Exploiting the Endogenous Ubiquitin Proteasome System in Targeted Cancer Treatment

SIMPLE SUMMARY: The design of specific targeting reagents/drugs still remains a major goal in cancer treatment, with the aim of directing therapeutic agents into cancer cells, while avoiding damage to normal tissues and without evoking an immune response. One way to deal with this problem is to use a chimeric protein composed of two components, one to specifically target the cancer cells and the other to kill them. In this study the targeting component is IL2, targeting activated T-cells that over-express the IL2 receptor, and the killing component is Smurf2, a component of the endogenous cellular protein degradation system. When the IL2-Smurf2 chimeric protein enters cancer cells in a dose- and time-dependent manner, it creates a large number of downstream degradation processes, which lead to apoptotic cell death. In this way, the cancer cells program their own death in a non-inflammatory pathway. ABSTRACT: To overcome the lack of specificity of cancer therapeutics and thus create a more potent and effective treatment, we developed a novel chimeric protein, IL2-Smurf2. Here, we describe the production of this chimeric IL2-Smurf2 protein and its variants, with inactive or over-active killing components. Using Western blots, we demonstrated the chimeric protein’s ability to specifically enter target cells alone. After entering the cells, the protein showed biological activity, causing cell death that was not seen with an inactive variant, and that was shown to be apoptotic. The chimeric protein also proved to be active as an E3 ligase, as demonstrated by testing total ubiquitination levels along with targeted ubiquitination for degradation. Finally, we tested IL2-Smurf2 and its variants in an in vivo mouse model of leukemia and demonstrated its potential as a drug for the targeted treatment of cancer cells. In the course of this work, we established for the first time the feasibility of the use of Smurf2 as a killing component in chimeric targeting proteins. Utilizing the IL2 cytokine to target cells overexpressing IL-2R and Smurf2 to cause protein degradation, we were able to produce a chimeric protein with dual functionality which causes targeted cell death.