Endogenous protein tagging coupled with a CRISPR screening approach identifies UBE3C as a potential MYC oncogene regulator

The transcription factor MYC is a key regulator of cellular proliferation and metabolism and is frequently dysregulated in malignancies such as multiple myeloma (MM). Despite its clinical relevance, direct therapeutic targeting of MYC remains limited, emphasizing the need to identify upstream regulators that control endogenous MYC expression. To systematically uncover such regulators, we developed a genome-wide CRISPR-Cas9 loss-of-function screening approach, employing a custom-engineered MM reporter cell line (RPMI8226-F11), in which oncogenic MYC protein was endogenously tagged with EGFP (referred to as GFP). This fluorescent readout enabled a direct, quantitative assessment of endogenous MYC expression levels. A pooled genome-wide sgRNA library was introduced, and cells were sorted based on GFP fluorescent intensity to reflect varying MYC levels. Next-generation sequencing of sgRNA distributions across sorted populations enabled the identification of candidate MYC regulators. Validation of screen hits, including the established MYC activator IRF4 and repressor FBXW7, confirmed the reliability of our system. To further dissect regulatory networks, we performed an overrepresentation analysis of target genes, which revealed the enrichment of Mediator complex subunits among MYC activators and ubiquitin-proteasome pathway components among MYC repressors. Functional validation of prioritized hits-MED30 (Mediator complex) and UBE3C (E3 ubiquitin ligase)-demonstrated a strong impact on endogenous MYC levels. Notably, the knockout of UBE3C markedly increased MYC expression, whereas its paralogs, UBE3A and UBE3B, showed no measurable effect, suggesting a specific regulatory role for UBE3C in MM cells. Together, our study provides a comprehensive CRISPR screen-based resource for the discovery of MYC regulators and highlights UBE3C as a potential therapeutic node for modulating MYC expression in MM.