Zhongzhong Chen

An Atlas of the Human Kinome Reveals the Mutational Landscape Underlying Dysregulated Phosphorylation Cascades in Cancer

Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular subnetworks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks that drive tumorigenesis. Cancer Res; 76(7); 1733-45. ©2016 AACR.

472 Do we hit the target? Phospho-reactome measurements determine efficacy of targeted therapies

negative regulator of CD133. In addition, p53 directly binds on the CD133 promoter and suppresses its promoter activity, showing that CD133 could be a novel target gene of p53. Furthermore, CD133 is necessary for tumor formation and growth of cancer cells, and these functions of CD133 are obstructed by overexpression of p53. Conclusions: These results suggest that p53 is a key modulator for CD133 expression in CSC, and provide novel perspective on targeting CSCs for the cancer therapy.

Abstract P4-05-02: Phospho-reactome measurements reveal heterogenic kinase signatures

Background: Treating cancer increasingly relies on targeting kinases, because their oncogenic activity drive tumorigenesis. Discovering which active mechanisms of disease progression can be efficiently targeted, and knowing whether kinase networks circumvent therapeutic interventions, are challenges researchers and clinicians face. Surprisingly however, measuring the phosphorylating activity of kinases, and potentially monitoring the functionality of the entire human phospho-reactome at once, remains largely unexplored. We developed a semi-high throughput assay to monitor the phospho-catalytic activity of kinase enzymes, using their biological targets as phospho-sensors. We successfully used this assay to identify oncogenic phospho-signatures prevalent in breast cancer, and can be used to establish drug-sensitivity profiles in models of kinase-targeted therapies. Methods: We first defined how to computationally build a library of peptide sensors established from confirmed kinase substrates’ phosphorylation sites. Precisely, we used computational methods to create a unique phospho-repertoire cataloguing 3,408 peptide sequences established from validated human proteins’ phosphorylation sites, curated from 38 public databases. Second, we experimentally used these biologically relevant probes in multiplex assays to quantify the catalytic state of kinases. Specifically, a kinome-representative 242-peptide set was developed into an ATP-consumption screen to identify the activity signatures of EGFR, MAPK, AKT, ABL and SRC family kinases, and explore 642 kinase/substrate nodes. Next, we described analysis methods to derive phospho-signatures from semi-high throughput ATP-consumption measurements. We validated the assay using isogenic culture model of basal-like breast cancer (HMT-3522 S1 and T4-2), and cell lines harboring EGFR/HER2-oncogenic alterations such as MDA-MB-231, MCF7 or T47D. Results: The differential phosphorylation activity of 25 recombinant, active kinase enzymes was successfully captured. In cancer cell extracts, hyper-activated EGFR, ERK, MEK, AKT, and SRC kinases originally identified by immuno-detection were reliably and specifically detectable using the peptide-based kinase-activity assay. The phospho-sensing assay revealed the heterogeneity of active kinase signaling circuits among different breast cancer cells. Conclusion: This unique strategy and resources allow to comprehensively measure the catalytic activity of multitude kinases at once, representing a new molecular dimension to characterize biological samples. We will use such new phospho-reactome profiling system to determine the efficacy of new combinatorial therapies, and define how chemotherapeutic interventions lead to the reprogramming of phospho-circuits. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-02.

Kinase activity signature of breast cancer using peptide phosphosensors.

27 Background: Improper control of phosphorylation networks represents one of the most relevant regulator and biomarkers of cell dysfunctions leading to cancer. Surprisingly however, measuring the phosphorylation activity of kinases, and potentially monitoring the functionality of the entire kinome at once, remains largely unexplored. Specifically, a highly convenient tool for clinical applications or research purposes would be the advent of a microarray-like chip to comprehensively map phosphorylation networks. While developing such a kinase-sensing platform, we had to establish a library of probes capable of reporting on the catalytic activity of kinases. We present here strategies and results supporting such methodology. METHODS The devised kinase activity-sensing system we are currently exploiting, relies on peptide probes that act as beacons to evaluate enzymes functionality, which emit distinctive signals before and after (de-)phosphorylation. As presented below, computational and molecular biology methods were used to create and test such (phospho-)peptide sensors. RESULTS First, in order to use baits relevant to human cancer etiology, we created a protein and peptide repertoire from thousands of publications and >30 public databases computationally curated, filtered and merged. Such library catalogues 6173 distinctive kinase-substrate active nodes and compiles 2702 unique biological kinase peptide targets that all represent latent nano-sensors usable to comprehensively track kinase signaling networks. Second, real-time kinetics, kinase inhibitor and dilution experiments using 5 recombinant enzymes and cell extracts in ATP consumption assays demonstrated peptide 50 probes can directly and specifically assess the functionality of kinases. A panel of 10 peptides shows that the increased presence of activated Src kinase protein in breast cancer cell extracts is related to a significantly elevated kinase activity signature. CONCLUSIONS Biological peptides can serve as phospho-sensing probes to evaluate the activity of kinases. Our efforts will now focus on expending this approach into a functional kinomic-screening platform to map phosphorylation signatures relevant for breast cancer.

Abstract C89: Functional kinomic-screening platform to explore oncogenic kinase networks.

Treating cancer increasingly relies on targeting kinases, because their oncogenic activity harnesses myriad signalling cascades that drive tumorigenesis. Surprisingly however, measuring the phosphorylation activity of kinases, and potentially monitoring the functionality of the entire kinome at once to identify molecular signatures causing cancers and guide therapeutic interventions, remains largely unexplored. Specifically, a highly convenient tool for clinical applications or research purposes would be the advent of a microarray-like chip to map phosphorylation networks. We undertook to develop such a kinase-sensing platform, and present here unique strategies, technical advances and data -including the detection of oncogenic Src kinase activity in biological samples-supporting such potential breakthrough. The devised kinase activity-sensing system relies on peptide probes that act as beacons to evaluate enzymes functionality, and emit distinctive optical signals before and after (de-)phosphorylation. The following key steps have been achieved. First, in order to use baits relevant to human (patho-)physiology, we created a protein and peptide repertoire cataloguing 6173 distinctive kinase-substrate active nodes and compiling 2702 unique biological kinase peptide targets that all represent latent nano-sensors usable to comprehensively track kinase signaling networks. Second, we established that our label-free detection approach (namely Raman scattering of probes monitored using surface-enhanced Raman spectroscopy (SERS)) is a functional system that reliably and robustly identifies the phosphorylation state of peptide probes. Specifically, peptide-tethered arrays demonstrated high sensitivity, repeatability, and specificity to discriminate between presence or absence of phosphate group on tyrosine-containing peptides densely printed on biocompatible chips. Third, real-time kinetics and kinase inhibitor experiments using biochemical samples and biological extracts from mammalian cells demonstrated that our approach could directly assess the functionality of kinases. Finally, in an attempt to identify hyperactive Src kinase in tumor cells, our assay discerned normal from oncogenic Src-mutated cells (wild type vs oncogenic v-Src vs Src knock-down). Together, our strategy allows for highly sensitive, specific, rapid, one-step, label-free, multiplex capture and measurement of phosphorylation events. Our efforts will now focus on expending this approach into a fully functional kinomic-screening platform to explore oncogenic kinase networks in human cancers. We will translate such device into the clinic, and map phosphorylation signatures that cause mammary gland malignancies to eventually guide therapeutic interventions that best match breast cancer patients9 disease characteristics and predicted responses to (chemo-)therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C89.