Cloud P. Paweletz

Sensitive multiplexed analysis of kinase activities and activity-based kinase identification

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.

Isolation and Characterization of SATB2, a Novel AT-rich DNA Binding Protein Expressed in Development- and Cell-Specific Manner in the Rat Brain

AT-rich DNA elements play an important role in regulating cell-specific gene expression. One of the AT-rich DNA binding proteins, SATB1 is a novel type of transcription factor that regulates gene expression in the hematopoietic lineage through chromatin modification. Using DNA-affinity purification followed by mass spectrometry we identified and isolated a related protein, SATB2 from the developing rat cerebral cortex. SATB2 shows homology to SATB1 and the rat protein is practically identical to the mouse and human SATB2. Using competitive EMSA, we show that recombinant SATB2 protein binds with high affinity and specificity to AT-rich dsDNA. Using RT-PCR, Western analysis and immunohistochemistry we demonstrate that SATB2 expression is restricted to a subset of postmitotic, differentiating neurons in the rat neocortex at ages E16 and P4. We suggest that similar to its homologue SATB1, SATB2 is also involved in regulating gene expression through altering chromatin structure in differentiating cortical neurons.

De Novo Biosynthetic Profiling of High Abundance Proteins in Cystic Fibrosis Lung Epithelial Cells

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210–2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [35S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFκB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that ∼30% of the 51-member hybrid high abundance CF proteome interacts with the NFκB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Protein microarray platforms for clinical proteomics

Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2‐DE and/or MS need to be complemented by different kinds of technologies. The well‐known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody‐centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label‐free detection technologies, human genome‐scale numbers of analytes, and increases in analytic and bioinformatic speeds.

Application of an End-to-End Biomarker Discovery Platform to Identify Target Engagement Markers in Cerebrospinal Fluid by High Resolution Differential Mass Spectrometry

The rapid identification of protein biomarkers in biofluids is important to drug discovery and development. Here, we describe a general proteomic approach for the discovery and identification of proteins that exhibit a statistically significant difference in abundance in cerebrospinal fluid (CSF) before and after pharmacological intervention. This approach, differential mass spectrometry (dMS), is based on the analysis of full scan mass spectrometry data. The dMS workflow does not require complex mixing and pooling strategies, or isotope labeling techniques. Accordingly, clinical samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis in which each subject acts as its own control. As a proof of concept, we performed multifactorial dMS analyses on CSF samples drawn at 6 time points from n = 6 cisterna magna ported (CMP) rhesus monkeys treated with 2 potent gamma secretase inhibitors (GSI) or comparable vehicle in a 3-way crossover study that included a total of 108 individual CSF samples. Using analysis of variance and statistical filtering on the aligned and normalized LC-MS data sets, we detected 26 features that were significantly altered in CSF by drug treatment. Of those 26 features, which belong to 10 distinct isotopic distributions, 20 were identified by MS/MS as 7 peptides from CD99, a cell surface protein. Six features from the remaining 3 isotopic distributions were not identified. A subsequent analysis showed that the relative abundance of these 26 features showed the same temporal profile as the ELISA measured levels of CSF A beta 42 peptide, a known pharmacodynamic marker for gamma-secretase inhibition. These data demonstrate that dMS is a promising approach for the discovery, quantification, and identification of candidate target engagement biomarkers in CSF.

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.

High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer's Disease in Human Cerebrospinal Fluid.

Disease modifying treatments for Alzheimer’s disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.

Abstract PR3: Preclinical and molecular profiling data suggest LumB breast cancer as a potential indication for co-targeting IGF1R and mTOR with MK-0646 and MK-8669

There has been increasing interest in developing cancer therapies targeting PI3K pathway nodes. However, inhibition of a single node in PI3K pathway such as mTOR by rapamycin analogues results in compensatory activation of survival signaling pathway such as AKT and thereby limiting monotherapy activity. Therefore, in most tumor types, multipathway inhibition, guided by an in-depth understanding of feedback and crosstalk between pathways, may be required for tumor regression. It is well known that that mTOR inhibition upregulates pAKT via the S6K-IRS2 negative feedback loop. Inhibiting both mTOR and IGF1R may therefore ablate such feedback upregulation and lead to clinical response. However, it is unclear which tumor types will respond to the combination in the clinic. Here, we present preclinical and molecular profiling data that supports LumB breast cancer as a potential indication for MK-8669/MK-0646 (mTOR/IGF1 R inhibitor combination). When a panel of over 60 breast cancer cell lines was treated with MK-8669, ER+ cell lines and HER2+ cell lines were clearly more responsive than the triple negative ones. Meanwhile, the status of the MAPK pathway, as captured by a RAS gene expression signature, correlated with resistance to MK-8669 across the panel. Moreover, MK-8669 treatment unregulated multiple key nodes of the IGF1R pathway in the breast cell line panel, especially IRS2, whose upregulation correlated with the response to MK-8669. Furthermore, IGF1R, whose mRNA level correlates with the response to MK-0646 (IGF1 Ri) in multiple internal and external studies, is the highest in a subset of LumB tumors, suggesting a potential dependency on the IGF1R pathway in those tumors. Finally, a comparison between the gene expression profiles of breast cancer cell lines and tumors revealed that the ER+ breast cancer cell lines only represented the LumB, not LumA, subtype of human breast tumors. Taken together, these findings allow us to hypothesize that LumB breast cancer may be enriched in responders to the MK-0646/MK-8669 combination. In 2009, Merck initiated a phase I trial of MK-8669/MK-0646 in which clinical response was observed in LumB breast cancer patients. A phase II clinical trial has been initiated to test the hypothesis retrospectively. This talk is also presented as Poster A52. Citation Information: Clin Cancer Res 2010;16(14 Suppl):PR3.

Abstract 4151: Mutual feedback activation of PI3K and MAPK pathways support combining inhibitors of the two pathways to enhance efficacy

There has been increasing interest in developing cancer therapies targeting PI3K pathway nodes. Unfortunately, the number of tumor types responding to PI3K pathway inhibitors as monotherapy has been limited, since most cancer cells accumulate genetic alterations that result in the constitutive activation of a complex network of proliferation and survival signals. As such, the inhibition of a single survival/proliferation pathway or node within this broader network may be circumvented by constitutive activation (or feedback activation) of an alternative survival pathway, and thus be insufficient for inducing a clinical response. Therefore, in most tumor types, dual pathway inhibition, guided by an in-depth understanding of feedback and cross-talk between pathways, may be required to induce tumor regression. Here we present a nonclinical study exploring mutual feedback activation of MAPK and PI3K pathways. Inhibition of the PI3K pathway by MK-2206 (AKT inhibitor) or MK-8669 (mTOR inhibitor) resulted in feedback activation of MAPK pathway nodes in subsets of lung cell lines. Similarly, inhibition of the MAPK pathway by a MEK inhibitor resulted in feedback activation of the PI3K pathway. Notably, a lack of such feedback activation was associated with sensitivity to the mono inhibitors. The strength of the feedback activation correlated with changes of several receptor tyrosine kinases, suggesting them as a part of the feedback mechanism. Finally, combined inhibition of both MAPK and PI3K pathways synergistically inhibited the feedback activation of both pathways and led to improved in vitro efficacy. Taken together, our findings identify mutual feedback activation of the PI3K and MAPK pathways in response to specific inhibitors and underscore the importance of combined therapeutic approaches with inhibitors to both pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4151.

Abstract 4953: Identification of direct target engagement biomarkers for kinase drug discovery using quantitative mass spectrometry: PDK1 case study

The human genome encodes some 500 protein kinases and an estimated twenty percent of drugs currently in development target this enzyme class. Because kinase inhibitors frequently bind to the conserved ATP-binding pocket, in vitro kinase selectivity profiling of drug candidates are an integral part of the small-molecule drug discovery process. Likewise, the co-development of pharmacodynamic (PD) biomarkers is critical to drug development as they link drug-target inhibition in cells with biological activity. As such PD biomarkers are essential for the interpretation of in vivo PK/PD/efficacy studies, which ultimately inform medicinal chemistry. Here, we present a universal mass-spectrometry-based approached to discover kinase drug-target engagement biomarkers using the phosphoinositide dependent protein kinase 1 (PDK1) as an example. Western blot analysis using a commercial available phosphosite-specific PDK1 antibody (Ser241) revealed that classical, ATP-competitive PDK1 inhibitors do not dynamically impact PDK1 T-loop phosphorylation in cells, despite cell biochemical inhibition of phospho-AKT(Thr308), a bona fide PDK1 substrate phosphorylation site. However, because receptor tyrosine kinases (RTKs), lipid kinases and other pathways often signal via AKT, the monitoring of phospho-AKT as a PD biomarker for PDK1 is not sufficient for delineating the specificity of small-molecule inhibitors and ascertain the degree of PDK1 target engagement in cells. Here, we used stable isotope labeling by amino acids (SILAC) in cell culture combined with immunoprecipitation of PDK1 protein from cells for quantitative mass-spectrometry-based identification of phosphorylation sites modulated by PDK1 inhibitor treatment. We mapped 10 Serine/Threonine phosphorylation sites on PDK1 including several novel phosphorylation sites. Of these, two PDK1 phosphorylation sites (Ser410 and Thr513) were modulated by pharmacological PDK1 inhibition and the degree of dephosphorylation correlated with inhibitor potency in PDK1 enzymatic assays. By contrast, pharmacological inhibition of PDK1 did not quantitatively modulate PDK1 T-loop phosphorylation (Ser241) consistent with the initial western blot analysis. Altogether, our study provides proof-of-concept for identifying novel kinase target engagement PD biomarkers for PDK1, complementing pathway biomarkers such p-AKT and p-S6RP, which are modulated broadly by PI3K-pathway targeting agents including inhibitors of PI3K/mTOR and RTKs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4953.