Research Article

A compact synthetic pathway rewires cancer signaling to therapeutic effector release

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Science  03 May 2019:
Vol. 364, Issue 6439, eaat6982
DOI: 10.1126/science.aat6982

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Seeking and destroying cancer cells

Rather than inhibiting aberrant signaling in cancer cells, what if that signal was put to work in detecting and destroying the cancer cells? Such an anticancer strategy could be based on the ErbB family of receptors that is activated in many cancers. Chung et al. developed a cell-killing circuit that is activated by excessive ErbB signaling and used a viral delivery system to add it to cells. The ErbB receptor proteins are tyrosine kinases that autophosphorylate. The authors designed a protease that would be recruited to such over-phosphorylated receptors. Once in place, the protease cleaves a protein anchored to the cell membrane, releasing it into the cytoplasm where it causes death of the transformed cells.

Science, this issue p. eaat6982

Structured Abstract

INTRODUCTION

The specific identification and ablation of cancer cells is a long-standing problem in medicine that has not been fully solved. Cancer cells differ from normal cells in their ability to proliferate and survive in an uncontrolled manner, a consequence of mutations that drive the constitutive activation of intracellular signaling pathways. Signals commonly activated in cancer have been targeted for suppression by drugs, but these drugs are limited by toxic effects from inhibiting normal signaling as well. We considered a fundamentally different approach to cancer treatment in which oncogenic signals are detected and then, instead of being suppressed, are co-opted to trigger a therapeutic program. Here, we describe a method, Rewiring of Aberrant Signaling to Effector Release (RASER), in which a compact synthetic signaling pathway detects an oncogenic signal with high specificity and then rewires it to a variety of customizable responses.

RATIONALE

As oncogenic signaling differs from normal signaling in its constitutive nature, we hypothesized that signal integration over time should provide a measurement specific for cancer states. We also hypothesized that proteolytic release from sequestration could provide a mechanism for both irreversible signal integration and activation of a variety of effector proteins. We chose ErbB proteins, which include EGFR and HER2 and are constitutively activated in a large fraction of solid tumors, as targets for detection and rewiring of oncogenic states. We then designed ErbB-specific RASER on the basis of recruitment of a sequence-specific viral protease to release effector domains from sequestration at the plasma membrane. Modular design of these synthetic proteins facilitated tuning of sensitivity over a broad range and allowed output functions to be generalized to a variety of effectors. Finally, we developed a complete mathematical model of RASER to accurately predict system behavior, enabling the rational identification of strategies for system optimization.

RESULTS

Three rounds of model-guided optimization resulted in a RASER system with three mechanisms contributing to ErbB-induced effector release: protease recruitment to active ErbB, substrate recruitment to active ErbB, and ErbB-dependent stabilization of the protease. The final ErbB RASER system successfully released a reporter protein in a variety of ErbB-driven cancer cells but not in tissue-matched ErbB-normal cells. The fold induction of reporter release in the presence of constitutive ErbB exceeded that of Akt or ERK activation, two endogenous signaling outputs of ErbB. As desired, ErbB RASER output was similar to baseline after activation of ErbB by treatment with its natural ligand, epidermal growth factor (EGF), whereas Akt and ERK were well activated by EGF, indicating that ErbB RASER was specifically responsive to oncogenic ErbB states. We then successfully programmed RASER to link ErbB signaling to a variety of biological outcomes, including apoptosis and activation of endogenous genes of choice via catalytically dead Cas9–mediated RNA-directed transcription. These responses occurred robustly in ErbB-hyperactive cancer cells in an ErbB activity-dependent manner and were again absent in ErbB-normal cells. Finally, we used nonintegrating adeno-associated virus (AAV) to deliver ErbB RASER with an apoptotic cargo to cocultures of ErbB-hyperactive pancreatic cancer cells and ErbB-normal hepatocytes. As desired, AAV-delivered ErbB RASER selectively ablated the pancreatic cancer cells but spared the ErbB-normal hepatocytes.

CONCLUSION

RASER introduces a new concept for cancer detection and treatment, in which specific oncogenic signals are detected in cancer cells and then used to trigger a programmable therapeutic response. The performance of RASER demonstrates that synthetic signaling pathways based on first principles can detect an oncogenic protein activity with specificity matching or exceeding that of natural signaling pathways. RASER also serves as an example of the ability of mathematical models to accurately predict the behavior of compact synthetic signaling systems. Finally, the ability of AAV-delivered ErbB RASER to selectively ablate ErbB-hyperactive tumor cells suggests the possibility of using RASER-expressing viruses to treat cancer in vivo. Further generalization of RASER to other inputs and outputs could enable the development of a panel of active biological therapies targeted to specific cancerous states.

RASER in cancer cells.

(Left) In response to ErbB (EGFR or HER2) activity, RASER proteins (green and blue) release a programmable effector to carry out therapeutic responses. (Right) RASER transforms normal signaling, which is transient, to low and transient accumulation of effector. In contrast, with constitutive oncogenic signaling, effector accumulates until a therapeutic threshold is reached.

Abstract

An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9–mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.

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