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-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 B lymphocytes, 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 hyper-responsive 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-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.
Highlights
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
The observation that phospholipase C-␥2 (PLC␥2) variants are, instead, hypersensitive in intact cells to the Rho GTPase RAC2, as well as the upstream protein-tyrosine kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN) [39, 40], has led us to suggest that the PLCG2 mutations found in BTK inhibitor–resistant cells may cause a rerouting of the transmembrane signals emanating from B-cell receptor (BCR) to converge on and activate phospholipase C (PLC)␥2
Stimulation of PLC␥2 variants mediating BTK inhibitor resistance can be explained by their hypersensitivity to the noncatalytic actions of BTK. We suggest that this mechanism contributes to the acquired BTK inhibitor resistance observed in CLL patients with PLCG2 mutations and, potentially, to preexisting reduced sensitivity or even primary resistance of clonal populations of CLL cells with hyperactivated BCR
Summary
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. The observation that PLC␥2 variants are, instead, hypersensitive in intact cells to the Rho GTPase RAC2, as well as the upstream protein-tyrosine kinases SYK and LYN [39, 40], has led us to suggest that the PLCG2 mutations found in BTK inhibitor–resistant cells may cause a rerouting of the transmembrane signals emanating from BCR to converge on and activate PLC␥2. Stimulation of PLC␥2 variants mediating BTK inhibitor resistance can be explained by their hypersensitivity to the noncatalytic actions of BTK We suggest that this mechanism contributes to the acquired BTK inhibitor resistance observed in CLL patients with PLCG2 mutations and, potentially, to preexisting reduced sensitivity or even primary resistance of clonal populations of CLL cells with hyperactivated BCR
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