The molecular glue anagrelide induces proximity between phosphodiesterase 3 A (PDE3A) and Schlafen-12 (SLFN12) in cancers with high PDE3A expression, triggering SLFN12 tRNase activity, tRNA-Leu-TAA degradation, and ultimately, translation inhibition and cell death. However, other mechanisms, such as the GCN2-eIF2α-mediated stress response, also contribute to the anticancer effects of molecular glues, suggesting multiple parallel mechanisms. Here, we aimed to elucidate these mechanisms by using an interactomics approach to identify the altered protein-protein interactions (PPIs) in the PDE3-SLFN12 complex.

We used proximity dependent biotin labeling (Bio-ID) to discover the PDE3A-SLFN12 complex PPIs. SLFN12, PDE3A, and GFP-containing MAC-Tag-N expression plasmids were generated and transfected into SA-4 and HeLa cells. After biotin and either DMSO or anagrelide treatment, cells were lysed and analyzed via LC-MS. High-confidence interactors were filtered with Significance Analysis of INTeractome (SAINT) and Contaminant Repository for Affinity Purification (CRAPome). The results were validated with immunofluorescence, and new complexes were modeled with AlphaFold.

Peripheral eIF3 subunit PPIs were induced, while core eIF3 subunit PPIs were suppressed. As the peripheral eIF3 complex localizes to the opposite ribosomal face relative to the core eIF3 complex, these results suggest a shift in PDE3-SLFN12 localization to the ribosomal mRNA entry channel upon complex formation. Immunofluorescence confirmed increased colocalization of PDE3 and SLFN12 with ribosomes and each other following anagrelide treatment. AlphaFold modeling of the PDE3-SLFN12-eIF3 PPIs further supported these findings. Functional enrichment analysis via the STRING database revealed significant enrichments in ribonucleoprotein biogenesis, translation initiation and aminoacyl tRNA biosynthesis, reinforcing the complex’s role in translation control. Conversely, suppressed PPIs linked to protein folding, antiviral defence, and proteasome function indicated impaired nascent polypeptide folding, loss of SLFN12’s innate immune role, and disrupted PDE3A/SLFN12 degradation, respectively.

Anagrelide-induced remodeling of the PDE3-SLFN12 interactome localizes the complex to the ribosomal mRNA entry channel, inducing ribosomal and peripheral eIF3 PPIs while suppressing proteasomal, chaperonal, and core eIF3 PPIs. This provides insights into complex stabilization, altered translation dynamics and affected signaling pathways. Suppressed PPIs suggest impaired protein folding and disrupted PDE3A/SLFN12 turnover, contributing to translational stress and cell death. Together, these findings enhance our understanding of molecular glue mechanisms and support the design of improved therapeutics.