Quantification of protein-protein interactions and activation dynamics: A new path to predictive biomarkers

Abstract

Oncogene dysregulation is a driver of neoplasia development and progression. The use of quantitative molecular imaging to quantify oncogene activation will be crucial in developing companion diagnostics which can identify personalised patient regimens. However, the evaluation of oncogene activation does not necessarily correlate with oncoprotein activation. Post-translational modifications, such as phosphorylation, lipidation and methylation, may enhance oncoprotein functionality. It is this functionality that progresses neoplasia and may correlate with patient outcome. Advanced molecular imaging may be used to directly quantify oncoprotein, as opposed to oncogene, activation. Time-resolved Förster Resonance Energy Transfer (TR-FRET) involves the non-radiative transfer of energy from one chromophore to another over distances of 1-10 nm; allowing FRET to be used as a “chemical ruler”. TR-FRET can be utilised to directly elucidate spatial oncoprotein activation in single cells and patient tissues. In single cells, TR-FRET has uncovered the mechanisms by which PKCβ1 is trafficked to the nucleus and cleaved. Additionally it has revealed the mechanism of activation of Akt/PKB, whereby Akt/PKB undergoes a conformational change, allowing the Thr308 site to be phosphorylated by PDK1. Moreover TR-FRET has been utilised to quantify HER2-HER heterodimerisation and Akt/PKB activation states in patient biopsies, where it is shown to be predictive of outcome/relapse. The role of TR-FRET is not solely limited to intracellular signalling events. A study has used TR-FRET to measure intercellular immune-checkpoint receptor-ligand interactions. Within this study it was seen that PD-L1 expression was not indicative of PD-1/PD-L1 interaction states in a range of solid tumours. Crucially, in melanoma and NSCLC, PD-1/PD-L1 interaction was a predictive of an improved patient outcome. PD-L1 expression did not predict patient outcome. Several groups have worked to improve Fluorescence lifetime imaging microscopy (FLIM) acquisition times, including the use of: window-galvanometers; multifocal multiphoton FLIM and parallel pixel excitation coupled with wide-field time-gated FLIM. The development of novel quantitative molecular imaging will be critical in the development of personalised patient therapies in the future.