A Protein Therapeutic Modality Founded On Molecular Regulation

The exquisite specificity of proteins is a key feature driving their application to anti-cancer therapies. The therapeutic potential of another fundamental property of proteins, their ability to be regulated by molecular cues in their environment, is unknown.  We propose a synthetic biology strategy for designing protein therapeutics that autonomously activate a therapeutic function in response to a specific cancer marker of choice.  We demonstrate this approach by creating a switchable prodrug-activating enzyme that selectively kills human cancer cells that accumulate the cancer marker hypoxia-inducible factor 1a (HIF-1a). HIF-1a accumulates to a high level in many solid tumors including breast, prostate, colorectal, and pancreatic cancers but is virtually undetectable in normal, well-oxygenated tissues.  When HIF-1a accumulates in the cytoplasm it interacts with the CH1 domain of p300 and is transported to the nucleus, where it activates the transcription of many genes.  We created a switchable enzyme that is a fusion of this CH1 domain and the enzyme cytosine deaminase from yeast, which can convert the non-toxic prodrug 5-fluorocytosine (5FC) to the chemotherapeutic 5-fluorouracil (5FU).  This switch couples the production of 5FU to cellular HIF-1a levels. Thus, expression of the switch in RKO colorectal cancer cells confers susceptibility to 5FC in a HIF-1a dependent manner.  Our strategy offers a platform for the development of inherently selective protein therapeutics for cancer and other diseases.