Project Summary Covalent inhibitors of Bruton’s tyrosine kinase (BTK) are effective in multiple B-cell malignancies but patients discontinue these agents due to resistance and intolerance. Highly selective and reversible non-covalent BTK inhibitors have been developed to overcome these problems and are currently being tested in clinical trials in patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Despite these beneficial features of non-covalent BTK inhibitors and a high overall response rate seen in early clinical trials, some patients did not respond to treatment or relapsed after initial response to monotherapy. This project’s broad and long-term goals are to better understand these resistance mechanisms leading to relapse and design better combination strategies or use of novel drugs to overcome this resistance. Our specific aims are to elucidate the molecular mechanisms of resistance to this novel class of BTK inhibitors by interrogating the signaling pathways in mutant BTK cells that are kinase deficient. We plan to comprehensively evaluate the dependence of kinase dead BTK mutations on potential surrogate kinases activated by mutant BTK. We are also testing the requirement for BTK for survival and signaling in cells with PLCG2 mutations. Lastly, we will identify the genomic basis for BTK degrader resistance. Given the widespread use of BTK inhibitors across CLL and B-cell malignancies, and the increasing incidence of patients with acquired resistance to non-covalent BTK inhibitors, the results of this study will have major therapeutic importance for patients with a variety of B- cell malignancies. Our findings will inform future application of disease monitoring on non-covalent BTK inhibitors and about the development of resistance mechanisms with BTK degraders that are now being used in clinical trials.