Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells

Abstract

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK , most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2 , encoding phospholipase C-γ 2 (PLCγ 2 ). The PLCγ 2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases s pleen-associated t y rosine k inase (SYK) and L ck/ Y es-related n ovel tyrosine kinase (LYN). The sensitivity of the PLCγ 2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ 2 -mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca 2+ ] i ), we show that various CLL-specific PLCγ 2 variants such as PLCγ 2 S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ 2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL pa…