Peter Blume-Jensen

Oncogenic kinase signalling

Protein-tyrosine kinases (PTKs) are important regulators of intracellular signal-transduction pathways mediating development and multicellular communication in metazoans. Their activity is normally tightly controlled and regulated. Perturbation of PTK signalling by mutations and other genetic alterations results in deregulated kinase activity and malignant transformation. The lipid kinase phosphoinositide 3-OH kinase (PI(3)K) and some of its downstream targets, such as the protein-serine/threonine kinases Akt and p70 S6 kinase (p70S6K), are crucial effectors in oncogenic PTK signalling. This review emphasizes how oncogenic conversion of protein kinases results from perturbation of the normal autoinhibitory constraints on kinase activity and provides an update on our knowledge about the role of deregulated PI(3)K/Akt and mammalian target of rapamycin/p70S6K signalling in human malignancies.

THE KIT RECEPTOR PROMOTES CELL SURVIVAL VIA ACTIVATION OF PI 3-KINASE AND SUBSEQUENT AKT-MEDIATED PHOSPHORYLATION OF BAD ON SER136

The c-kit-encoded receptor protein tyrosine kinase for stem cell factor (Kit/SCF-R) is essential for the development of cells within the hematopoietic, melanogenic and gametogenic lineages. SCF stimulation induces activation of phosphatidylinositol (PI) 3-kinase, which is required for SCF-induced mitogenesis and cell survival, and for activation of the serine/threonine, we found that, in response to SCF Akt became activated and mediated phosphorylation of Bad, a pro-apoptotic molecule, in a PI-3-kinase-dependent manner. Phosphorylation of Bad was restricted to Ser112 and Ser136 in vivo, but only the Akt phosphorylation sit Ser136 was essential for SCF-promoted cell survival. Furthermore, Bad and Akt interacted and colocalized in intact cells. A Kit/SCF-R gain-of-function mutant that has increased mitogenic and PI 3-kinase activation potential, due to the absence of the two protein kinase C negative feedback phosphorylation site, enhanced both Akt activation and Bad phosphorylation and also resulted in increased cell survival. Such a mechanism may account for how deregulated PI 3-kinase activity and naturally occurring gain-of-function point mutants of Kit/SCF-R lead to cellular transformation and fatal malignancies in man.

Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3′-kinase is essential for male fertility

The c-kit-encoded transmembrane tyrosine kinase receptor for stem cell factor (Kit/SCF-R) is required for normal haematopoiesis, melanogenesis and gametogenesis. However, the roles of individual Kit/SCF-R-induced signalling pathways in the control of developmental processes in the intact animal are completely unknown. To examine the function of SCF-induced phosphatidylinositol (PI) 3′-kinase activation in vivo, we employed the Cre-loxP system to mutate the codon for Tyr719, the PI 3′-kinase binding site in Kit/SCF-R, to Phe in the genome of mice by homologous recombination. Homozygous (Y719F/Y719F) mutant mice are viable. The mutation completely disrupted PI 3′-kinase binding to Kit/SCF-R and reduced SCF-induced PI 3′-kinase-dependent activation of Akt by 90%. The mutation induced a gender- and tissue-specific defect. Although there are no haematopoietic or pigmentation defects in homozygous mutant mice, males are sterile due to a block in spermatogenesis, with initially decreased proliferation and subsequent extensive apoptosis occurring at the spermatogonial stem-cell level. In contrast, female homozygotes are fully fertile. This is the first report so far demonstrating the role of an individual signalling pathway downstream of Kit/SCF-R in the intact animal. It provides the first in vivo model for male sterility caused by a discrete signalling pathway defect affecting early germ cells.

Epidermal Growth Factor-Induced Tumor Cell Invasion and Metastasis Initiated by Dephosphorylation and Downregulation of Focal Adhesion Kinase

ABSTRACT Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less affected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C

The product of the c‐kit proto‐oncogene, denoted Kit/SCF‐R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF‐R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF‐R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF‐induced tyrosine autophosphorylation of Kit/SCF‐R. In vitro studies showed that PKC phosphorylated the Kit/SCF‐R directly on serine residues and inhibited autophosphorylation of Kit/SCF‐R, as well as its kinase activity towards an exogenous substrate. The PKC‐induced phosphorylation did not affect Kit/SCF‐R ligand binding affinity. Inhibition of PKC led to increased SCF‐induced tyrosine autophosphorylation, as well as increased SCF‐induced mitogenicity. In contrast, PKC was necessary for SCF‐induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF‐R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells CHARACTERIZATION OF A SELECTIVE ALLOSTERIC KINASE INHIBITOR

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1–5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

Identification of direct target engagement biomarkers for kinase-targeted therapeutics.

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3′-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1Ser410 and p-PDK1Thr513 are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1Ser241), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.