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Katharine Ellwood-Yen,
Heike Keilhack,
Kaiko Kunii,
Brian Dolinski,
Yamicia Connor,
Kun Hu, Kumiko Nagashima,
Erin O'Hare,
Yusuf Erkul,
Alessandra Di Bacco, [......],
Ekaterina V Bobkova,
Shailaja Kasibhatla,
Roderick T Bronson,
Martin L Scott,
Giulio Draetta,
Victoria Richon,
Nancy Kohl,
Peter Blume-Jensen,
Jannik N Andersen,
Manfred Kraus
[show abstract]
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ABSTRACT: PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.
Cancer Research 04/2011; 71(8):3052-65. · 7.86 Impact Factor
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Kumiko Nagashima,
Stuart D. Shumway,
Sriram Sathyanarayanan,
Albert H. Chen,
Brian Dolinski,
Youyuan Xu,
Heike Keilhack,
Thi Nguyen,
Maciej Wiznerowicz,
Lixia Li, [......],
Sujal Deshmukh,
Zangwei Xu,
Uwe Mueller,
Alexander A. Szewczak,
Bo-Sheng Pan,
Victoria Richon,
Roy Pollock,
Peter Blume-Jensen,
Alan Northrup,
Jannik N. Andersen
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 02/2011; 286(8):6433-6448. · 4.77 Impact Factor
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Kumiko Nagashima,
Stuart D Shumway,
Sriram Sathyanarayanan,
Albert H Chen,
Brian Dolinski,
Youyuan Xu,
Heike Keilhack,
Thi Nguyen,
Maciej Wiznerowicz,
Lixia Li, [......],
Sujal Deshmukh,
Zangwei Xu,
Uwe Mueller,
Alexander A Szewczak,
Bo-Sheng Pan,
Victoria Richon,
Roy Pollock,
Peter Blume-Jensen,
Alan Northrup,
Jannik N Andersen
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 02/2011; 286(8):6433-48. · 4.77 Impact Factor
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Cloud P Paweletz,
Jannik N Andersen,
Roy Pollock, Kumiko Nagashima,
Mansuo L Hayashi,
Shangshuan U Yu,
Hongbo Guo,
Ekaterina V Bobkova,
Zangwei Xu,
Alan Northrup,
Peter Blume-Jensen,
Ronald C Hendrickson,
An Chi
[show abstract]
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ABSTRACT: 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-PDK1(Ser410) and p-PDK1(Thr513) 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-PDK1(Ser241)), 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.
PLoS ONE 01/2011; 6(10):e26459. · 4.09 Impact Factor
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Andrey Loboda,
Michael Nebozhyn,
Rich Klinghoffer,
Jason Frazier,
Michael Chastain,
William Arthur,
Brian Roberts,
Theresa Zhang,
Melissa Chenard,
Brian Haines,
Jannik Andersen, Kumiko Nagashima,
Cloud Paweletz,
Bethany Lynch,
Igor Feldman,
Hongyue Dai,
Pearl Huang,
James Watters
[show abstract]
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ABSTRACT: Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence.
We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets.
The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer.
These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.
BMC Medical Genomics 01/2010; 3:26. · 3.69 Impact Factor
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[show abstract]
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ABSTRACT: The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of approximately 21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein.
Journal of Biomolecular Screening 10/2009; 14(10):1257-62. · 2.05 Impact Factor
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Kazuishi Kubota,
Rana Anjum,
Yonghao Yu,
Ryan C Kunz,
Jannik N Andersen,
Manfred Kraus,
Heike Keilhack, Kumiko Nagashima,
Stefan Krauss,
Cloud Paweletz,
Ronald C Hendrickson,
Adam S Feldman,
Chin-Lee Wu,
John Rush,
Judit Villén,
Steven P Gygi
[show abstract]
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ABSTRACT: Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.
Nature Biotechnology 10/2009; 27(10):933-40. · 29.50 Impact Factor
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Kumiko Nagashima,
Vito G Sasseville,
Danyi Wen,
Andrew Bielecki,
Hua Yang,
Chris Simpson,
Ethan Grant,
Michael Hepperle,
Gerry Harriman,
Bruce Jaffee,
Tim Ocain,
Yajun Xu,
Christopher C Fraser
[show abstract]
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ABSTRACT: The transcription factor NF-kappaB plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKKbeta, an Ikappa-B kinase that regulates NF-kappaB. IKKbeta and NF-kappaB are required in vivo for prevention of TNFalpha-mediated apoptosis. ML120B sensitized mouse bone marrow progenitors and granulocytes, but not mature B cells to TNFalpha killing in vitro, and induced apoptosis in vivo in the bone marrow and spleen within 6 hours of a single oral dose. In vivo inhibition of IKKbeta with ML120B resulted in depletion of thymocytes and B cells in all stages of development in the bone marrow but did not deplete granulocytes. TNF receptor-deficient mouse thymocytes and B cells were resistant to ML120B-induced depletion in vivo. Surprisingly, surviving bone marrow granulocytes expressed TNFR1 and TNFR2 after dosing in vivo with ML120B. Our results show that inhibition of IKKbeta with a small molecule in vivo leads to rapid TNF-dependent depletion of T and B cells. This observation has several implications for potential use of IKKbeta inhibitors for the treatment of inflammatory disease and cancer.
Blood 07/2006; 107(11):4266-73. · 9.90 Impact Factor
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[show abstract]
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ABSTRACT: Rip2 (Rick, Cardiak, CCK2, and CARD3) is a serine/threonine kinase containing a caspase recruitment domain (CARD) at the C terminus. Previous reports have shown that Rip2 is involved in multiple receptor signaling pathways that are important for innate and adaptive immune responses. However, it is not known whether Rip2 kinase activity is required for its function. Here we confirm that Rip2 participates in lipopolysaccharide (LPS)/Toll-like receptor (TLR4) signaling and demonstrate that its kinase activity is not required. Upon LPS stimulation, Rip2 was transiently recruited to the TLR4 receptor complex and associated with key TLR signaling mediators IRAK1 and TRAF6. Furthermore, Rip2 kinase activity was induced by LPS treatment. These data indicate that Rip2 is directly involved in the LPS/TLR4 signaling. Whereas macrophages from Rip2-deficient mice showed impaired NF-kappaB and p38 mitogen-activated protein kinase activation and reduced cytokine production in response to LPS stimulation, LPS signaling was intact in macrophages from mice that express Rip2 kinase-dead mutant. These results demonstrate that Rip2-mediated LPS signaling is independent of its kinase activity. Our findings strongly suggest that Rip2 functions as an adaptor molecule in transducing signals from immune receptors.
Journal of Biological Chemistry 05/2005; 280(16):16278-83. · 4.77 Impact Factor
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ABSTRACT: Glycoprotein VI is a type I membrane protein identified as a key platelet receptor for collagen. In vitro binding of the GPVI receptor with collagen leads to activation and ultimately to aggregation of platelets. In vivo, GPVI-collagen interactions could cause formation of occlusive thrombi within vessels with damaged endothelial barriers. GPVI antagonists are therefore important therapeutics in patients suffering from collagen-mediated ischemic disorders such as myocardial infarction or stroke. Polyclonal antibodies to GPVI prepared from one patient serum have previously been described. However, only their monovalent Fab fragments, incapable of receptor crosslinking, were found to protect platelets from collagen-mediated aggregation. Here we describe GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies. By selecting phage on GPVI-expressing U937 cells, we isolated five specific antibodies - A4, A9, A10, C3 and C9. Of the set A10 and C3 specifically blocked GPVI binding to collagen-rich adventitial layers in aorta sections. The higher affinity antibody A10 inhibited binding of snake-venom convulxin to GPVI. It also specifically protected human platelets from collagen-induced aggregation in vitro. A10-bound platelets could still be activated by ADP or thrombin suggesting that this human scFv may represent an original anti-platelet agent for the treatment of collagen-mediated thrombotic diseases.
Human antibodies 02/2002; 11(3):97-105.
