Dissecting BCR-ABL Variant Signaling Pathways Using Novel Interactome Identification Strategies

Abstract

Cell signaling is an essential function of cells and tissues. Understanding cell signaling necessitates technologies that can identify protein-protein interactions as well as post translational modifications to proteins within protein complexes. The goals of this study are (1) to understand how BCR-ABL variants differentially signal to produce different clinical/experimental phenotypes and (2) to develop novel interactome detection strategies to understand signaling. This dissertation describes an integrated approach of the use of proximity dependent labeling protein-protein interaction analysis assays coupled with global phosphorylation analysis to investigate the differences in signaling between two variants the oncogenic fusion protein, BCR-ABL. Two major types of leukemogenic BCR-ABL fusion proteins are p190BCR-ABL and p210BCR-ABL. Although the two fusion proteins are closely related, they can lead to different clinical outcomes. A thorough understanding of the signaling programs employed by these two fusion proteins is necessary to explain these clinical differences. Our findings suggest that p190BCR-ABL and p210BCR-ABL differentially activate important signaling pathways, such as JAK-STAT, and engage with molecules that indicate interaction with different subcellular compartments. In the case of p210BCR-ABL, we observed an increased engagement of molecules active proximal to the membrane and in the case of p190BCR-ABL, an engagement of molecules of the cytoskeleton. These differences in signaling could underlie the distinct leukemogenic process induced by these two protein variants. Additionally, this dissertation also describes the development of a novel interactome detection strategy, called Biotinylation Site Identification Technology (BioSITe), which increases the sensitivity and specificity of proximity dependent biotin labeling technologies. When applied to BCR-ABL variants, BioSITe provides structural information about BCR-ABL interacting proteins and the degree of proximity these proteins are to BCR-ABL. Finally, this thesis demonstrates the use of isotopically labeled biotin for quantitative BioSITe experiments, applied to BCR-ABL variants, simplifies differential interactome analysis