Small molecule inhibitor combination treatment effectively represses global B-cell signaling in diffuse large B-cell lymphoma

Cells sense their environment via signaling networks and malignant cells often hijack signaling pathways for their growth, which complicates defining the effects of drugs. Here, we map the state of the signaling network of B-cell lymphoma cells via simultaneous quantification of 111 (phospho-)proteins. We demonstrate that the B-cell signaling network can be disrupted with specific clinical small molecule inhibitors (iBTK, iSYK, iNF?B), thereby inducing a repressed state of the network. Principal component analysis identifies how the three inhibitors work along subtly different repression axes through the signaling state landscape. Finally, we observe that 1 µM combination treatment with all three inhibitors is more effective in inducing the repressed state than 10 µM of the single inhibitors. These results emphasize that cellular signaling occurs in complex networks, and underscore how quantification of the signaling state landscape provides insights into how combination drug treatments can bring cells in a desired network state. Overall design: We performed comprehensive ID-seq experiments to define a DLBCL-specific signaling state landscape. The HBL1 cell line (RRID: CVCL_4213) was used as model for ABC-DLBCL. Cells were treated with different doses and combinations of the inhibitors ibrutinib (iBTK), R406 (iSYK), and QNZ (iNFkB) for 60 min (0, 0.1, 1, 10, 100 uM) and subsequently activated with anti-Ig + hydrogen peroxide for 20 min (triplicate samples). Samples were then measured with ID-seq to quantify 111 intracellular (phospho-)proteins.