Isoform- and Phosphorylation-specific Multiplexed Quantitative Pharmacodynamics of Drugs Targeting PI3K and MAPK Signaling in Xenograft Models and Clinical Biopsies

Abstract

Ras/Raf/MEK/ERK (MAPK) and PI3K/AKT signaling pathways influence several cell functions involved in oncogenesis, making them attractive drug targets. We describe a novel multiplex immunoassay to quantitate isoform-specific phosphorylation of proteins in the PI3K/AKT and MAPK pathways as a tool to assess pharmacodynamic changes. Isoform-specific assays measuring total protein and site-specific phosphorylation levels of ERK1/2, MEK1/2, AKT1/2/3, and rpS6 were developed on the Luminex platform with validated antibody reagents. The multiplex assay demonstrated satisfactory analytic performance. Fit-for-purpose validation was performed with xenograft models treated with selected agents. In PC3 and HCC70 xenograft tumors, the PI3Kβ inhibitor AZD8186 suppressed phosphorylation of AKT1, AKT2, and rpS6 for 4 to 7 hours post single dose, but levels returned to baseline by 24 hours. AKT3 phosphorylation was suppressed in PC3 xenografts at all doses tested, but only at the highest dose in HCC70. The AKT inhibitor MK-2206 reduced AKT1/2/3 phosphorylation in SW620 xenograft tumors 2 to 4 hours postdose, and the MEK inhibitor selumetinib reduced MEK1/2 and ERK1/2 phosphorylation by up to 50% and >90%, respectively. Clinical utility was demonstrated by analyzing biopsies from untreated patients with plexiform neurofibromas enrolled in a clinical trial of selumetinib (NCT02407405). These biopsies showed MEK and ERK phosphorylation levels sufficient for measuring up to 90% inhibition, and low AKT and rpS6 phosphorylation. This validated multiplex immunoassay demonstrates the degree and duration of phosphorylation modulation for three distinct classes of drugs targeting the PI3K/AKT and MAPK pathways.

Figure 1:

Overview of Luminex multiplex assay. (A) Quantitative isoform-specific Luminex sandwich immunoassays were developed with isoform-specific monoclonal antibodies conjugated to MagPlex© microspheres and biotinylated pan-isoform monoclonal antibodies for detection of total and site-specific phosphorylated protein. The assays are divided into 5 panels for validation: MEK/ERK Total, MEK/ERK Phospho, AKT/rpS6 Total, AKT/rpS6 Phospho A, and AKT/rpS6 Phospho B. Standard curves were fit for the MEK/ERK panels (B) and the AKT/rpS6 panels (total rpS6 only) (C). Phosphorylation sites assayed in the MEK/ERK phospho panel are shown in the respective tables.

Figure 2:

Pharmacodynamic effects on selected PD endpoints in HCC70 xenografts following a single dose of AZD8186 (n = 4 mice/time point). Isoform-specific phosphorylation was measured relative to vehicle levels following doses of 25 mg/kg (panels A, C, and E) or 50 mg/kg (panels B, D, F). Data are presented as the mean of vehicle-normalized percent-phosphorylation at 2, 4, 7, and 24 hours post-single dose (psd). Error bars are standard errors of the mean.

Figure 3:

Pharmacodynamic effects on phosphorylation of measured sites on AKT isoforms and rpS6 in PC3 xenografts following a single 25 mg/kg or 50 mg/kg dose of AZD8186 (n=4 mice/time point). Data are presented as the mean of vehicle-normalized percent-phosphorylation at 2, 4, 7, and 24 hours psd. Error bars are standard errors of the mean.

Figure 4:

Pharmacodynamic changes following the last dose of a week-long course of 50 mg/kg AZD8186 every 12 hours in PC3 xenograft-bearing mice (n = 4 mice/time point). Three time points post-last dose are shown for each analyte and are colored as indicated. Data are presented as the mean of vehicle-normalized percent-phosphorylation; error bars are standard errors of the mean.

Figure 5:

Pharmacodynamic changes following a single 20 mg/kg dose of selumetinib in SW620 xenografts (n=5 mice/time point). Data are presented as the mean of vehicle-normalized percent phosphorylation at 2, 4, 24, and 48 hours psd. Error bars are standard errors of the mean.

Figure 6:

Total protein levels and percent phosphorylation in biopsies from untreated patients with neurofibromas. Total protein levels are reported as pg of analyte per μg of total protein in dermal (A) and core needle (B) biopsies. Phosphorylation levels in dermal (C) and core needle (D) biopsies are reported as the percent of total analyte phosphorylated at sites assayed with this multiplex immunoassay. AKT samples refer to the Phospho A panel (Ser473/474/472-AKT1/2/3 and Ser235-rpS6). Patients are identified by number and distinct biopsy sites are lettered (e.g., 20A is from patient 20, biopsy site A). Error bars are standard deviations.