Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3

Abstract

Decoy-oligodeoxynucleotides (ODN) allow for targeting undruggable transcription factors, such as STAT3, but their limited potency and lack of delivery methods hampered translation. To overcome these challenges, we conjugated STAT3-specific decoy to thalidomide, a ligand to cereblon in E3 ubiquitin ligase complex, to generate a proteolysis-targeting chimera (STAT3D^PROTAC). STAT3D^PROTAC downregulated STAT3 in target cells, but not STAT1 or STAT5. Computational modeling of the STAT3D^PROTAC 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 STAT3D^PROTAC effect. Next, we conjugated STAT3D^PROTAC to a CpG oligonucleotide targeting Toll-like receptor 9 (TLR9) to generate myeloid/B-cell-selective C-STAT3D^PROTAC. Naked C-STAT3D^PROTAC was spontaneously internalized by TLR9+ myeloid cells, B-cells, human and mouse lymphoma cells but not by T cells. C-STAT3D^PROTAC effectively decreased STAT3 protein levels and also STAT3-regulated target genes critical for lymphoma cell proliferation and/or survival (BCL2L1, CCND2, MYC). Finally, local C-STAT3D^PROTAC administration to human Ly3 lymphoma-bearing mice triggered tumor regression while control C-STAT3D and C-SCR treatments had limited effects. Our results underscore feasibility of using PROTAC strategy for cell-selective, decoy oligonucleotide-based STAT3 targeting of and potentially other tumorigenic transcription factors for cancer therapy.