The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein

Abstract

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...

Overcoming ibrutinib resistance by targeting phosphatidylinositol-3-kinase signaling in diffuse large B-cell lymphoma

“…24 BTK mutation C481S and others (e.g., PLCγ2) have been reported to be associated with ibrutinib resistance. 25,26 However, in the IR-DLBCL cell lines examined in this study, targeted sequencing for BTK and PLCγ2 genes confirmed the lack of these mutations (data not shown). We anticipated that dysregulated signaling pathways could lead to development of ibrutinib resistance so we assayed the protein expression in our parental-and IR-DLBCL pairs by reverse phase protein array (RPPA).…”

AreBTKandPLCG2mutations necessary and sufficient for ibrutinib resistance in chronic lymphocytic leukemia?

“…Mutations within the SH2 domain inactivate its inhibitory function, leading to hyperfunctional PLCγ2 [41, 42]. Prior work had demonstrated the hypermorphic activity of germline S707Y mutations [41], and work with the DT40 cell line and primary CLL cells has shown that PLCG2 R665W and L845F mutations are also hypermorphs [7, 43]. With these mutations present, calcium influx upon BCR stimulation is sustained in the presence of ibrutinib [7].…”

“…In primary patient samples with PLCG2 mutations, PLCγ2 remains in an active phosphorylated state even upon treatment of the samples with ibrutinib [7]. These mutants are also hyper-responsive to upstream agonist signals, such as those provide by the GTPase Rac2 [43]. However, over a dozen different mutations in PLCG2 have been described, many lying outside of the SH2 domain, and the effect of many of these mutations on protein function are unclear (Table 2) [35].…”

Ibrutinib-resistant CLL: unwanted and unwonted!

“…Two of these amino acid changes resulted in a hyperactivated form of PLCG2 via the GTPase RAC. 7 An unresolved issue is the oftentimes low allelic fraction of mutations of BTK and PLCG2 that has been observed in patients with resistant CLL. 1,8 Concurrent mutation of BTK and PLCG2 suggests either that the mutations are present in different subclones or that an additional activation of PLCG2 is advantageous for bypassing BTK inhibition.…”

“…This equilibrium is probably dependent on the complex interactions between genetic, epigenetic, telomere biology, and HSC niche determinants. 7 The identification of factors that promote clonal expansion and break this equilibrium is of high clinical relevance. It may be key to understanding why some aging individuals develop clonal hematopoiesis, 8 which has been associated with risk of hematological cancer progression.…”