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

Mutations in the gene encoding phospholipase C-γ2 (PLCγ2) 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 PLCγ2 an ∼2–3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca2+ 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 PLCγ2 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 PLCγ2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLCγ2F897Q mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLCγ2 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 PLCγ2 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.

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 determines the clinical course of CLL, with patients carrying mutated IGHV genes generally following a more indolent course (10). In CLL, the BCR repertoire is characterized by subsets of closely homologous ("stereotyped") immunoglobulin V(D)J sequences, which are directly involved in antigen binding. This, together with the finding that most malignant B cells thrive only poorly in vitro, further supports the notion of a role of antigenic drive in B cell tumorigenicity (11). Recent evidence suggests that, at least in CLL, BCRs also induce cell-autonomous signaling independent of extrinsic antigens that is caused by intra-or inter-BCR interactions (12). Finally, there is evidence for the existence of constitutively activated protein kinases and transcription factors downstream of PLC␥ 2 in leukemic B cells of certain CLL cells (13)(14)(15). The observation that some of the signaling components upstream of PLC␥ 2 , such as the protein-tyrosine kinases Syk and Btk, can promote B cell proliferation and/or survival, either along the pathway of normal B cell development or at specific stages following malignant transformation, is well in line with this concept (16 -18).
The orally bioavailable irreversible Btk inhibitor ibrutinib has recently undergone a remarkably successful evolution as a second-line treatment of patients with relapsed or refractory CLL or mantle cell lymphoma and as a first-line treatment of patients with CLL carrying a del(17p) or TP53 mutation (19,20). Currently, the drug is being evaluated for treatment of other diseases, including other malignancies, autoimmune disease, inflammatory diseases, osteoclast-associated bone diseases, and ischemic stroke (21)(22)(23)(24)(25)(26). As is the case for other targeted tumor therapies (27), ibrutinib treatment is characterized, in some cases, by the development of acquired drug resistance (28). Thus, whole-exome sequencing of six CLL patients with late relapses revealed C481S mutations in BTK of five patients and three distinct mutations in PLCG2 of two patients as follows: L845F, R665W, and S707Y in one patient with tumor cells also harboring a BTK C481S mutation and PLCG2 R665W representing the sole mutation in the other patient (29). Although the resistance mechanism conferred by the BTK C481S mutation is immediately apparent from the fact that the thiol group of Cys-481 is the site of covalent linkage of ibrutinib to Btk close to its ATP-binding site, the mechanisms of action of the mutations found in PLCG2 remained less well understood. Whereas S707Y had previously been reported as a constitutively activating mutation in the dominantly inherited human disease APLAID (autoinflammation and PLC␥ 2associated antibody deficiency and immune dysregulation) (30), the R665W and L845F mutants of PLC␥ 2 appeared to be functionally normal in reconstituted DT40 chicken B cells in the absence of BCR stimulation, but to mediate moderately enhanced and markedly prolonged ibrutinib-resistant increases in [Ca 2ϩ ] i following BCR ligation with anti-IgM (29). Very recent evidence showed Btk-independent activation of the overexpressed R665W PLC␥ 2 mutant after B cell receptor engagement in Btk-deficient DT40 cells, suggesting Btk independency of this mutant (31). When the same mutant was expressed in PLC␥ 2 -deficient DT40 cells containing endogenous wild-type Btk, BCR-mediated PLC␥ 2 activation was resistant to ibrutinib, but sensitive to pharmacologic inhibitors of Syk and Lyn. These results suggested the existence of proteintyrosine kinase mechanisms emanating from BCR and bypassing Btk to activate R665W to mediate ibrutinib resistance even in tumor cells lacking BTK mutations (31).
We have previously shown that PLC␥ 2 is specifically activated by Rac GTPases by a mechanism independent of PLC␥ 2 tyrosine phosphorylation, but dependent on the direct interaction of activated Rac with the bipartite split PH domain (spPH) juxtaposed between the two halves, X and Y, of the PLC␥ 2 catalytic domain (32,33). Studies using a Rac-resistant mutant of PLC␥ 2 , F897Q, reconstituted into PLC␥ 2 -deficient DT40 B cells recently showed that Rac-mediated stimulation of PLC␥ 2 amplifies BCR-mediated Ca 2ϩ signaling (34). The fact that failure to proliferate in response to immunoglobulin receptor stimulation had also been observed in mice carrying deletions in all three genes encoding Vav guanine nucleotide exchange factors of Rac GTPases, Vav1, Ϫ2, and Ϫ3 (35), prompted us to examine, in this work, the effect of the PLCG2 mutations R665W and L845F on the Rac-PLC␥ 2 interaction in intact cells and in a cell-free system in vitro. The results show that the two mutations take marked stimulatory effects on this interaction. These stimulatory effects may not only contribute to the mechanism(s) of ibrutinib resistance caused by mutations in PLCG2 rather than BTK, but they also provide novel strategies to tackle ibrutinib resistance in CLL and other debilitating human diseases.

Results
The first experiment was designed to determine whether the two PLC␥ 2 mutants R665W and L845F exhibit constitutive activity in intact cells. To this end, the two mutants were expressed in COS-7 cells to be radiolabeled with [ 3 H]inositol for measurement of [ 3 H]inositol phosphate formation. Wildtype PLC␥ 2 and PLC␥ 2 carrying an M28L germ line mutation identified in an ibrutinib-resistant patient were analyzed for comparison. Fig. 1A shows that, in contrast to wild-type PLC␥ 2 and PLC␥ 2 M28L , the mutants R665W and L845F caused marked, up to 18-fold, increases in basal inositol phosphate formation when expressed in increasing amounts (Fig. 1, A, left  panel, and B). Only slight, 1.4-fold, increases were apparent at the highest amounts of wild-type and PLC␥ 2 M28L . There was no difference between wild-type and M28L mutant PLC␥ 2 in terms of their stimulatory responses to constitutively active Rac2 G12V (Fig. 1A, right panel), indicating that PLC␥ 2 M28L did not harbor a defect in enzyme activation.  To determine and compare the sensitivity of wild-type PLC␥ 2 to stimulation by constitutively active Rac2 to the sensitivities of the mutants R665W and L845F, the PLC␥ 2 isozymes were coexpressed with increasing amounts of Rac2 G12V . Fig. 2A shows that there were striking increases in inositol phosphate formation in response to increasing amounts of Rac2 G12V . Specifically, the maximal increase in Rac2 G12V efficacy was ϳ6.7and 35-fold for PLC␥ 2 R665W and PLC␥ 2 L845F , respectively. In addition, we consistently observed that the two point mutations caused an increase in the potency of Rac2 G12V , which was ϳ4.5and 6.5-fold for PLC␥ 2 R665W and PLC␥ 2 L845F , respectively. The increase in Rac2-stimulated PLC activity caused by the PLC␥ 2 mutations was not caused by changes in PLC␥ 2 protein production in transfected cells (Fig. 2B).
Most interestingly, enhanced sensitivity of PLC␥ 2 R665W and PLC␥ 2 L845F to Rac2 was not limited to constitutively active Rac2 G12V but was also observed for wild-type Rac2 (Fig. 3A). Specifically, although there was no effect of increasing amounts of Rac2 on the activity of wild-type PLC␥ 2 , the mutants R665W and L845F were activated up to 5.1-and 4.2-fold, respectively. There was little, if any, change in the expression of wild-type or mutant PLC␥ 2 in the presence of increasing amounts of Rac2 (Fig. 3B).
We have previously shown that PLC␥ 2 is sensitive to stimulation by exogenous, constitutively active Vav1, Vav1⌬N, presumably via activation of endogenous Rac GTPases present in COS-7 cells (33). Fig. 4A shows that the two ibrutinib resistance mutations imparted on PLC␥ 2 a marked increase in its responsiveness to stimulation by Vav1⌬N, which amounted to ϳ9.3and 12-fold for the mutants R665W and L845F, respectively. As observed for Rac2 G12V , there was an increase in the apparent potency of Vav1⌬N by the mutations, which was ϳ5.3and 5.7-fold for PLC␥ 2 R665W and PLC␥ 2 L845F , respectively. The results shown in Fig. 4B suggest that the increase in inositol phosphate formation by wild-type PLC␥ 2 , but not by its mutants may have, at least in part, been due to a slight increase in the production of the protein at increasing concentrations of Vav1⌬N. The ability of activated Vav1 to catalyze activation of Rho GTPases (36) is dependent on an intact interaction network between the DH, PH, and cysteine-rich domain regions of the Vav1 protein and is abolished by an Asp to Ala mutation at position 376, D376A (37). Fig. 4C shows that the inactivating Vav1 mutation caused an almost complete (ϳ85%) and a complete loss in the stimulatory effect of Vav1⌬N on the activity of R665W and L845F, respectively.
The abilities of wild-type and constitutively active Rac2 to cause enhanced activation of PLC␥ 2 R665W in comparison with wild-type PLC␥ 2 were dependent on the C-terminal isoprenylation of the exogenous Rac2 proteins (Fig. 5A). Specifically, their stimulatory effect on PLC␥ 2 R665W activity was completely (R665W) (left panel) or with 50 ng/well vector encoding wild-type PLC␥ 2 (WT) or PLC␥ 2 L845F (L845F) (right panel) and increasing amounts of vector encoding Rac2 G12V . Note that the amounts of vector DNA encoding mutant PLC␥ 2 was different in the left and right panels to observe full-range stimulation of the two mutants by Rac2 G12V without running out of available phospholipid substrate. Twenty four hours after transfection, the cells were incubated for 20 h with myo-[2-3 H]inositol, and inositol phosphate formation was then determined. The ED 50 values of vector encoding Rac2 G12V for the stimulation of wild-type or mutant PLC␥ 2 activity obtained by non-linear curve fitting are shown above the graphs in nanograms/well. B, homogenates from cells functionally analyzed in A were subjected to SDS-PAGE and immunoblotting using an antibody reactive against the c-Myc epitope. Co., control. lost upon replacement of the cysteine residue at position Ϫ4 from the Rac2 C terminus, which normally serves as a substrate for geranylgeranylation, by a serine residue, C189S (38). This was despite the fact that the C189S mutants were expressed at similar, if not somewhat enhanced levels, as compared with their respective counterparts (Fig. 5B).
Figs. 3A, 4, A and C, and 5A also confirm the increase in basal activity of PLC␥ 2 R665W and PLC␥ 2 L845F even in the absence of exogenous PLC␥ 2 stimuli such as Rac2 and Vav1⌬N observed in Fig. 1. The fact that COS-7 cells are transformed cells prompted us to investigate the following possibilities: (i) that enhanced basal activity is caused by cell-autonomous activation of the mutant PLC␥ 2 isoforms, e.g. by spontaneously active cell surface receptors, and (ii) that Rac participates in this activation. To this end, F897Q mutants of wild-type PLC␥ 2 and its mutants R665W and L845F were generated and functionally characterized (Fig. 6A). We have previously shown that the F897Q substitution blocks activation of PLC␥ 2 by constitutively active Rac2 and abolishes binding of GTP␥S-activated Rac2 to PLC␥ 2 spPH, while leaving the overall fold of PLC␥ 2 spPH unaffected (33). Fig. 6A, left panel, shows that the F897Q mutation, as expected, caused a complete or near complete loss of activation of wild-type PLC␥ 2 by Rac2 G12V and of PLC␥ 2 R665W by wild-type and G12V mutant Rac2. More importantly, however, the increased "basal" PLC␥ 2 R665W activity, determined in the absence of exogenous Rac2, was reduced by  about 74% (Fig. 6A, left panel). Fig. 6A, right panel, shows that F897Q mutation caused similar reductions of basal activity of the R665W and L845F variants of PLC␥ 2 (ϳ68 and ϳ78%, respectively). This indicates that the basal activities of the R665W and L845F mutants are strongly dependent on Rac1 endogenously present in COS-7 cells. These reductions in PLC␥ 2 activity were not related to reduced PLC␥ 2 protein synthesis in transfected cells (Fig. 6B).
To obtain further independent evidence for an involvement of active Rac in the enhanced "basal" activities of PLC␥ 2 R665W and PLC␥ 2 L845F , we employed the Rac-specific pharmacologic inhibitor EHT 1864 and its inactive analog EHT 4063 (39). EHT 1864 is known to bind with high affinity to Rac1, Rac1b, Rac2, and with somewhat lower affinity to Rac3. The inhibitor has been suggested to place Rac in an inactive state by promoting the loss of bound guanine nucleotide, rather than interfering with RhoGEF-induced Rac activation, as described for other Rac inhibitors such as NSC23766 (40,41). Fig. 7A, left panel, shows that EHT 1864, but not EHT 4063, caused a clear (ϳ55%) inhibition of basal inositol phosphate formation by PLC␥ 2 R665W and PLC␥ 2 L845F . There was a smaller (ϳ25%) not quite statistically significant (p ϭ 0.0676) inhibitory effect for wild-type PLC␥ 2 . No effect of EHT 1864 was observed in the absence of exogenous PLC isozyme and in the presence of PLC␦ 1 ⌬44, a constitutively active variant of PLC␦ 1 . PLC␦ 1 is an evolutionarily divergent relative to PLC␥ 2 and insensitive to stimulation by Rac (32,42). The inhibitory effect of EHT 1864 on basal inositol phosphate formation by PLC␥ 2 L845F was concentration-dependent with an IC 50 of about 1 M (Fig. 7A, right panel), which is slightly lower than, but still in line with, the previously reported value of about 5 M for modulation of ␥-secretase-mediated amyloid precursor protein (APP) processing (39). There was no effect of EHT 1864 and EHT 4063 on the expression of the various recombinant PLC isozymes in transfected cells (Fig. 7B).
The two PLC␥ isoforms are distinct in their response to activated Rac, with PLC␥ 1 , in marked contrast to PLC␥ 2 , showing In the right panel, COS-7 cells were transfected with 500 ng/well (from left to right) empty vector (Co.) or vector encoding either wild-type PLC␥ 2 , PLC␥ 2 F897Q , PLC␥ 2 R665W , PLC␥ 2 R665W/F897Q, PLC␥ 2 L845F , or PLC␥ 2 L845F/F897Q. Note that the vectors encoding mutant PLC␥ 2 were used at the same maximal amount (500 ng/well) to observe the stimulation by wild-type Rac2 (left panel) and enhanced basal activity of the PLC␥ 2 mutants (right panel).Twenty four hours after transfection, the cells were incubated for 20 h with myo- [2-3 H] inositol, and inositol phosphate formation was determined. B, homogenates from cells functionally analyzed in A were subjected to SDS-PAGE and immunoblotting using an antibody reactive against the c-Myc epitope. little if any stimulation (32). We therefore set out to examine the functional effects of the PLC␥ 2 R665W mutation in the PLC␥ 1 context. The two isozymes are very similar in the overall structure and amino acid sequence in the region close to the point mutation R665W in PLC␥ 2 and the corresponding residue, Arg-687, in PLC␥ 1 (cf. Fig. 8A, right panel, inset; PLC␥ 1 , LMRVPR 687 DGAFL; PLC␥ 2 LMRIPR 665 DGAFL, single divergent residue underlined). Fig. 8A, left panel, shows that overexpression of wild-type PLC␥ 1 and wild-type PLC␥ 2 led to only minor, if any, changes in basal inositol phosphate formation. Introduction of the point mutations R687W and R665W into PLC␥ 1 and PLC␥ 2 , respectively, led to no change in activity at lower expression levels. Only at the highest amount of cDNA used for transfection, 500 ng/well, an ϳ2-fold increase in activ-ity was evident in either case. Fig. 8A, right panel, shows that neither wild-type nor the R687W mutant PLC␥ 1 responded to wild-type Rac2, in contrast to the ϳ5.8-fold enhancement witnessed for the R665W mutation in PLC␥ 2 . Although PLC␥ 1 R687W exhibited an ϳ2.3-fold statistically significant (p Ͻ 0.01) stimulatory response to Rac2 G12V , this response was by far less prominent than the ϳ18.4-fold increase observed for PLC␥ 2 R665W . The enhanced basal activity of PLC␥ 1 R687W was insensitive to the Rac inhibitor EHT 1864 (Fig. 8B), unlike those of the PLC␥ 2 mutants associated with ibrutinib resistance, PLC␥ 2 R665W and PLC␥ 2 L845F (cf. Fig. 7A), indicating that the basal activity of PLC␥ 1 R687W is independent of Rac activity, despite its low but statistically significant sensitivity to exogenous Rac2 G12V . The latter was also evident, as described before (32), for wild-type PLC␥ 1 (Fig. 8A, right panel). Hypersensitivity to protein tyrosine phosphorylation offers a possible explanation for the increased basal activity of PLC␥ 1 R687W . Fig. 8C shows that although there were noticeable differences in the expression levels of the PLC␥ 1 versus PLC␥ 2 isozymes, in particular at low transfection levels, these differences did not explain the marked functional differences observed in Fig. 8A, right panel. There were no effects of the EHT compounds on the expression of wild-type or R687W mutant PLC␥ 1 or endogenous Rac in the experiment shown in Fig. 8, B and D. Next, wild-type and R665W mutant PLC␥ 2 were produced as recombinant polypeptides in baculovirus-infected insect cells and purified to near homogeneity by sequential column chromatography (Fig. 9A). The two purified preparations were adjusted to contain the same amounts of PLC␥ 2 by label-free quantitative mass spectrometry and then used for cell-free determination of inositol phosphate formation from artificial lipid vesicles containing radiolabeled PtdInsP 2 as a substrate. Fig. 9B, left panel, shows that wild-type and R665W mutant PLC␥ 2 displayed a similar dependence on free Ca 2ϩ for PtdInsP 2 hydrolysis under these conditions, with half-maximal and maximal hydrolysis occurring at ϳ1 and 20 M free Ca 2ϩ , respectively. Maximal activity was slightly (ϳ1.4-fold) higher for PLC␥ 2 R665W than for the wild-type enzyme. Upon functional reconstitution of the two PLC␥ 2 isoforms with isoprenylated Rac2 that had also been produced in and purified to near homogeneity from baculovirus-infected insect cells, the R665W mutant PLC␥ 2 exhibited a response to increasing concentrations of the poorly hydrolysable Rac2-activating guanine nucleotide analog GTP␥S that was clearly different from that of its wild-type counterpart. Specifically, GTP␥S showed a higher potency (EC 50 ϳ155 nM versus ϳ830 nM) and a higher efficacy (stimulation by ϳ57 versus ϳ21 pmol inositol phosphates ϫ min Ϫ1 ) to activate the R665W mutant PLC␥ 2 in comparison with its wild-type counterpart. Thus, functional differences between wild-type and R665W mutant PLC␥ 2 were particularly striking at limited activation of Rac2. A maximal, almost 7-fold difference in Rac2-stimulated activity was observed between wild-type and R665W mutant PLC␥ 2 at about 100 nM GTP␥S (cf. dotted lined in Fig. 9B, right panel).
COS-7 cells exhibit endogenous expression of EGF receptors, known to be coupled to activation of several intracellular signaling intermediates, including Rac (43). Upon heterologous expression of PLC␥ 2 in COS-7 cells, the enzyme is phosphorylated at tyrosine residues and translocated to the plasma membrane to mediate enhanced PtdInsP 2 hydrolysis (44,45). These previous findings led us to compare the activation of wild-type and L845F mutant PLC␥ 2 by endogenously expressed EGF receptors and to examine the relative contribution of tyrosine phosphorylation-mediated and Rac-mediated activation by also studying the two PLC␥ 2 isoforms carrying either replacements of four tyrosines known to be phosphorylated by upstream tyrosine kinases during enzyme activation by phenylalanines (4F) or the F897Q mutation blocking activation by Rac. Fig. 10A, left panel, shows that there was a concentrationdependent increase of wild-type and L845F mutant PLC␥ 2 stimulation by EGF, which was half-maximal at ϳ13 and 6.9 ng/ml EGF, respectively, and maximal at about 50 ng/ml in both cases. Maximal EGF-stimulated PLC␥ 2 activity was about 4.5fold higher in the presence of PLC␥ 2 L845F in comparison with wild-type PLC␥ 2 . The results obtained with the 4F, F897Q, and 4F/F897Q mutants of the two variants suggest that about half of the responses of both wild-type and L845F mutant PLC␥ 2 were due to tyrosine phosphorylation-and Rac-mediated activation. Similar findings were obtained for PLC␥ 2 R665W and its F897Q variant. Interestingly, PLC␥ 2 L845F was sensitive to activation by EGF even in the additional presence of the 4F and F897Q mutations. Wild-type and mutant PLC␥ 2 isozymes were present at equal amounts throughout the experiment shown in Fig. 10A,  left panel, and B. The ϳ12.5-fold enhancement of PLC␥ 2 L845Fmediated inositol phosphate formation by EGF was almost completely blocked (Ϫ95%) by the EGF receptor inhibitor cetuximab (Fig. 10A, right panel).
At least two ibrutinib-resistant patients have been described thus far harboring more than one PLCG2 mutations, including one with the coexistence of R665W and L845F (46). This prompted us to determine the effects of a compound R665W/ L845F mutation on the functions of PLC␥ 2 . Fig. 11A shows that basal activity of PLC␥ 2 R665W/L845F was much higher than that of either PLC␥ 2 R665W or PLC␥ 2 L845F , despite similar levels of protein expression (cf. Fig. 11D). Specifically, when 150 ng of DNA encoding mutant PLC␥ 2 was used per well for transfection, the enhancement was ϳ1.1-, 2.6-, and 70-fold over the activity observed for mock-transfected control cells for PLC␥ 2 R665W , PLC␥ 2 L845F , and PLC␥ 2 R665W/L845F , respectively. In addition to FIGURE 9. Purified PLC␥ 2 R665W displays a slight enhancement of basal activity and a marked increase in the sensitivity to purified Rac2 in a reconstituted system in vitro. A, recombinant wild-type PLC␥ 2 and PLC␥ 2 R665W were purified from baculovirus-infected insect cells. Aliquots of the two purified preparations were analyzed by label-free quantitative mass spectrometry to determine the relative abundance of PLC␥ 2 -specific tryptic peptides in the two purified protein preparations. Samples adjusted to contain the same quantities of wild-type and R665W mutant PLC␥ 2 were subjected to SDS-PAGE and Coomassie Blue staining. B, aliquots of the two samples analyzed in A containing equal quantities of purified recombinant wild-type PLC␥ 2 and PLC␥ 2 R665W were incubated at increasing concentrations of free Ca 2ϩ and 2.5 mM sodium deoxycholate with phospholipid vesicles containing [ 3 H]PtdInsP 2 . There was no difference between wild-type and R665W mutant PLC␥ 2 in the concentrations of free Ca 2ϩ required to observe half-maximal stimulatory effects (left panel). The EC 50 value of Ca 2ϩ for the stimulation of wild-type or mutant PLC␥ 2 activity obtained by non-linear curve fitting is shown above the graphs in nanomolar. In the right panel, equal quantities of purified recombinant wild-type PLC␥ 2 and PLC␥ 2 R665W were reconstituted with purified Rac2 in the presence of 30 nM free Ca 2ϩ and 1 mM sodium deoxycholate (32) and incubated at increasing concentrations of GTP␥S, as indicated at the abscissa, with phospholipid vesicles containing [ 3 H]PtdInsP 2 . The EC 50 values of GTP␥S for the stimulation of wild-type or mutant PLC␥ 2 activity obtained by non-linear curve fitting is shown above the graphs in nanomolar.

Discussion
The functions of PLC␥ 2 mutants mediating ibrutinib resistance in CLL patients have previously mostly been characterized following their reconstitution into PLC␥ 2 -deficient DT40 B cells (29). Even in those cells, expressing only the mutant PLC␥ 2 rather than a combination of wild-type and mutant PLC␥ 2 isozymes, there was no evidence of autonomous PLC␥ 2 signaling. Instead, the PLC␥ 2 mutants R665W and L845F still relied on BCR activation. The main change was an ϳ2-3-fold increase in the level of cytosolic Ca 2ϩ upon BCR ligation, which was insensitive to inhibition by ibrutinib and, interestingly, did not return to baseline within the time frame of the experiment. Subsequent experiments in reconstituted DT40 cells showed that BCR-mediated activation of PLC␥ 2 R665W was enhanced even in Btk Ϫ/Ϫ cells in comparison with the wild-type enzyme, suggesting that the mutant functionally bypasses Btk upon BCR activation (31). In cells expressing PLC␥ 2 R665W , the BCR-mediated increase in Ca 2ϩ was sensitive to pharmacologic inhibitors of Syk and Lyn, signaling components previously known to be essential for BCR-mediated InsP 3 generation and rapid Ca 2ϩ mobilization, respectively, in DT40 cells (47). These results suggested that the R665W mutation renders PLC␥ 2 independent of Btk and therefore capable of mediating ibrutinib resistance in CLL cells.
Using the same experimental model, reconstituted PLC␥ 2 Ϫ/Ϫ DT40 cells, we have previously shown that interaction of PLC␥ 2 with Rac amplifies the BCR-induced Ca 2ϩ signaling by increasing the sensitivity of the cells to BCR ligation, augmenting the BCR-mediated Ca 2ϩ release from intracellular stores, enhancing the Ca 2ϩ entry from the extracellular compartment, and facilitating the nuclear translocation of the Ca 2ϩ -regulated nuclear factor of activated T cells (34). Although performed in a different cellular system, the results presented here suggest that the bypass of Btk exploited by the PLC␥ 2 mutant R665W may also be based on an increased sensitivity of this and another mutant, L845F, to enhanced activation by Rac.
Our results showing marked increases in basal inositol phosphate formation upon expression of the two PLC␥ 2 mutants R665W and L845F at increasing levels, suggest, at first glance, that the mutants exhibit constitutively enhanced intrinsic activity. Given that constitutive protein activity is a hallmark of hypermorphic mutations (48), this is consistent with the designation of the two PLC␥ 2 mutations as belonging to the hypermorphic class (31). However, several lines of evidence presented in this work suggest that enhanced constitutive PLC activity, such as that observed for other mutants of several PLC isozymes (42), including PLC␥ 2 (49), is unlikely to be the main molecular mechanism of ibrutinib resistance. Specifically, both mutants are strikingly hypersensitive to activation by Rac2 and its upstream regulator Vav1, such that even wild-type Rac2 suffices to activate the mutant enzymes, but not their wild-type counterpart, upon its introduction into intact COS-7 cells (Fig.  3A). This view is strongly supported by the fact that enhanced basal activity of the mutant enzymes is markedly reduced by the F897Q mutation of PLC␥ 2 (Fig. 6A), which has previously been shown to block PLC␥ 2 activation by Rac but not by loss of SH-  (68). The data on PLC␥ 2 L845F and its mutants are from one experiment; the data on wild-type PLC␥ 2 and its mutants are from three experiments, each comparing the activity of wild-type PLC␥ 2 and one of the mutants. The latter activities were normalized to the maximal activity of PLC␥ 2 L845F/4F/F897Q used as an internal control in one of the latter experiments (shown as a fraction of 1.0 on the right y axis). The EC 50 values of EGF for the stimulation of wild-type or L845F mutant PLC␥ 2 activity obtained by non-linear curve fitting are shown above the graphs in nanograms/ml (left panel). In the right panel, COS-7 cells were transfected with 150 ng/well vector encoding PLC␥ 2 L845F (L845F) and then incubated as described above and treated for 60 min in the absence or presence of 20 g/ml cetuximab without or with 10 ng/ml EGF in medium containing 20 mM LiCl prior to determination of inositol phosphate formation. B, homogenates from cells functionally analyzed in A, left panel, were subjected to SDS-PAGE and immunoblotting using an antiserum reactive against PLC␥ 2 or antibody reactive against ␤-actin. OCTOBER 14, 2016 • VOLUME 291 • NUMBER 42 mediated autoinhibition or Ca 2ϩ (34). Further support stems from the observations that this activity is subject to specific inhibition by the small molecule Rac inhibitor EHT 1864 (Fig.  7A) and that the purified PLC␥ 2 R665W displays only a subtle increase of its basal activity (Fig. 9B). Hence, it appears likely that the mutants are hypersensitive to activated Rac2 rather than simply constitutively active.

Rac2 Hypersensitivity of PLC␥ 2 Ibrutinib Resistance Mutants
Several lines of evidence have been presented suggesting that Rac is activated by BCR ligation. Thus, several elements of the canonical BCR signaling cascade, e.g. Syk (50), Btk (51), and BLNK (52,53), are known to physically interact with and activate the Rac activator Vav, by processes not necessarily involving the protein kinase activity of Btk. BCR cross-linking caused activation of both Rac1 and Rac2 within minutes (54). Total internal reflection fluorescence microscopy has shown that PLC␥ 2 , Vav, BLNK, and Btk synergize to form highly coordinated microsignalosomes. Very interestingly, efficient assembly of the latter is absolutely dependent on Lyn and Syk (55). Therefore, it appears likely that one of the major functional consequences of the two PLC␥ 2 mutations conferring ibrutinib resistance to intact cells, including B lymphocytes, is hypersensitivity of PLC␥ 2 to activated Rac. The finding that the enhanced PLC␥ 2 stimulation by ligation of endogenous EGF receptors requires the replacement by phenylalanines of the tyrosine residues involved in enzyme activation in addition to a F897Q mutation to be maximally reduced (Fig. 10A, left panel) suggests that tyrosine phosphorylation may be involved, in addition to direct PLC␥ 2 -Rac interaction in mediating hyper- sensitivity of PLC␥ 2 to Rac. In intact B cells, this hypersensitivity is likely to be the molecular basis of a relatively focused rewiring of the signaling pathways immediately downstream of the BCR, such that PLC␥ 2 loses its dependence on activated Btk (56) and gains sensitivity to the pathway made up of Lyn, Syk, Vav, and Rac. Hence, in our opinion, the two PLC␥ 2 ibrutinib resistance mutations, R665W and L845F, are not simply and solely hypermorphic. We suggest that they would be better termed allomorphic, according to the Greek word ␣ for other, different.
In B cells, activation of Rac is not limited to BCR activation, but also occurs upon activation of BCR coreceptors, such as CD19/CD21, integrins, as well as certain G-protein-coupled chemokine and Toll-like receptors (cf. discussion and references in Ref. 34). It is thus possible that the rewiring process induced by the ibrutinib resistance mutations also enhances the sensitivity of PLC␥ 2 to extracellular ligands of these cell surface proteins, such as cleavage fragments of the third complement component, pathogen-derived molecules, extracellular matrix proteins, and chemokines. This may ultimately allow costimulatory signals to become stimulatory in their own right and as such to alter the interactions of the ibrutinib-resistant tumor cells with their protective microenvironments, for example (57). Interestingly, integrin-mediated adhesion and migration in response to the chemokines CXCL12 or CXCL13, as well as in vivo homing to lymphoid organs, was impaired in Btk-deficient (pre-)B cells, whereas CXCL12-mediated activation of Rac was intact. Deficiency of PLC␥ 2 also curtailed the CXCL12mediated migratory response (8). Independence of Btk and increased sensitivity of the PLC␥ 2 mutants to Rac by rewiring may provide CLL cells with the ability to home to and remain in protective microenvironments for survival and expansion even in the presence of ibrutinib-mediated Btk inhibition.
Enhanced sensitivity of PLC␥ 2 by signaling mechanisms emanating from BCR or other B cell surface receptors bypassing Btk may provide novel mechanisms for targeted treatment of CLL and, possibly, B cell lymphomas. Thus, inhibitors of Syk and Lyn have been shown to oppose ibrutinib resistance mediated by PLCG2 mutations (31). Although the exact position of PI3K␦ relative to other components within the BCR signalosome is still controversial, some evidence puts class IA PI3Ks, at least in part, upstream of Vav and Rac (58). Hence, inhibition of PI3K␦ with idelalisib may also interfere with Rac-mediated activation of wild-type and, even more so, R665W or L845F mutant PLC␥ 2 isozymes. Pharmacologic interventions at the level of Rac itself or other upstream activators also appear to be a viable option. These include inhibition of Rac C-terminal modification or of Rac protein-protein interaction, e.g. by small molecules like EHT 1864, or prevention of integrin, CD19, or chemokine receptor activation (59 -61). Very recent results suggest that ibrutinib therapy of CLL patients favors selection and expansion of rare subclones already present before ibrutinib treatment, including subclones containing mutations in PLCG2 (62). Hence, it may be worthwhile to investigate combining ibrutinib with adjuvant drugs of this type ab initio to suppress this selection and expansion.

Experimental Procedures
Materials-The mouse monoclonal antibody 9B11 reactive against the c-Myc epitope (EQKLISEEDL) and the rabbit polyclonal antiserum reactive against human PLC␥ 2 (sc-407) were obtained from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. The rabbit polyclonal antiserum reactive against human Rac2 (sc-96) was purchased from Santa Cruz Biotechnology. The anti-␤-actin antibody (clone AC-15) and the anti-Rac1 antibody (clone 23A8) were obtained from Sigma and Merck Millipore, respectively. The Rac inhibitor EHT 1864 and its inactive analog EHT 4063 were synthesized as described previously (63). Human epidermal growth factor (EGF) (E9644) was from Sigma. ProGreen baculovirus vector DNA (A1) was purchased from AB Vector. GTP␥S (catalog no. 10220647001) was purchased from Roche Applied Bioscience. Cetuximab is marketed by Merck.
Construction of Vectors-The construction of complementary DNAs encoding c-Myc epitope-tagged human PLC␥ 1 (1291 amino acids, accession number ABB84466), human PLC␥ 2 (1265 amino acids, accession number NP_002652), and F897Q mutant of PLC␥ 2 was described previously (34). The construction of all other vectors and of the baculoviruses was outlined in Refs. 32, 33. Complementary DNAs encoding mutants of PLC␥ 1 and PLC␥ 2 were constructed by in vitro mutagenesis using the QuikChange II XL site-directed mutagenesis kit (200521, Agilent Technologies). The primer sequences and PCR protocols are available from the authors upon request. A vector encoding c-Myc epitope-tagged human PLC␦ 1 ⌬44 was kindly supplied by J. Sondek (42).
Cell Culture and Transfection-COS-7 cells were maintained at 37°C in a humidified atmosphere of 90% air and 10% CO 2 in Dulbecco's modified Eagle's medium (DMEM) (catalog no. 41965-039, Gibco) supplemented with 10% (v/v) fetal calf serum (catalog no. 10270-106, Gibco), 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin (all from PAA Laboratories). Prior to transfection, COS-7 cells were seeded into 24-well plates at a density of 0.75 ϫ 10 5 cells/well and grown for 24 h in 0.5 ml of medium/well. For transfection, plasmid DNA (500 -800 ng/well) was diluted in 50 l of jetPRIME buffer, and 1-1.6 l of jetPRIME was added according to the manufacturer's instructions. The total amount of DNA was maintained constant by adding empty vector. Four hours after the addition of the DNA-jetPRIME complexes to the dishes, the medium was replaced by fresh medium, and the cells were incubated for a further 20 h at 37°C and 10% CO 2 .
Radiolabeling of Inositol Phospholipids and Analysis of Inositol Phosphate Formation-Twenty four hours after transfection, COS-7 cells were washed once with 0.3 ml/well Dulbecco's PBS (PAA Laboratories) and then incubated for 18 h in 0.2 ml/well DMEM containing supplements as described above, supplemented with 2.5 Ci/ml myo-[2-3 H]inositol (NET1156005MC, PerkinElmer Life Sciences) and 10 mM LiCl. The cells were then washed once with 0.2 ml/well of Dulbecco's PBS and lysed by addition of 0.2 ml/well 10 mM ice-cold formic acid. The analysis of inositol phosphate formation was performed as described previously (33).
To examine EGF-mediated PLC␥ 2 stimulation, COS-7 cells were radiolabeled for 24 h in serum-free DMEM as described previously (45). Briefly, cells were washed twice with 0.3 ml/well DMEM containing the above supplements except serum and then incubated for 24 h in 0.2 ml/well of the same medium supplemented with 0.25% fatty-acid-free bovine serum albumin (catalog no. A8806, Sigma) and 2.5 Ci/ml myo- [2-3 H]inositol. The cells were then washed with 0.3 ml/well Dulbecco's PBS and incubated for 1 h in 0.2 ml/well DMEM without serum containing the above supplements, 20 mM LiCl, and increasing concentrations of EGF. After removal of the medium, the cells were lysed by addition of 0.2 ml/well of 10 mM ice-cold formic acid for analysis of inositol phosphate formation.
Expression and Purification of Proteins-Post-translationally modified Rac2 was expressed as a glutathione S-transferase fusion protein in baculovirus-infected insect cells and solubilized from the particulate fraction. The Rac2 portion of the fusion protein was purified as detailed previously (33). c-Myc epitope-tagged PLC␥ 2 and PLC␥ 2 R665W were purified from soluble fractions of baculovirus-infected High Five TM insect cells grown in suspension culture by sequential chromatography on HiTrap TM Heparin HP and Resource Q (GE Healthcare) as described before for PLC␤ 2 ⌬ (64). Our attempts at purifying PLC␥ 2 L845F for functional analysis have thus far been unsuccessful, mostly due to the lower expression of the enzyme in baculovirus-infected insect cells and to its lower stability during purification.
Label-free Quantitative Mass Spectrometry-Equal volumes (5 l each) of purified wild-type or R665W mutant PLC␥ 2 were mixed with 300 fmol of the Pierce TM peptide retention time calibration mixture (PRTC, catalog no. 88320, ThermoFisher) containing 15 known synthetic tryptic peptides. Samples were reduced for 20 min at room temperature with 5 mM DTT and subsequently alkylated for 20 min at 37°C with 50 mM iodoacetamide. The samples were then subjected to tryptic digestion overnight at 37°C. The resulting peptides were identified by LC/MS analysis. Using the SEQUEST search engine within the Proteome Discoverer TM software suite (1.4.1.14, Thermo Scientific), mass spectra were correlated with a database containing the sequences of wild-type and mutant PLC␥ 2 , a concatenation of the PRTC peptide sequences, and sequences of common contaminants commonly encountered in proteomic experiments, as used in the MaxQuant software (65). For relative quantitation, the precursor ions area detector node within Proteome Discoverer TM was used; preceding event detection was set to 4 ppm.
Measurement of PLC Activity in Vitro-Phospholipase C activity was determined as described (64,66) with minor modifications. In brief, aliquots (10 l) of purified PLC␥ 2 proteins appropriately diluted in buffer containing 60 mM Tris/maleate, pH 7.3, 84 mM KCl, 3.6 mM EGTA, 2.4 mM dithiothreitol, 2 mg/ml bovine serum albumin were incubated for 45 min at 30°C in a volume of 60 l containing 50 mM Tris/maleate, pH 7.3, 70 mM KCl, 3 mM EGTA, 2 mM dithiothreitol, 536 M phosphatidylethanolamine, 33.4 M [ 3 H]PtdInsP 2 (185 GBq/ mmol), 0.33 mg/ml bovine serum albumin, and the concentrations of sodium deoxycholate and free Ca 2ϩ specified in the figure legends. For reconstitution of wild-type and mutant PLC␥ 2 with Rac2, purified PLC␥ 2 was reconstituted with 5 l of purified isoprenylated Rac2 and incubated with the phospholipid substrate as described above. The concentration of CaCl 2 required to adjust the concentration of free Ca 2ϩ to the desired value was calculated using the program EqCal for Windows (Biosoft, Ferguson, MO). The reaction was terminated, and the samples were analyzed for inositol phosphates, as described (64).
Miscellaneous-SDS-PAGE and immunoblotting were performed according to standard protocols using antibodies reactive against the c-Myc epitope for wild-type and mutant PLC␥ 2 . Immunoreactive proteins were visualized using the ECL Western blotting detection system (GE Healthcare). All experiments were performed at least three times. Similar results and identical trends were obtained each time. Data from representative experiments are shown as means Ϯ S.E. of triplicate determinations. In Figs. 2A, 3A, 4A, 7A, 9B, 10A, and 11C, the data were fitted by nonlinear least squares curve fitting to three-or fourparameter dose-response equations using GraphPad Prism, version 5.04. In certain cases, the global curve fitting procedure contained in Prism was used to determine whether the best fit values of selected parameters differed between data sets. The simpler model was selected unless the extra sum of squares F-test had a p value of less than 0.05. Repeated measures analysis of variance with Tukey's post test contained in the GraphPad InStat software package (version 3.10; GraphPad Software, La Jolla, CA) was used for the statistical analysis of the data shown in Figs. 1A and 8A. Statistically significant effects are denoted by ***, p Ͻ 0.001; **, p Ͼ 0.001 and p Ͻ 0.01; and *, p Ͼ 0.01 and p Ͻ 0.05. Non-significant changes are denoted by ns, p Ͻ 0.05.
Author Contributions-C. W., E. H., A. S., S. W., J. D., and M. Z. performed the experiments and analyzed the data. P. G. provided overall direction and wrote the manuscript with input from L. D., D. M., S. S., and the other authors.