Kinomic profiling of glioblastoma cells reveals PLCG1 as a target in restricted glucose
Kiera Walker1, Nathaniel H Boyd1, Joshua C Anderson2
11Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294 USA.
Background:
For glioblastoma (GBM) treatments to be effective in vivo, understanding the effects of the tumor microenvironment is imperative. In traditional cell culture conditions, glucose concentrations do not model physiologic levels, nor the diminished concentrations found in tumor niches. We therefore sought to profile the differences in kinase activity in GBM cells cultured in restricted glucose to identify pathways that could be targeted with small molecule inhibitors.
Methods:
Using the PamStation12 platform, we examined the ability of GBM lysates from cells cultured in standard or low glucose conditions to phosphorylate 144 tyrosine and 144 serine/threonine peptides that correspond to known protein phosphorylation sites. Potential kinase targets were identified and validated using small molecule kinase inhibitors in GBM spheroid cultures.
Results:
Using results from two GBM patient-derived xenografts, we determined common changes to peptides derived from Phospholipase C, Gamma 1 (PLCG1) and Raf-1. Using PLC and Raf inhibitors, we found a significantly stronger growth inhibitory effect of the PLC inhibitor U73122 under restricted glucose conditions. In contrast, Raf inhibitors were significantly growth inhibitory regardless of the nutrient level tested.
Conclusions:
Together, our data demonstrate that kinase activity is altered in low glucose conditions and that kinomic profiling can assist with the identification of effective strategies to target GBM growth. Our data further suggest the importance of accurately modeling the tumor microenvironment to reproduce cancer cell signaling and develop drug screens for anti-cancer agents.
Insights
Understanding glioblastoma (GBM) kinase activity in low glucose conditions is key for effective treatment. Kinomic profiling identified specific targets, like Phospholipase C, showing promise for novel GBM therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Glioblastoma (GBM) treatment efficacy depends on understanding the tumor microenvironment.
- Standard cell culture glucose levels do not reflect physiological or tumor niche conditions.
- Investigating kinase activity under restricted glucose is crucial for identifying therapeutic targets.
Purpose of the Study:
- To profile kinase activity differences in GBM cells cultured in restricted glucose conditions.
- To identify specific kinase pathways amenable to small molecule inhibitor targeting.
- To validate potential therapeutic targets using GBM spheroid models.
Main Methods:
- Utilized the PamStation12 platform to assess GBM cell lysate phosphorylation of 144 tyrosine and 144 serine/threonine peptides.
- Compared kinase activity in GBM cells cultured in standard versus low glucose environments.
- Validated identified kinase targets using small molecule inhibitors in GBM spheroid cultures.
Main Results:
- Identified common peptide changes in Phospholipase C, Gamma 1 (PLCG1) and Raf-1 from two GBM patient-derived xenografts.
- Demonstrated a significantly stronger growth inhibitory effect of the PLC inhibitor U73122 under restricted glucose conditions.
- Observed that Raf inhibitors exhibited significant growth inhibition irrespective of nutrient levels.
Conclusions:
- Kinase activity is altered in low glucose conditions, and kinomic profiling aids in identifying GBM growth strategies.
- Accurate modeling of the tumor microenvironment is essential for understanding cancer cell signaling.
- This study highlights the importance of microenvironment modeling for developing anti-cancer drug screens.
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