A new multi-pathway kinase activity assay applied to compound library screening in cancer biology

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A new multi-pathway kinase activity assay applied to compound library screening in cancer biology Project Abstract Dysregulation of protein kinase signaling pathways is a hallmark of most human cancers. Although targeting kinases has been an effective therapeutic strategy in diverse cancer types, limited druggable targets, tumor heterogeneity and drug resistance remain problems in kinase-targeted cancer therapies. Finding new modulators of kinase activity in complex cancer models is therefore an urgent need, but current kinase activity measurement methods suffer from being low throughput and only provide surrogates for kinase activity. This proposal aims to apply the latest sample multiplexing technologies using Tandem Mass Tag (TMT) reagents and mass spectrometry to create a novel, multi-pathway, high-throughput, direct kinase activity readout assay and apply it to profile kinase activity for drug screening. An in vitro peptide phosphorylation assay provides a way to directly measure kinase activities for many pathways, while TMT increases throughput by sample multiplexing. First, I will select peptide substrates to represent specific kinase pathways (e.g., AKT, SRC, MAPK, CDK, etc.) and create a high-throughput in vitro kinase assay based on the TMT reagents. This assay will then be adapted to a 96-well plate format (Aim1). In Aim2, I will expand the assay to a three-dimensional (3D) cell culture model, profile and compare the kinase activities in response to 137 known kinase inhibitors in both standard two-dimensional (2D) and 3D cell culture models. In Aim3, I will perform high-throughput profiling of kinase activities across a library of 3200 compounds in both 2D and 3D ovarian cancer models using the 96-well plate platform for drug screening. Upon completion of these Aims, I will have developed and applied a highly optimized method for direct kinase activity measurement. This novel assay will result in the ability to quantitatively measure the activation state of dozens of signaling pathways directly from cellular lysates and, therefore, identify kinase modulators for future drug development. This proposal draws on my training in mass spectrometry-based method development and kinase and cancer biology. However, to ensure completion of the goals and successful transition to independence, I have a detailed training plan, under the guidance of my mentors Dr. Steven Gygi and Dr. Joan Brugge, as well as my advisory team, Dr. Peter Sicinski, Dr. Jennifer Smith and Dr. Joao Paulo. During the mentored phase, I plan to (1) deepen my understanding of mass spectrometry instrumentation, (2) develop new expertise in drug screening, (3) strengthen my knowledge base in cancer biology, especially in 3D cell culture and cellular signaling, and (4) enhance my scientific writing and communication skills while gaining experience in lab management and leadership. The environment at Harvard Medical School presents an excellent opportunity for training in drug screening and cancer biology. The proposed research strategy combined with the career development training outlined herein will provide the necessary elements for my successful transition to an independent investigator in the fields of mass spectrometry and cancer biology.