诱骗寡脱氧核苷酸(ODN)允许靶向不可药物的转录因子,如STAT3,但其有限的效力和缺乏传递方法阻碍了翻译。为了克服这些挑战,我们将STAT3特异性诱饵与沙利度胺结合,E3泛素连接酶复合物中ceblon的配体,以产生蛋白水解靶向嵌合体(STAT3DPROTAC)。STAT3DPROTAC下调靶细胞中的STAT3,但不是STAT1或STAT5。STAT3DPROTAC三元复合物的计算模型预测了两个表面赖氨酸,K601和K626,在STAT3中作为潜在的泛素化位点。因此,STAT3中的K601/K626点突变,以及蛋白酶体抑制或小脑缺失,缓解STAT3DPROTAC效应。接下来,我们将STAT3DPROTAC与靶向Toll样受体9(TLR9)的CpG寡核苷酸缀合,以产生髓样/B细胞选择性C-STAT3DPROTAC.裸C-STAT3DPROTAC被TLR9+骨髓细胞自发内化,B细胞,以及人类和小鼠淋巴瘤细胞,但不是T细胞。C-STAT3DPROTAC可有效降低STAT3蛋白水平,也可降低STAT3调节对淋巴瘤细胞增殖和/或存活至关重要的靶基因(BCL2L1、CCND2和MYC)。最后,对携带人类Ly3淋巴瘤的小鼠进行局部C-STAT3DPROTAC,引发肿瘤消退,而对照C-STAT3D和C-SCR治疗效果有限。我们的结果强调了使用PROTAC策略进行细胞选择性的可行性,基于诱饵寡核苷酸的STAT3靶向和潜在的其他肿瘤转录因子用于癌症治疗。
Decoy oligodeoxynucleotides (ODNs) allow targeting undruggable transcription factors, such as STAT3, but their limited potency and lack of delivery methods hampered translation. To overcome these challenges, we conjugated a STAT3-specific decoy to thalidomide, a ligand to cereblon in E3 ubiquitin ligase complex, to generate a proteolysis-targeting chimera (STAT3DPROTAC). STAT3DPROTAC downregulated STAT3 in target cells, but not STAT1 or STAT5. Computational modeling of the STAT3DPROTAC ternary complex predicted two surface lysines, K601 and K626, in STAT3 as potential ubiquitination sites. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibition or cereblon deletion, alleviated STAT3DPROTAC effect. Next, we conjugated STAT3DPROTAC to a CpG
oligonucleotide targeting Toll-like receptor 9 (TLR9) to generate myeloid/B cell-selective C-STAT3DPROTAC. Naked C-STAT3DPROTAC was spontaneously internalized by TLR9+ myeloid cells, B cells, and human and mouse lymphoma cells but not by T cells. C-STAT3DPROTAC effectively decreased STAT3 protein levels and also STAT3-regulated target genes critical for lymphoma cell proliferation and/or survival (BCL2L1, CCND2, and MYC). Finally, local C-STAT3DPROTAC administration to human Ly3 lymphoma-bearing mice triggered tumor regression, while control C-STAT3D and C-SCR treatments had limited effects. Our results underscore the feasibility of using a PROTAC strategy for cell-selective, decoy
oligonucleotide-based STAT3 targeting of and potentially other tumorigenic transcription factors for cancer therapy.