A phase I, pharmacokinetic and pharmacodynamic study of GSK2256098, a focal adhesion kinase inhibitor, in patients with advanced solid tumors.

BACKGROUND: Focal adhesion kinase (FAK) is important in cancer growth, survival, invasion, and migration. The purpose of this study was to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), and pharmacodynamics (PD) of the FAK inhibitor, GSK2256098, in cancer patients. PATIENTS AND METHODS: The dose of GSK2256098 was escalated, in cohorts of patients with advanced cancer, from 80 to 1500 mg, oral twice daily (BID), until the MTD was determined. Serial blood samples were obtained from all patients, and the PK was determined. Paired tumor biopsies were obtained in select patients, and the level of phospho-FAK (pFAK) was determined. RESULTS: Sixty-two patients (39 males, 23 females; median age 61 y.o., range 21-84) received GSK2256098. Dose-limiting toxicities of grade 2 proteinuria (1000 mg BID), grade 2 fatigue, nausea, vomiting (1250 mg BID), and grade 3 asthenia and grade 2 fatigue (1500 mg BID) were reported with the MTD identified as 1000 mg BID. The most frequent adverse events (AEs) were nausea (76%), diarrhea (65%), vomiting (58%), and decreased appetite (47%) with the majority of AEs being grades 1-2. The PK was generally dose proportional with a geometric mean elimination half-life range of 4-9 h. At the 750, 1000, and 1500 mg BID dose levels evaluated, the pFAK, Y397 autophosphorylation site, was reduced by ∼80% from baseline. Minor responses were observed in a patient with melanoma (-26%) and three patients with mesothelioma (-13%, -15%, and -17%). In the 29 patients with recurrent mesothelioma, the median progression-free survival was 12 weeks with 95% CI 9.1, 23.4 weeks (23.4 weeks merlin negative, n = 14; 11.4 weeks merlin positive, n = 9; 10.9 weeks merlin status unknown, n = 6). CONCLUSIONS: GSK2256098 has an acceptable safety profile, has evidence of target engagement at doses at or below the MTD, and has clinical activity in patients with mesothelioma, particularly those with merlin loss.

Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization.

The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub((n)) is qualitatively similar to p53-Ub((n)) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.

Thermodynamics of p53 binding to hdm2(1-126): effects of phosphorylation and p53 peptide length.

Upon exposure to DNA-damaging agents, the p53 tumor suppressor protein is stabilized and activated, leading to cell cycle arrest, DNA repair, or apoptosis. One of the major factors that regulates the level and the transcriptional activity of p53 is the hdm2 oncoprotein. hdm2 binds to the N-terminal transactivation domain of p53 to block the transcriptional activity of p53 directly. hdm2 also functions as the E3 ligase that ubiquitinates p53 for proteasome degradation. Fluorescence anisotropy was employed to measure directly the binding of hdm2(1-126) to a p53 N-terminal peptide labeled with Oregon Green (an analogue of fluorescein). Phosphorylation of Ser15 and Ser2O did not affect the binding of the p53 peptide to hdm2. Thrl8 phosphorylation, on the other hand, reduced the binding by at least 20-fold. This suggests that phosphorylation of Thr18 could be a regulatory mechanism that disrupts the hdm2-p53 complex, thus activating p53 in response to DNA damage. The effect of p53 peptide length on binding to hdm2 was also measured quantitatively. Interestingly, p53(18-26) exhibits 10-fold higher affinity to hdm2 than do longer peptides (20- or 35-mer). This result may reflect a strong entropic barrier to binding for the longer peptides.

SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.

NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536

Constitutive cellular expression of PI 3-kinase is distinct from transient expression.

The discovery that the PTEN tumor suppressor encodes a phosphoinositide 3-phosphatase has raised interest in the effects of constitutive activation of PI 3-kinase. To gain insight into PI 3-kinase function, we have stably expressed a myristoylated form of the catalytic subunit p110alpha (myr-p110) in cells. The myr-p110 associated with the endogenous p85 regulatory subunit and retained lipid and protein kinase activity. Stable lines expressing myr-p110 had 2- to 4-fold more PI 3-kinase activity than controls. Expression of myr-p110 altered cellular morphology and increased the saturation density in culture. These clones were morphologically transformed but Akt and pp70(s6k) were not constitutively activated in contrast to transient assays and from tumor cell lines deficient in PTEN. In addition, the ability of PDGF to induce activation of Akt and pp70(s6k) was diminished. Therefore, expression of a myristoylated PI 3-kinase in murine fibroblasts induces a morphological transformation of the cells.

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate.

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).

Platelet-derived growth factor stimulation of monocyte chemoattractant protein-1 gene expression is mediated by transient activation of the phosphoinositide 3-kinase signal transduction pathway.

Platelet-derived growth factor (PDGF) stimulates transcription of an immediate-early gene set in Balb/c 3T3 cells. One cohort of these genes, typified by c-fos, is induced within minutes following activation of PDGF receptors. A second cohort responds to PDGF only after a significant time delay, although induction is still a primary response to receptor activation as shown by "superinduction" in the presence of the protein synthesis inhibitor cycloheximide. PDGF-receptor activated signaling pathways for the "slow" immediate-early genes are poorly resolved. Using gain-of-function mutations together with small molecule inhibitors of kinase activity, we show that activation of PI 3-kinase is both necessary and sufficient for the induction of the prototype slow immediate-early gene, monocyte chemoattractant-1 (MCP-1). Following activation of PDGF receptors, MCP-1 mRNA does not begin to accumulate for at least 90 min. However, only a brief (10 min) interval of PI 3-kinase activity is required to trigger this delayed response. The serine/threonine protein kinase, Akt/PKB, likely functions as a downstream affector of PI 3-kinase for this induction.

Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase.

The avian sarcoma virus 16 (ASV 16) is a retrovirus that induces hemangiosarcomas in chickens. Analysis of the ASV 16 genome revealed that it encodes an oncogene that is derived from the cellular gene for the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase). The gene is referred to as v-p3k, and like its cellular counterpart c-p3k, it is a potent transforming gene in cultured chicken embryo fibroblasts (CEFs). The products of the viral and cellular p3k genes have PI 3-kinase activity. CEFs transformed with either gene showed elevated levels of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate and activation of Akt kinase.

Platelet-derived growth factor-induced formation of tensin and phosphoinositide 3-kinase complexes.

Tensin is an SH2 domain-containing cytoskeletal protein that binds to and caps actin filaments. Investigation of signal transduction mechanisms associated with tensin revealed the presence of phosphoinositide 3-kinase (PI 3-kinase) activity in tensin immunoprecipitates from platelet-derived growth factor-treated cells. Association of PI 3-kinase activity with tensin was transitory, and the amount of activity was approximately 1% of the total PI 3-kinase activity found in anti-phosphotyrosine (anti-pY) immunoprecipitates. In vitro, PI 3-kinase activity associated with the SH2 domain of tensin in a platelet-derived growth factor-dependent manner. The optimal phosphopeptide binding specificity of the SH2 domain of tensin was determined to be phospho-Y (E or D), N, (I, V, or F). Synthetic phosphopeptides containing the sequence YENI could specifically block the association of PI 3-kinase activity with tensin in a dose-dependent manner. These results suggest that PI 3-kinase interacts with the cytoskeleton via the SH2 domain of tensin and may play an important role in platelet-derived growth factor-induced cytoskeletal reorganization that is concomitant with cell migration and proliferation.

Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A.

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex.

Direct association of Grb2 with the p85 subunit of phosphatidylinositol 3-kinase.

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play a key role in growth factor signaling pathways, although its signaling mechanism has not been fully elucidated. Using the yeast interaction trap system, we have identified Grb2 as a PI 3-kinase interacting protein. Our experiments demonstrate that p85, the regulatory subunit of PI 3-kinase, interacts with Grb2 in vivo, and this interaction is independent of growth factor stimulation. The direct association between Grb2 and p85 was reconstituted in vitro with glutathione S-transferase fusion proteins. Domain analyses and peptide competition indicate that the association is mediated by the SH3 domains of Grb2 and the proline-rich motifs of p85 and that only one SH3 domain is required for minimal binding. The interaction does not displace the catalytic subunit of PI 3-kinase but is exclusive of Sos. Signaling through PI 3-kinase, therefore, may involve the ubiquitous adapter Grb2, which serves as a convergence point for multiple pathways.

Polyoma middle tumor antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr) motif in middle tumor antigen.

Polyomavirus middle tumor antigen (MT) transforms a large number of cell types by binding to and modulating the activities of cellular proteins. Previous genetic analysis defined in MT an independent motif, NPTY (Asn-Pro-Thr-Tyr), required for transformation. This report demonstrates that NPTY is required for interaction between MT and SHC protein, a Src homology 2 (SH2)-containing protooncogene product implicated in activating Ras via association with GRB2 protein. SHC is phosphorylated on tyrosine and associates with GRB2 in MT-transformed cells. These effects require an intact NPTY motif in MT. SHC immunoprecipitates from MT-transformed cells possess kinase activity that phosphorylates not only SHC and MT but also the 85-kDa subunit of phosphatidylinositol 3-kinase. This result suggests that a complex exists that contains, at a minimum, MT, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and SHC.

Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit.

Tyrosine-phosphorylated peptides based on the regions of polyoma virus middle t antigen and the platelet-derived growth factor receptor that bind phosphoinositide 3-kinase are shown to activate this enzyme 2-3-fold in vitro. The concentrations of the peptides required to activate the enzyme are at least 10-1000-fold higher than the dissociation constants of these peptides for the individual SH2 domains of the 85-kDa subunit (KD < 100 nM). Doubly phosphorylated peptides are more effective than singly phosphorylated peptides. The results suggest that a fraction of the cellular phosphoinositide 3-kinase has SH2 domains with relatively low affinity for phosphopeptides and that binding of phosphopeptides to these enzymes causes activation. Thus, SH2 domains may be involved not only in recruiting the enzyme but also in regulating activity.

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.

Inhibition of SH2 domain/phosphoprotein association by a nonhydrolyzable phosphonopeptide.

Using the association between the pp60c-src/polyoma virus middle T antigen (mT) complex and phosphatidylinositol 3'-kinase (PI 3-kinase) as a prototype for phosphoprotein-SH2 domain interactions, we tested whether a nonhydrolyzable phosphonopeptide would inhibit association. (Phosphonomethyl)-phenylalanine (Pmp) is a nonnatural analogue of phosphotyrosine in which the > C-O-PO3H2 moiety is replaced by > C-CH2-PO3H2. We synthesized a 13 amino acid phosphonopeptide (mT-Pmp315), a related phosphopeptide (mT-pY315), and an unmodified sequence (mT-Y315), all corresponding to the pp60c-src-phosphorylated site of the mT which is within a YMXM motif common to proteins that bind to and activate PI 3-kinase. Only the phosphonopeptide persistently blocked the in vitro association of the baculovirus-expressed pp60c-src/mT complex with cytosolic PI 3-kinase activity. Sustained inhibition of association by the phosphopeptide required the additional presence of vanadate, a potent protein tyrosine phosphatase (PTPase) inhibitor. The phosphopeptide and L-phosphonopeptide bound tightly (KD approximately 10-20 nM) and specifically to isolated SH2 domains of PI 3-kinase p85, demonstrating that the mechanism of inhibited association is competitive binding to PI 3-kinase SH2 domains. We conclude that the appropriate phosphonopeptide sequence inhibits the interaction between a tyrosine-phosphorylated protein and a cognate SH2 domain-containing protein and is resistant to the actions of PTPases. Proteolytically stable phosphonopeptide derivatives should be useful inhibitors of protein-protein interactions when introduced into cells and may provide a basis for the rational design of a new class of chemotherapeutic agent.

Polyoma virus middle T antigen-pp60c-src complex associates with purified phosphatidylinositol 3-kinase in vitro.

Reconstitution of the polyoma virus middle T antigen (mT)-pp60-src complex and phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been accomplished in vitro with immunopurified baculovirus-expressed mT-pp60c-src and PtdIns 3-kinase purified from rat liver. Both the 110- and 85-kDa subunits of the PtdIns 3-kinase associated with the mT-pp60c-src complex. The association of PtdIns 3-kinase with the mT-pp60c-src complex was dependent on the protein-tyrosine kinase activity of pp60c-src as a kinase-inactive mutant (pp60(295c-src)) still complexed with mT, but the mT-pp60(295c-src)) complex was unable to bind PtdIns 3-kinase. The mT-pp60c-src complex phosphorylated both subunits of PtdIns 3-kinase on tyrosine residues. The immunopurified mT-pp60c-src complex also associated with PtdIns 3-kinase activity from whole cell lysates, and this association was dependent upon the protein-tyrosine kinase activity of pp60c-src. Comparison of 35S-labeled proteins from whole cell lysates which associated with immunopurified mT-pp60c-src and mT-pp60(295c-src) revealed proteins of 110 and 85 kDa as the major peptides dependent on protein-tyrosine kinase activity for association with the complex. In addition, a synthetic phosphopeptide (13-mer) containing sequences conserved between the major tyrosine phosphorylation site of murine polyoma virus mT, hamster polyoma virus mT, and the insulin receptor substrate (IRS-1) specifically blocked the association of the 85- and 110-kDa polypeptides with the mT-pp60c-src complex. The ability to block the association was dependent on the tyrosine phosphorylation of the peptide. Association of PtdIns 3-kinase activity was blocked concurrently. This is the first demonstration that the 110-kDa subunit of PtdIns 3-kinase can associate with mT-pp60c-src. This association in vitro is a step toward understanding protein-protein interactions important in the signal transduction pathway of oncogenic proteins.

Novel polyphosphoinositides in cell growth and activation.

The "conventional" polyphosphoinositide pathway is important for the transmission and amplification of signals across the cell membrane. Ligand-induced activation of phospholipase C results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the well-characterized second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, three novel polyphosphoinositides have been implicated as important signaling molecules for cell proliferation and activation. These lipids are phosphorylated in the D-3 position of the inositol ring and appear to represent branch points from the conventional polyphosphoinositide pathway.