Dual Pharmacological Inhibition of PAK1 and CaMKII Cooperatively Suppresses Malignant Phenotypes in Glioblastoma Cells

Dual inhibition of PAK1 and CaMKII shows promise in enhancing anti-tumor effects in glioblastoma cell lines, but further research is needed to assess its clinical relevance.

Summary and findings

This study examined the effects of dual pharmacological inhibition of PAK1 and CaMKII in glioblastoma cell lines U251, U87, and T98G. The combination treatment with G-5555 and KN-93 enhanced growth inhibition compared to either agent alone. The findings suggest potential vulnerabilities in glioblastoma signaling pathways.

Abstract

<h4>Background: </h4> Glioblastoma is a highly aggressive brain tumor characterized by rapid proliferation, diffuse invasion, and resistance to therapy. Although p21-activated kinase 1 (PAK1) and calcium/calmodulin-dependent protein kinase II (CaMKII) have each been independently implicated in malignant signaling, the biological consequences of their simultaneous pharmacological inhibition in glioblastoma remain poorly understood. Methods. U251, U87, and T98G glioblastoma cells lines were used to characterize basal PAK1 and CaMKII expression and activation by Western blotting and immunofluorescence. Public transcriptomic datasets were analyzed to assess the prognostic associations of PAK1 and CAMK2A expression and their transcriptional correlation in glioblastoma. Cells were treated with the group I PAK inhibitor G-5555 and the CaMKII inhibitor KN-93, either alone or in combination. Drug interactions were evaluated using the ZIP, Loewe, Bliss, and HSA reference models. Functional effects were assessed by cell viability and growth kinetics, cell-cycle analysis, caspase-3 cleavage, clonogenic assays, Transwell migration, wound healing assays, and MMP2/MMP9 expression. Exploratory phos-pho-signaling profiling was performed in U251 cells. Results. Higher PAK1 and CAMK2A expression was associated with poorer survival, and their transcript levels were positively correlated in glioblastoma samples (r = 0.54, p = 0.0001). Total and phosphorylated PAK1 and CaMKII were detected across all three cell lines, with heterogeneous basal abundance and activation patterns. Combined G-5555 and KN-93 treatment consistently enhanced growth inhibition relative to either single agent. Drug-interaction estimates varied across reference models, with the strongest positive interactions observed using HSA (mean synergy scores: 12.07, U251; 13.91, U87; and 14.25, T98G). Dual inhibition also suppressed cell expansion and clonogenicity, altered cell-cycle progression, increased caspase-3 cleavage, and impaired migration and invasion-associated phenotypes, accompanied by reduced MMP2 and MMP9 expression. Exploratory phospho-signaling profiling further revealed broad alterations in signaling networks associated with proliferation, survival, migration, cellular stress, and apoptosis. Conclusions. Combined pharmacological inhibition of PAK1 and CaMKII produces cooperative anti-tumor effects across multiple glioblastoma cell models despite inter-cell-line heterogeneity. These findings identify PAK1 and CaMKII signaling as potentially co-targetable vulnerabilities and provide a rationale for further mechanistic and in vivo evaluation of this combination.

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