Many enzymes catalyzing ubiquitin-like (Ubl) modifications have been identified as targets for the therapeutic development of life-threatening human diseases that lack a cure. However, Ubl modifications are poorly addressed by FDA approved drugs, highlighting the immense potential to exploit this type of post-translational modification to address unmet medical needs. Conjugation of Ubls to target proteins begins by activation of the C-terminus of a Ubl, a step catalyzed by enzymes generally known as activating enzyme or E1. Several E1 enzymes, including the SUMO-activating enzyme (SAE), have been validated as therapeutic targets by animal models and by early phase clinical studies. Our recent discovery of a conserved cryptic site on the SAE provides a paradigm-shifting allosteric approach to inhibit Ubl activating enzymes. In the next funding period, we will further elucidate the mechanism of the structure-activity relationship. Additionally, we will validate that the allosteric inhibition approach we discovered reduces cancer drug resistance for targeting Ubl activating enzymes. Furthermore, we will define the molecular mechanism of how SUMOylation regulates type I IFN expression and validate that SAE inhibition is an effective approach to induce anti-tumor immunity for immune cold colorectal cancers. The proposed studies build on our scientific progress in the previous funding cycle and our more than 20-year experience in studying SUMOylation. The proposed studies are expected to result in long- lasting impacts that will spur innovation in targeting Ubl for therapeutic development and result in new immune therapeutic strategies for immune cold tumors that do not respond to current immune therapies and are high unmet needs, such as colorectal cancers.