Functional Analysis of BrutonÕs Tyrosine Kinase: A Bottom-up Proteomics Approach

Abstract

BrutonÕs tyrosine kinase (BTK) is a non-receptor tyrosine protein kinase indispensable in B lymphocyte development. After being activated by the B cell receptor (BCR), BTK subsequently signals downstream to promote B cell survival and development. In humans, loss of BTK function results in X-linked agammaglobulinemia (XLA), an immunodeficiency characterized by a defect in B cell development that leads to a dearth of circulating B cells. In line with its role in normal B cells, BTK is also essential for malignant B cell survival . Hence, BTK has become an attractive target for treatment of B cell malignancies. Targeting BTK with small molecular inhibitors has achieved impressive clinical outcomes in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cases. However, primary and acquired resistance to BTK inhibitors is common and often leads to clinical failure. To address these challenges, a comprehensive understanding of BTK function would be highly informative. In this project, we developed a novel bottom-up proteomic approach, termed the Native In-gel Kinase-Substrate Assay (NIKSA), to analyze BTK function by globally identifying its physiological substrates. A BTK NIKSA performed on a K562 cell extract found 926 proteins as potential direct BTK substrates that could be ordered into distinct cellular pathways by pathway analysis. The top pathway found as a target of BTK control was the ubiquitin-proteasome pathway. In subsequent analyses, we found multiple proteasome subunits that are robust in vitro BTK substrates and can physically interact with BTK in vivo. Furthermore, we demonstrated that modulating BTK activity in vivo led to concomitant changes in proteasome activity. To exploit this finding in a therapeutically meaningful manner, we demonstrated that combining BTK activation with proteasome inhibition effectively killed malignant B cells in cell culture and diminished B cell tumor growth in a xenograft setting in mice. In addition, we demonstrated that BTK phosphorylates the oncogenic kinase ABL and increases its catalytic activity, providing a rationale for combining BTK and ABL inhibitors to treat chronic lymphocytic leukemia (CLL). Taken together, these data demonstrate that proteomic profiling of kinase substrates is an effective strategy to illuminate pathways under kinase control. Furthermore, we demonstrate how this new knowledge can be exploited to develop new rational combinatorial therapies to treat hematological malignancies to reduce the morbidity and mortality of these devastating diseases. Application of this general methodology to other oncogenic kinases holds great promise to inform therapeutic approaches to treat other malignancies.