iSH2 Domain Research Articles

Structural insights into the activation mechanism of phosphoinositide 3-kinase alpha

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases known to regulate important cellular functions by phosphorylating the inositol ring of inositol-phospholipids (PtdIns) at 3’ position. The PI3Kα is a heterodimer and the activation of the catalytic subunit (p110α) is regulated by its regulatory subunit (p85α). The current work deals with studying the activation mechanism of the PI3Kα using multi micro-second molecular dynamic simulations. Structural changes involved in activation mechanism is studied by gradually releasing the inhibitory effects of different domains of regulatory subunit namely, n-terminal SH2 (nSH2) and inter SH2 (iSH2). The observation shows that even in the presence of n-terminal and inter SH2 domain (niSH2) of regulatory subunit, the catalytic domain has some intrinsic activation activity and the presence of c-terminal SH2 (cSH2) domain may be required for complete inhibition. The release of nSH2 domain leads to loss of interactions between iSH2 domain (regulatory subunit) and C2 and kinase domain (catalytic subunit). The study shows that early events in the activation mechanism involve the movement of the ABD domain of the catalytic subunit along with the linker region between ABD and RBD region which may lead to movement of ABD closer to the CLobe of the kinase domain. This movement is essentially as it triggers the rearrangement of CLobe especially the catalytic loop and activation loop which bring catalytic important residues closer to ATP and PIP2(phosphatidylinositol-4,5-bisphosphate). Water mediated interaction analysis reveal that water may be playing an important role in the transfer of phosphate from ATP to PIP2. The study shows that initial signal for release of inhibitory effect of the regulatory subunit might be propagated through the linker region between ABD and RBD through allosteric effect to different regions of the protein. These understanding of early events during the activation mechanism may help in the design of better therapeutic targeting PI3K.

A somatic splice-site variant in PIK3R1 in a patient with vascular overgrowth and low immunoglobulin levels: A case report.

The PI3K/AKT pathway, extensively studied in cancer, is vital for regulating cell metabolism, differentiation, and proliferation. Pathogenic variants in the PIK3R1 gene, which encodes three regulatory units of class IA PI3Ks, have been found in affected tissue of individuals with vascular lesions. These variants predominantly occur in the iSH2 domain, disrupting inhibitory contacts with the catalytic unit and leading to PI3K activation. Germline variants in this gene are also linked to an immunological condition called Activated PI3K delta syndrome type 2 (APDS2). This is a case report and literature review. Clinical data were retrieved from medical records. A male patient presented with extensive vascular malformation covering over 90% of his body, along with complete 2-3 toe syndactyly, suggesting a vascular malformation syndrome called PROS. Low levels of IgA and IgG were detected. The patient achieved his developmental milestones and had above-average weight, height, and head circumference. Exome sequencing of skin and blood DNA revealed a de novo variant in PIK3R1 (c.1746-2A>G, p.?) in 9% of the patient's blood cells and 25% of cultured fibroblasts. Initially, classified as a variant of uncertain significance, this variant was later confirmed to be the cause. This is the first intronic SNV in a canonical splice site within iSH2 described, highlighting the importance of iSH2 in the regulation of the PI3K/AKT pathway and its involvement in the development of vascular overgrowth and antibody deficiency.

Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus

PIK3CA encoding the phosphoinositide 3-kinase (PI3K) p110α catalytic subunit is frequently mutated in cancer, with mutations occurring widely throughout the primary sequence. The full set of mechanisms underlying how PI3Ks are activated by all oncogenic mutations on membranes are unclear. Using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS), we reveal unique regulatory mechanisms underlying PI3K activation. Engagement of p110α on membranes leads to disengagement of the ABD of p110α from the catalytic core, and the C2 domain from the iSH2 domain of the p85 regulatory subunit. PI3K activation also requires reorientation of the p110α C-terminus, with mutations that alter the inhibited conformation of the C-terminus increasing membrane binding. Mutations at the C-terminus (M1043I/L, H1047R, G1049R, and N1068KLKR) activate p110α through distinct mechanisms, with this having important implications for mutant selective inhibitor development. This work reveals unique mechanisms underlying how PI3K is activated by oncogenic mutations, and explains how double mutants can synergistically increase PI3K activity.

Abstract 671: Cryo-electronmicroscopy of PI3K reveals additional regulatory features

Abstract Many solid tumors have alterations in phosphoinositide 3-kinase (PI3K) signaling. These can occur through mutation of the p110alpha and p85alpha proteins of PI3Kalpha itself as well as through alterations to upstream receptor tyrosine kinases (RTKs) and phosphatases of PIP3, including PTEN. As a whole, PI3K signaling is one of the most frequently altered in cancer. These mutations lead to activation of the PI3K complex and to constitutive downstream signaling. This signaling allows cells to enter a pro-growth and pro-survival state which is beneficial to the cancer cells. The structure of PI3K has been extensively studied by crystallography and NMR. Here, we present our work on the full-length complex of p110alpha with p85alpha using cryo-EM. When compared with the previous structural studies, cryo-EM shows a similar structure and major structural features are unchanged. However, our cryo-EM studies revealed several additional structural features of the PI3K complex. Cryo-EM showed that SH3, BH and cSH2 domains of p85alpha can bind to p110alpha. When inhibited with Alpelisib, these domains form a stable cover of the active site of the kinase. These domains interact with the helical and kinase domains of p110alpha and the iSH2 domain of p85alpha. The position of these domains restricts access to the kinase active site and may contribute to the inhibition of PI3K activity by Alpelisib. When the PI3K complex binds to phosphorylated RTKs, the nSH2 binds to phosphotyrosine and relieves inhibitory interactions with the helical domain of p110alpha. Using cryo-EM, we have studied this activation by incubating the full-length PI3K complex with synthetic phosphopeptides. 2D cryo-EM class averages showed a dramatic rearrangement of the PI3K complex. In addition to the expected displacement of the nSH2 domain, the ABD and iSH2 domains undergo structural changes. Acknowledgments: This work was supported by the National Cancer Institute of the National Institutes of Health under awards R35 CA197582 (PKV) and R50 CA243899 (JRH). Citation Format: Jonathan R. Hart, Su Yang, Xiao Liu, Yingna Xu, Xinyu Zou, Huibing Zhang, Qingtong Zhou, Tian Xia, Yan Zhang, Dehua Yang, Ming-Wei Wang, Peter K. Vogt. Cryo-electronmicroscopy of PI3K reveals additional regulatory features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 671.

Disordered Region in PI3K Regulatory Subunit p85 Drives Clathrin‐dependent Endocytosis and Regulates Cell Motility

Class 1A PI3Ks are lipid kinases composed of catalytic subunit p110 and regulatory subunit p85. Besides its catalytic activity of PI(3,4,5)P3 production, PI3K has been implicated in the process of receptor endocytosis. However, it remained unclear if and how directly class 1A PI3K engages endocytosis machinery. We found that a disordered region in the inter-SH2 (iSH2) domain of regulatory subunit p85 has binding motifs for AP-2, an adaptor protein complex for clathrin, and its plasma membrane targeting drives clathrin-mediated endocytosis. Remarkably, this endocytosis induction is independent of lipid kinase activity. Mutation of the endocytosis motifs in p85 caused its focal adhesion accumulation and increased random migration of mouse embryonic fibroblasts (MEFs). This newly discovered aspect of p85 would be a central piece to comprehensively understand the regulation of PI3K and its effect on cell dynamics.

Defining How Oncogenic and Developmental Mutations of PIK3R1 Alter the Regulation of Class IA Phosphoinositide 3-Kinases

The class I phosphoinositide 3-kinases (PI3Ks) are key signaling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders. Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations alter the activity of p110α/p110δ catalytic subunits. We find that the oncogenic Q572∗ truncation of PIK3R1 disrupts all p85-inhibitory inputs, with p110α being hyper-activated compared with p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals unique insight into isoform-specific regulation of p110s by p85α.

838P - PI3K/AKT pathway deleterious mutations in lethal prostate cancer

Background: PI3K/AKT signaling is commonly hijacked in prostate cancer (PC), associating with poorer prognosis and worse response to next generation hormonal treatment (NGHT) including abiraterone and enzalutamide. We studied deleterious pathway mutations in metastatic castration resistant PC (mCRPC) patients correlating these to prognosis and response to NGHT in a case-control study. Methods: Patients with mCRPC and available treatment-naïve and/or CRPC tumor samples were evaluated. Mutation analyses involved MiSeqTM based targeted next generation sequencing (NGS); PTEN was assessed by immunohistochemistry (IHC) with the control cohort having no PTEN IHC loss. Correlations between pathway mutations and outcomes and response to NGHT utilized Cox and Kaplan-Meier analyses. Results: Overall 418 samples (from Feb/15 to Dec/17) were sequenced by targeted NGS; 46 (11%) had mutated pathway genes of which 26 (6%) (PIK3CA, n = 18; AKT1, n = 6; PIK3R1, n = 2) were pathogenic pathway activating mutations. Among these 25 were previously described, while an undescribed PIK3R1 mutation (R543*), located in the iSH2 domain, was also identified. We then randomly selected 56 tumour samples with normal PTEN expression by IHC as a control group without detected pathway aberrations. Overall, 43% (10/23) of patients with mutated cancers had NGHT in pre-, and 57% (13/23) in post-chemotherapy settings, while 3 were NGHT naïve. In the control group, 14 patients (25%) were pre-chemotherapy while 42 (75%) post-chemotherapy. Deleterious pathway mutations were associated with a shorter median overall survival (2.8 vs 4.3 years; HR: 2.73; p < 0.001) and median duration of NGHT (5.9 vs 10.0 mo; p < 0.001), despite a higher proportion of post-chemotherapy patients in the control group. Conclusions: PI3K/AKT pathway deleterious mutations impart a poor prognosis from mCRPC and are associated with shorter responses to NGHT. We envision that these data can impact treatment selection in a targeted treatment approach for mCRPC. Legal entity responsible for the study: The Institute of Cancer Reserach. Funding: CRUK, Movember, PCF, PCUK. Disclosure: All authors have declared no conflicts of interest.

Abstract 682: The mechanism of PI3Kα activation by SH2 release

Abstract Phosphoinositide 3-kinase α (PI3Kα) is a ubiquitous kinase mediating signaling cascades important for cell survival, proliferation and protein synthesis. It phosphorylates PIP2 to PIP3, to which downstream Akt kinase binds to execute singling. PI3Kα dysfunction has been extensively implicated in cancers. The kinase activity of PI3Kα is controlled by the interactions between its catalytic p110 and regulatory p85 subunits. The interactions of the SH2 domains in the p85 subunit, particularly nSH2, with p110 are key factors in PI3Kα inhibition. Release of the SH2 domains from the p110 subunit, is promoted by factors including phosphotyrosine peptides (RTK), calmodulin (CaM), and oncogenic mutations, thereby activating PI3Kα. Our recent data show that both phosphorylated and unphosphorylated CaMs are capable of binding to the SH2 domains, nSH2 and cSH2, thus promoting the PI3K activation. Despite numerous seminal publications over the past two decades, the mechanism of PI3Kα activation is still a mystery. The large distance between the PIP2 binding sites and the ATP pocket in the crystal structure suggests that the solved PI3Kα structure represents the inactive conformation. What is the activated conformation of PI3Kα, and how PI3Kα switches from the inactive to the active conformation in response to stimulus are the significant mechanistic questions. We employ the large-scale molecular dynamics (MD) simulations to visualize the PI3Kα activation stimulated by the release of SH2 domains in the p85 subunit at the atomic resolution. Our trajectories show that release of the nSH2 domain initiates a “switch-like” conformational change of the iSH2 domain, extracting the ATP from its pocket to the PIP2 binding sites, eventually leading to an activated conformation. This dynamic process is initiated by the break of salt-bridges between iSH2 and helical domains induced by the nSH2 release, and further facilitated by the various residues in activation loop of p110 subunit including His-917, Lys-802 and Lys-776 and in iSH2 of p85 subunit including Arg-461 and Arg-465. The release of the cSH2 domain in PI3Kα has a minor effect. This work, which for the first time uncovers the mechanism of PI3Kα activation at atomic resolution, is expected to promote cancer drug design from a mechanistic dynamic standpoint view. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Mingzhen Zhang, Hyunbum Jang, Ruth Nussinov. The mechanism of PI3Kα activation by SH2 release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 682.

Domain analysis reveals striking functional differences between the regulatory subunits of phosphatidylinositol 3-kinase (PI3K), p85α and p85β.

Our understanding of isoform-specific activities of phosphatidylinositol 3-kinase (PI3K) is still rudimentary, and yet, deep knowledge of these non-redundant functions in the PI3K family is essential for effective and safe control of PI3K in disease. The two major isoforms of the regulatory subunits of PI3K are p85α and p85β, encoded by the genes PIK3R1 and PIK3R2, respectively. These isoforms show distinct functional differences that affect and control cellular PI3K activity and signaling [1–4]. In this study, we have further explored the differences between p85α and p85β by genetic truncations and substitutions. We have discovered unexpected activities of the mutant proteins that reflect regulatory functions of distinct p85 domains. These results can be summarized as follows: Deletion of the SH3 domain increases oncogenic and PI3K signaling activity. Deletion of the combined SH3-RhoGAP domains abolishes these activities. In p85β, deletion of the cSH2 domain reduces oncogenic and signaling activities. In p85α, such a deletion has an activating effect. The deletions of the combined cSH2 and iSH2 domains and also the deletion of the cSH2, iSH2 and nSH2 domains yield results that go in the same direction, generally activating in p85α and reducing activity in p85β. The contrasting functions of the cSH2 domains are verified by domain exchanges with the cSH2 domain of p85β exerting an activating effect and the cSH2 domain of p85α an inactivating effect, even in the heterologous isoform. In the cell systems studied, protein stability was not correlated with oncogenic and signaling activity. These observations significantly expand our knowledge of the isoform-specific activities of p85α and p85β and of the functional significance of specific domains for regulating the catalytic subunits of class IA PI3K.

TGF-β promotes PI3K-AKT signaling and prostate cancer cell migration through the TRAF6-mediated ubiquitylation of p85α.

Transforming growth factor-β (TGF-β) is a pluripotent cytokine that regulates cell fate and plasticity in normal tissues and tumors. The multifunctional cellular responses evoked by TGF-β are mediated by the canonical SMAD pathway and by noncanonical pathways, including mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol 3'-kinase (PI3K)-protein kinase B (AKT) pathway. We found that TGF-β activated PI3K in a manner dependent on the activity of the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). TRAF6 polyubiquitylated the PI3K regulatory subunit p85α and promoted the formation of a complex between the TGF-β type I receptor (TβRI) and p85α, which led to the activation of PI3K and AKT. Lys63-linked polyubiquitylation of p85α on Lys513 and Lys519 in the iSH2 (inter-Src homology 2) domain was required for TGF-β-induced activation of PI3K-AKT signaling and cell motility in prostate cancer cells and activated macrophages. Unlike the activation of SMAD pathways, the TRAF6-mediated activation of PI3K and AKT was not dependent on the kinase activity of TβRI. In situ proximity ligation assays revealed that polyubiquitylation of p85α was evident in aggressive prostate cancer tissues. Thus, our data reveal a molecular mechanism by which TGF-β activates the PI3K-AKT pathway to drive cell migration.

Insights into the pathological mechanisms of p85α mutations using a yeast-based phosphatidylinositol 3-kinase model.

In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, β, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85–p110 interactions typical of cancer.

Structural characterization of ANGPTL8 (betatrophin) with its interacting partner lipoprotein lipase

Angiopoietin-like protein 8 (ANGPTL8) (also known as betatrophin) is a newly identified secretory protein with a potential role in autophagy, lipid metabolism and pancreatic beta-cell proliferation. Its structural characterization is required to enhance our current understanding of its mechanism of action which could help in identifying its receptor and/or other binding partners. Based on the physiological significance and necessity of exploring structural features of ANGPTL8, the present study is conducted with a specific aim to model the structure of ANGPTL8 and study its possible interactions with Lipoprotein Lipase (LPL). To the best of our knowledge, this is the first attempt to predict 3-dimensional (3D) structure of ANGPTL8. Three different approaches were used for modeling of ANGPTL8 including homology modeling, de-novo structure prediction and their amalgam which is then proceeded by structure verification using ERRATT, PROSA, Qmean and Ramachandran plot scores. The selected models of ANGPTL8 were further evaluated for protein–protein interaction (PPI) analysis with LPL using CPORT and HADDOCK server. Our results have shown that the crystal structure of iSH2 domain of Phosphatidylinositol 3-kinase (PI3K) p85β subunit (PDB entry: 3mtt) is a good candidate for homology modeling of ANGPTL8. Analysis of inter-molecular interactions between the structure of ANGPTL8 and LPL revealed existence of several non-covalent interactions. The residues of LPL involved in these interactions belong from its lid region, thrombospondin (TSP) region and heparin binding site which is suggestive of a possible role of ANGPTL8 in regulating the proteolysis, motility and localization of LPL. Besides, the conserved residues of SE1 region of ANGPTL8 formed interactions with the residues around the hinge region of LPL. Overall, our results support a model of inhibition of LPL by ANGPTL8 through the steric block of its catalytic site which will be further explored using wet lab studies in future.

Abstract PR02: Targeting PI3K: The PIP2 binding site

Abstract Structural biology has yielded many significant insights into inhibitor binding and isoform selectivity, directing the design of many important clinical candidates. To date, structural studies have focused on the ATP-binding site and ATP-competitive inhibitors. We have recently determined the structure of PI3Kα; in complex with the soluble lipid substrate mimetic, diC4-PIP2. The substrate binds in a positively charged pocket, defined by the activation and P-loops of the kinase domain, and the iSH2 domain of p85. The positively charged residues responsible for binding PIP2 that are present on the activation loop of Class I PI3K are missing in Class II and III PI3K. This explains why these two classes have lower affinity for PIP2. The complex structure provides insights into the catalysis and regulation of PI3K. Key interactions between the activation loop and nSH2 domain may modulate active and inactive conformations of the enzyme upon the binding of phosphorylated receptor tyrosine kinases. In addition, a second lipid substrate was identified in the structure. It binds in a hydrophobic pocket between the Adaptor Binding Domain (ABD), kinase domain and iSH2 domain of p85. Fluorescence quenching studies confirm the ability of PI3K to bind an additional PIP2 molecule. This abstract is also being presented as Poster B17. Citation Format: Michelle Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Targeting PI3K: The PIP2 binding site. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR02.

Structural basis of nSH2 regulation and lipid binding in PI3Kα.

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.

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.

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects.

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP2) to phosphoinositide-3,4,5-trisphosphate (PIP3), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP2, a membrane lipid.

Abstract 2738: PIK3CA and APC mutations are synergistic in the development of intestinal cancers.

Abstract Background: Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been thoroughly investigated. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas in the proximal colon. These cancers form via a non-canonical mechanism without aberrations in Wnt signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the vast majority of human colon cancers and often occurs concurrently with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine using novel murine models. Methods: B6 females carrying a transgene encoding for a chimeric protein with the iSH2 domain of the p85 subunit of PI3K fused to the N-terminus of p110 (PIK3ca*) were crossed to B6 males carrying the Min allele of Apc (ApcMin/+). The resulting B6 PIK3ca* ApcMin/+ mice were then crossed to mice carrying a transgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3ca* ApcMin/+ progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon, as well as having lost one allele of Apc. An additional novel model system was developed to induce mutations in the cells of the mid to distal colon using a minimally invasive technique to instil adenovirus expressing the Cre recombinase. These tumors could then be monitored over time with colonoscopy with biopsies taken for histological and molecular analyses. Results: FC PIK3ca* ApcMin/+ mice develop multiple small intestinal and colon tumors, including invasive adenocarcinomas with lymphatic and mesenteric metastases. Tumor multiplicity and size were increased. Moreover, the tumors appeared less differentiated and were more invasive as compared to those in controls. Advanced adenomas and adenocarcinomas were easily detectible with a novel near-infrared phospholipid ether analog, CLR1502, that selectively accumulates in tumors. In the proximal colon, mucinous adenocarcinomas that were initiated by the dominant active PI3K rarely exhibited nuclear β-catenin and when observed, this localization was limited to select areas. This indicates that in these tumors loss of APC in these cells was a late event and can be a source of clonal outgrowth. The tumors that form in this model are microsatellite stable. Interestingly this synergy appears to be independent of PI3K mediated regulation of GSK-3β and β-catenin signaling. Conclusion: Expression of a dominant active PI3K synergizes with loss of APC activity resulting in dramatic changes in tumor multiplicity, size, morphology, and invasiveness. Citation Format: Dustin Deming, Alyssa Leystra, Laura Nettekoven, Chelsie Kohns, Linda Clipson, Dawn Albrecht, Jeff Bacher, Mary Kay Washington, Christopher Pazoles, Jamey Weichert, Richard Halberg. PIK3CA and APC mutations are synergistic in the development of intestinal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2013-2738

Allosteric activation of PI3Kα by oncogenic mutations

The gene encoding the p110α catalytic subunit of class IA phosphoinositide 3-kinase (PIK3CA) is one of the most frequently mutated genes in human cancer [1]. Somatic mutations are widely dispersed throughout every domain of the catalytic subunit (Fig ​(Fig1A).1A). Two “hot spot” mutants have been shown biochemically and structurally to activate lipid kinase activity by distinct mechanisms [2]. Rare mutations, which have been shown to play a role in endometrial cancers [3], also increase lipid kinase activity. The mechanism of activation of rare mutations that are distant from both the active site and membrane interface have remained ambiguous. We have recently determined a set of allosteric conformational events that occur upon activation of wild type PI3Kα using hydrogen/deuterium exchange mass spectrometry (HDX-MS), and we find that all oncogenic mutants studied activated lipid kinase activity by mimicking or enhancing these specific events [4]. Figure 1 Oncogenic mutants in PI3K The lipid kinase activity of the class IA PI3Ks is tightly regulated by the p85 regulatory subunit which stabilizes, inhibits, and allows activation downstream of receptor tyrosine kinases (RTKs) (Fig. 1A, 1B). The interaction between the catalytic and regulatory subunit is mediated by the interaction of the adaptor-binding domain (ABD) of p110 and the coiled coil iSH2 domain of p85 [5]. The enzyme is inhibited by contacts between the N-terminal SH2 domain (nSH2) of p85 and the C2, helical and lipid kinase domains of the p110 subunit [5, 6]. The binding of phosphorylated tyrosine residues in RTKs and their associated proteins by the nSH2 disrupts this inhibitory interface and activates the enzyme. The enzyme is also inhibited by contacts between the C2 of p110 and the iSH2 of p85 [7]. The C-terminal SH2 (cSH2) domain of p85 which inhibits the p110δ and p110β isoforms of PI3K has no inhibitory influence on the p110α isoform [8]. To determine the mechanism of activation by oncogenic mutants we first investigated conformational changes that occur in the wild type upon binding to tyrosine-phosphorylated peptides derived from RTKs (pY), and upon binding to phospholipid membranes [4]. We identified four distinct conformational events that occur in the wild type PI3Kα upon activation (Fig. ​(Fig.1C):1C): (i) Disruption of the nSH2/helical interface, (ii) Disruption of the C2/iSH2 interface, (iii) Movement of the ABD relative to the kinase domain, and (iv) Interaction of the lipid kinase domain with membranes. We propose that these events are involved in the transition of a cytosolic, inhibited form of p110α to an open, activated form on the membrane surface. Oncogenic mutants in the ABD (G106V), ABD-RBD (Ras-binding domain) linker (G118D), C2 (N345K, C420R), and the hot spot mutants in the helical (E545K) and kinase domains (H1047R) upregulated lipid kinase activity by mimicking or enhancing one or more of the conformational events identified in the wild type. Mutations in the ABD and ABD-RBD linker caused both enhanced movement of the ABD domain relative to the kinase domain, and disruption of the C2/iSH2 interface compared to the WT enzyme. This same relationship was seen in mutations in the C2 domain, which also caused disruption of the C2/iSH2 interface along with movement of the ABD relative to the kinase domain. This shows that these two regions are allosterically linked to each other. These mutations can be activated by RTK derived pY, and are hyperactivated compared to the WT enzyme. The presence of the helical domain hotspot mutation located at the nSH2/helical interface fully disrupted this contact and made the mutant insensitive to pY activation, as predicted from previous biochemical and structural data [5, 6]. The kinase domain hotspot mutation caused conformational changes in the C-lobe of the kinase domain located at the membrane interface. Interestingly, the regions of the kinase domain that were exposed by this mutation are the exact same regions that were protected by the cSH2 domain in the p110β and p110δ isoforms [8]. This raises an interesting possibility that p110α has acquired inhibitory mutations in the kinase domain that enable it to bypass cSH2 mediated inhibition that occurs in the p110β and p110δ isoforms. The important role of oncogenic mutants of PI3Kα in the development of a variety of human cancers makes the development of novel inhibitory strategies an important goal. The discovery of these unexpected allosteric conformational changes that are enhanced in oncogenic mutants opens up a novel strategy towards designing small molecule inhibitors that function outside of the conserved ATP binding pocket.

Somatic Mutations of PIK3R1 Promote Gliomagenesis

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.

Abstract 1341: Mice expressing activated PI3K develop advanced colon cancer

Abstract Background: Mutations in PIK3CA are a common feature of human cancers and multiple new PI3K inhibitors are in clinical development. These mutations are present in 20-30% of colorectal cancers in humans; however, the effects of such mutations on the intestinal mucosa have not been previously studied in animal models. Here we demonstrate that the expression of a dominant active form of the PI3K protein in the mouse intestine results not only in hyperplasia but also advanced neoplasia. Methods: Mice carrying a transgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC) were crossed to mice carrying a transgene encoding a chimeric protein with the iSH2 domain of the p85 subunit fused to the N-terminus of p110 (PIK3ca*). Progeny carrying both transgenes (FC PIK3ca*) express this constitutively active form of p110 in epithelial cells of the distal small bowel and colon. Results: Invasive mucinous adenocarcinomas develop in the proximal colon by only 50 days of age. These tumors are very aggressive, spreading into the mesentery and adjacent organs, and their histological characteristics are strikingly similar to those of invasive colon cancers in humans. Neoplastic cells exhibit a high rate of cell proliferation as seen by increased Ki67 immunohistochemical staining and phosphorylation of AKT, pS6, and p4E-BP1 by western blot demonstrating downstream signaling of PI3K. Interestingly, these cells did not exhibit perturbations in ERK and WNT signaling. Consistent with the lack of aberrant WNT signaling, no benign polypoid intermediaries were identified. Together, the results indicate that tumors in this model form via a non-canonical mechanism. A cohort of FC PIK3ca* mice were imaged by FDG dual hybrid PET/CT colonography and mice with similar sized tumors were divided into control and experimental arms. The experimental arm was treated with rapamycin (6mg/kg/day) for 14 days. Both groups were re-imaged 14 days later. The full results of the rapamycin treatment will be presented. Conclusion: This unique model of advanced colonic invasive adenocarcinomas has the potential to further our understanding of human disease and facilitate the development of therapeutics, especially inhibitors targeting downstream of PI3K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1341. doi:1538-7445.AM2012-1341