Abstract LB037: E3 pairing and structural mechanisms underlying anti-tumor activity of clinical STAT3 degrader KT-333
Authors: Sharma Kirti , Fei Xue , Shi Yatao , Browne Christopher , Walther Dirk , Daigle Caroline , Ramanathan Anand , Miller Richard , Yuan Karen , Mahasenan Kiran , Zhu Sean , Huang Xin , Yang Bin 
Published: April 2024

Abstract
To elucidate the underlying mechanisms of STAT3 degradation and the suitability of VHL as an E3 ligase partner for targeting STAT3 in cancer using our clinical degrader, KT-333.
Signal transducer and activator of transcription 3 (STAT3) is an undrugged oncogenic transcription factor and its role as cancer driver and tumor microenvironment modulator has been validated in a multitude of studies. Based on it’s potential as a target for cancer therapeutics and limitations of prior approaches, we developed KT-333, a first-in-class, potent, highly selective, heterobifunctional STAT3 degrader currently in Phase 1 clinical trials. Here, based on STAT3 degradation by multiple E3s based degraders, structure of STAT3-KT333-VHL and a lysine site-resolved target ubiquitination model, we provide evidence for VHL as the ideal partner E3 for targeting STAT3 in cancer. We successfully identified potent and selective STAT3 degraders using either CRBN or VHL as the E3 ligase, but ultimately preferred VHL based degraders for their increased potency, and consistent STAT3 degradation across multiple cancer lines. KT-333, our VHL based clinical STAT3 degrader, induces a strong ternary complex between STAT3 and VHL. We present a high resolution cryo-electron microscopy based ternary complex structure of STAT3-KT333-VHL which provides mechanistic insights further validating VHL as the E3 of choice for deep, selective and fast STAT3 degradation. Specifically, KT-333 enables favorable protein-protein interactions between STAT3 and VHL, burying a large protein interface in the ternary complex to the extent typically observed only in native protein complexes. Additionally, the residues forming the STAT3-VHL interface are not conserved in other STAT family members corroborating with degradation selectivity observed with KT-333. Next, we leveraged this structure and generated a ubiquitination super-complex model by deploying a combination of biochemical and proteomics techniques and reveal that KT-333-induced ubiquitination occurs by precise targeting of specific lysine resides on STAT3. Furthermore, to decode the mechanism for KT-333 activity against SUDHL1 cell line both in vitro and in a mouse xenograft model, we measured temporal changes at protein level by discovery proteomics. Reduced expression of canonical STAT3 targets and down-regulation of cytokine-mediated signaling and cell cycle signature genes indicated that cell cycle arrest and subsequent apoptosis are the main drivers of efficacy in vitro and in vivo. In summary, we show that our clinical STAT3 degrader, KT-333, is designed and optimally paired with VHL resulting in a very stable ternary complex exhibiting properties of native protein complexes. Our data provides precise structural and molecular mechanisms behind potent, selective, consistent, and fast degradation of STAT3, and downstream mechanism of action observed in vitro and in vivo.
Citation Format: Kirti Sharma, Xue Fei, Yatao Shi, Christopher Browne, Dirk Walther, Caroline Daigle, Anand Ramanathan, Richard Miller, Karen Yuan, Kiran Mahasenan, Sean Zhu, Xin Huang, Bin Yang. E3 pairing and structural mechanisms underlying anti-tumor activity of clinical STAT3 degrader KT-333 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB037.