[PMC free article] [PubMed] [Google Scholar] 19
[PMC free article] [PubMed] [Google Scholar] 19. and treatment. One Sentence Summary: RASER is usually a rationally designed synthetic pathway that specifically detects an intracellular oncogenic state and rewires it to programmable therapeutic outputs. Synthetic biology, the engineering of new functions into living cells, has the potential to produce novel solutions to hard medical problems (1). One challenging problem is the specific identification ARN-3236 and treatment of malignancy cells. Malignancy cells differ fundamentally from normal cells in constitutively activating signaling pathways promoting cell growth, proliferation, or survival (2). For example, constitutive activation of ErbB-family receptor tyrosine kinases (RTKs), which include ErbB1 (HER1, EGFR) and ErbB2 (HER2, Neu), occurs in a substantial fraction of brain, esophageal, head and neck, lung, and breast cancers (3). Treatments have been developed that are specific for ErbB receptors, but not for their constitutive state. These treatments, which include small-molecule drugs (4), antibodies (4), viruses (5), and cells (6), attempt to inhibit ErbB receptors or eliminate the cells expressing them. However, because ErbB receptors are also required in normal cells for physiological signaling (Fig. 1A), they cannot be fully inhibited, or the cells expressing them completely eliminated, without causing toxicity to healthy tissues. Open in a separate windows Fig. 1. Concept and model of a molecular integrator of ErbB signaling. (A) Pharmacological approaches to malignancy therapy that aim at blocking tumor-promoting signals. (B) Signal-induced proteolysis can integrate transmission activity over time and function as a generalizable activation mechanism for multiple effectors. (C) Molecular modeling suggests the OFP-substrate-CAAX protein should be able to be cleaved by PTB-pro bound to ErbB. (D) Predicted concentration of released cargo at numerous occasions in ErbB-inhibited and ErbB-hyperactive says, using ErbB figures from BT-474 breast malignancy cells. (E) Predicted percent substrate cleavage after 24 h of protein expression. Note percent substrate cleavage is not the same as concentration of cleaved cargo, because the model accounts for the observation that ~50% less total substrate is usually expressed in ErbB-inhibited conditions. Predicted percent substrate cleavage normalizes for this expression CCM2 difference whereas the predicted concentration of released cargo does not. (F) Observed cleavage efficiency by protease and substrate variants. BT-474 cells, in which ErbB2 (HER2) is usually overexpressed and constitutively active, were transfected with the indicated constructs. Cells were then incubated with 0.5 M lapatinib to inhibit ErbB or without lapatinib to leave ErbB signaling on. After 24 h, cells were lysed for immunobloting against the V5 epitope tag fused to OFP. GAPDH served as a loading control. (G) Observed percent substrate cleavage. Error bars represent standard error of the mean (s.e.m.) of three biological replicates. We considered a new theory for malignancy therapy where oncogenic signaling is not blocked but instead is detected and then co-opted to trigger therapeutic responses via transmission rewiring (Fig. 1B). We propose that synthetic proteins could be launched into cells to query the state of a specific signaling pathway and execute a therapeutic program only if an oncogenic state is detected, preventing undesired toxicities in normal tissues. For therapeutic versatility, the ability ARN-3236 to activate any launched protein or transcribe any gene of choice would be ideal. While signaling pathways have been engineered within immune cells to customize responses upon binding antigens enriched on malignancy cells (7C9), the approach we propose would be conceptually different in sensing ARN-3236 and rewiring oncogenic signals within malignancy cells, targeting the fundamental biological difference between malignancy and normal cells (10). In this study, we statement a synthetic system, composed of only two modular proteins, that functions as a molecular integrator to discern oncogenic ErbB signals from normal signals. The simplicity of this system, named Rewiring of Aberrant Signaling to Effector Release (RASER), allowed its behavior to be comprehensively simulated by a mathematical model, facilitating rational optimization. We demonstrate that RASER is usually more dependent on constitutive ErbB signaling than native growth- and survival-promoting kinase pathways, and can be very easily programmed to produce a variety of therapeutic outputs, including apoptosis and transcription of endogenous genes. RASER represents the first method to detect a specific oncogenic transmission in living cells, and may enable precision molecular therapeutics for malignancy. A simple synthetic system for rewiring aberrant signaling to effector release An ideal system for linking oncogenic transmission transduction to therapeutic outputs would have to meet two general requirements. First, it should be specific; and of the PTB-pErbB conversation (11), and of the NS3-substrate reaction (16, 17), and the juxtamembrane volume in which ErbB cytosolic domains reside (fig. S1B). While we could obtain or estimate some.