Cryo-EM analysis of PI3K signaling complexes in glioblastoma

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PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is a lethal disease for which there is no known cure. Following ~2 years of initial treatment, which includes surgical resection, radiation, and chemotherapy, more than 90% of GBM patients succumb to disease progression. Inhibitors of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) have been used clinically to treat original and recurrent GBMs with modest benefits. Our recent research finds that PIK3CB (PI3K catalytic subunit β, encoding p110β), but not other PI3K catalytic subunits, shows a strong association with GBM progression. Moreover, depletion or pharmaceutical inhibition of p110β induces growth inhibition/cell death in GBM cells highly expressing this subunit. In contrast, blocking other PI3K catalytic subunits fails to do so. Hence, PIK3CB/p110β is a selective survival factor for GBM. Our results strongly support that targeting one PI3K isoform that is dominant in GBM may be a more effective approach to treat GBM. While past research on PI3K isoforms has identified PI3K isoform-selective inhibitors, the clinical benefits of these chemical compounds are limited. The lack of molecular details pertaining to p110β selective activation and structural information of native p110 complexes likely contributes to the poor outcomes of current therapies. Understanding the molecular/structural details of p110β will permit a better design of more selective and effective p110β-based therapies for GBM. To this end, we will complete the following two specific aims. In aim 1, we will acquire high-resolution 3D conformations of p110β native protein complexes using immuno-capture cryoEM. Resolution of cryo-EM images we acquired previously was not high enough to provide 3D conformations of p110β/p85 complexes at a greater detail. Access to a FEI Titan Krios G2 electron microscope has rendered high resolution 3D conformations of native p110β/p85 complexes possible. To acquire more clinically relevant protein structures, primary GBM xenograts derived from patient specimens will be used. In aim 2, we will test the hypothesis that p110βC2in changes 3D conformations of p110β native protein complexes, thus inactivating p110β. Primary GBM cells will be treated with p110βC2in or a control scramble peptide. 3D conformations of p110β native complexes will be revealed by immuno-capture cryo-EM. Structural differences between p110β native complexes with or without p110βC2in will then be determined. Results from this R21 application will be highly impactful, particularly to our future research in PI3K signaling and on the therapeutic intervention for GBM. The molecular details of p110β native complexes revealed by cryo-EM will encourage us to further our understanding of molecular mechanisms underlying selective activation of p110β in GBM and to identify novel vulnerabilities of p110β at atomic levels. Visualization of p110β native complexes with or without p110βC2in will not only help us understand how this peptide acts but also will facilitate a further development of p110βC2in into a GBM treatment.