Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Let-7a targets Rsf-1 to modulate radiotherapy response of non-small cell lung cancer cells through Ras-MAPK pathway.

PURPOSE: Radiotherapy is the most commonly selective medical treatment for non-small cell lung cancer (NSCLC) and the multiple underlying mechanisms are considered as the effectively theoretical foundation. Herein, we investigated the effects of let-7a targets Rsf-1 on modulating the radiotherapy response in NSCLC cells by Ras-MAPK pathway. METHODS: A549 cells were divided into different groups to investigate the role of let-7a and Rsf-1 on the radiotherapy response. The expression of let-7a and Rsf-1 were detected by RT-PCR. Bioinformatic analysis indicated that Rsf-1 is the target of let-7a. The binding site of let-7a in the Rsf-1 3'UTR was detected based on double luciferase reporter assay and Western blot. The cell variability and proliferation were assessed by MTT and colony formation assay. The expression levels of Ras-MARK signaling pathway related proteins were assessed by RT-PCR. RESULTS: RT-PCR results showed that radiotherapy could up-regulate the expression of let-7a, thereby reducing the expression of Rsf-1, and the correlation between the two factors was negatively correlated. At the same time, let-7a overexpression and Rsf-1 silencing could further reduce the activity of A549 cells after radiotherapy, have an inhibitory effect on cell proliferation and inhibit the expression of related proteins in the Ras-MAPK pathway. CONCLUSIONS: Rsf-1 is the target of Let-7a. The present study provides evidence that let-7a targeting Rsf-1 can modulate radiotherapy response in NSCLC cells through Ras-MAPK pathway.

Rheb1 promotes glucose-stimulated insulin secretion in human and mouse ß-cells by upregulating GLUT expression.

Reduced ß-cell mass and impaired ß-cell function are primary causes of all types of diabetes. However, the intrinsic molecular mechanism that regulates ß-cell growth and function remains elusive. Here, we demonstrate that the small GTPase Rheb1 is a critical regulator of glucose-stimulated insulin secretion (GSIS) in ß-cells. Rheb1 was highly expressed in mouse and human islets. In addition, ß-cell-specific knockout of Rheb1 reduced the ß-cell size and mass by suppressing ß-cell proliferation and increasing ß-cell apoptosis. However, tamoxifen-induced deletion of Rheb1 in ß-cells had no significant effect on ß-cell mass and size but significantly impaired GSIS. Rheb1 facilitates GSIS in human or mouse islets by upregulating GLUT1 or GLUT2 expression, respectively, in a mTORC1 signaling pathway-dependent manner. Our findings reveal a critical role of Rheb1 in regulating GSIS in ß-cells and identified a new target for the therapeutic treatment of diabetes mellitus.

Emerging Roles of Small GTPases in Islet ß-Cell Function.

Several small guanosine triphosphatases (GTPases) from the Ras protein superfamily regulate glucose-stimulated insulin secretion in the pancreatic islet ß-cell. The Rho family GTPases Cdc42 and Rac1 are primarily involved in relaying key signals in several cellular functions, including vesicle trafficking, plasma membrane homeostasis, and cytoskeletal dynamics. They orchestrate specific changes at each spatiotemporal region within the ß-cell by coordinating with signal transducers, guanine nucleotide exchange factors (GEFs), GTPase-activating factors (GAPs), and their effectors. The Arf family of small GTPases is involved in vesicular trafficking (exocytosis and endocytosis) and actin cytoskeletal dynamics. Rab-GTPases regulate pre-exocytotic and late endocytic membrane trafficking events in ß-cells. Several additional functions for small GTPases include regulating transcription factor activity and mitochondrial dynamics. Importantly, defects in several of these GTPases have been found associated with type 2 diabetes (T2D) etiology. The purpose of this review is to systematically denote the identities and molecular mechanistic steps in the glucose-stimulated insulin secretion pathway that leads to the normal release of insulin. We will also note newly identified defects in these GTPases and their corresponding regulatory factors (e.g., GDP dissociation inhibitors (GDIs), GEFs, and GAPs) in the pancreatic ß-cells, which contribute to the dysregulation of metabolism and the development of T2D.

Oncogenic RAS drives the CRAF-dependent extracellular vesicle uptake mechanism coupled with metastasis.

Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with extracellular vesicles (EVs). This is important as intercellular EV trafficking plays a key role in cancer invasion and metastasis. Here we report that overexpression of mutant RAS drives the EV internalization switch from endocytosis (in non-transformed cells) to macropinocytosis (in cancer cells) resulting in enhanced EV uptake. This process depends on the surface proteoglycan, fibronectin and EV engulfment mechanism regulated by CRAF. Both mutant RAS and activated CRAF expression is associated with formation of membrane ruffles to which they colocalize along with actin, sodium-hydrogen exchangers (NHEs) and phosphorylated myosin phosphatase (pMYPT). RAS-transformed cells internalize EVs in the vicinity of ruffled structures followed by apparent trafficking to lysosome and degradation. NHE inhibitor (EIPA) suppresses RAS-driven EV uptake, along with adhesion-independent clonal growth and experimental metastasis in mice. Thus, EV uptake may represent a targetable step in progression of RAS-driven cancers.

Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system.

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

The ERK mitogen-activated protein kinase signaling network: the final frontier in RAS signal transduction.

The RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK-MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK-MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK-MAPK network and identify unresolved issues for ongoing and future study.

Predicted 'wiring landscape' of Ras-effector interactions in 29 human tissues.

Ras is a plasma membrane (PM)-associated signaling hub protein that interacts with its partners (effectors) in a mutually exclusive fashion. We have shown earlier that competition for binding and hence the occurrence of specific binding events at a hub protein can modulate the activation of downstream pathways. Here, using a mechanistic modeling approach that incorporates high-quality proteomic data of Ras and 56 effectors in 29 (healthy) human tissues, we quantified the amount of individual Ras-effector complexes, and characterized the (stationary) Ras "wiring landscape" specific to each tissue. We identified nine effectors that are in significant amount in complex with Ras in at least one of the 29 tissues. We simulated both mutant- and stimulus-induced network re-configurations, and assessed their divergence from the reference scenario, specifically discussing a case study for two stimuli in three epithelial tissues. These analyses pointed to 32 effectors that are in significant amount in complex with Ras only if they are additionally recruited to the PM, e.g. via membrane-binding domains or domains binding to activated receptors at the PM. Altogether, our data emphasize the importance of tissue context for binding events at the Ras signaling hub.

RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target for high-risk acute lymphoblastic leukemia.

Leukemias are routinely sub-typed for risk/outcome prediction and therapy choice using acquired mutations and chromosomal rearrangements. Down syndrome acute lymphoblastic leukemia (DS-ALL) is characterized by high frequency of CRLF2-rearrangements, JAK2-mutations, or RAS-pathway mutations. Intriguingly, JAK2 and RAS-mutations are mutually exclusive in leukemic sub-clones, causing dichotomy in therapeutic target choices. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. On primary clinical DS-ALL samples, we show that wtRAS-activation is an obligatory consequence of mutated/hyperphosphorylated JAK2. We further prove that CRLF2-ligand TSLP boosts the direct binding of active PTPN11 to wtRAS, providing the molecular mechanism for the wtRAS activation. Pre-inhibition of RAS or PTPN11, but not of PI3K or JAK-signaling, prevented TSLP-induced RAS-GTP boost. Cytotoxicity assays on primary clinical DS-ALL samples demonstrated that, regardless of mutation status, high-risk leukemic cells could only be killed using RAS-inhibitor or PTPN11-inhibitor, but not PI3K/JAK-inhibitors, suggesting a unified treatment target for up to 80% of DS-ALL. Importantly, protein activities-based principal-component-analysis multivariate clusters analyzed for independent outcome prediction using Cox proportional-hazards model showed that protein-activity (but not mutation-status) was independently predictive of outcome, demanding a paradigm-shift in patient-stratification strategy for precision therapy in high-risk ALL.

The lipid phosphatase-like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory molecules.

Phospholipid Phosphatase-Related Protein Type 1 (PLPPR1) is a member of a family of lipid phosphatase related proteins, integral membrane proteins characterized by six transmembrane domains. This family of proteins is enriched in the brain and recent data indicate potential pleiotropic functions in several different contexts. An inherent ability of this family of proteins is to induce morphological changes, and we have previously reported that members of this family interact with each other and may function co-operatively. However, the function of PLPPR1 is not yet understood. Here we show that the expression of PLPPR1 reduces the inhibition of neurite outgrowth of cultured mouse hippocampal neurons by chondroitin sulfate proteoglycans and the retraction of neurites of Neuro-2a cells by lysophosphatidic acid (LPA). Further, we show that PLPPR1 reduces the activation of Ras homolog family member A (RhoA) by LPA in Neuro-2a cells, and that this is because of an association of PLPPR1with the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI1). These results establish a novel signaling pathway for the PLPPR1 protein.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability.

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.

Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase.

Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.

Comparison of hair manifestations in cardio-facio-cutaneous and Costello syndromes highlights the influence of the RAS pathway on hair growth.

BACKGROUND: Abnormal hair growth is a defining feature of RASopathies, syndromes caused by germline mutations in the RAS pathway. However, detailed hair manifestations and the mechanisms of altered hair growth in RASopathies are poorly delineated. OBJECTIVES: To identify distinguishing clinical features and investigate how the RAS pathway influences hair growth by performing a systematic and detailed side-by-side comparison of hair manifestations in cardio-facio-cutaneous syndrome (CFCS) and Costello syndrome (CS), two RASopathies caused by mutations in the downstream and upstream elements of the RAS pathway, respectively. METHODS: Sixteen individuals with CFCS and 23 individuals with CS were enrolled. Mutation data were recorded. Scalp hair, eyebrows and eyelashes of individuals with CFCS or CS were examined for texture, colour, density and morphology. Scalp hairs were examined by light microscopy. RESULTS: While both syndromes displayed abnormal hair, striking differences were observed, including darker and thicker scalp hair and sparse eyebrows and eyelashes in CFCS. By contrast, synophrys, trichomegaly and abnormalities of the scalp hair shafts were observed in CS. Possible correlation with straight hair and genotype was observed in CS. CONCLUSION: The results emphasize the role of the RAS pathway in hair growth, improve accuracy of clinical diagnosis of CFCS and CS and provide a foundation for identification of therapeutic targets.

Suppression of macrophages- Induced inflammation via targeting RAS and PAR-4 signaling in breast cancer cell lines.

Tumor associated macrophages (TAMs) have a crucial role in cancer progression, metastasis and drug response. Piroxicam and sulindac sulfide are non-steroidal anti-inflammatory drugs (NSAID) that decrease the incidence and progression of several types of cancer. However, their role in suppressing the interactions between TAMs and cancer cells remain unclear. Herein, we studied the impact of human monocytes conditioned media (CM) on cellular proliferation of ER-dependent MCF-7 and ER-independent MDA-MB-231 cells, and the effects of piroxicam and sulindac sulfide on the expression levels of RAS, COX-2, IL-6, IL-1ß and PAR-4 (qRT-PCR), BCL-2 and BAX (western blot), Caspase-3, VEGF-a and PGE2 (ELISA), MMP-2 and -9 (zymography) in the stimulated cells. Our results showed that CM caused a significant increase in cells survival through significant increase in RAS expression which resulted in upregulation of COX-2, PGE2, BCL-2, IL-6, IL-1ß, VEGF-A and MMP-9 and down regulation of PAR-4. Treatment with one of the NSAIDs used in this study produced a time and concentration dependent growth inhibition of stimulated cells by inhibiting RAS expression. Suppression of RAS was accompanied by downregulation of its downstream signaling of IL-1ß, IL-6, COX-2 and PGE2, activation of apoptotic machinery through upregulation of PAR-4 and caspase-3, as well as, inhibition of BCL-2, VEGF-A, MMP-2 and MMP-9. In conclusion, our data support the role of piroxicam and sulindac sulfide in suppressing inflammation-driven breast cancer progression and identifies promising novel target in RAS and PAR-4 signaling.

Function, Regulation and Biological Roles of PI3Kγ Variants.

Phosphatidylinositide 3-kinase (PI3K) γ is the only class IB PI3K member playing significant roles in the G-protein-dependent regulation of cell signaling in health and disease. Originally found in the immune system, increasing evidence suggest a wide array of functions in the whole organism. PI3Kγ occur as two different heterodimeric variants: PI3Kγ (p87) and PI3Kγ (p101), which share the same p110γ catalytic subunit but differ in their associated non-catalytic subunit. Here we concentrate on specific PI3Kγ features including its regulation and biological functions. In particular, the roles of its non-catalytic subunits serving as the main regulators determining specificity of class IB PI3Kγ enzymes are highlighted.

Enhanced antitumor effect of binimetinib in combination with capecitabine for biliary tract cancer patients with mutations in the RAS/RAF/MEK/ERK pathway: phase Ib study.

BACKGROUND: A phase Ib study of binimetinib and capecitabine for gemcitabine-pretreated biliary tract cancer (BTC) patients was conducted. METHODS: Binimetinib and capecitabine were dosed twice daily on days 1-14, in 3-week cycles. In the dose-escalation (DE) part, three dose levels (DL) were tested (DL1: binimetinib/capecitabine, 15 mg/1000 mg/m2; DL2: 30 mg/1000 mg/m2; DL3: 30 mg/1250 mg/m2). RESULTS: In the DE part, nine patients were recruited and no dose-limiting toxicity was noted. Therefore, the recommended phase 2 dose was determined as DL3. In the expansion part, 25 patients were enrolled. In total, 34 patients, 25 (73.5%) and 9 patients (26.5%) were second-line and third-line settings, respectively. The 3-month progression-free survival (PFS) rate was 64.0%, and the median PFS and overall survival (OS) were 4.1 and 7.8 months. The objective response rate and disease control rate were 20.6% and 76.5%. In total, 68.4% of stable diseases were durable (> 12 weeks). Furthermore, patients with RAS/RAF/MEK/ERK pathway mutations (38.5%) showed significantly better tumour response (p = 0.028), PFS (5.4 vs. 3.5 months, p = 0.010) and OS (10.8 vs. 5.9 months, p = 0.160) than wild type. Most of the adverse events were grade 1/2 and manageable. CONCLUSIONS: A combination of binimetinib and capecitabine shows acceptable tolerability and promising antitumor efficacy for gemcitabine-pretreated BTC, especially in patients with RAS/RAF/MEK/ERK pathway mutations. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (Identifier: NCT02773459).

RETRACTED: Ras-ERK1/2 signaling accelerates the progression of colorectal cancer via mediation of H2BK5ac.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article "… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Effect of Cyclophilin from Pyropia Yezoensis on the Proliferation of Intestinal Epithelial Cells by Epidermal Growth Factor Receptor/Ras Signaling Pathway.

Cyclophilin (Cyp) is peptidyl-prolyl isomerase (PPIase), and it has many biological functions, including immune response regulation, antioxidants, etc. Cyp from red algae is known for its antioxidant and antifungal activity. However, the other biological effects of Cyp from Pyropia yezoensis are unclear. In this study, we synthesized Cyp from P. yezoensis (pyCyp) and examined its biological activity on IEC-6 cells. First, the MTS assay showed that pyCyp increased cell proliferation in a dose-dependent manner. pyCyp activated the EGFR signaling pathway that regulates cell growth, proliferation, and survival. It induced intracellular signaling pathways, including the Ras signaling pathway. In addition, we observed cell cycle-related proteins. pyCyp increased the expression of cyclin A, cyclin E, and Cdk2, and decreased the expression of p27 and p21 proteins. These results indicate that pyCyp stimulates cell proliferation via the EGFR signaling pathway and promotes cell cycle progression in intestinal epithelial cells. Therefore, we suggest pyCyp as a potential material to promote the proliferation of intestinal epithelial cells.

Insulin-Driven PI3K-AKT Signaling in the Hepatocyte Is Mediated by Redundant PI3Kα and PI3Kß Activities and Is Promoted by RAS.

Phosphatidylinositol-3-kinase (PI3K) activity is aberrant in tumors, and PI3K inhibitors are investigated as cancer therapeutics. PI3K signaling mediates insulin action in metabolism, but the role of PI3K isoforms in insulin signaling remains unresolved. Defining the role of PI3K isoforms in insulin signaling is necessary for a mechanistic understanding of insulin action and to develop PI3K inhibitors with optimal therapeutic index. We show that insulin-driven PI3K-AKT signaling depends on redundant PI3Kα and PI3Kß activities, whereas PI3Kδ and PI3Kγ are largely dispensable. We have also found that RAS activity promotes AKT phosphorylation in insulin-stimulated hepatocytes and that promotion of insulin-driven AKT phosphorylation by RAS depends on PI3Kα. These findings reveal the detailed mechanism by which insulin activates AKT, providing an improved mechanistic understanding of insulin signaling. This improved model for insulin signaling predicts that isoform-selective PI3K inhibitors discriminating between PI3Kα and PI3Kß should be dosed below their hyperglycemic threshold to achieve isoform selectivity.