Cancer remains one of the leading causes of death worldwide, responsible for millions of deaths each year. One widely used therapeutic strategy involves kinase inhibitors: a class of drugs that block the activity of protein kinases, which drive tumor growth and survival. Although these drugs have transformed cancer care, their clinical impact is limited by the emergence of drug-resistant mutations, which allow cancer cells to avoid inhibition. The first kinase inhibitor, imatinib (Gleevec), targets Abl kinase and revolutionized treatment of chronic myeloid leukemia. However, clinical use of imatinib revealed a wide range of mutations within Abl that confer resistance to imatinib and other inhibitors, including the T315I gatekeeper mutation. Understanding the molecular basis of resistance is essential for improving current therapies and guiding the development of next-generation inhibitors like ponatinib (Iclusig), which is effective against the T315I mutation. Therefore, we will use Abl kinase as a model for understanding how resistance mutations emerge. This project seeks to systematically map drug-resistant mutations in Abl kinase. To accomplish our goals, we need a high-throughput system that can measure Abl activity. Here, we will use a bacterial two-hybrid (B2H) system that relies on Abl-dependent phosphorylation allowing us to detect Abl activity. Once we validate this system, we will construct a comprehensive deep mutational scanning (DMS) library encompassing all possible single Abl mutants. We will then use fluorescence-activated cell sorting (FACS) to sort cells that contain Abl mutants that maintain activity in the presence of imatinib and use next-generation sequencing (NGS) to find enriched mutants. This process will allow us to generate a mutational heat map, highlighting mutations that confer resistance. Finally, we will individually express, purify, and subject these resistance mutations to fluorescence assays to confirm their resistance. Our work will result in a detailed map of resistance-conferring mutations in Abl kinase. Future work would include studying the resistance of Abl kinase to different drugs to learn more about drug-protein interactions. Ultimately, our findings have the potential to inform rational drug design strategies aimed at overcoming resistance and improving the efficacy of cancer therapeutics.
