Class I PI3K in Oncogenic Cellular Transformation

L Zhao and PK Vogt

Division of Oncovirology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA

Class I phosphoinositide 3-kinase (PI3K) is a dimeric Class I PI3Ks phosphorylate phosphatidylinositol 4,5 enzyme, consisting of a catalytic and a regulatory subunit. bisphosphate (PIP2) at the 3 position of the inositol ring. The catalytic subunit occurs in four isoforms designated The product, phosphatidylinositol 3,4,5-trisphosphate as p110a, p110b, p110c and p110d. These isoforms (PIP3), functions as a second cellular messenger that combine with several regulatorysubunits; for p110 a, b controls cell growth, survival, proliferation, motility and and d, the standard regulatorysubunit is p85, for p110 c, morphology (Vanhaesebroeck and Waterfield, 1999; it is p101. PI3Ks playimportant roles in human cancer. Katso et al., 2001; Vanhaesebroeck et al., 2001; Cantley, PIK3CA, the gene encoding p110a, is mutated frequently 2002; Vivanco and Sawyers, 2002; Okkenhaug and in common cancers, including carcinoma of the breast, Vanhaesebroeck, 2003; Deane and Fruman, 2004; Bader prostate, colon and endometrium. Eightypercent of these et al., 2005; Engelman et al., 2006; Hawkins et al., 2006). mutations are represented byone of the three amino-acid The phosphatase PTEN (phosphatase and TENsin substitutions in the helical or kinase domains of the homolog deleted on chromosome 10) hydrolyzes PIP3 enzyme. The mutant p110a shows a gain of function in to PIP2, thus acting as the catalytic antagonist of enzymatic and signaling activity and is oncogenic in cell PI3K (Maehama and Dixon, 1998; Myers et al., 1998; culture and in animal model systems. Structural and Stambolic et al., 1998). Mutational activation and genetic data suggest that the mutations affect regulatory overexpression of class I PI3K and genetic or epigenetic inter- and intramolecular interactions and support the inactivation of PTEN result in enhanced PI3K signaling, conclusion that there are at least two molecular mechan- which is associated with oncogenic cellular transforma- isms for the gain of function in p110a. One of these tion and cancer (Ali et al., 1999; Maehama et al., 2001; mechanisms operates largelyindependentlyof binding to Simpson and Parsons, 2001; Cantley, 2002; Wishart and p85, the other abolishes the requirement for an interaction Dixon, 2002; Eng, 2003; Bachman et al., 2004; with Ras. The non-a isoforms of p110 do not show cancer- Broderick et al., 2004; Campbell et al., 2004; Fruman, specific mutations. However, theyare often differentially 2004; Leslie and Downes, 2004; Samuels et al., 2004; expressed in cancer and, in contrast to p110a, wild-type Bader et al., 2005; Hartmann et al., 2005; Lee et al., non-a isoforms of p110 are oncogenic when overexpressed 2005; Levine et al., 2005; Li et al., 2005; Saal et al., 2005; in cell culture. The isoforms of p110 have become Wang et al., 2005; Kang et al., 2005b; Cully et al., 2006; promising drug targets. Isoform-selective inhibitors have Vogt et al., 2007; Salmena et al., 2008). Because of the been identified. Inhibitors that target exclusivelythe enzymatic antagonism of PI3K and PTEN, it is cancer-specific mutants of p110a constitute an important tempting to equate loss of PTEN with gain in PI3K goal and challenge for current drug development. function. However, there is mounting evidence that a Oncogene (2008) 27, 5486–5496; doi:10.1038/onc.2008.244 loss of PTEN results in cellular changes that are quite different from those induced by a gain of function in Keywords: PI3K; PTEN; Akt; Ras; p85 PI3K (Blanco-Aparicio et al., 2007). The enzymatic antagonism is not the only determining factor that characterizes the balance of PTEN and PI3K in the cell. The cellular distribution of the two proteins is different, and these differences can be enhanced by external and Introduction internal stimuli. Interaction with other proteins could also gravely affect the balance between PTEN and This contribution will present a brief review of class I PI3K. Tumors that have lost PTEN often show drug phosphoinositide 3-kinases (PI3Ks) and their oncogenic sensitivities that are different from those that have a activities, focusing on cancer-specific mutations and on direct gain of PI3K function (Salmena et al., 2008). differential expression of the four catalytic subunits of Only class I PI3Ks are involved in cancer; there are no this enzyme family. data linking class II PI3Ks or class III PI3K (Vps34p) to oncogenesis. This fact probably reflects the different product and substrate specificities of the three classes of PI3K. Only class I PI3Ks can use PIP2 to generate PIP3, Correspondence: Dr L Zhao, Division of Oncovirology, Department of Molecular and Experimental Medicine, 10550 North Torrey Pines class II PI3Ks produce the 3,4-bisphosphate and the Road, BCC-239, La Jolla, 92037CA, USA. 3-monophosphate of inositol lipids, and class III can E-mail: [email protected] only make the 3-monophosphate. PIP3 is a critical PI3K in oncogenic cellular transformation L Zhao and PK Vogt 5487 component in the control of cell growth and replication, phosphorylates and thereby activates Akt at threonine and the ability to produce this important second 308 (Alessi et al., 1997). Signals originating from Akt messenger molecule confers oncogenic potential to the control the initiation of protein synthesis through a lipid kinase. Class I PI3Ks have both lipid and protein cascade of interactions that proceeds through the TSC kinase activities (Dhand et al., 1994; Foukas et al., 2004; (tuberous sclerosis complex), Rheb (Ras homolog Foukas and Shepherd, 2004). Genetic experiments have enriched in brain) and TOR (target of rapamycin) to shown that lipid kinase is essential for oncogenic two critical downstream targets, S6K (p70 S6 kinase) activity; p110 engineered to have only protein kinase and 4EBP (eukaryotic initiation factor 4E-binding activity is nononcogenic (Kang et al., 2006). Whether protein; Inoki et al., 2002, 2003; Garami et al., 2003; protein kinase plays a role in conjunction with lipid Tee et al., 2003; Zhang et al., 2003; Bader and Vogt, kinase is not known. 2004). Akt signals also regulate transcription, inducing phosphorylation-dependent degradation of FOXO1 (forkhead box O transcription factor; Biggs et al., The canonical PI3K signaling pathway 1999; Brunet et al., 1999; Kops et al., 1999; Takaishi et al., 1999; Tang et al., 1999) and inactivation of In normal cells, the activity of class I PI3Ks is tightly GSK3b (glycogen synthase kinase-3b; Cross et al., controlled. Upstream signals recruit the cytosolic PI3Ks 1995). Important targets of FOXO1 are the growth- to the plasma membrane. This relocation is mediated by attenuating p27(Kip1; Medema et al., 2000) and interactions with receptor tyrosine kinases (Skolnik p21(Cip1; Seoane et al., 2004) and proapoptotic BIM et al., 1991) or G-protein-coupled receptors (Stephens (Bcl-2 interacting mediator of cell death) proteins (Stahl et al., 1994). Interaction with Ras also contributes to the et al., 2002; Gilley et al., 2003; Arden, 2004). GSK3b activation of PI3K (Rodriguez-Viciana et al., 1994, regulates the potentially oncogenic transcription factors 1996; Chan et al., 2002; Figure 1). The product of class I Jun (cellular homolog of the Jun oncoprotein of avian PI3K, PIP3, recruits proteins that contain a pleckstrin retrovirus ASV17) and Myc (cellular homolog of the homology domain to cellular membranes (Corvera and avian myelocytoma retroviral oncogene; Nikolakaki Czech, 1998). Among these are the serine-threonine et al., 1993; de Groot et al., 1993; Sears et al., 2000; kinase Akt (cellular homolog of murine thymoma virus Gregory et al., 2003; Wei et al., 2005). In a positive Akt8 oncoprotein) as well as its activating kinase feedback loop, TOR, in complex with the Rictor PDK1 (3-phosphoinositide-dependent kinase 1). PDK1 (rapamycin-insensitive companion of TOR) protein

Figure 1 The canonical PI3K signaling pathway. PI3Ks can be activated by RTKs (with or without adaptors such as IRS1) or GPCRs. Ras is an additional positive regulator of PI3K, probably by facilitating membrane localization. The phosphatase PTEN dephosphorylates the product of PI3K, PIP3 at the 3 position and thus acts as the exact enzymatic antagonist of PI3K. PIP3 initiates downstream signaling by recruiting the serine-threonine kinases Akt and PDK1. PDK1 phosphorylates and thereby activates Akt. Three major signaling branches originate from Akt. Akt-mediated phosphorylation of GSK3b and of FOXO directly and indirectly controls transcriptional activities and cellular growth and survival (blue icons). The signal proceeding through the TSC complex, RHEB and TOR affects primarily protein synthesis (beige icons). A positive feedback loop extends from the TOR–RICTOR complex to Akt, resulting in additional activating phosphorylation of Akt. A negative feedback loop consists of the S6K-mediated phosphorylation of IRS1. GPCRs, G-protein-coupled receptors; RTKs, receptor tyrosine kinases; Raf, V-Raf murine leukemia viral oncogene homolog; MEK, Map/Erk kinase; ERK, extracellular signal-regulated kinase; Map, mitogen-activated protein.

Oncogene PI3K in oncogenic cellular transformation L Zhao and PK Vogt 5488 phosphorylates and thereby additionally activates Akt p85 at serine 473 (Sarbassov et al., 2005). S6K can introduce an inhibitory phosphorylation on IRS1 (insulin receptor C C-SH2 iSH2 N-SH2 BH SH3 N substrate 1), mediating a negative feedback loop (Harrington et al., 2004). Ras is also linked to the PI3K pathway. Activated Ras enhances the activities of PI3K; in turn, the product of PI3K, PIP3, stimulates Ras activation (Rodriguez-Viciana et al., 1994, 1996; Chan N ABD RBD C2 HELICAL KINASE C et al., 2002). The overall effect of the combined PI3K signals is to enhance the stimulation of cellular replication and survival and to reduce the growth inhibition and apoptosis. p110α Class I PI3Ks are heterodimeric proteins that consist of a catalytic subunit and a regulatory subunit, also Figure 2 PI3K is a dimeric enzyme. The figure shows the domain referred to as an adaptor (Figure 2). There are four structure and domain interaction map of the standard regulatory subunit, p85 and the catalytic subunit, p110 (Walker et al., 1999; isoforms of the catalytic subunit: p110a, p110b,p110d Pacold et al., 2000; Shekar et al., 2005; Huang et al., 2007; Miled and p110g. They share the same domain composition: et al., 2007). PI3K, phosphoinositide 3-kinase. an amino-terminal adaptor-binding domain (ABD) that provides the principal interaction surface with the kinase domain. The hot spot mutations induce a gain of regulatory subunit, a Ras-binding domain (RBD) that function in p110a. The lipid kinase activity of the mediates the interaction between p110 and Ras-GTP mutant protein is significantly upregulated (Samuels and contributes to the stimulation of PI3K and to the et al., 2004; Ikenoue et al., 2005; Kang et al., 2005a). Ras-driven signaling pathway, a C2 (protein-kinase-C Mutant-expressing cells show constitutive downstream homology-2) domain with affinity for lipid membranes, signaling detectable by the elevated phosphorylation of a helical domain acting as a scaffold for other domains Akt, S6K, 4EBP and GSK3b. p110a carrying one of the of p110 and a carboxyl-terminal kinase domain (Walker hot spot mutations shows oncogenic activity. It can et al., 1999). Class I PI3Ks are further subdivided transform primary fibroblasts in culture, induce ancho- according to their regulatory subunits (Vanhaesebroeck rage-independent growth and cause tumors in animal et al., 1997a). Class IA, encompassing p110a, p110b and model systems (Ikenoue et al., 2005; Isakoff et al., 2005; p110d associates with the regulatory subunits p85a, Zhao et al., 2005; Kang et al., 2005a; Bader et al., 2006). p85b, p55a, p55g and p50a. Class IB, consisting only of This oncogenic potential probably contributes to the p110g, binds the regulatory subunits p101, p84 and neoplastic phenotype of the human cancer cells carrying p87PIKAP. The representative regulatory subunit, mutant p110a; the mutations can therefore be regarded p85a, contains several modular protein–protein inter- as ‘driver’ mutations. The clustering of p110a mutations action domains: a Src-homology 3 (SH3) domain, a in hot spots suggests that the mutations provide a breakpoint clustered homology (BH) domain, two Src- selective growth advantage to the cell. In addition to the homology 2 (SH2) domains, and an inter-SH2 (iSH2) three hot spot mutations, which account for four fifths domain. The iSH2 domain is the primary p110-binding of the p110a mutations, numerous different rare cancer- domain (Dhand et al., 1994; Klippel et al., 1994). specific mutations have been identified (for selected Regulatory subunits link p110 to upstream signals, examples, see Figure 3). They are widely distributed over interacting with receptor tyrosine kinases and G- the coding sequence and occur in all domains of p110a protein-coupled receptors . In the absence of upstream except the RBD. Most of these rare mutations also show signals, the regulatory subunits stabilize p110 and a gain of function (Ikenoue et al., 2005; Gymnopoulos suppress its catalytic activities (Yu et al., 1998; Luo et al., 2007). However, quantitative measurements of et al., 2005). oncogenic activity show that these rare mutants are far less potent than the hot spot mutants. This lower level of oncogenic activity may translate into a weaker selective Cancer-specific mutations in PIK3CA advantage for the mutant-carrying cell and may explain the rarity of these marginally oncogenic mutations. A recurring The gene coding for p110a, PIK3CA, is mutated in theme in the gain-of-function mutations of p110a is a various human cancers (Samuels et al., 2004; Samuels substitution of an acidic or neutral residue with a basic and Ericson, 2006). The mutations are nonsynonymous, residue and location of the substituted residue on the arising from single-nucleotide substitutions. They occur surface of the protein. Indeed, some of the engineered point in around 30% of several common cancers, including mutations of p110a that meet these criteria show a gain of carcinoma of the breast, colon, endometrium and function (Gymnopoulos et al., 2007). prostate (Catalogue of Somatic Mutations in Cancer, http://www.sanger.ac.uk/genetics/CGP/cosmic). These cancers carry a single p110a mutation, and 80% of Structural data the mutated proteins contain one of three ‘hot spot’ mutations. Two of these hot spot mutations map to the The X-ray crystal structure of p110a in complex with a helical domain of p110a, and the third resides in the portion of p85a has been solved (Huang et al., 2007,

Oncogene PI3K in oncogenic cellular transformation L Zhao and PK Vogt 5489

Figure 3 A map of selected cancer-speciﬁc gain-of-function mutations in p110a. Suggested mechanisms for the gain of function are listed at the right. The three hot spot mutations are in red.

2008). The data show that the ABD of p110a not only The C2 domain has been postulated to facilitate the binds to the iSH2 domain of p85 (the region responsible recruitment of p110 to the plasma membrane (Nalefski for high-affinity binding between p85 and p110), but and Falke, 1996; Newton and Johnson, 1998; Rizo and also interacts with the kinase domain and a linker region Sudhof, 1998). This function is evident in the crystal between ABD and RBD. R38 and R88 of p110a form structure of p110g (Walker et al., 1999). Positively hydrogen bonds with Q738, D743 and D746 of the charged amino acids are critically involved in membrane N-terminal lobe of the kinase domain. The rare cancer- binding (Heo et al., 2006). Cancer-specific mutations specific mutations, R38H, R38C and R88Q possibly (N345K and C420R) in the C2 domain of p110a disrupt these interactions, resulting in a conformational increase the positive surface charge of the domain and change of the kinase domain. An ABD deletion mutant were thought to mediate improved binding to the cell of wild-type p110a shows the enhanced lipid kinase membrane, making lipid kinase activity independent of activity compared to its full-length counterpart (Zhao signals transmitted through the regulatory subunit et al., 2005; Zhao and Vogt, 2008). This increase in (Gymnopoulos et al., 2007). However, in the cocrystal activity may be due to the loss of an inhibitory structure of p110a and p85, N345 of p110a forms a interaction between ABD and the kinase domain of hydrogen bond with N564 and D560 in the iSH2 p110a, or it may reflect a relief of the inhibition that is domain of p85. Therefore, N345K is likely to disrupt caused by the binding of the N-SH2 (N-terminal SH2) this interaction of the C2 and iSH2 domains and thus domain of p85 to the helical domain of p110 (see below). alter the regulatory effect of p85 on p110a (Huang et al., Deletion of part of the p85-binding domain in wild-type 2007). The electron density of the C420 residue in the C2 p110a also reveals a low level of oncogenic transforming domain of p110a is not seen in the cocrystal structure. activity that is readily demonstrable in cell culture (Zhao The C420R mutation may function to increase the and Vogt, 2008). affinity of p110a for lipid membranes as previously

Oncogene PI3K in oncogenic cellular transformation L Zhao and PK Vogt 5490 proposed (Gymnopoulos et al., 2007). Another C2 domain mutation, E453Q, also disrupts the interaction of the C2 domain with iSH2, similar to the N345K mutation (Huang et al., 2007). Biochemical and structural modeling studies provide evidence for an interaction between the helical domain of p110a and the N-SH2 domain of p85 (Shekar et al., 2005; Miled et al., 2007; Wu et al., 2007). In the cocrystal structure of p110a/p85, the N-SH2 domain is not highly ordered. However, a structural model can be generated, if biochemically identified interactions are taken into account. In this model, the N-SH2 domain of p85 binds to the interface between the kinase and the helical domains of p110a (Huang et al., 2007). These Figure 4 The interactions with p85 and with Ras define two interactions may be responsible for the p85-induced distinct molecular mechanisms for the gain of function seen in the inhibition of p110a. The helical domain mutations hot spot mutations in p110a. The helical domain mutations are (E542K and E545K) could interfere with this p85- largely but not completely independent of binding to p85 but p110a interaction and thus could relieve the inhibition. require the interaction with Ras. The kinase domain mutation completely depends on the interaction with p85 but is not affected The phosphorylated insulin receptor substrate activates by a loss of Ras binding. However, the kinase domain mutation the lipid kinase activity of wild-type p110a, presumably still shows residual signaling activity in the absence of p85 binding. by engaging the N-SH2 domain of p85 and thus lifting the inhibitory hold of N-SH2 on the helical domain. This activation is not seen in the helical domain mutants p110a interaction by the K227E mutation in the RBD suggesting that the inhibitory interaction with the N- has the opposite effect on the hot spot mutants of SH2 domain of p85 has been weakened or interrupted p110a. Interaction of GTP-bound Ras with wild-type by the mutations (Carson et al., 2008). However, helical p110a is known to augment the activity of p110a (Chan domain mutations that carry an ABD truncation show et al., 2002), possibly by inducing a conformational significantly higher oncogenic activity than the trun- change in the substrate-binding site (Pacold et al., 2000). cated wild-type p110a (Zhao and Vogt, 2008). As both In turn, PI3K is an important Ras effector, mediating have lost p85 binding, the difference in oncogenic the proliferative and survival functions of Ras (Rodri- potency suggests an effect of the helical domain guez-Viciana et al., 1994, 1996). Introducing the Ras- mutations that goes beyond interference with p85 binding mutation into the hot spot mutants causes a binding. The relevant intra- and intermolecular inter- complete loss of oncogenic potency in the helical actions are schematically summarized in Figure 2. domain mutations together with a cessation of signaling, whereas the kinase domain mutant is unaffected by the absence of Ras-binding (Figures 3 and 4). The kinase The genetics of cancer-specific mutations in PIK3CA domain mutant is even able to rescue helical domain mutants that were incapacitated by the absence of Ras- Genetic experiments provide insight into the molecular binding, restoring oncogenic and signaling activities to mechanisms of mutant-induced gain of function in the synergistic levels seen with helical-kinase domain p110a (Zhao et al., 2005; Kang et al., 2005a; Liu and double mutants. The kinase domain mutation maps Roberts, 2006; Zhao and Vogt, 2008). The location of close to the activation loop and may affect the the hot spot mutations in two different domains of the conformation of the loop, altering the interaction with protein, E542K and E545K in the helical and H1047R in the substrate (Huang et al., 2007, 2008). Previous the kinase domain, suggests that they operate by structural studies on the p110g-Ras complex have different mechanisms. This proposal is supported by demonstrated a change in the conformation of the the observation that combining helical and kinase substrate-binding site as a result of the interaction with domain hot spot mutations in the same molecule has a Ras (Pacold et al., 2000). The kinase domain mutation strongly synergistic effect on downstream signaling and H1047R may induce a similar conformational change in on oncogenic potency. The double mutant, E545K/ the absence of Ras and thus gain Ras independence. The H1047R, has also been found in human cancer (Lee data on p85 and Ras interaction strongly support the et al., 2005). The case for mechanistic differences existence of two distinct molecular mechanisms for the between helical and kinase domain mutations is further mutation-induced gain of function in p110a. The data strengthened by the interactions with p85 and Ras are compatible with the suggestion that the helical (Zhao and Vogt, 2008). A truncation of the p85-binding domain mutations lift the inhibitory interaction between domain that eliminates the interaction with p85 does not N-SH2 of p85 and the helical domain of hot spot silence oncogenic and signaling activities of the helical mutants of p110a and that the kinase domain mutation domain mutants, but completely abolishes oncogenicity mimics the conformational change that is triggered by in the kinase domain mutant. Curiously, the thus the interaction with Ras (Zhao and Vogt, 2008). These incapacitated kinase domain mutant still signals through straightforward interpretations ascribe the effect of the Akt and TOR, albeit at lower levels. Disabling the Ras- mutations in the p85 and Ras interacting domains to the

Oncogene PI3K in oncogenic cellular transformation L Zhao and PK Vogt 5491 specific elimination of p85 and Ras binding, respec- Rodriguez-Borlado et al., 2003; Ali et al., 2004). tively. However, the possibility that these mutant effects Conditional and tissue-specific mutations of the are caused by some conformational change that is p110 isoforms and experiments with isoform-specific independent of the targeted protein–protein interactions antibodies have generated a steadily increasing catalog has not been ruled out. For instance, the cocrystal of diverse isoform-specific activities (Vanhaesebroeck structure of p110a and p85 also reveals an unexpected et al., 1999, 2005; Hooshmand-Rad et al., 2000; Bony interaction between p85-binding domain and kinase et al., 2001; Leverrier et al., 2003; Yip et al., 2004; domain, which would be affected by the truncation of Foukas et al., 2006; Suire et al., 2006; Ji et al., 2007; Ali the p85-binding region (Huang et al., 2007). The et al., 2008; Graupera et al., 2008). The general ultimate test of these ideas will be the cocrystal structure conclusions emerging from this work place p110g and of mutant p110a bound to the full-length p85 regulatory d firmly in the realm of the immune system, assign p110a subunit. to cell growth and reveal an interesting connection between p110b and blood clotting (Ono et al., 2007; van der Meijden et al., 2008). Class I p110a has attracted The non-a isoforms of class I PI3K much attention because of its involvement in cancer, documented by the frequent occurrence of gain-of- Although the four isoforms of class I PI3K have function, cancer-specific mutations. No such cancer- identical enzymatic activities, they have different, specific mutations have been identified in the non-a nonredundant cellular functions. Their patterns of isoforms. Yet there is evidence that non-a isoforms of expression are distinct, ubiquitous for the p110a and p110 are involved in the development and progression of p110b isoforms and largely leukocyte-specific for p110g malignancies. Consistent overexpression of p110d is seen and p110d (Vanhaesebroeck et al., 1997b; Sawyer et al., in acute myeloblastic leukemia (Sujobert et al., 2005). 2003). Genetic inactivation of p110a and p110b in mice Inhibitors of p110d specifically interfere with the growth leads to early embryonic lethality (Bi et al., 1999, 2002); of the leukemic cells, suggesting that p110d can function p110g and p110d knockout mice are viable but show as an oncoprotein (Sadhu et al., 2003). Elevated defective immune responses (Hirsch et al., 2000; Li et al., expression of p110g is observed in chronic myeloid 2000; Sasaki et al., 2000; Clayton et al., 2002; Jou et al., leukemia (Skorski et al., 1997; Hickey and Cotter, 2006). 2002; Laffargue et al., 2002; Okkenhaug et al., 2002; Further data suggest a role of non-a isoforms in cancers

Figure 5 Cells transformed by the four isoforms of class I p110 show distinct patterns of constitutive downstream signaling that group p110aH1047R together with p110d, as both constitutively activate Akt and downstream components of the pathway. In contrast, p110b- and p110g-transformed cells do not show this constitutive activation of Akt, but this deﬁciency can be remedied by linking a myristylation signal to the N-terminus of p110.

Oncogene PI3K in oncogenic cellular transformation L Zhao and PK Vogt 5492 of the bladder, brain and colon (Benistant et al., 2000; is the recruitment of p110b and p110g to the cell Mizoguchi et al., 2004; Knobbe et al., 2005). Over- membrane. This recruitment function of Ras may also expression of non-a isoforms in cancer is significant in explain the Ras independence of p110d.p110d has a view of observations in cell culture. Unlike wild-type unique accumulation of basic residues in its C2 domain, p110a, which lacks oncogenic activity when expressed in which probably mediate a direct interaction with the cell primary fibroblasts, the wild-type non-a isoforms are membrane. Mutating these basic residues results in an oncogenic. This surprising activity of p110b, g and d was inactive protein. The dependence of p110b and p110g on first documented in avian cells (Kang et al., 2006), but Ras is also seen in their sensitivity to inhibitors of the has now been observed in rodent cells as well (Ueno MAP kinase (mitogen-activated protein kinase) path- and Vogt, unpublished). The absence of cancer-specific way. The Raf inhibitor BAY43-9006 and the MEK1/2 mutations in the non-a isoforms may therefore reflect an inhibitor U0126 interfere with oncogenicity and signal- inherent oncogenic potential that can be activated by ing of p110b and p110g (Figure 6b). differential expression in the absence of mutation. A study of cells transformed by p110 isoforms has revealed striking isoform-specific activities that group p110b and p110g together, placing them apart from Evolving small molecule inhibitors of p110 p110a and p110d (Figure 5; Denley et al., 2008). In PI3K-transformed cells, wild-type p110d signals consti- The distinctive properties of the p110 isoforms lead to tutively as does the H1047R mutant of p110a. Trans- the question of small molecule inhibitors and their formation by p110b and p110g does not result in specificity. The standard PI3K inhibitors for experi- constitutive downstream signaling. This deficiency can mental work, wortmannin and LY294002, are not be remedied by the addition of a myristylation signal to isoform specific. However, there are several inhibitors p110b and p110g, which also results in an enhancement that show significant selectivity for one of the isoforms of oncogenic activity (Figure 5). Additional criteria also (Denley et al., 2008; Figure 7). However, they are ATP- show similarities between p110b and p110g: oncogeni- competitive inhibitors, and because ATP-binding pock- city and signaling of these isoforms require interactions ets of different kinases are structurally similar, such with Ras. The disabling mutation of the RBD K227E inhibitors usually show activities against several kinases. eliminates transforming and signaling activities of p110b Of the ones shown in the figure, PI 103 also inhibits and p110g. In contrast, the activities of p110a H1047R TOR, and TGX221 is effective against class III PI3K. and of wild-type p110d are not affected by this mutation All four p110 isoforms are promising targets for small in the RBD. Again, myristylation of p110b and p110g molecule inhibitors, each isoform is linked to a specific can substitute for Ras binding, restoring oncogenic and set of clinical indications. The identification and signaling potential (Figure 6a). This observation sug- development of such inhibitors with drug-like properties gests that an essential function of Ras in PI3K signaling and therapeutic potential has proceeded at a rapid pace in recent years (for example, see Knight et al., 2006; Knight and Shokat, 2007; Raynaud et al., 2007; Yaguchi et al., 2006; Patent 2007-EP4501; Patent 2007-US9866; Patent 2006-GB4554; Patent 2007-US18057 and Patent 2007-EP50618). The success of these efforts and the initiation of clinical trials will require a careful examination of side effects, important especially in long-term use. For p110a, the cancer-specific mutations offer a solution to this potential problem: the identification

Isoform-selective inhibitors of class I p110

IC50 (µM) for oncogenic transformation in cell Selectivity culture. Inhibitor for α-H1047R wt-β wt-γ wt-δ

PI 103 α 0.01 >1 >5 >1

TGX-221 β >1 0.035 >0.5 0.3

AS 604850 γ >10 9 1.2 >20 Figure 6 (a) Loss of Ras binding inactivates wild-type p110b and IC87114 δ >10 8 >10 0.6 p110g, but not p110aH1047R and p110d. A myristylation signal can substitute for Ras binding in p110b and p110g, suggesting that Ras functions as membrane anchor. (b) The dependence on Ras is Figure 7 Isoform-selective inhibitors of PI3K. The IC50 values also reﬂected by the sensitivity of p110b and p110g to inhibitors of were determined by measuring oncogenic activity in cell culture the MAP kinase (mitogen-activated protein kinase) pathway. (Denley et al., 2008). PI3K, phosphoinositide 3-kinase.

Oncogene PI3K in oncogenic cellular transformation L Zhao and PK Vogt 5493 and development of mutant-speciﬁc inhibitors. These Acknowledgements could interfere with the oncogenic versions of p110a and leave the important normal functions of wild-- This work was supported by grants from the National Cancer Institute and by The Stein Foundation. Dr Li Zhao is the type p110a untouched. The genetic and biochemical recipient of a postdoctoral fellowship from the National data on the p110a mutants suggest that mutant- Cancer Institute (F32CA130304). This is manuscript no. speciﬁcity could be attainable. A crystal structure of 19561-MEM from The Scripps Research Institute. We thank the mutant proteins would provide decisive guidance in Dr Jonathan Hart and Dr Marco Gymnopoulos for thought- this effort. ful discussions and critical information.

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