Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras

Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as the cation-independent mannose 6–phosphate receptor (CI-M6PR), to direct extracellular proteins to lysosomes. In this work, we used a genome-wide CRISPR knockout approach to identify modulators of LYTAC-mediated membrane protein degradation in human cells. We found that disrupting retromer genes improved target degradation by reducing LYTAC recycling to the plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was a predictive marker for LYTAC efficacy. A substantial fraction of cell surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Thus, inhibition of M6P biosynthesis increased the internalization of LYTAC-target complexes. Our findings inform design strategies for next-generation LYTACs and elucidate aspects of cell surface receptor occupancy and trafficking. Description Editor’s summary Delivery to lysosomes of various biotherapeutics such as lysosomal enzyme replacement therapies, antibody-drug conjugates, and oligonucleotide therapeutics is crucial for their activity. Extracellular protein degraders, including lysosome-targeting chimeras (LYTACs), enable the precise depletion of proteins from the extracellular space through lysosomal targeting. Ahn et al. used unbiased genetic screens and proteomics to map cellular determinants of extracellular degrader activity. Activation of the E3 Cullin-3 enabled LYTAC target complexes to reach lysosomes and was a predictive marker for cell susceptibility to degraders. Cell surface CI-M6PR, a prototypical lysosome-targeting receptor, was found to be blocked by endogenous mannose-6-phosphate (M6P)–modified glycoproteins. The retromer complex counteracted this degradation activity through recycling LYTAC-CI-M6PR complexes. Accordingly, disruption of M6P biosynthesis or CI-M6PR recycling increased LYTAC activity. Detailed mechanistic study of membrane protein degradation by LYTACs can thus elucidate endocytic biology and could help in the rational deployment of degraders against disease. —Stella M. Hurtley A genome-wide screen reveals cellular factors involved in targeted membrane protein degradation by lysosome-targeting chimeras. INTRODUCTION Repurposing endogenous cellular machinery to degrade disease-driving proteins is a promising therapeutic strategy, as evidenced by the rapid development of intracellular degraders over the past two decades. Extracellular degraders, including lysosome-targeting chimeras (LYTACs), have expanded the scope of protein degradation to secreted and plasma membrane targets. LYTACs harness endogenous cell surface receptors, such as cation-independent mannose 6–phosphate receptor (CI-M6PR), to traffic specific proteins between the plasma membrane and lysosomes. However, the cellular features that affect the ability to hijack lysosomal trafficking for membrane protein degradation are largely unknown. An approach to map features that potentiate or attenuate the ability to degrade membrane proteins is needed to further understand extracellular degradation mechanisms. RATIONALE The factors that affect membrane protein degradation could facilitate rational therapeutic development and reveal biological insights crucial for numerous therapeutic modalities. Membrane proteins, unlike soluble secreted proteins, can retain their own distinct trafficking behavior through the endosomal system, which must be overcome or enhanced for targeted degradation by extracellular degraders. We deploy an unbiased genome-wide CRISPR knockout screening approach complemented by proteomics to identify and characterize cellular components that modulate LYTAC-mediated membrane protein degradation. This approach combines modular chemically defined LYTAC ligands, scalable cell separation through magnetic beads, immunoprecipitation, and cell surface proteomics to identify key molecular players. RESULTS This genome-wide screen confirmed the importance of several endolysosomal genes for LYTAC-mediated degradation. We characterized the effect of additional genes distinct from the canonical endolysosomal pathway or genes whose knockout can enhance LYTAC activity. We identified that the retromer complex recycles LYTAC–CI-M6PR complexes from endosomes to the cell surface before dissociation of LYTAC from the receptor. Disruption of retromer complex genes enhanced degradation mediated by LYTAC by inhibiting this competitive recycling pathway. Genes involved in the neddylation of cullin 3 (CUL3), and therefore activation of its E3 ligase activity, were crucial for the trafficking of LYTAC-target complexes to lysosomes. We determined that levels of neddylated CUL3 are correlated with degradation efficacy, thereby serving as a potential predictive marker for LYTAC activity. In addition, we revealed that genes involved in the mannose 6–phosphate (M6P) biosynthesis pathway counteract LYTAC activity through synthesizing competitive ligands for CI-M6PR. We observed that cell surface CI-M6PR receptors are occupied by distinct M6P-modified lysosomal glycoproteins. Disruption of M6P biosynthesis increased the fraction of unoccupied receptors on the cell surface, allowing enhanced LYTAC-receptor engagement and internalization. CONCLUSION Mechanistic studies driven by extracellular degraders can reveal previously unknown aspects of receptor and endosomal biology. This study uncovered drivers of extracellular degradation using CI-M6PR as a lysosome trafficking receptor. The findings of this study motivate new design strategies for lysosome-targeted protein degraders. Furthermore, the magnetic enrichment–based genomic screen approach can be expanded to any other extracellular degraders, allowing the identification of common and distinct mechanisms across extracellular degradation platforms. We anticipate that the impacts of receptor recycling, occupancy, and ability to traverse the endolysosomal system revealed in our work will have broader implications for other therapeutics that rely on cell surface–to–lysosome trafficking, such as enzyme-replacement therapies, nucleic acid delivery, and antibody-drug conjugates. Identifying cellular determinants of LYTAC-mediated degradation of membrane proteins. A genome-wide CRISPR screen was conducted to identify factors regulating LYTAC-mediated degradation of epidermal growth factor receptor (EGFR) via magnetic cell sorting. Knockout (KO) of retromer complex resulted in reduced recycling of LYTAC-target complex. CUL3 neddylation was essential for lysosomal trafficking and degradation. Disruption of M6P biosynthesis pathway resulted in increased unoccupied CI-M6PR on the cell surface, enhancing LYTAC-receptor internalization. Ctx, cetuximab.