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

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 inPLCG2, encoding phospholipase C-γ₂ (PLCγ₂). The PLCγ₂ 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 spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ₂ variants to BTK itself has remained unknown. Here, using genetically-modified DT40 Blymphocytes, along with various biochemical assays, including analysis of PLCγ₂-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ₂ variants such as PLCγ₂S707Y are hyperresponsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ₂ phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically- inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactiveBTKis insensitive to active-siteBTKinhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.