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Published on: September 16, 2019
The Pyk2 FERM domain as a target to inhibit glioma migration
Joseph C Loftus1, Zhongbo Yang, Nhan L Tran
1Mayo Clinic Arizona, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA. loftus.joseph@mayo.edu
Abstract:
The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH(2)-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the beta5C-alpha1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression.
Insights
Targeting the Pyk2 FERM domain inhibits malignant glioma cell migration. A novel antibody fragment targeting this domain reduced glioma progression and extended survival in preclinical models, offering a new therapeutic strategy.
Area of Science:
- Neuro-oncology
- Molecular Cell Biology
- Biochemistry
Background:
- Malignant glioma cell invasion into normal brain tissue presents a significant challenge for effective clinical treatment.
- The protein tyrosine kinase Pyk2 plays a role in cell migration, but its regulatory mechanisms are not fully understood.
- The NH(2)-terminal FERM domain of Pyk2 is implicated in regulating its promigratory function.
Purpose of the Study:
- To investigate the role of the NH(2)-terminal FERM domain in regulating Pyk2's promigratory function.
- To identify specific regions within the FERM domain critical for Pyk2 activity.
- To develop and evaluate novel therapeutic strategies targeting Pyk2 for glioma treatment.
Main Methods:
- Generated a monoclonal antibody (12A10) targeting the Pyk2 FERM domain.
- Utilized site-directed mutagenesis to alter residues within the FERM domain's F3 module.
- Conjugated the antibody to a membrane transport peptide and expressed a single-chain Fv fragment intracellularly.
- Assessed the impact on Pyk2 phosphorylation, glioma cell migration in vitro, and survival in a glioma xenograft model.
Main Results:
- Amino acid substitutions in the FERM domain's F3 module abolished Pyk2 phosphorylation and antibody binding.
- The 12A10 antibody, when conjugated to a transport peptide, inhibited glioma cell migration.
- Intracellular expression of the 12A10 single-chain Fv fragment reduced Pyk2 phosphorylation and glioma cell migration.
- Stable intracellular expression of 12A10 scFv significantly improved survival in a glioma xenograft model.
Conclusions:
- The FERM domain, particularly the F3 module, is crucial for regulating Pyk2 activity.
- The 12A10 antibody epitope is located at a site critical for Pyk2 function.
- Targeting the Pyk2 FERM domain represents a promising novel strategy for inhibiting glioma progression and improving patient survival.
