APT1-Mediated Cross-Talk Between Palmitoylation and Phosphorylation Events of the BCR Pathway Sensitizes CLL Cells Towards BCR-Associated Kinase Inhibitors

Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Regulation of phosphorylation events of the BCR is critical for survival and proliferation of CLL cells. Palmitoylation, a common post-translational modification defined as the addition of palmitic acid to internal cysteins, was shown recently to regulate phosphorylation of proteins by controlling their localization and activity. Many proteins in the B cell receptor (BCR) signaling pathway in CLL cells are primarily cytosolic, but upon activation transiently located to the plasma membrane to fulfill their functions. Some of these proteins, like Src kinase family members Lyn, Yes and Fyn, are already reported to be palmitoylated. Previous studies by our group showed that global protein palmitoylation is deregulated in CLL cells and primarily caused by overexpression of the depalmitoylating enzyme APT1. To investigate, if overexpressed APT1 directly targets BCR signaling, we inhibited (genetically and pharmacologically) APT1 in CLL cells and analyzed occurring changes in 45 different phosphorylation-sites of major signaling pathways. Interestingly, we found that APT1 controls the central phosphorylation events within Akt/mTOR/p70S6 signaling. For example, phosphorylation of Akt (T308, S473) and p70S6 (T389, T421, S424) was significantly decreased after interference with protein depalmitoylation. By biochemical dissection of these pathways with acyl-biotin exchange (ABE) assays we identified novel palmitoylation candidates particularly within the PI3K/Akt axis, which are indispensable for phosphorylation of kinases of the Akt/mTOR/p70S6 axis. Functionally, pharmacological inhibition of APTs and genetic knockdown of APT1 sensitizes CLL cells towards BCR-associated KIs like Ibrutinib and Idelalisib. Our data uncovers that central phosphorylation events within the BCR pathway are dependent on palmitoylation controlled by APT1. Future studies should therefore investigate more in detail how addition of APT1 inhibitors can improve clinical outcome of patients treated with Idelalisib or Ibrutinib-based regimens.