Discovery of a New CaMKII-Targeted Synthetic Lethal Therapy against Glioblastoma Stem-like Cells

Abstract

Glioblastoma stem-like cells (GSCs) drive tumor initiation, cancer invasion, immune evasion, and therapeutic resistance and are thus a key therapeutic target for improving treatment for glioblastoma multiforme (GBM). We previously identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an emerging molecular target for eliminating GSCs. In this study, we aim to explore a new CaMKII-targeted synthetic lethal therapy for GSCs. Through high-throughput drug combination screening using CaMKII inhibitors and a bioactive compound library in GSCs, neurokinin 1 receptor (NK1R) inhibitors such as SR 140333 and aprepitant are found to be potential anticancer agents that exhibit chemical synthetic lethal interactions with CaMKII inhibitors, including hydrazinobenzoylcurcumin (HBC), berbamine, and KN93. Combined treatment with NK1R and CaMKII inhibitors markedly suppresses the viability and neurosphere formation of U87MG- and U373MG-derived GSCs. In addition, the combination of HBC and NK1R inhibitors significantly inhibits U87MG GSC tumor growth in a chick embryo chorioallantoic membrane (CAM) model. Furthermore, the synthetic lethal interaction is validated using RNA interference of CaMKIIγ and NK1R. Notably, the synthetic lethal effects in GSCs are associated with the activation of caspase-mediated apoptosis by inducing p53 expression and reactive oxygen species generation, as well as the suppression of stemness marker expression by reducing nuclear factor-kappa B (NF-κB) activity. This follows the downregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling and a decrease in intracellular calcium concentration. Moreover, NK1R affects CaMKIIγ activation. These findings demonstrate that NK1R is a potential synthetic lethal partner of CaMKII that is involved in eradicating GSCs, and they suggest a new CaMKII-targeted combination therapy for treating GBM.

Atorvastatin inhibits the proliferation of MKN45-derived gastric cancer stem cells in a mevalonate pathway-independent manner

“…The cells were grown in RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin–amphotericin B. To propagate GCSCs, MKN45 cells were cultured in DMEM/F12 containing 1× B-27, 5 µg/ml heparin, 2 mM L-glutamine, 20 ng/ml EGF, 20 ng/ml bFGF, and 1% penicillin/streptomycin as described previously [ 16 , 17 ]. Tumorspheres grown in serum-free media were subcultured by dissociation using Accutase.…”

“…MKN45-derived GCSCs (5 × 10 3 cells/well) were seeded in a 96-white-well culture plate using serum-free media and treated with the different concentrations of each compound for 7 days. Cell proliferation was measured using the CellTiter-Glo 2.0 Cell Viability Assay as described previously [ 16 , 17 ]. Luminescence was detected using a multimode microplate reader (BioTek, Inc., Winooski, VT, USA).…”

“…MKN45-derived GCSCs (1 × 10 5 cells/well) were seeded in a 60-mm culture plate using serum-free media and treated with the different concentrations of each compound for 4 days. The cells were harvested, fixed with 70% ethanol, and stained with 200 µl of Muse Cell Cycle reagent as described previously [ 16 , 17 ]. Cell cycle plots were obtained using the Guava Muse Cell Analyzer (MuseSoft_V1.8.0.3; Luminex Corporation).…”

“…MKN45-derived GCSCs (1 × 10 5 cells/well) were seeded in a 60-mm culture plate using serum-free media and treated with the different concentrations of atorvastatin for 4 days. The cells were collected and stained with 100 µl of Muse Annexin V & Dead Cell reagent as described previously [ 16 , 17 ]. Cellular apoptosis was quantitatively analyzed using the Guava Muse Cell Analyzer.…”

Neurotransmitters: Potential Targets in Glioblastoma

“…Of note, the effects of NK-1R antagonists on cell growth and apoptosis have been identified and summarized recently [ 73 ]. In addition, an innovative co-treatment with NK1R antagonists and CaMKII inhibitors has shown synergistic inhibition on the growth of GSCs and their derivatives both in vitro and in vivo [ 231 ]. Further experiments on animal models in the future will make efforts to translate this combination therapy to the clinic.…”