Effective implementation of novel MET pharmacodynamic assays in translational studies

Abstract

MET tyrosine kinase (TK) dysregulation is significantly implicated in many types of cancer. Despite over 20 years of drug development to target MET in cancers, a pure anti-MET therapeutic has not yet received market approval. The failure of two recently concluded phase III trials point to a major weakness in biomarker strategies to identify patients who will benefit most from MET therapies. The capability to interrogate oncogenic mutations in MET via circulating tumor DNA (ctDNA) provides an important advancement in identification and stratification of patients for MET therapy. However, a wide range in type and frequency of these mutations suggest there is a need to carefully link these mutations to MET dysregulation, at least in proof-of-concept studies. In this review, we elaborate how we can utilize recently developed and validated pharmacodynamic biomarkers of MET not only to show target engagement, but more importantly to quantitatively measure MET dysregulation in tumor tissues. The MET assay endpoints provide evidence of both canonical and non-canonical MET signaling, can be used as "effect markers" to define biologically effective doses (BEDs) for molecularly targeted drugs, confirm mechanism-of-action in testing combination of drugs, and establish whether a diagnostic test is reporting MET dysregulation. We have established standard operating procedures for tumor biopsy collections to control preanalytical variables that have produced valid results in proof-of-concept studies. The reagents and procedures are made available to the research community for potential implementation on multiple platforms such as ELISA, quantitative immunofluorescence assay (qIFA), and immuno-MRM assays.

Volatilome Analyses and In Vitro Antimicrobial Activity of the Essential Oils from Five South African Helichrysum Species

“…The statistical analysis was done on each VOC and EO composition. The principal component analysis (PCA) was performed selecting the two highest principal components (PCs) of a variance/covariance matrix, methods aimed at reducing the dimensionality of the multivariate data of the matrices, while preserving most of the variance [ 58 ]. The hierarchical cluster analysis (HCA) was performed using paired group (UPGMA) algorithm and Bray-Curtis as a similarity index for both the headspace analyses and the EO contents.…”

Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells

“…To illustrate the value of our novel EMT TF antibodies for examining tumor cell EMT in a complex histological context, we used these antibodies to assess EMT TF expression in a human HGF ( hHGF ) knock-in mouse xenograft model in which implanted human H596 non-small cell lung cancer (NSCLC) tumors undergo induction of EMT due to: 1) constitutive expression and secretion of hHGF by mouse stromal cells in the tumor microenvironment [ 11 ] and 2) potentiated activity of the human MET receptor in H596 tumors resulting from a MET exon 14−skipping mutation that suppresses MET proteasomal degradation [ 12 ]. Relative to H596 NSCLC tumors implanted in wild-type SCID mice, H596 tumors implanted in homozygous human HGF knock-in ( hHGF ki/ki ) mice exhibit enhanced growth rates and mesenchymal-like phenotypes, the latter assessed via immunofluorescence assays for E-cadherin and vimentin, markers of the epithelial and mesenchymal states, respectively [ 11 ]. We first confirmed that H596 tumor−bearing hHGF ki/ki mice exhibited substantially higher plasma hHGF levels compared to H596 tumor−bearing SCID mice, which had plasma hHGF levels below the lower limit of quantitation ( S4 Fig ).…”

“…In the latter case, we took advantage of the known role of HGF/Met signaling in inducing EMT [ 10 ] and compared EMT transcription factor (TF) expression in human non-small cell lung cancer (NSCLC) H596 tumors grown in severe combined immunodeficiency (SCID) mice to those implanted in SCID mice containing a homozygous human HGF ( hHGF ) knock-in mutation. hHGF knock-in H596 models exhibit rapid tumor growth and constitutive tumor EMT due to hHGF secretion by mouse stromal cells, thereby enhancing tumor HGF/MET signaling [ 11 ]; H596 tumors are particularly receptive to enhanced HGF secretion due to the presence of a MET exon 14−skipping mutation that suppresses proteasome-mediated MET degradation [ 12 ]. To validate the in vivo use of our antibody for the CSC marker CD133, we compared SUM149PT triple-negative breast cancer (TNBC) xenograft models treated with vehicle or the CSC-targeting focal adhesion kinase (FAK) inhibitor VS-6063 (defactinib; formerly PF-04554878) [ 13 ].…”

Extracellular Vesicles and Their Current Role in Cancer Immunotherapy

“…In the recent years, the use of vesicles as biological carriers has also been extended to cancer immunotherapy [ 18 ]. This kind of cancer treatment involves the use of extracellular vesicles to transport molecules capable of triggering an immune response to damage cancer cells.…”