The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein
Open accessAbstract
Mutations in the gene encoding phospholipase C-γ 2 (PLCγ 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 PLCγ 2 an ∼2–3-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 PLCγ 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 PLCγ 2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLCγ 2 F897Q mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLCγ 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 PLCγ 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.
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Written from the paper’s full text.
TL;DR
The PLCγ2 mutants R665W and L845F, found in ibrutinib-resistant CLL, are not simply constitutively active but are hypersensitive to activation by Rac2, including wild-type Rac2. The authors propose calling them allomorphic rather than hypermorphic mutations.
Key findings
- R665W and L845F caused marked basal inositol phosphate increases in COS-7 cells, up to 18-fold, whereas wild-type PLCγ2 and PLCγ2 M28L showed only slight 1.4-fold increases.
- The mutants were hypersensitive to Rac2: Rac2 G12V gave maximal increases of ~6.7-fold for R665W and ~35-fold for L845F, with potency increases of ~4.5- and ~6.5-fold; wild-type Rac2 activated R665W and L845F up to 5.1- and 4.2-fold but had no effect on wild-type PLCγ2.
- The enhanced basal activity was Rac-dependent: F897Q reduced basal R665W activity by ~74% and R665W/L845F variants by ~68% and ~78%; EHT 1864 inhibited basal R665W/L845F activity by ~55%, with IC50 ~1 µM, while the inactive analog EHT 4063 had no effect.
- Purified R665W PLCγ2 showed only slightly higher maximal Ca2+-dependent activity (~1.4-fold) but much greater Rac2 sensitivity: GTPγS EC50 ~155 nM versus ~830 nM, efficacy ~57 versus ~21 pmol inositol phosphates·min−1, and an almost 7-fold difference at ~100 nM GTPγS. The compound R665W/L845F mutant showed ~70-fold basal enhancement at 150 ng DNA, versus ~1.1-fold for R665W and ~2.6-fold for L845F.
Methods
- COS-7 cells were transfected with wild-type or mutant PLCγ2, Rac2, Rac2 G12V, Vav1ΔN, and related constructs; inositol phospholipids were radiolabeled with myo-[2-3H]inositol, and inositol phosphate formation was measured after LiCl treatment.
- Recombinant wild-type PLCγ2 and PLCγ2 R665W, plus isoprenylated Rac2, were expressed in baculovirus-infected insect cells, purified, adjusted by label-free quantitative mass spectrometry, and assayed for PtdInsP2 hydrolysis in lipid vesicles at controlled free Ca2+ and GTPγS concentrations.
- Additional experiments used EGF receptor stimulation, cetuximab, EHT 1864/EHT 4063, immunoblotting, and nonlinear curve fitting/statistical analysis; experiments were performed at least three times unless otherwise noted.
Limitations
- The paper states that attempts to purify PLCγ2 L845F for functional analysis were unsuccessful, mostly due to lower expression in baculovirus-infected insect cells and lower stability during purification.
- The figure legend for the EGF experiments states that data on PLCγ2 L845F and its mutants are from one experiment, while wild-type PLCγ2 data are from three experiments.
- The paper does not include a formal limitations section; evident from the text, most mechanistic conclusions rely on overexpression in COS-7 cells rather than primary CLL cells, and proposed therapeutic implications for CLL are largely speculative.
Model-written summary. Check it against the paper before relying on it.
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Claims the paper makes
17 of 28 cited sources checked · 11 supported · 0 flagged · accuracy not yet measured
- Premisecitation
Mutations in PLCG2 are associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib.
“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.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [29] Woyach, 2014, Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib, N. Engl. J. Med source
- Premisecitation
Inhibitors of Syk and Lyn have been shown to oppose ibrutinib resistance mediated by PLCG2 mutations.
“Thus, inhibitors of Syk and Lyn have been shown to oppose ibrutinib resistance mediated by PLCG2 mutations (31).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [31] Liu, 2015, Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation, Blood source
- Premisecitation
Btk-independent activation of the overexpressed R665W PLCγ2 mutant after B cell receptor engagement in Btk-deficient DT40 cells suggests Btk independency of this mutant.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [31] Liu, 2015, Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation, Blood source
- Premisecitation
Ibrutinib therapy of CLL patients favors selection and expansion of rare subclones already present before ibrutinib treatment, including subclones containing mutations in PLCG2.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [62] Burger, 2016, Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition, Nat. Commun source
- Findingcitation
The predicted structures of the wild-type and mutant SH2c domains of PLCγ1 and PLCγ2 and the localizations of residues 687 (γ1) and 665 (γ2) are virtually identical to each other.
“The predicted structures of the wild-type and mutant SH2c domains of PLC␥ 1 and PLC␥ 2 and the localizations of the residues in positions 687 (␥ 1 ) and 665 (␥ 2 ) are virtually identical to each other.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [67] Bunney, 2012, Structural and functional integration of the PLCγ interaction domains critical for regulatory mechanisms and signaling deregulation, Structure source
The Results sections describe crystal structures of PLCγ1 SH2 domains and experiments with PLCγ1 mutants, but do not compare predicted wild-type and mutant cSH2 structures across PLCγ1 and PLCγ2. Searches for residues 687 and 665 found no passage establishing their corresponding locations or the claimed structural similarity. No page numbers are available in our copy. (LLM, conf=0.75)
Experimental — accuracy not yet measured
- Findingown data
Enhanced basal activity of the PLCγ2 mutant enzymes is markedly reduced by the F897Q mutation of PLCγ2.
“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)”
Quote in the paperSubstantial claim - Findingown data
The increase in Rac2-stimulated PLC activity caused by the PLCγ2 mutations was not caused by changes in PLCγ2 protein production in transfected cells.
“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).”
Quote in the paperSubstantial claim - Findingown data
Upon functional reconstitution with isoprenylated Rac2, R665W mutant PLCγ2 exhibited a response to increasing concentrations of GTPγS clearly different from that of its wild-type counterpart.
“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”
Quote in the paperSubstantial claim - Findingown data
Basal activity of PLCγ2 R665W/L845F was much higher than that of either PLCγ2 R665W or PLCγ2 L845F, despite similar levels of protein expression.
“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).”
Quote in the paperSubstantial claim - Findingown data
Inositol phosphate formation in the presence of Vav1ΔN was enhanced about 1.2-fold, 3.1-fold, 9.9-fold, and 61-fold for wild-type PLCγ2, PLCγ2 R665W, PLCγ2 L845F, and PLCγ2 R665W/L845F when compared with expression of Vav1ΔN alone.
“For example, inositol phosphate formation in the presence of Vav1⌬N was enhanced ϳ1.2-, 3.1-, 9.9-, and 61-fold for wild-type PLC␥ 2 , PLC␥ 2 R665W , PLC␥ 2 L845F , and PLC␥ 2 R665W/L845F when compared with expression of Vav1⌬N alone (Fig. 11C, right panel).”
Quote in the paperSubstantial claim - Findingown data
The abilities of wild-type and constitutively active Rac2 to cause enhanced activation of PLCγ2 R665W in comparison with wild-type PLCγ2 were dependent on the C-terminal isoprenylation of the exogenous Rac2 proteins.
“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).”
Quote in the paperSubstantial claim - Findingown data
The enhanced PLCγ2 stimulation by ligation of endogenous EGF receptors requires replacement by phenylalanines of the tyrosine residues involved in enzyme activation in addition to a F897Q mutation to be maximally reduced.
“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)”
Quote in the paperSubstantial claim - Findingown data
The inhibitory effect of EHT 1864 on basal inositol phosphate formation by PLCγ2 L845F was concentration-dependent with an IC50 of about 1 μM.
“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)”
Quote in the paperSubstantial claim - Findingown data
No effect of EHT 1864 was observed in the absence of exogenous PLC isozyme and in the presence of PLCδ1Δ44, a constitutively active variant of PLCδ1.
“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 .”
Quote in the paperSubstantial claim - Findingown data
Wild-type and R665W mutant PLCγ2 displayed similar dependence on free Ca2+ for PtdInsP2 hydrolysis, with half-maximal and maximal hydrolysis occurring at about 1 and 20 μM free Ca2+, respectively.
“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.”
Quote in the paperNot a substantial claim - Findingown data
EHT 1864, but not EHT 4063, caused a clear (about 55%) inhibition of basal inositol phosphate formation by PLCγ2 R665W and PLCγ2 L845F.
“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 .”
Quote in the paperSubstantial claim - Findingown data
The about 12.5-fold enhancement of PLCγ2 L845F-mediated inositol phosphate formation by EGF was almost completely blocked (−95%) by the EGF receptor inhibitor cetuximab.
“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).”
Quote in the paperNot a substantial claim - Findingown data
The two ibrutinib resistance mutations imparted on PLCγ2 a marked increase in responsiveness to stimulation by Vav1ΔN, about 9.3-fold for R665W and about 12-fold for L845F.
“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.”
Quote in the paperSubstantial claim - Findingown data
Basal phospholipase C activity, Rac2-, Rac2 G12V-, and Vav1ΔN-stimulated activity was markedly enhanced for PLCγ2 R665W/L845F in comparison with the other two mutants.
“basal phospholipase C activity, Rac2-, Rac2 G12V -, and Vav1⌬ N-stimulated activity was also markedly enhanced for PLC␥ 2 R665W/L845F in comparison with the other two mutants (Fig. 11, B and C).”
Quote in the paperSubstantial claim - Findingown data
The compound mutation also markedly enhanced the apparent potency of Rac2 G12V and Vav1ΔN to activate PLCγ2.
“The compound mutation also markedly enhanced the apparent potency of Rac2 G12V and Vav1⌬N to activate PLC␥ 2 .”
Quote in the paperSubstantial claim - Findingown data
Maximal EGF-stimulated PLCγ2 activity was about 4.5-fold higher in the presence of PLCγ2 L845F compared with wild-type PLCγ2.
“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 .”
Quote in the paperSubstantial claim - Findingown data
Only slight, 1.4-fold, increases in basal inositol phosphate formation were apparent at the highest amounts of wild-type PLCγ2 and PLCγ2 M28L.
“Only slight, 1.4-fold, increases were apparent at the highest amounts of wild-type and PLC␥ 2 M28L .”
Quote in the paperSubstantial claim - Findingown data
There was a smaller (about 25%) not quite statistically significant (p = 0.0676) inhibitory effect of EHT 1864 for wild-type PLCγ2.
“There was a smaller (ϳ25%) not quite statistically significant (p ϭ 0.0676) inhibitory effect for wild-type PLC␥ 2 .”
Quote in the paperNot a substantial claim - Findingown data
A maximal, almost 7-fold difference in Rac2-stimulated activity was observed between wild-type and R665W mutant PLCγ2 at about 100 nM GTPγS.
“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).”
Quote in the paperSubstantial claim - Findingown data
Purified PLCγ2 R665W displays only a subtle increase of its basal activity.
“and that the purified PLC␥ 2 R665W displays only a subtle increase of its basal activity (Fig. 9B).”
Quote in the paperSubstantial claim - Findingown data
The inactivating Vav1 mutation D376A caused an almost complete (about 85%) loss and a complete loss in the stimulatory effect of Vav1ΔN on the activity of R665W and L845F, respectively.
“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.”
Quote in the paperSubstantial claim - Findingown data
PLCγ2 L845F was sensitive to activation by EGF even in the additional presence of the 4F and F897Q mutations.
“Interestingly, PLC␥ 2 L845F was sensitive to activation by EGF even in the additional presence of the 4F and F897Q mutations.”
Quote in the paperNot a substantial claim - Findingown data
The enhanced basal activity of the PLCγ2 mutants is subject to specific inhibition by the small molecule Rac inhibitor EHT 1864.
“Further support stems from the observations that this activity is subject to specific inhibition by the small molecule Rac inhibitor EHT 1864 (Fig. 7A)”
Quote in the paperSubstantial claim - Findingown data
The F897Q mutation caused similar reductions of basal activity of the R665W and L845F variants of PLCγ2, about 68% and about 78%, respectively.
“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).”
Quote in the paperNot a substantial claim - Findingown data
The two PLCγ2 mutants R665W and L845F 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.
“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.”
Quote in the paperSubstantial claim - Findingown data
Expression of PLCγ2 mutants R665W and L845F at increasing levels causes marked increases in basal inositol phosphate formation.
“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.”
Quote in the paperSubstantial claim - Findingown data
In COS-7 cells, R665W and L845F caused marked, up to 18-fold, increases in basal inositol phosphate formation when expressed in increasing amounts, in contrast to wild-type PLCγ2 and PLCγ2 M28L.
“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”
Quote in the paperSubstantial claim - Findingcitation
The stimulatory effect on PLCγ2 R665W activity was completely lost upon replacement of the cysteine residue at position -4 from the Rac2 C terminus by a serine residue, C189S.
“Specifically, their stimulatory effect on PLC␥ 2 R665W activity was completely 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).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [38] Gutierrez, 1989, Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis, EMBO J source
- Findingown data
Overexpression of wild-type PLCγ1 and wild-type PLCγ2 led to only minor, if any, changes in basal inositol phosphate formation.
“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.”
Quote in the paperNot a substantial claim - Findingown data
There is no difference between wild-type and R665W mutant PLCγ2 in the concentrations of free Ca2+ required to observe half-maximal stimulatory effects.
“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).”
Quote in the paperNot a substantial claim - Findingown data
Purified PLCγ2 R665W displays a slight enhancement of basal activity in a reconstituted system in vitro.
“Purified PLC␥ 2R665W displays a slight enhancement of basal activity and a marked increase in the sensitivity to purified Rac2 in a reconstituted system in vitro.”
Quote in the paperNot a substantial claim - Findingown data
PLCγ2 ibrutinib resistance mutations R665W and L845F synergize to enhance basal enzyme activity.
“PLC␥ 2 ibrutinib resistance mutations R665W and L845F synergize to enhance basal enzyme activity and to sensitize the enzyme to stimulation by activated Vav1 and Rac2.”
Quote in the paperSubstantial claim - Findingown data
PLCγ2 L845F is hypersensitive to activation by EGF receptor(s) endogenously expressed in COS-7 cells.
“PLC␥ 2 L845F is hypersensitive to activation by EGF receptor(s) endogenously expressed in COS-7 cells by a mechanism dependent on both protein tyrosine phosphorylation and activation by Rac.”
Quote in the paperSubstantial claim - Findingown data
PLCγ2 ibrutinib resistance mutations R665W and L845F synergize to sensitize the enzyme to stimulation by activated Vav1.
“PLC␥ 2 ibrutinib resistance mutations R665W and L845F synergize to enhance basal enzyme activity and to sensitize the enzyme to stimulation by activated Vav1 and Rac2.”
Quote in the paperSubstantial claim - Findingown data
PLCγ1 R687W, corresponding to PLCγ2 R665W, causes enhanced basal activity of PLCγ1 in intact cells.
“Point mutation in PLC␥ 1 , corresponding to R665W in PLC␥ 2 , R687W, causes enhanced basal activity of PLC␥ 1 in intact cells that is insensitive to the Rac inhibitor EHT 1864.”
Quote in the paperNot a substantial claim - Findingown data
The enhanced basal activity of PLCγ1 caused by R687W is insensitive to the Rac inhibitor EHT 1864.
“Point mutation in PLC␥ 1 , corresponding to R665W in PLC␥ 2 , R687W, causes enhanced basal activity of PLC␥ 1 in intact cells that is insensitive to the Rac inhibitor EHT 1864.”
Quote in the paperSubstantial claim - Findingown data
PLCγ2 ibrutinib resistance mutations R665W and L845F synergize to sensitize the enzyme to stimulation by activated Rac2.
“PLC␥ 2 ibrutinib resistance mutations R665W and L845F synergize to enhance basal enzyme activity and to sensitize the enzyme to stimulation by activated Vav1 and Rac2.”
Quote in the paperSubstantial claim - Findingown data
Purified PLCγ2 R665W displays a marked increase in sensitivity to purified Rac2 in a reconstituted system in vitro.
“Purified PLC␥ 2R665W displays a slight enhancement of basal activity and a marked increase in the sensitivity to purified Rac2 in a reconstituted system in vitro.”
Quote in the paperSubstantial claim - Interpretationown data
Enhanced constitutive PLC activity is unlikely to be the main molecular mechanism of ibrutinib resistance for the R665W and L845F mutants.
“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.”
Quote in the paperSubstantial claim - Interpretationown data
About half of the responses of both wild-type and L845F mutant PLCγ2 to EGF were due to tyrosine phosphorylation- and Rac-mediated activation.
“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.”
Quote in the paperSubstantial claim - Interpretationreasoning
One of the major functional consequences of the two PLCγ2 mutations conferring ibrutinib resistance to intact cells, including B lymphocytes, is hypersensitivity of PLCγ2 to activated Rac.
“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.”
Quote in the paperSubstantial claim- Rests on an assumption: The behavior of PLCγ2 mutants in COS-7 cells reflects their behavior in intact B cells/CLL cells.
- Rests on an assumption: Rac2 is the relevant Rac isoform for PLCγ2 hypersensitivity in intact B cells.
- Premisecitation
In cells expressing PLCγ2 R665W, the BCR-mediated increase in Ca2+ was sensitive to pharmacologic inhibitors of Syk and Lyn.
“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).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [47] Takata, 1994, Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways, EMBO J source
- Interpretationreasoning
The stimulatory effects of the two mutations on the Rac-PLCγ2 interaction may contribute to the mechanism(s) of ibrutinib resistance caused by mutations in PLCG2 rather than BTK.
“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.”
Quote in the paperSubstantial claim - Interpretationreasoning
The PLCγ2 mutants are hypersensitive to activated Rac2 rather than simply constitutively active.
“Hence, it appears likely that the mutants are hypersensitive to activated Rac2 rather than simply constitutively active.”
Quote in the paperSubstantial claim- Rests on an assumption: Rac2 is the relevant Rac isoform for PLCγ2 hypersensitivity in intact B cells.
- Interpretationown data
The basal activities of the R665W and L845F mutants are strongly dependent on Rac1 endogenously present in COS-7 cells.
“This indicates that the basal activities of the R665W and L845F mutants are strongly dependent on Rac1 endogenously present in COS-7 cells.”
Quote in the paperSubstantial claim - Interpretationreasoning
The bypass of Btk exploited by PLCγ2 mutant R665W may also be based on increased sensitivity of R665W and L845F to enhanced activation by Rac.
“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.”
Quote in the paperSubstantial claim- Rests on an assumption: The behavior of PLCγ2 mutants in COS-7 cells reflects their behavior in intact B cells/CLL cells.
- Interpretationown data
Tyrosine phosphorylation may be involved, in addition to direct PLCγ2-Rac interaction, in mediating hypersensitivity of PLCγ2 to Rac.
“suggests that tyrosine phosphorylation may be involved, in addition to direct PLC␥ 2 -Rac interaction in mediating hyper- sensitivity of PLC␥ 2 to Rac.”
Quote in the paperSubstantial claim - Interpretationown data
The hypersensitivity of PLCγ2 L845F to activation by EGF receptors in COS-7 cells depends on activation by Rac.
“PLC␥ 2 L845F is hypersensitive to activation by EGF receptor(s) endogenously expressed in COS-7 cells by a mechanism dependent on both protein tyrosine phosphorylation and activation by Rac.”
Quote in the paperSubstantial claim - Interpretationown data
The hypersensitivity of PLCγ2 L845F to activation by EGF receptors in COS-7 cells depends on protein tyrosine phosphorylation.
“PLC␥ 2 L845F is hypersensitive to activation by EGF receptor(s) endogenously expressed in COS-7 cells by a mechanism dependent on both protein tyrosine phosphorylation and activation by Rac.”
Quote in the paperSubstantial claim
- May combine two claims. 22
- Classified as probably not a substantial claim. 18
May combine two claims.
PremisecitationWhole-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: L845F, R665W, and S707Y in one patient with tumor cells also harboring a BTK C481S mutation, and PLCG2 R665W as the sole mutation in the other patient.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [29] Woyach, 2014, Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib, N. Engl. J. Med source
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationRac-mediated stimulation of PLCγ2 amplifies BCR-mediated Ca2+ signaling.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [34] Walliser, 2015, Rac-mediated stimulation of phospholipase Cγ2 amplifies B cell receptor-induced calcium signaling, J. Biol. Chem source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationInteraction of PLCγ2 with Rac amplifies BCR-induced Ca2+ signaling by increasing sensitivity to BCR ligation, augmenting BCR-mediated Ca2+ release from intracellular stores, enhancing Ca2+ entry from the extracellular compartment, and facilitating nuclear translocation of the Ca2+-regulated nuclear factor of activated T 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”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [34] Walliser, 2015, Rac-mediated stimulation of phospholipase Cγ2 amplifies B cell receptor-induced calcium signaling, J. Biol. Chem source
May combine two claims.
PremisecitationBCR-mediated activation of PLCγ2 R665W was enhanced even in Btk-/- cells compared with the wild-type enzyme, suggesting that the mutant functionally bypasses Btk upon BCR activation.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [31] Liu, 2015, Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation, Blood source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSeveral elements of the canonical BCR signaling cascade, e.g. Syk, Btk, and BLNK, are known to physically interact with and activate the Rac activator Vav, by processes not necessarily involving the protein kinase activity of Btk.
“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.”
Quote in the paperNot a substantial claim1 of 4 sources checked- cites [50] Deckert, 1996, Functional and physical interactions of Syk family kinases with the Vav proto-oncogene product, Immunity source
- cites [51] Guinamard, 1997, The SH3 domain of Bruton's tyrosine kinase interacts with Vav, Sam68 and EWS, Scand. J. Immunol source
- cites [52] Fu, 1998, BLNK: a central linker protein in B cell activation, Immunity source
- cites [53] Wienands, 1998, SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation, J. Exp. Med source
The Results and Discussion (page not provided) report that SLP-65 coprecipitates with Vav and is constitutively associated with it, supporting the physical-interaction part of the BLNK citation. The paper does not show that SLP-65 activates Vav. The separate claim about Btk kinase activity is attached to the Btk citation, not specifically to this source. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationIn B cells, activation of Rac is not limited to BCR activation but also occurs upon activation of BCR coreceptors such as CD19/CD21, integrins, and certain G-protein-coupled chemokine and Toll-like receptors.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [34] Walliser, 2015, Rac-mediated stimulation of phospholipase Cγ2 amplifies B cell receptor-induced calcium signaling, J. Biol. Chem source
The discussion (page unavailable in our copy) says that receptors for integrins, chemokines, and pathogen-derived ligands can activate Rac in B cells; it specifically discusses CXCR4/CXCR5 and TLR4. It discusses CD19/CD21 coreceptor signaling only as a possible setting for Rac–PLCγ2 activity, not as evidence that activation of CD19/CD21 activates Rac. (LLM, conf=0.86)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationThe R665W and L845F mutants of PLCγ2 appeared functionally normal in reconstituted DT40 chicken B cells in the absence of BCR stimulation, but mediated moderately enhanced and markedly prolonged ibrutinib-resistant increases in [Ca2+]i following BCR ligation with anti-IgM.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [29] Woyach, 2014, Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib, N. Engl. J. Med source
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured
Classified as probably not a substantial claim. May combine two claims.
PremisecitationPLCγ2 is specifically activated by Rac GTPases by a mechanism independent of PLCγ2 tyrosine phosphorylation, but dependent on direct interaction of activated Rac with the bipartite split PH domain (spPH) juxtaposed between the two halves, X and Y, of the PLCγ2 catalytic domain.
“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).”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [32] Piechulek, 2005, Isozyme-specific stimulation of phospholipase C-γ2 by Rac GTPases, J. Biol. Chem source
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured - cites [33] Walliser, 2008, Rac regulates its effector phospholipase Cγ2 through interaction with a split pleckstrin homology domain, J. Biol. Chem source
May combine two claims.
PremisecitationWhen R665W was expressed in PLCγ2-deficient DT40 cells containing endogenous wild-type Btk, BCR-mediated PLCγ2 activation was resistant to ibrutinib but sensitive to pharmacologic inhibitors of Syk and Lyn.
“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.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [31] Liu, 2015, Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation, Blood source
Classified as probably not a substantial claim.
PremisecitationEHT 1864 is a Rac inhibitor.
“Rac inhibitor EHT 1864”
Quote in the paperSubstantial claim1 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationEfficient assembly of the microsignalosomes is absolutely dependent on Lyn and Syk.
“Very interestingly, efficient assembly of the latter is absolutely dependent on Lyn and Syk (55).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [55] Weber, 2008, Phospholipase C-γ2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen, J. Exp. Med source
May combine two claims.
Findingown dataThere was no difference between wild-type and M28L mutant PLCγ2 in their stimulatory responses to constitutively active Rac2 G12V, indicating that PLCγ2 M28L did not harbor a defect in enzyme activation.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe enhanced basal activity of PLCγ1 R687W was insensitive to the Rac inhibitor EHT 1864, unlike the PLCγ2 mutants associated with ibrutinib resistance, PLCγ2 R665W and PLCγ2 L845F.
“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)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIntroduction of R687W into PLCγ1 and R665W into PLCγ2 led to no change in activity at lower expression levels; only at the highest amount of cDNA used for transfection, 500 ng/well, an about 2-fold increase in activity was evident in either case.
“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.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataWhen 150 ng of DNA encoding mutant PLCγ2 was used per well for transfection, the enhancement was about 1.1-fold, 2.6-fold, and 70-fold over mock-transfected control cells for PLCγ2 R665W, PLCγ2 L845F, and PLCγ2 R665W/L845F, respectively.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataEnhanced sensitivity of PLCγ2 R665W and PLCγ2 L845F to Rac2 was not limited to constitutively active Rac2 G12V but was also observed for wild-type Rac2.
“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).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe two point mutations caused an increase in the potency of Rac2 G12V, about 4.5-fold for PLCγ2 R665W and about 6.5-fold for PLCγ2 L845F.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataNeither wild-type nor R687W mutant PLCγ1 responded to wild-type Rac2, in contrast to the about 5.8-fold enhancement witnessed for the R665W mutation in PLCγ2.
“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 .”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe F897Q mutation caused a complete or near complete loss of activation of wild-type PLCγ2 by Rac2 G12V and of PLCγ2 R665W by wild-type and G12V mutant Rac2.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe mutations increased the apparent potency of Vav1ΔN, about 5.3-fold for PLCγ2 R665W and about 5.7-fold for PLCγ2 L845F.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAlthough increasing amounts of Rac2 had no effect on the activity of wild-type PLCγ2, the mutants R665W and L845F were activated up to 5.1-fold and 4.2-fold, respectively.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGTPγS showed higher potency (EC50 about 155 nM versus about 830 nM) and higher efficacy (stimulation by about 57 versus about 21 pmol inositol phosphates × min−1) to activate R665W mutant PLCγ2 compared with wild-type.
“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.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataMaximal activity was slightly (about 1.4-fold) higher for PLCγ2 R665W than for the wild-type enzyme.
“Maximal activity was slightly (ϳ1.4-fold) higher for PLC␥ 2 R665W than for the wild-type enzyme.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataBoth PLCγ2 mutants R665W and L845F 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 introduction into intact COS-7 cells.
“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).”
Quote in the paperSubstantial claim- Rests on an assumption: Rac2 is the relevant Rac isoform for PLCγ2 hypersensitivity in intact B cells.
May combine two claims.
Findingown dataThe maximal increase in Rac2 G12V efficacy was about 6.7-fold for PLCγ2 R665W and about 35-fold for PLCγ2 L845F.
“Specifically, the maximal increase in Rac2 G12V efficacy was ϳ6.7and 35-fold for PLC␥ 2 R665W and PLC␥ 2 L845F , respectively.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe basal activity of PLCγ1 R687W is independent of Rac activity, despite its low but statistically significant sensitivity to exogenous Rac2 G12V.
“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 .”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationSome evidence puts class IA PI3Ks, at least in part, upstream of Vav and Rac.
“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).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [58] Hawkins, 2006, Signalling through Class I PI3Ks in mammalian cells, Biochem. Soc. Trans source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationS707Y had previously been reported as a constitutively activating mutation in the dominantly inherited human disease APLAID.
“Whereas S707Y had previously been reported as a constitutively activating mutation in the dominantly inherited human disease APLAID”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [30] Zhou, 2012, A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency, Am. J. Hum. Genet source
May combine two claims.
InterpretationreasoningThe R665W mutation renders PLCγ2 independent of Btk and therefore capable of mediating ibrutinib resistance in CLL cells.
“These results suggested that the R665W mutation renders PLC␥ 2 independent of Btk and therefore capable of mediating ibrutinib resistance in CLL cells.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
Study design and conductcitationBackground inositol phosphate formation in response to EGF was determined in parallel on empty-vector-transfected cells and subtracted from individual values, with consideration of error propagation.
“Background inositol phosphate formation in response to addition of EGF at increasing concentrations was determined in parallel on cells transfected with empty vector and subtracted from the individual values, with appropriate consideration of error propagation (68).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [68] Meyer, 1975
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe simpler model was selected unless the extra sum of squares F-test had a p value of less than 0.05.
“The simpler model was selected unless the extra sum of squares F-test had a p value of less than 0.05.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationBCR cross-linking caused activation of both Rac1 and Rac2 within minutes.
“BCR cross-linking caused activation of both Rac1 and Rac2 within minutes (54).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [54] Arana, 2008, Activation of the small GTPase Rac2 via the B cell receptor regulates B cell adhesion and immunological-synapse formation, Immunity source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenData from representative experiments are shown as means ± S.E. of triplicate determinations.
“Data from representative experiments are shown as means Ϯ S.E. of triplicate determinations.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenThe data on PLCγ2 L845F and its mutants are from one experiment; the data on wild-type PLCγ2 and its mutants are from three experiments, each comparing wild-type PLCγ2 and one mutant.
“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.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenIn 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.
“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.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationDeficiency of PLCγ2 curtailed the CXCL12-mediated migratory response.
“Deficiency of PLC␥ 2 also curtailed the CXCL12mediated migratory response (8).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [8] de Gorter, 2007, Bruton's tyrosine kinase and phospholipase Cγ2 mediate chemokine-controlled B cell migration and homing, Immunity source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataAttempts at purifying PLCγ2 L845F for functional analysis have been unsuccessful, mostly due to lower expression in baculovirus-infected insect cells and lower stability during purification.
“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.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The behavior of PLCγ2 mutants in COS-7 cells reflects their behavior in intact B cells/CLL cells.
“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.”
The paper relies on this without support.
Rac2 is the relevant Rac isoform for PLCγ2 hypersensitivity in intact B cells.
“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).”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The missense PLCG2 M28L variant (rs61749044) is associated with increased Alzheimer's disease risk.
“Here, we identified a loss-of-function PLCG2 variant, PLCG2 M28L , that confers an increased AD risk.”
From Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease (2023) · cites this paper as [23]
The Results section reports that PLCγ2 M28L responded to activated Rac2 like wild-type PLCγ2 and concludes that it had no defect in enzyme activation, contrary to the citing sentence’s loss-of-function description. The paper identifies M28L in an ibrutinib-resistant patient but does not address whether it increases Alzheimer’s disease risk; a mention of amyloid precursor protein processing does not establish that association. (LLM, conf=0.82)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredPLCγ2 mutations R665W and L845F in Ibrutinib-resistant CLL were shown to be hypersensitive to the Rho GTPase Rac2 protein in vitro.
“Furthermore, PLCγ2 mutations R665W and L845F in Ibrutinib-resistant CLL were shown to be hypersensitive to the Rho GTPase, Rac2 protein in vitro, which may suggest Rac2 as a potential target of refractory disease (135).”
From The role of PLCγ2 in immunological disorders, cancer, and neurodegeneration (2021) · cites this paper as [135]
M28L variant and WT enzyme activity demonstrate no differences.
“whereas M28L variant and WT enzyme activity demonstrate no differences 23 .”
From Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease (2023) · cites this paper as [23]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Nonsignificant results are assigned a significant p-value
MinorThe methods define p-values below 0.05 as statistically significant, but also state that nonsignificant changes are denoted by ns when p is below 0.05.
“Non-significant changes are denoted by ns, p Ͻ 0.05.”
“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.”
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Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase Cγ2
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Rac-mediated stimulation of phospholipase Cγ2 amplifies B cell receptor-induced calcium signaling
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Self-citationRegulation of Phospholipase C‐γ2 Networks in B Lymphocytes
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Ibrutinib for the treatment of Waldenström macroglobulinemia
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Structural Basis of Promiscuous Guanine Nucleotide Exchange by the T-Cell Essential Vav1
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Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia
Ahn, I. E., Underbayev, C., Albitar, A., Herman, S. E. M., Tian, X., Maric, I., Arthur, D. C., Wake, L., Pittaluga, S., Yuan, C. M., et al. (2017) · Blood · doi:10.1182/blood-2016-06-719294 · Cited by 341 · see citing works
“Further, R665W and L845F mutations were shown to increase sensitivity of PLCγ2 to Rac2 and reconstitute downstream BCR signaling independent of BTK activation.(37) Ibrutinib resistant CLL often harbored several subclonal mutations.”
BackgroundThe role of PLCγ2 in immunological disorders, cancer, and neurodegeneration
Jackson, J. T., Mulazzani, E., Nutt, S. L., & Masters, S. L. (2021) · Journal of Biological Chemistry · doi:10.1016/j.jbc.2021.100905 · Cited by 113 · see citing works
Ibrutinib Resistance Mechanisms and Treatment Strategies for B-Cell Lymphomas
George, B., Chowdhury, S. M., Hart, A., Sircar, A., Singh, S. K., Nath, U. K., Mamgain, M., Singhal, N. K., Sehgal, L., & Jain, N. K. (2020) · Cancers · doi:10.3390/cancers12051328 · Cited by 96 · see citing works
“have shown that PLCG2R665W and PLCG2L485F mutants are hypersensitive to Rac2 (a Rho family of GTPase) activation, therefore, targeting Rac with specific inhibitors could provide a beneficial therapeutic regimen for PLCG-mutant-dependent ibrutinib-resistant cases [110].”
BackgroundPhospholipase C families: Common themes and versatility in physiology and pathology
Katan, M., & Cockcroft, S. (2020) · Progress in Lipid Research · doi:10.1016/j.plipres.2020.101065 · Cited by 89 · see citing works
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“These results suggest that once active, the mutant forms of PLC-g2 are stabilized at membranes by binding to Rac2.”
BackgroundInfluentialGenetic and Non-Genetic Mechanisms of Resistance to BCR Signaling Inhibitors in B Cell Malignancies
Ondrišová, L., & Mráz, M. (2020) · Frontiers in Oncology · doi:10.3389/fonc.2020.591577 · Cited by 77 · see citing works
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From Biology to Therapy: The CLL Success Story
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Revolution of Chronic Lymphocytic Leukemia Therapy: the Chemo-Free Treatment Paradigm
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“2 ml/well DMEM-containing supplements, as described previously (38), supplemented with 2.”
BackgroundMethodResultInfluentialSelf-citationJain, N. K., Singh, S. V., Laliotis, G., Hart, A., Muhowski, E. M., Kupcová, K., Chrbolkova, T., Khashab, T., Chowdhury, S. M., Sircar, A., et al. (2020) · Blood Advances · doi:10.1182/bloodadvances.2020001685 · Cited by 35 · see citing works
“The BTK mutation C481S and mutations in other genes (eg, PLCg2) have been reported to be associated with ibrutinib resistance.(25,26) However, in the ibrutinib-resistant DLBCL cell lines examined in this study, targeted sequencing for BTK and PLCg2 genes confirmed a lack of these mutations (supplemental Figure 2C).”
BackgroundPhospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells
Jing, H., Reed, A., Ulanovskaya, O. A., Grigoleit, J.-S., Herbst, D. M., Henry, C. L., Li, H., Barbas, S., Germain, J., Masuda, K., et al. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2112971118 · Cited by 24 · see citing works
“The PLCγ2_S707Y and _L845F variants have been shown to be further stimulated by interactions with the small GTPase Rac2 (32, 35), and we found that all PLCγ2 variants, as well as PLCγ2_WT, produced greater IP1 elevations in HEK293T cells coexpressing a constitutively active Rac2 (Fig.”
BackgroundInfluentialBlombery, P. A., Chatzikonstantinou, T., Gerousi, M., Rosenquist, R., Gaïdano, G., Pospı́šilová, Š., Roberts, A. W., Birkinshaw, R. W., Rossi, D., Scarfò, L., et al. (2025) · Leukemia · doi:10.1038/s41375-025-02662-y · Cited by 26 · see citing works
“1) [24].”
Self-citationSchmid, V., & Hobeika, E. (2024) · Frontiers in Oncology · doi:10.3389/fonc.2024.1339620 · Cited by 26 · see citing works
Pepe, F., & Balatti, V. (2020) · Journal of Clinical Medicine · doi:10.3390/jcm9020593 · Cited by 24 · see citing works
“In addition, mutations of PLCγ2 are also associated with ibrutinib resistance [98].”
BackgroundRac GTPases in Hematological Malignancies
Durand-Onaylı, V., Haslauer, T., Härzschel, A., & Hartmann, T. N. (2018) · International Journal of Molecular Sciences · doi:10.3390/ijms19124041 · Cited by 22 · see citing works
“Rac2 was found to activate constitutively two known mutants of PLCγ2 occurring in CLL, namely R665W and L845F [104].”
BackgroundArthur, R., Valle‐Argos, B., Steele, A. J., & Packham, G. (2020) · Exploration of Targeted Anti-tumor Therapy · doi:10.37349/etat.2020.00009 · Cited by 24 · see citing works
Rac-mediated stimulation of phospholipase Cγ2 amplifies B cell receptor-induced calcium signaling
Walliser, C., Tron, K., Clauss, K., Gutman, O., Kobitski, A. Y., Retlich, M., Schade, A., Röcker, C., Henis, Y. I., Nienhaus, G. U., et al. (2015) · Journal of Biological Chemistry · doi:10.1074/jbc.m115.645739 · Cited by 21 · see citing works
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“Building on prior published fi ndings (29, 37), our study provides further evidence that catalytically inhibited BTK can coordinate with RAC2 to activate PLC g 2, establishing RAC2/PLC g 2 mediated BTK scaffolding function as a bypass mechanism for BCR signaling in the absence of BTK kinase activity.”
Li, K., Ran, B., Wang, Y., Liu, L., & Li, W. (2022) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2022.999061 · Cited by 18 · see citing works
Walliser, C., Wist, M., Hermkes, E., Zhou, Y., Schade, A., Haas, J., Deinzer, J., Désiré, L., Li, S. S. C., Stilgenbauer, S., et al. (2018) · Oncotarget · doi:10.18632/oncotarget.26173 · Cited by 17 · see citing works
“This effect is presumably due to activation of Rac2 by active Rac guanine nucleotide exchange factors (GEFs) endogenously present in COS-7 cells [17].”
BackgroundMethodInfluentialSelf-citationLarribère, L., Kuphal, S., Sachpekidis, C., Sachindra, Hüser, L., Bosserhoff, A., & Utikal, J. S. (2018) · Cancers · doi:10.3390/cancers10110451 · Cited by 16 · see citing works
Targeted and cellular therapies in lymphoma: Mechanisms of escape and innovative strategies
Deshpande, A. J., & Munoz, J. L. (2022) · Frontiers in Oncology · doi:10.3389/fonc.2022.948513 · Cited by 15 · see citing works
Abdoul‐Azize, S., Hami, R., Riou, G., Derambure, C., Charbonnier, C., Vannier, J., Guzmán, M. L., Schneider, P., & Boyer, O. (2024) · Nature Communications · doi:10.1038/s41467-024-48818-9 · Cited by 13 · see citing works
“Among the six PLC isoforms 18 , PLC γ 2 is a master regulator of B cell signaling, maturation, and function 10,18,19 .”
InfluentialBravo-González, A., Alasfour, M., Soong, D., Noy, J., & Pongas, G. N. (2024) · Cancers · doi:10.3390/cancers16203434 · Cited by 14 · see citing works
“Notably, mutations of the PCL γ 2 SH2 domain have been shown to activate the PCL γ 2 independently of the BTK, and reconstitute the BCR signaling, which can explain their acquisition in the context BTK inhibition [64,65].”
BackgroundGhaderi, A., Okhovat, M. A., Wikanthi, L. S. S., et al. (2021) · eJHaem · doi:10.1002/jha2.232 · Cited by 12 · see citing works
“Even though most CLL patients initially respond to ibrutinib, an increasing number of patients develop resistance to the drug which is associated with mutation in the BTK gene at the ibrutinib binding site as C481S as well as mutations in the immediate downstream effector phospholipase Cγ2 (PLCγ2) (R665W and L845F) do…”
BackgroundHuber, H., Edenhofer, S., Estenfelder, S., & Stilgenbauer, S. (2017) · OncoTargets and Therapy · doi:10.2147/ott.s102646 · Cited by 10 · see citing works
“potential gain of function, allowing activation in the presence of persistent BTK inhibition.(24,57,58) In contrast to ibru-”
Self-citationDynamic Allostery in PLCγ1 and Its Modulation by a Cancer Mutation Revealed by MD Simulation and NMR
Koss, H., Bunney, T. D., Esposito, D., Martins, M., Katan, M., & Driscoll, P. C. (2018) · Biophysical Journal · doi:10.1016/j.bpj.2018.05.031 · Cited by 10 · see citing works
The Conformational State of the BTK Substrate PLCγ Contributes to Ibrutinib Resistance
Joseph, R. E., Lowe, J., Fulton, D. B., Engen, J. R., Wales, T. E., & Andreotti, A. H. (2021) · Journal of Molecular Biology · doi:10.1016/j.jmb.2021.167422 · Cited by 6 · see citing works
Ibrutinib-resistant CLL: unwanted and unwonted!
Mertens, D., & Stilgenbauer, S. (2017) · Blood · doi:10.1182/blood-2017-01-761536 · Cited by 7 · see citing works
“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.”
BackgroundSelf-citationArthur, R., Wathen, A., Lemm, E. A., Stevenson, F. K., Forconi, F., Linley, A. J., Steele, A. J., Packham, G., & Valle‐Argos, B. (2022) · Cellular Signalling · doi:10.1016/j.cellsig.2022.110358 · Cited by 5 · see citing works
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Kubuschok, B., & Trepel, M. (2017) · Expert Opinion on Drug Discovery · doi:10.1080/17460441.2017.1329293 · Cited by 4 · see citing works
“The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor signaling [66].”
BackgroundVisvanathan, R., Utsuki, T., Beck, D. E., Lendy, E. K., Sun, K.-L., Liu, Y., Hering, K. W., Mesecar, A. D., Zhang, Z., & Putt, K. S. (2023) · SLAS DISCOVERY · doi:10.1016/j.slasd.2023.03.003 · Cited by 3 · see citing works
Parigger, T., Drothler, S., Scherhäufl, C., Gassner, F. J., Schubert, M., Steiner, M. J., Höpner, J. P., Hödlmoser, A., Schultheis, L., Bakar, A. A., et al. (2024) · Acta Haematologica · doi:10.1159/000537791 · Cited by 4 · see citing works
“Most of the refractory patients have mutated BTK, which impairs covalent binding of ibrutinib, or mutated phospholipase C gamma 2 (PLCG2), which compensates for BTK inhibition [9, 10].”
Genetik der Alzheimer-Krankheit
Bajaj, T., Ramı́rez, A., & Wagner-Thelen, H. (2018) · Medizinische Genetik · doi:10.1007/s11825-018-0193-3 · Cited by 3 · see citing works
“DarüberhinauswurdenPLCG2Mutationenmit der Ibrutinib-resistenten chronischen lymphatischen Leukämie in Verbindung gebracht [64].”
BackgroundGenomics of Resistance to Targeted Therapies
Thangavadivel, S., & Woyach, J. A. (2021) · Hematology/Oncology Clinics of North America · doi:10.1016/j.hoc.2021.03.004 · Cited by 3 · see citing works
Advances and resistance adaptation in BTK-targeted therapy for B-cell malignancies
Yang, S., Oh, J., Hwang, S.-Y., Paik, J., & Hwang, I. (2026) · Drug Resistance Updates · doi:10.1016/j.drup.2026.101383 · Cited by 3 · see citing works
Visvanathan, R., Utsuki, T., Beck, D. E., Clayton, B., Lendy, E. K., Sun, K.-L., Liu, Y., Hering, K. W., Mesecar, A. D., Zhang, Z., et al. (2024) · PLoS ONE · doi:10.1371/journal.pone.0299541 · Cited by 2 · see citing works
Pharmacotherapy of relapsed/refractory chronic lymphocytic leukemia
Zahr, A. A., Bose, P., & Keating, M. J. (2017) · Expert Opinion on Pharmacotherapy · doi:10.1080/14656566.2017.1324420 · Cited by 2 · see citing works
“PLCγ2 mutations lead to a stimulatory effect on its interaction with activator Rac GTPases [81].”
PLCG2 as a Risk Factor for Alzheimer's DiseasePreprint
Tsai, A. P., Dong, C., Preuß, C., Moutinho, M., Lin, P. B., Hajicek, N., Sondek, J., Bissel, S. J., Oblak, A. L., Carter, G. W., et al. (2020) · doi:10.21203/rs.3.rs-33082/v1 · Cited by 2 · see citing works
PLCG2 signaling and genetic resilience in Alzheimer’s disease
Tsai, A. P., Martin, A. K., Mi, A., Yeh, A. E., Ramirez Lopez, E., & Wyss-Coray, T. (2026) · Molecular Neurodegeneration · doi:10.1186/s13024-026-00935-3
“In contrast, the missense M28L variant (PLCG2 M28L ; rs61749044; allele frequency: 1.48%), which maintains normal basal activity following Rac2 GTPase stimulation [41], is associated with increased AD risk [15, 31].”
InfluentialTherapy-driven clonal dynamics in chronic lymphocytic leukemia
Mansouri, L., Chatzikonstantinou, T., Scarfò, L., & Rosenquist, R. (2026) · Seminars in Cancer Biology · doi:10.1016/j.semcancer.2026.04.001 · Cited by 1 · see citing works
Ibrutinibrezisztencia krónikus limfocitás leukémiában
Aczél, D., Mátrai, Z., Kiss, R., Balogh, A., Illés, S., Bödör, C., & Alpár, D. (2019) · Hematológia–Transzfuziológia · doi:10.1556/2068.2019.52.2.8 · Cited by 1 · see citing works
Matriptase as a Novel Therapeutic Target in Non-Hodgkin Lymphoma
Chiu, Y. (2018) · DigitalGeorgetown (Georgetown University Library)
Mougeot, J.-L., Beckman, M. F., Alexander, A. S., Hovan, A. J., Hasséus, B., Legert, K. G., Johansson, J., Bültzingslöwen, I. V., Brennan, M. T., & Mougeot, F. B. (2024) · Supportive Care in Cancer · doi:10.1007/s00520-024-08408-3
“Multiple studies have shown that in CLL mutations in PLCG2 cause Bruton’s Tyrosine Kinase inhibitors to lose efficacy during treatment [46–49].”
La stimulation cellulaire liée aux glucocorticoïdes
Abdoul‐Azize, S. (2025) · médecine/sciences · doi:10.1051/medsci/2025030
PLCG2 exon-skipped variants: insights into their potential role in chronic lymphocytic leukemia
Qi, J., Li, W., Dheenadayalan, R. P., Yosifov, D. Y., Drewes, C. C., Wist, M., Wieczorek, M., Schmid, V., Hobeika, E., Gao, X., et al. (2026) · Blood Advances · doi:10.1182/bloodadvances.2025019077
“…a widespread expression of 2 additional variants: Δ 22 and Δ 20-22 in the absence of PLCG2 SSDs or SSMs. Similar to PLCG2 point mutations, 6,36 the Δ 21 and Δ 20-22 16 variants were previously described to exhibit enhanced phospholipase activity, potentially enabling cells to bypass BTK…”
Self-citation
- Authors
- Walliser, C., Hermkes, E., Schade, A., Wiese, S., Deinzer, J., Zapatka, M., Désiré, L., Mertens, D., Stilgenbauer, S., & Gierschik, P.
- Affiliations
- Universität Ulm (DE)
- German Cancer Research Center (DE)
- Corresponding author
- Gierschik, Peter
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2016-10
- Volume
- 291
- Issue
- 42
- Pages
- 22136-22148
- ISSN
- 0021-9258 (ISSN-L)
- PMC
- PMC5063995
- OpenAlex
- W2508465076
- Cited by
- 51
- References
- 70
- Citations by year
- 2026: 4 · 2025: 2 · 2024: 6 · 2023: 3 · 2022: 3 · 2021: 5 · 2020: 10 · 2019: 4 · 2018: 8 · 2017: 5 · 2015: 1
- FWCI
- 3.58
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (submittedVersion)
- PubMed Central (submittedVersion)
- Funders
- Deutsche Forschungsgemeinschaft (SFB1074, TP A8)
- Topics
- Health Sciences › Medicine › Genetics › Chronic Lymphocytic Leukemia Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Health Sciences › Medicine › Pathology and Forensic Medicine › Lymphoma Diagnosis and Treatment
- Keywords
- PLCγ2, PLCG2 mutations, ibrutinib resistance, chronic lymphocytic leukemia, Rac2, Rho GTPases, B cell receptor signaling, calcium signaling, BTK inhibitors, signal transduction
- MeSH terms
- RAC2 GTP-Binding Protein, Adenine, Animals, Chlorocebus aethiops, Humans, Neoplasm Proteins, Piperidines, Pyrazoles, Pyrimidines, Pyrones, Quinolines, Receptors, Antigen, B-Cell, Signal Transduction, Leukemia, Lymphocytic, Chronic, B-Cell, Drug Resistance, Neoplasm, COS Cells, Amino Acid Substitution, Mutation, Missense, rac GTP-Binding Proteins, Phospholipase C gamma
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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