Protein complexes have a wide range of functions that can be modulated through both enzymatic activity and protein-protein interactions. In efforts to design small molecule therapeutics that target specific protein functions, molecules with diverse mechanisms of action have been developed. Traditionally, drug design has focused on inhibiting an enzyme’s catalytic activity, either by directly blocking its active site or by designing an allosteric modulator. However, beyond catalytic function, enzymes often serve as scaffolds that mediate interactions with other proteins—interactions that influence the overall function of a protein complex within cells. Discovering new small molecule modalities that selectively target one function of a protein can optimize therapies for various disease indications, while also offering insight into novel mechanisms of protein regulation and disease pathogenesis. In this thesis, I investigate the mechanisms of action of two recently developed small molecule modalities targeting the LSD1-HDAC1/2-CoREST (LHC) complex and uncover how mutations in an E3 ligase substrate adaptor lead to a new mode of LHC complex dysregulation in cells. In Chapter 1, I provide an overview of the LHC complex and key structural studies that have elucidated its fundamental functions and biological roles in cells and human development. I then review its involvement in disease and therapeutic applications, which has driven efforts to identify small molecules that target the complex. Finally, I introduce the concepts of targeted protein degradation—a therapeutic strategy recently applied to targeting the LHC complex—and genomic screening methodologies that we have used to evaluate novel mechanisms of protein regulation. In Chapter 2, I describe our work elucidating the mechanism of action of the small molecule T-448, an LSD1 inhibitor that selectively targets enzymatic activity while preserving LSD1’s interactions with transcription factors. Through mass spectrometry and structure-activity relationship studies with T-448 analogs, we found that T-448 forms a covalent drug-FAD adduct in LSD1’s active site, which subsequently v undergoes Grob fragmentation to yield a compact formyl-FAD adduct. This adduct preserves LSD1’s scaffolding function with transcription factors such as GFI1/GFI1B, thereby reducing hematological toxicity and making T-448 a more promising candidate for treating neurological disorders. Additionally, we show that this conversion from drug-FAD to formyl-FAD can serve as a resistance mechanism in AML cells. Using CRISPR suppressor scanning, we previously identified a loop deletion mutation distal to the catalytic site that confers resistance to certain LSD1 inhibitors through this mechanism. Altogether, this work highlights how small molecule design can target specific LSD1 functions and how distal loop mutations can impact drug mechanism of action. In Chapter 3, I detail our investigation into the mechanism of action of another LSD1-targeting molecule, UM171. It was initially identified in a phenotypic screen and later shown to induce degradation of LSD1 and CoREST via the E3 ubiquitin ligase substrate adaptor KBTBD4, however the direct binding partners of UM171 remained unknown. Using fluorescence-based cellular assays and biochemical binding studies, we determined that HDAC1/2 is the direct binding partner of UM171 within the LHC complex. UM171 acts as a molecular glue and increases the affinity between LHC and KBTBD4. We validated this by solving a cryo-EM structure of the KBTBD4-UM171-LHC complex and unexpectedly identified a second molecular glue—inositol hexakisphosphate—at the binding interface. The structure revealed that the KBTBD4 dimer engages a single copy of the HDAC1/2-CoREST complex asymmetrically. Additionally, base editing scanning was performed and confirmed UM171’s binding sites. This was the first study to elucidate the binding mode of a molecular glue that engages a Cullin3 E3 ligase substrate adaptor, revealing the mechanism behind LSD1 and CoREST degradation. In Chapter 4, I move beyond small molecule regulation to explore how mutations can also modulate protein-protein interactions. In medulloblastoma, complex insertion-deletion and substitution mutations have been found in a single loop in the Kelch domain of KBTBD4. In patient-derived xenograft models harboring these mutations, CoREST and LSD1 levels are depleted and knockout of mutant KBTBD4 rescues these protein levels. Through a series of biochemical assays, we also discover that the mutant KBTBD4 have increased binding affinities to LHC. To understand the scope of these gain-of-function mutations, we performed a deep mutational scan of the loop mutated in medulloblastoma. Surprisingly, a wide array of mutations promoted CoREST degradation and further analysis was performed to determine vi the types of mutations that had the strongest phenotype. Structural studies were performed to determine how these KBTBD4 variants were engaging the LHC substrate and revealed that mutant KBTBD4 mimics the UM171-induced binding mode observed in Chapter 3. This structural insight prompted us to test known HDAC active-site inhibitors as a potential therapeutic strategy to disrupt the mutant KBTBD4-LHC interaction. Through this study, we show that KBTBD4 cancer mutations chemically and functionally mimic UM171 and that deep mutational scanning can identify mutations that drive substrate degradation. We also demonstrate that active-site inhibitors can be repurposed to disrupt pathogenic protein-protein interactions. Overall, this thesis showcases multiple ways to perturb the LHC protein complex, via distinct small molecule modalities and neomorphic mutations that induce novel protein interactions leading to degradation. These studies not only teach us how we can differentially regulate the LHC complex for different therapeutic applications but also serve as an instructive example of how we can use both chemical and genetic methodologies to induce similar phenotypic effects. This work opens the door to applying high-throughput genomics to discover novel modulators for other protein complexes and cellular pathways in various disease contexts.