Lumit-based profiling of degrader dynamics reveals signaling-dependent, cell context-specific sensitivity to degraders

While targeted protein degradation (TPD) offers a transformative approach for refractory disease, testing degraders in disease relevant models remains challenging. Determining degrader kinetics and potency often involves engineered cell systems, which despite their merits, are resource intensive and low throughput. To alleviate these issues, we used the Lumit® Immunoassay Cellular Systems (ICS) platform to develop quantitative, high-throughput luminescent immunoassays for TPD targets. These assays measure degrader potency, selectivity, and cell-line variability. Multiple target-degrader pairs have been validated, including BRD4-ARV-771, IKZF1-Iberdomide, and STAT3-SD-36. These validated assays demonstrate the platforms versatility and robustness in capturing diverse degradation mechanisms. Using Lumit to profile the STAT3 degrader, SD-36, across numerous cells lines, we uncovered an unexpected potency shift in A431 cells. This was evidenced by DC₅₀ values in the µM range, compared to nM range for other cell lines tested. Multiple avenues were explored to uncover the cause of this shift, including discrepancies in degrader mechanism or the target protein. When looking across multiple cell lines including A431, no discrepancies in degrader membrane permeability, target ubiquitination, cereblon expression, STAT3 expression, or STAT3 turnover were found. However, we uncovered that higher expression of epidermal growth factor receptor (EGFR) correlated with reduced degrader efficacy. Furthermore, we found that degrading EGFR or inhibiting EGFR phosphorylation in A431 cells restored the potency of SD-36. Combined, these data establish EGFR signaling as a key modulator of STAT3 degradation response. This phenomenon was specific to STAT3 degradation, as degraders for other targets maintained comparable nanomolar DC₅₀ values across cell lines, including A431. Further exploration also indicated that the PARylation and PERK pathways appear to contribute to the protection of STAT3, with the effect being more pronounced in A431 cells. The PARylation and PERK pathways have previously been shown to affect degradation of certain targets, while our findings point to an additional signaling pathway that seems to specifically regulate STAT3 degradation. While it has been traditionally thought that resistance to TPD therapies can occur due to changes in degrader mechanics or the target protein within the cell, we have demonstrated that intrinsic cellular pathways can impact degrader potency. This discovery may redefine TPD limitations to include signaling-regulated phenotypes.