PO.ET09.04 · 实验与分子治疗
基于Lumit的降解剂动力学分析揭示对降解剂的信号依赖性、细胞背景特异性敏感性
Lumit-based profiling of degrader dynamics reveals signaling-dependent, cell context-specific sensitivity to degraders
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
尽管靶向蛋白降解（TPD）为难治性疾病提供了一种变革性方法，但在疾病相关模型中测试降解剂仍具挑战性。确定降解剂的动力学和效力通常涉及工程化细胞系统，这些系统尽管有其优点，但资源消耗大且通量低。为缓解这些问题，我们利用Lumit® 免疫分析细胞系统（ICS）平台，为TPD靶点开发了定量、高通量的发光免疫分析方法。这些分析可测量降解剂的效力、选择性和细胞系变异性。已验证了多个靶点-降解剂配对，包括BRD4-ARV-771、IKZF1-Iberdomide和STAT3-SD-36。这些经验证的分析证明了该平台在捕获多样化降解机制方面的通用性和稳健性。利用Lumit在众多细胞系中分析STAT3降解剂SD-36时，我们在A431细胞中发现了一个出人意料的效力变化。这表现为DC₅₀值处于µM范围，而在测试的其他细胞系中为nM范围。我们探索了多种途径以揭示这一变化的原因，包括降解剂机制或靶蛋白的差异。在包括A431在内的多个细胞系中考察时，未发现降解剂膜通透性、靶点泛素化、cereblon表达、STAT3表达或STAT3周转方面的差异。然而，我们发现表皮生长因子受体（EGFR）的高表达与降解剂效力降低相关。此外，我们发现降解EGFR或抑制A431细胞中EGFR的磷酸化可恢复SD-36的效力。综合来看，这些数据确立了EGFR信号是STAT3降解应答的一个关键调节因子。这一现象是STAT3降解所特有的，因为针对其他靶点的降解剂在包括A431在内的各细胞系中维持了相当的纳摩尔级DC₅₀值。进一步探索还表明，PARylation和PERK通路似乎参与了对STAT3的保护，且该效应在A431细胞中更为显著。PARylation和PERK通路此前已被证明会影响某些靶点的降解，而我们的发现指向了一条似乎专门调控STAT3降解的额外信号通路。虽然传统上认为对TPD疗法的耐药可源于细胞内降解剂机制或靶蛋白的变化，但我们已证明内在细胞通路可影响降解剂效力。这一发现可能重新定义TPD的局限性，将信号调控的表型纳入其中。
查看英文原文 English abstract
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.
利益披露 Disclosure
M. Swiatnicki, None..
L. Engel, None..
H. Zegzouti, None.