Summary/Abstract The objective of this proposal is to develop a molecular toolbox for non-invasive methods to kill cells in situ via specific cell death pathways using safe, penetrant, and tunable temperature stimuli. There is an urgent need for new technology that can engage specific forms of cell death in vivo in a manner that is controlled in space, time, and magnitude. Such technology would enable researchers to systematically interrogate the distinct physiological consequences of programmed cell death through apoptosis, necroptosis, or pyroptosis, and to harness immunogenic types of cell death for cancer therapy. However, current methods to stimulate cell death are either not specific to individual types of death, lack spatiotemporal precision, or are limited by poor tissue penetration. Our proposed molecular tools overcome these limitations by leveraging the unique advantages of temperature as a control signal, including its ability to be modulated non-invasively with high spatial and temporal resolution even deep within tissue. We will generate single protein constructs that can trigger apoptosis, necroptosis, or pyroptosis with either gentle heating or cooling, and validate their efficacy in mouse models of human cancer. The rationale for our work is that this toolset will open powerful new avenues of discovery by enabling the study of programmed cell death pathways in living animals with a high degree of control. Moreover, the ability to selectively engage immunogenic cell death in a dose-dependent manner directly within tumors would establish a novel strategy for targeted, personalized cancer immunotherapy with the potential to be more efficacious and less toxic than current approaches. In preliminary studies, we engineered a temperature-responsive protein called Melt that clusters upon cooling and can induce cell death when fused to caspase-1. We demonstrated that Melt-caspase1 efficiently eliminates cancer cells in vitro and in mouse xenografts with high spatiotemporal precision. Building upon this promising foundation, we will pursue three specific aims: 1) Generate a suite of Melt fusions for cold-induced control of apoptosis, necroptosis and pyroptosis; 2) Test the ability of Melt-induced pyroptosis to stimulate immunogenic cell death and protect against rechallenge in an immune-competent mouse model; and 3) Develop proteins that induce cell death upon gentle heating by fusing effectors to proteins that self-assemble between 37-42C. We will achieve these aims using molecular engineering, live cell imaging, custom devices for feedback-controlled temperature regulation in cells and in mice, and syngeneic mouse models of human cancer. Success in our work will open new horizons for both fundamental discovery and therapeutic translation. Beyond cell death, this technology will enable remote control of a wide array of cell and molecular events directly within living mammals, a capability that promises to broadly impact both basic and applied biomedical research.

Project Narrative This project will develop new technology to control cell death in living organisms using mild temperature changes as a precise and non-invasive stimulus, enabling researchers to uncover how different cell death pathways impact human health and disease. This technology could ultimately establish a new approach to cancer therapy that precisely stimulates the body's own immune system to selectively eliminate tumors in a targeted and safe manner. In the shorter term the technology can be broadly applied for non-invasive control over a wide array of cell behaviors in animals, thus demonstrating broad impact for both basic and applied studies.