Functional characterization of phospholipase C-γ2 mutant protein causing both somatic ibrutinib resistance and a germline monogenic autoinflammatory disorder

Abstract

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

Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes

“…Overall, our comprehensive analyses in a standard cell assay have revealed that the majority of tested mutations in the selected parts of PLCg enzymes, irrespective of the disease and frequency of observations, resulted in an increase in enzyme activity. This functional analysis is consistent with previous findings reported for some of the mutations [6,8,15] and demonstrates the importance of the structural features with clustered mutations in controlling the activation state of the enzyme. The functional analyses also support the conclusion that the interacting surfaces revealed by structural studies have a role in autoinhibition.…”

“…Several frequently mutated residues in Ibrutinib-resistant CLL, including PLCg2 S707, L845, D993 and M1141, are likely to impact on PLC activity via the same mechanism. Interestingly, some of the somatic mutations in the resistant CLL are identical to genetic lesions found in APLAID and include mutations of PLCg2 S707 and M1141 residues [6,15,32,33]. However, not all mutations reside within, or in the vicinity, of the intramolecular inhibitory interactions, suggesting other mechanistic classes.…”

“…PLCG2 variants have been also associated with inflammatory bowel disease (IBD) and one rare PLCG2 variant has been reported to strongly associate with the protection from the development of Alzheimer's disease (AD). Interestingly, in a number of cases where functional characterization of the effect of genetic changes on PLC activity has been performed, these alterations (predominantly single amino-acid substitutions) result in an increase of PLCg activity [6,8,14,15].…”

Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells

“…While the molecular basis of clinical resistance to BTK inhibitors, such as ibrutinib and acalabrutinib, caused by mutations in position C481 of BTK are relatively clear, the mechanism of resistance caused by the other BTK and the PLCG2 mutations is less understood. While PLCG2 mutations have been suggested to constitute, as a whole, gain-offunction mutations (40,45,57), we have previously found that several PLC 2 variant enzymes carrying mutations in various regions are not constitutively active when assayed in cell-free systems (38,39). The observation that these PLC 2 variants are, instead, hypersensitive in intact cells to the Rho GTPase RAC2 led us to suggest that the PLCG2 mutations found in BTK-inhibitor-resistant CLL cells may cause a rerouting of the transmembrane signals emanating from BCR and converging on as well as activating PLC 2 (38,39).…”

“…PLC 2 is immediately downstream of BTK (26) leading to the suggestion that the variant proteins may be constitutively active. However, we have previously found that several PLC 2 variant enzymes associated with ibrutinib resistance carrying mutations in various regions are not constitutively active when assayed, even as purified proteins, in a cell-free system employing artificial lipid vesicles as substrate (38,39). The observation that PLC 2 variants are, instead, hypersensitive in intact cells to the Rho GTPase RAC2, as well as the upstream protein tyrosine kinases SYK and LYN (39,40), has led us to suggest that the PLCG2 mutations found in BTK-inhibitor-resistant cells may cause a rerouting of the transmembrane signals emanating from BCR to converge on and activate PLC 2 .…”