

PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition.

PIK3CA hotspot mutation is well established as an oncogenic driver event in cancer and its durable and efficacious inhibition is a focus in the development and testing of clinical cancer therapeutics. However, hundreds of cancer-associated PIK3CA mutations remain uncharacterized, their sensitivity to PI3K inhibitors unknown. Here, we describe a series of PIK3CA C-terminal mutations, primarily nucleotide insertions, that produce a frame-shifted protein product with an extended C terminus. We report that these mutations occur at a low frequency across multiple cancer subtypes, including breast, and are sufficient to drive oncogenic transformation in vitro and in vivo. We demonstrate that the oncogenicity of these mutant p110α proteins is dependent on p85 but not Ras association. P110α-selective pharmacologic inhibition blocks transformation in cells and mammary tumors characterized by PIK3CA C-terminal mutation. Taken together, these results suggest patients with breast and other tumors characterized by PIK3CA C-terminal frameshift mutations may derive benefit from p110α-selective inhibitors, including the recently FDA-approved alpelisib.

Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses.

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85ß subunit. Here we present the mechanism underlying the molecular recognition of the p85ß subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85ß of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85ß. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1ED exists in a dynamic equilibrium between p85ß-binding-competent and -incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85ß. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1ED can result in the faster hijacking of p85ß compared to Ud NS1ED, although the former has a lower affinity to p85ß than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure.

Chronic glucocorticoid exposure causes insulin resistance and muscle atrophy in skeletal muscle. We previously identified phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1) as a primary target gene of skeletal muscle glucocorticoid receptors involved in the glucocorticoid-mediated suppression of insulin action. However, the in vivo functions of Pik3r1 remain unclear. Here, we generated striated muscle-specific Pik3r1 knockout (MKO) mice and treated them with a dexamethasone (DEX), a synthetic glucocorticoid. Treating wildtype (WT) mice with DEX attenuated insulin activated Akt activity in liver, epididymal white adipose tissue, and gastrocnemius (GA) muscle. This DEX effect was diminished in GA muscle of MKO mice, therefore, resulting in improved glucose and insulin tolerance in DEX-treated MKO mice. Stable isotope labeling techniques revealed that in WT mice, DEX treatment decreased protein fractional synthesis rates in GA muscle. Furthermore, histology showed that in WT mice, DEX treatment reduced GA myotube diameters. In MKO mice, myotube diameters were smaller than in WT mice, and there were more fast oxidative fibers. Importantly, DEX failed to further reduce myotube diameters. Pik3r1 knockout also decreased basal protein synthesis rate (likely caused by lower 4E-BP1 phosphorylation at Thr37/Thr46) and curbed the ability of DEX to attenuate protein synthesis rate. Finally, the ability of DEX to inhibit eIF2α phosphorylation and insulin-induced 4E-BP1 phosphorylation was reduced in MKO mice. Taken together, these results demonstrate the role of Pik3r1 in glucocorticoid-mediated effects on glucose and protein metabolism in skeletal muscle.

ERG1 plays an essential role in rat cardiomyocyte fate decision by mediating AKT signaling.

ERG1, a potassium ion channel, is essential for cardiac action potential repolarization phase. However, the role of ERG1 for normal development of the heart is poorly understood. Using the rat embryonic stem cells (rESCs) model, we show that ERG1 is crucial in cardiomyocyte lineage commitment via interactions with Integrin ß1. In the mesoderm phase of rESCs, the interaction of ERG1 with Integrin ß1 can activate the AKT pathway by recruiting and phosphorylating PI3K p85 and focal adhesion kinase (FAK) to further phosphorylate AKT. Activation of AKT pathway promotes cardiomyocyte differentiation through two different mechanisms, (a) through phosphorylation of GSK3ß to upregulate the expression levels of ß-catenin and Gata4; (b) through promotion of nuclear translocation of nuclear factor-κB by phosphorylating IKKß to inhibit cell apoptosis, which occurs due to increased Bcl2 expression. Our study provides solid evidence for a novel role of ERG1 on differentiation of rESCs into cardiomyocytes.

MicroRNA-7 overexpression positively regulates the CD8+ SP cell development via targeting PIK3R1.

microRNA-7 (miR-7), a distinct miRNA family member, has been reported to be involved in the biological functions of immune cells. However, the potential role of miR-7 in the CD8+ T cell development remains to be elucidated. In this study, we estimated the potential effects of miR-7 overexpression in the thymic CD8+ SP cell development using miR-7 overexpression mice. Our results showed that compared with those in control wild type (WT) mice, the volume, weight and total cell numbers of thymus in miR-7 overexpression (OE) mice increased significantly. The absolute cell number of CD8+ SP cells in miR-7 OE mice increased and its ability of activation and proliferation enhanced. Futhermore, we clarified that miR-7 overexpression had an intrinsic promote role in CD8+ SP cell development by adoptive cell transfer assay. Mechanistically, the expression level of PIK3R1, a target of miR-7, decreased significantly in CD8+ SP cells of miR-7 OE mice. Moreover, the expression level of phosphorylated (p)-AKT and p-ERK changed inversely and indicating that miR-7 overexpression impaired the balance of AKE and ERK pathways. In summary, our work reveals an essential role of miR-7 in promoting CD8+ SP cell development through the regulation of PIK3R1 and balance of AKT and ERK pathways.

P300/CBP inhibition sensitizes mantle cell lymphoma to PI3Kδ inhibitor idelalisib.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder lacking reliable therapies. PI3K pathway contributes to the pathogenesis of MCL, serving as a potential target. However, idelalisib, an FDA-approved drug targeting PI3Kδ, has shown intrinsic resistance in MCL treatment. Here we report that a p300/CBP inhibitor, A-485, could overcome resistance to idelalisib in MCL cells in vitro and in vivo. A-485 was discovered in a combinational drug screening from an epigenetic compound library containing 45 small molecule modulators. We found that A-485, the highly selective catalytic inhibitor of p300 and CBP, was the most potent compound that enhanced the sensitivity of MCL cell line Z-138 to idelalisib. Combination of A-485 and idelalisib remarkably decreased the viability of three MCL cell lines tested. Co-treatment with A-485 and idelalisib in Maver-1 and Z-138 MCL cell xenograft mice for 3 weeks dramatically suppressed the tumor growth by reversing the unsustained inhibition in PI3K downstream signaling. We further demonstrated that p300/CBP inhibition decreased histone acetylation at RTKs gene promoters and reduced transcriptional upregulation of RTKs, thereby inhibiting the downstream persistent activation of MAPK/ERK signaling, which also contributed to the pathogenesis of MCL. Therefore, additional inhibition of p300/CBP blocked MAPK/ERK signaling, which rendered maintaining activation to PI3K-mTOR downstream signals p-S6 and p-4E-BP1, thus leading to suppression of cell growth and tumor progression and eliminating the intrinsic resistance to idelalisib ultimately. Our results provide a promising combination therapy for MCL and highlight the potential use of epigenetic inhibitors targeting p300/CBP to reverse drug resistance in tumor.

BRD7, a tumor suppressor, interacts with p85α and regulates PI3K activity.

Phosphoinositide 3-kinase (PI3K) activity is important for regulating cell growth, survival, and motility. We report here the identification of bromodomain-containing protein 7 (BRD7) as a p85α-interacting protein that negatively regulates PI3K signaling. BRD7 binds to the inter-SH2 (iSH2) domain of p85 through an evolutionarily conserved region located at the C terminus of BRD7. Via this interaction, BRD7 facilitates nuclear translocation of p85α. The BRD7-dependent depletion of p85 from the cytosol impairs formation of p85/p110 complexes in the cytosol, leading to a decrease in p110 proteins and in PI3K pathway signaling. In contrast, silencing of endogenous BRD7 expression by RNAi increases the steady-state level of p110 proteins and enhances Akt phosphorylation after stimulation. These data suggest that BRD7 and p110 compete for the interaction to p85. The unbound p110 protein is unstable, leading to the attenuation of PI3K activity, which suggests how BRD7 could function as a tumor suppressor.

SUMOylation modulates the stability and function of PI3K-p110ß.

Class I PI3K are heterodimers composed of a p85 regulatory subunit and a p110 catalytic subunit involved in multiple cellular functions. Recently, the catalytic subunit p110ß has emerged as a class I PI3K isoform playing a major role in tumorigenesis. Understanding its regulation is crucial for the control of the PI3K pathway in p110ß-driven cancers. Here we sought to evaluate the putative regulation of p110ß by SUMO. Our data show that p110ß can be modified by SUMO1 and SUMO2 in vitro, in transfected cells and under completely endogenous conditions, supporting the physiological relevance of p110ß SUMOylation. We identify lysine residue 952, located at the activation loop of p110ß, as essential for SUMOylation. SUMOylation of p110ß stabilizes the protein increasing its activation of AKT which promotes cell growth and oncogenic transformation. Finally, we show that the regulatory subunit p85ß counteracts the conjugation of SUMO to p110ß. In summary, our data reveal that SUMO is a novel p110ß interacting partner with a positive effect on the activation of the PI3K pathway.

Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α.

Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110ß, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment.

LPAR5 promotes thyroid carcinoma cell proliferation and migration by activating class IA PI3K catalytic subunit p110ß.

Lysophosphatidic acid receptor 5 (LPAR5) is involved in mediating thyroid cancer progression, but the underlying mechanism needs to be further revealed. In this study, we confirmed that LPAR5 is upregulated in papillary thyroid carcinoma (PTC), especially in BRAF-like PTC, by analyzing The Cancer Genome Atlas (TCGA) database and performing immunohistochemistry assay in human thyroid cancer tissues. LPAR5-specific antagonist TC LPA5 4 treatment inhibited CGTH-W3, TPC-1, B-CPAP, and BHT-101 cell proliferation, CGTH-W3 and TPC-1 cell migration significantly. In vivo, TC LPA5 4 treatment could delay CGTH-W3 xenograft growth in nude mice. We also found that LPAR5-specific antagonist TC LPA5 4, PI3K inhibitor wortmannin, or mTOR inhibitor rapamycin pretreatment abrogated phosphorylation of Akt and p70S6K1 stimulated by LPA in CGTH-W3 and TPC-1 cells. Stimulating CGTH-W3 cells transfected with pEGFPC1-Grp1-PH fusion protein with LPA resulted in the generation of phosphatidylinositol (3,4,5)-triphosphate, which indicates that PI3K was activated by LPA directly. The p110ß-siRNA instead of p110α-siRNA transfection abrogated the increase of levels of phosphorylated Akt and S6K1 stimulated by LPA. Furthermore, immunoprecipitation assay confirmed an interaction between LPAR5 and p110ß. Overall, we provide new insights that the downregulation of LPAR5 decreased the proliferation and migration phenotype via the PI3K/Akt pathway. Inhibition of LPAR5 or the PI3K/Akt signal may be a novel therapeutic strategy for treating thyroid cancer.

IGF1R as druggable target mediating PI3K-δ inhibitor resistance in a murine model of chronic lymphocytic leukemia.

Targeted therapy is revolutionizing the treatment of cancers, but resistance evolves against these therapies and derogates their success. The phosphatidylinositol 3-kinase delta (PI3K-δ) inhibitor idelalisib has been approved for treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, but the mechanisms conferring resistance in a subset of patients are unknown. Here, we modeled resistance to PI3K-δ inhibitor in vivo using a serial tumor transfer and treatment scheme in mice. Whole-exome sequencing did not identify any recurrent mutation explaining resistance to PI3K-δ inhibitor. In the murine model, resistance to PI3K-δ inhibitor occurred as a result of a signaling switch mediated by consistent and functionally relevant activation of insulin-like growth factor 1 receptor (IGF1R), resulting in enhanced MAPK signaling in the resistant tumors. Overexpression of IGF1R in vitro demonstrated its prominent role in PI3K-δ inhibitor resistance. IGF1R upregulation in PI3K-δ inhibitor-resistant tumors was mediated by functional activation and enhanced nuclear localization of forkhead box protein O1 transcription factors and glycogen synthase kinase 3ß. In human CLL, high IGF1R expression was associated with trisomy 12. CLL cells from an idelalisib-treated patient showed decreased sensitivity to idelalisib in vitro concomitant with enhanced MAPK signaling and strong upregulation of IGF1R upon idelalisib exposure. Thus, our results highlight that alternative signaling cascades play a predominant role in the resistance and survival of cancer cells under PI3K-δ inhibition. We also demonstrate that these pathway alterations can serve as therapeutic targets, because inhibition of IGF1R offered efficacious salvage treatment of PI3K-δ inhibitor-resistant tumors in vitro and in vivo.

Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.

Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.

Pathophysiology and therapeutic relevance of PI3K(p110α) protein in atrial fibrillation: A non-interventional molecular therapy strategy.

Genetically modified animal studies have revealed specific expression patterns and unequivocal roles of class I PI3K isoenzymes. PI3K(p110α), a catalytic subunit of class I PI3Ks is ubiquitously expressed and is well characterised in the cardiovascular system. Given that genetic inhibition of PI3K(p110α) causes lethal phenotype embryonically, the catalytic subunit is critically important in housekeeping and biological processes. A growing number of studies underpin crucial roles of PI3K(p110α) in cell survival, proliferation, hypertrophy and arrhythmogenesis. While the studies provide great insights, the precise mechanisms involved in PI3K(p110α) hypofunction and atrial fibrillation (AF) are not fully known. AF is a well recognised clinical problem with significant management limitations. In this translational review, we attempted a narration of PI3K(p110α) hypofunction in the molecular basis of AF pathophysiology. We sought to cautiously highlight the relevance of this molecule in the therapeutic approaches for AF management per se (i.e without conditions associate with cell proliferation, like cancer), and in mitigating effects of clinical risk factors in atrial substrate formation leading to AF progression. We also considered PI3K(p110α) in AF gene association, with the aim of identifying mechanistic links between the ever increasingly well-defined genetic loci (regions and genes) and AF. Such mechanisms will aid in identifying new drug targets for arrhythmogenic substrate and AF.

Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and ß bound equivalently to p110α or p110ß but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85ß, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110ß-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kß. Nevertheless, PI3Kß contributes substantially to acute PDGF-stimulation of PIP3 and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kß.