The human genome contains 556 kinase genes, commonly called the kinome. Kinases are enzymes that regulate nearly all aspects of cell signaling and their dysregulation leads to excess cell proliferation, a hallmark of cancer. Kinases are one of the most important targets in precision oncology, but the ideal kinase targets or kinase drug combinations for most cancers have not been established. Determining the cellular consequences of turning off kinase enzymes has immense potential to transform our understanding of kinases and kinase inhibitors. In this New Innovator Award application, I seek to decipher how kinase catalysis regulates cancer cell function by using cutting-edge base editing tools to turn off the enzyme activity in every human kinase across the kinome. This approach will identify novel kinases for therapeutic targeting and will act as a surrogate for small-molecule inhibition to enable drug discovery. Prior methods to perturb the kinome in cells use knock-out or knock-down, which are crude tools that remove the entire gene and do not necessarily translate to the effects of a small-molecule inhibitor. My base editing approach to inactivate kinase catalysis will solve two major issues that prevent existing mutagenesis and knock-in methods to both scale across the kinome and perform mutations at physiologic levels. Base editing can mutate hundreds of genes at physiologic levels in a pooled experiment due to its high editing efficiency. Here, I propose a base editing platform to turn off catalysis in in 556 kinases in cancer cells for pooled functional studies. As a case study, I will investigate breast cancer, where multiple kinase inhibitors are standard therapies but where new treatments are urgently needed. I have developed a suite of new technologies enabling whole kinome base editing in cells, supported by preliminary data. I will use this platform to interrogate how kinase activity regulates 1) cancer cell growth, 2) sensitivity to targeted therapies, and 3) druggable gene expression. This platform will compress the interval between kinome hit and lead discovery from decades to years by identifying the optimal mechanisms to inhibit kinases, transforming drug development and design of combination therapies. This proposal focuses on human kinases in cancer but once we establish this framework it will be applicable to studying any enzyme class in any physiologic process in mammals. This proposal is innovative conceptually, technically, and materially as the first experiment for precise, surgical inhibition of a large and diverse family of enzymes in a comprehensive fashion. My background studying structural biology, protein biochemistry, and kinase signaling; my clinical experience as a breast oncologist; and the strong institutional environment at Columbia University Irving Medical Center make me uniquely well-equipped to perform these investigations.

NARRATIVE Kinase proteins are druggable enzymes that regulate nearly all aspects of cell signaling in cancer cells, but the ideal kinase targets or kinase drug combinations to kill various types of cancers have not been established. We have developed new technologies to base edit all 556 human kinases and remove their enzyme activity at physiologic levels. Our proposed research will decipher the functional effects of how kinase inhibition alters cell growth, drug response, and gene expression which will accelerate kinase target validation and enable drug discovery.