Key Points

  • Basal protein phosphorylation is higher in UM-CLL

  • MARCKS regulates central signaling pathways and affects response to acalabrutinib

BTK inhibitors are highly active drugs for the treatment of CLL. To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines, threonines and tyrosines (pS:pT:pY). Expression of 83 proteins differed between unmutated IGHV (UM)-CLL and mutated IGHV (M)-CLL. Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition and survival. One protein, MARCKS, showed striking differences in expression and phosphorylation level in UM-CLL versus M-CLL. MARCKS sequesters PIP2, thereby affecting central signaling pathways and clustering of the B cell receptor. Genetically induced loss of MARCKSignificantly increased AKT signaling and the migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitiors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in CLL patients treated with acalabrutinib.

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