Abstract B081: Elucidating the pharmacodynamics of PI3K and Ras-Raf signaling through isoform-specific multiplexed quantification of downstream effector phosphorylation
Authors: Herrick William G. , Kilpatrick Casey L. , Hollingshead Melinda , Doroshow James H. , Parchment Ralph E. , Srivastava Apurva K. 
Published: August 2018

Abstract
Background: Ras-extracellular signal-regulated kinase (Ras-ERK) and phosphatidylinositol 3-kinase/AKT (PI3K/AKT) signaling pathways are signal transducers that influence many cell functions, making them attractive targets for therapeutic development. Several downstream kinases in these pathways (e.g., AKT, MEK, ERK, RSK, and S6K) converge on common substrates such as ribosomal protein S6 (rpS6) and play critical roles in feedback activation when individual nodes are suppressed by inhibitors. Cross-regulation and rebound activation of PI3K and Ras signaling contribute to known mechanisms of drug resistance. Therefore, monitoring the activation of downstream effectors in both pathways will provide critical information on mechanisms of drug action and resistance. Current assays for measurement of pathway proteins are qualitative and confounded by the lack of isoform specificity. We describe a novel multiplex approach to quantitatively measure phosphorylation status of individual isoforms of key enzymes in the PI3K and Ras pathways. Methods: We developed highly selective isoform- or phospho-specific antibodies for AKT1/2/3, MEK1/2, ERK1/2, and pRPS6(S235) at NCI or obtained them from commercial sources and validated their specificity using a combination of techniques involving isoform-specific CRISPR knockout cell lines, cancer cells treated with selective drugs, and epitope-peptide competition. Multiplex sandwich immunoassays were developed on the Luminex platform by using isoform-specific antibodies for capture and biotinylated antibodies for detection of total protein or phosphorylated residues. Recombinant proteins were used as calibrators. The first intended application of the assays is a clinical trial of a PI3Kβ inhibitor, AZD8186 (NCI NSC#777572), in PTEN-deficient tumors, and a preclinical study is under way to model this clinical trial using PTEN-null cancer cell lines (HCC70 and PC3 cell lines). Results: In the first phase, we multiplexed the analysis of biomarkers in the PI3K-AKT-rpS6 pathway including AKT1, AKT2, AKT3, rpS6, pT308-AKT1, pT309-AKT2, pT305-AKT3, pS473-AKT1, pS474-AKT2, pS472-AKT3, and pS235-rpS6. Using PTEN-null cancer cell lines (HCC70, MDA-MB-468, PC-3) treated with a PI3Kβ inhibitor, we have demonstrated that our multiplex assays measure 70-90% reduction in target phosphorylation with high precision (CV &lt; 15%). The AKT and rpS6 phospho-signals appear to be stable and quantifiable when biopsies are processed according to clinically validated biopsy collection and extraction protocols established previously for phospho c-MET (PMID 27001313). Data from the ongoing preclinical time-course study of molecular target responses to the PI3Kβ inhibitor will be presented. Conclusions: We have developed a multiplex approach for monitoring the pharmacodynamics of multiple classes of drugs that target the PI3K-AKT-mTOR-rpS6 pathway. The assays not only provide precise quantitative measurements of the magnitude of drug-induced target suppression but also indicate the effectiveness of a drug in suppressing downstream pathway signaling using a single tissue biopsy. Funded by contract No. HHSN261200800001E.
Citation Format: William G. Herrick, Casey L. Kilpatrick, Melinda Hollingshead, James H. Doroshow, Ralph E. Parchment, Apurva K. Srivastava. Elucidating the pharmacodynamics of PI3K and Ras-Raf signaling through isoform-specific multiplexed quantification of downstream effector phosphorylation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B081.