Pharmacodynamics
PD, tumour
LYSIS, TUMOUR
PD analysis for the proximal PD markers pPKC delta and phosphorylated myristoylated alanine-rich C-kinase substrate (MARCKS; pMARCKS) was performed in pre- and on-treatment tumour biopsies from patients who received LXS196 on a QD schedule. PD analysis was not performed on tumour samples from patients who received LXS196 on a BID schedule.Custom assays were developed to measure the status of the PKC pathway. In brief, tumour tissue was homogenised in lysis buffer, and total protein concentrations were determined using a bicinchoninic acid (BCA) assay (Pierce). The samples were then run on the Meso Scale Discovery platform to measure total and pMARCKS. A seven-point calibration curve based on the 92.1 UM cell line was used to determine the concentration of total and pMARCKS in patient samples. All samples were run in duplicate wells. Total and phosphorylated PKCdelta (PKCd; pPKCd, phosphorylated at S299) levels were determined using microcapillary electrophoresis on the Sally Sue platform (Protein Simple, San Jose, CA, USA). Area under the peaks corresponding to the target was measured and used for further calculation. All results were normalised for the amount of protein used in each assay, and the ratio of phosphorylated protein to the corresponding total protein was reported. The calculation of data for analysis are described in the supplementary appendix.