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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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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]
[hide abstract]
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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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]
[hide abstract]
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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Anita Lee, Cloud Paweletz,
Roy Pollock,
Robert Settlage,
Jonathan Cruz,
J Secrist,
Thomas Miller,
Matthew Stanton,
Astrid Kral,
Nicole Ozerova,
Fanyu Meng,
Nathan Yates,
Victoria Richon,
Ronald Hendrickson
[show abstract]
[hide abstract]
ABSTRACT: Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAC1 and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1-selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors.
Journal of Proteome Research 11/2008; · 5.11 Impact Factor
