Mutations in the gene encoding phospholipase C-␥ 2 (PLC␥ 2 ) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLC␥ 2 an ϳ2-3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca 2؉ following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLC␥ 2 mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells. Enhanced "basal" activity of PLC␥ 2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLC␥ 2 F897Q mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLC␥ 2 activity. We suggest that R665W and L845F be referred to as allomorphic rather than hypermorphic mutations of PLCG2. Rerouting of the transmembrane signals emanating from BCR and converging on PLC␥ 2 through Rac in ibrutinib-resistant CLL cells may provide novel drug treatment strategies to overcome ibrutinib resistance mediated by PLCG2 mutations or to prevent its development in ibrutinib-treated CLL patients.Inositol-phospholipid-specific phospholipases C (PLCs) 3 regulate many fundamental functions of normal and neoplastic B cells (1, 2). They catalyze the formation of inositol 1,4,5-trisphosphate (InsP 3 ) and diacylglycerol and, at the same time, decrease the local or general plasma membrane abundance of their substrate, phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) (3). Three members of the six mammalian PLC subfamilies, ␤, ␥, ␦, ⑀, , and , play important roles in B cells as follows: PLC␤ 2 , PLC␤ 3 , and PLC␥ 2 . PLC␤ 2 and PLC␤ 3 are important in mediating B cell responses to G-protein-coupled chemokine receptors (4). PLC␥ 2 serves as a key component of the B cell receptor (BCR) signalosome by interacting with cell surface receptor activation, e.g. by antigens (5), cleavage fragments of the third complement component (6), and bacterial, viral, or autoimmunity host DNA (7), and even certain chemokines (8). PLC␥ 2 activation results in InsP 3 -mediated increases in the concentration of free Ca 2ϩ , diacylglycerol-mediated activation of protein kinases C, and changes in transmembrane signaling directly mediated by PtdInsP 2 (9).Several lines of evidence point to an important contribution of enhanced BCR signaling in the pathogenesis, progression, and/or maintenance of B cell leukemias and lymphomas. For example, leukemic B cells of patients with chronic lymphocytic leukemia (CLL) specifically express a restricted immunoglobulin heavy variable (IGHV) gene repertoire, suggesting that CLL development represents an antigen-superantigen-driven process (10). Furthermore, the presence or absence of somatic mutations in rearranged IGHV genes determin...

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Background: Phospholipase C␥ 2 (PLC␥ 2 ) is stimulated by Rac GTPases through direct protein-protein interaction. Results: The Rac-PLC␥ 2 interaction markedly enhances B cell-receptor-mediated Ca 2ϩ mobilization and nuclear translocation of the Ca 2ϩ -regulated transcription factor NFAT in B cells. Conclusion: Rac-mediated stimulation of PLC␥ 2 activity amplifies B cell receptor-induced Ca 2ϩ signaling. Significance: A specific Rac-resistant PLC␥ 2 variant is used to determine the physiological cell signaling relevance of a functional Rac-PLC␥ 2 interaction in an appropriate cellular context.

Depending on its occurrence in the germline or somatic context, a single point mutation, S707Y, of phospholipase C-γ2 (PLCγ2) gives rise to two distinct human disease states: acquired resistance of chronic lymphocytic leukemia cells (CLL) to inhibitors of Brutons´s tyrosine kinase (Btk) and dominantly inherited autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation, APLAID, respectively. The functional relationships of the PLCγ2S707Y mutation to other PLCG2 mutations causing (i) Btk inhibitor resistance of CLL cells and (ii) the APLAID-related human disease PLCγ2-associated antibody deficiency and immune dysregulation, PLAID, revealing different clinical characteristics including cold-induced urticaria, respectively, are currently incompletely understood. Here, we show that PLCγ2S707 point mutants displayed much higher activities at 37° C than the CLL Btk inhibitor resistance mutants R665W and L845F and the two PLAID mutants, PLCγ2Δ19 and PLCγ2Δ20-22. Combinations of CLL Btk inhibitor resistance mutations synergized to enhance PLCγ2 activity, with distinct functional consequences for different temporal orders of the individual mutations. Enhanced activity of PLCγ2S707Y was not observed in a cell-free system, suggesting that PLCγ2 activation in intact cells is dependent on regulatory rather than mutant-enzyme-inherent influences. Unlike the two PLAID mutants, PLCγ2S707Y was insensitive to activation by cooling and retained marked hyperresponsiveness to activated Rac upon cooling. In contrast to the PLAID mutants, which are insensitive to activation by endogenously expressed EGF receptors, the S707Y mutation markedly enhanced the stimulatory effect of EGF, explaining some of the pathophysiological discrepancies between immune cells of PLAID and APLAID patients in response to receptor-tyrosine-kinase activation.