Pathway Phenotyping of B-Cell Receptor Activation and Inhibition: Translation to Novel Clinical Biomarkers

B-cell receptor (BCR) signaling has emerged as a vital and targetable component in a number of B-cell malignancies. The recent approval and numerous ongoing clinical trials of small molecule kinase inhibitors targeting this pathway has bolstered efforts to define the signaling proteins critical to BCR activity. To this end, we first mapped BCR signaling using 93 different activation state antibodies. These results provided a high level view of BCR signaling across more than 80 intracellular proteins covering canonical and non-canonical BCR effectors. The signaling responses were then modulated with known kinase inhibitors to associate kinase activity with pathway activation. Using these data as a backdrop, three BTK inhibitors (AVL-292, CNX-774, and ibrutinib) were tested to determine which components of BCR signaling are commonly inhibited by these molecules. These studies showed that ibrutinib was the most promiscuous of the inhibitors but that all three compounds blocked activation of the S6 signal transduction pathway. Interestingly, a number of other oncogenic pathways remained intact after inhibitor treatment including the MAPK-ERK and AKT signaling pathways. After identifying the S6 pathway as a commonly blocked effector of BCR signaling, we validated the pathway readouts for their use as ex vivo pharmacodynamic assays in whole blood patient samples. Our results show that the assays yield the appropriate precision and reproducibility necessary to be used as PD markers in a clinical trial setting. Thus, the workflow of pathway phenotyping to inhibitor testing and finally to clinical biomarker development further defined the activation patterns of BCR signaling and identified novel clinical readouts for measuring BCR inhibitor activity.