A Novel Cellular Class I Phosphoinositide-3-Kinase (Pi3k) Isoform-Specific Occupancy Assay Demonstrates A Direct Correlation Between Pi3k Isoform Activity And Phospho-Akt Levels

Abstract Background: AKT phosphorylation is a widely used pharmacodynamic indicator for Class I PI3K activity in cells and tumors; however, the direct correlation of pAKT with PI3K activity has not been carefully examined. We have developed a novel, direct isoform specific occupancy assay for PI3K to determine if intracellular isoform specific inhibition of PI3K is quantitatively equivalent to inhibition of pAKT formation. Methods: A biotin-conjugated derivative of the irreversible pan-PI3K inhibitor, wortmannin, was synthesized. All four class I PI3K isoforms could be pulled down using this biotin-probe. ATP-competitive PI3K inhibitor binding prevented probe binding in a dose dependent manner, and hence pull-down of PI3K. Inhibition isotherms monitoring the decrease in PI3K pulled down with increasing inhibitor concentrations were generated via western blot with PI3K isoform specific antibodies. The affinity of a given inhibitor against PI3K-β, -δ, and -γ was determined in various cell lines and compared to inhibition of pAKT levels measured by ELISA or western blotting in unstimulated 786.0 cells, IgM-stimulated Raji cells, and C5a-stimulated RAW264.7 cells. Results: We utilized the occupancy assay on these cell lines with compounds that have differential biochemical PI3K isoform inhibition profiles. All of these cell lines significantly express more than one PI3K isoform, with Raji and 786.0 cells expressing PI3K-δ and -β, and RAW264.7 cells expressing PI3K-δ and -γ. In unstimulated 786.0 cells, PI3K-β and PI3K-δ occupancy IC50s were obtained and compared with pAKT IC50s. PI3K-β occupancy IC50s, but not PI3K-δ occupancy IC50s, correlated well with pAKT IC50s, indicating that in 786.0 cells, PI3K-β is the main driver for pAKT formation. Similarly, PI3K-β and PI3K-δ occupancy IC50s were obtained in anti-IgM stimulated Raji cells. Here pAKT IC50s correlated with PI3K-δ, and not PI3K-β occupancy IC50s, indicating that PI3K-δ is the major signaling isoform responding to this stimulus. The PI3K-δ occupancy IC50s were similar in C5a-stimulated RAW264.7 cells, but here the PI3K-γ occupancy IC50 was equivalent to pAKT IC50, confirming that PI3K-γ is the major PI3K signaling isoform downstream of C5a in RAW264.7 cells. Interestingly, PI3K-δ and PI3K-γ occupancy IC50s were similar between stimulated or unstimulated Raji or RAW264.7 cells. Therefore, PI3K occupancy is unaffected by PI3K migration to the cell membrane after stimulation. Conclusions: To our knowledge, this is the first demonstration that occupancy of membrane-recruited Class I PI3K isoforms by PI3K inhibitors directly correlates with the inhibition of pAKT. These results also indicate a predominant PI3K signaling isoform is responsible for downstream pAKT formation in these cell lines, validating them for PI3K isoform specific cell based assays. Citation Format: Lina Gu, Erin Brophy, Erin Murphy, Bonnie Tillotson, Jonathan DiNitto, Louis Grenier, Janid Ali. A novel cellular class I phosphoinositide-3-kinase (PI3K) isoform-specific occupancy assay demonstrates a direct correlation between PI3K isoform activity and phospho-AKT levels. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4973. doi:10.1158/1538-7445.AM2015-4973