Leveraging the E3 Ligase RNF166 to Prevent T Cell Exhaustion in Solid Tumors - PROJECT SUMMARY Adoptive T-cell therapies (ACT) have emerged as a transformative approach in cancer immunotherapy, demonstrating remarkable success in hematological malignancies. However, their application in solid tumors is significantly limited by T-cell exhaustion (TEX), a dysfunctional state driven by chronic antigen stimulation and exacerbated by the environmental stresses of the tumor microenvironment (TME). TEX is marked by impaired proliferation, diminished effector function, and the upregulation of inhibitory receptors, ultimately curtailing the therapeutic potential of ACT. To overcome this barrier, innovative strategies are needed to engineer T-cells capable of maintaining their function and persistence within the challenging TME. E3 ubiquitin ligases (E3s) are critical regulators of protein stability and cellular adaptation under stress, yet their roles in TEX remain largely underexplored. Preliminary proteomic studies revealed significant global changes in protein turnover during TEX including the destabilization and loss of the T-cell receptor (TCR) complex, stabilization of inhibitory receptors, and the destabilization of costimulatory receptors. This discrepancy is partially attributed to the accumulation of unfolded proteins and the resulting compensatory increase in proteasomal activity. Through additional proteomic analysis modeling of TEX, I have revealed multiple E3s important to T-cell function. To determine which E3 has the greatest impact on T-cell function, I conducted an overexpression screen of multiple E3s in primary murine tumor-specific CD8+ T-cells. T-cells overexpressing each E3 were evaluated using serial in vitro cytotoxicity assays to assess their ability to control cancer growth. I identified the E3 ubiquitin ligase Ring Finger Protein 166 (RNF166) to enhance T-cell persistence, prolonging the cytotoxic function of the T-cells by 8 days. Further, elevated transcript levels of RNF166 in tumor infiltrating lymphocytes was found to correlate with responsiveness to immune checkpoint blockade (ICB) in metastatic melanoma. Additionally, I demonstrate that RNF166 overexpression reduces the expression of inhibitory receptors PD1, CTLA4, and LAG3 following chronic stimulation in primary human CD8+ T-cells. This proposal aims to elucidate the mechanisms by which RNF166 regulates T-cell function and explore its therapeutic potential to enhance ACT for solid tumors. Aim 1 will define the molecular targets of RNF166 using innovative proteomics approaches to investigate its impact on global proteome turnover and determine its targets for proteasomal degradation. Aim 2 will evaluate the efficacy of RNF166 overexpression in enhancing ACT using preclinical models, including murine syngeneic OT-1 T-cell and human CAR T-cell systems. This research represents the first comprehensive study of RNF166 in T-cell biology, providing critical insights into its role in TEX and its potential as a therapeutic target. By leveraging state-of-the- art mass spectrometry, bioinformatics, and in vivo modeling, this work aims to develop novel immunotherapeutic strategies to enhance ACT for solid tumors. This research proposal represents a unique training opportunity combining high-level mass spectrometry training, bioinformatics, systems biology, and cell engineering.
