Development of decoy oligonucleotide-warheaded chimeric molecules targeting STAT3

Abstract

Proteolysis-targeting chimera (PROTAC) technology is a disruptive innovation in the drug development community, and over 20 PROTAC molecules are currently under clinical evaluation. These PROTAC molecules contain small-molecule warheads that bind to target proteins. Recently, oligonucleotide-warheaded PROTACs have emerged as a promising new tool to degrade DNA-binding proteins such as transcription factors. In this study, we applied an oligonucleotide-warheaded PROTAC technology to induce the degradation of signal transducer and activator of transcription 3 (STAT3), which is a hard-to-target protein. A double-stranded decoy oligonucleotide specific to STAT3 was conjugated to E3 binders (pomalidomide, VH032, and LCL161) to generate PROTAC molecules that recruited different E3 ubiquitin ligases cereblon (CRBN), von Hippel-Lindau (VHL), and inhibitor of apoptosis protein (IAP), respectively. One of the resulting PROTAC molecules, POM-STAT3, which recruits CRBN, potently induces STAT3 degradation. STAT3 degradation by POM-STAT3 was abolished by scrambling the oligonucleotide sequences of POM-STAT3 and by adding a double-stranded decoy oligonucleotide against STAT3 in a competitive manner, suggesting the significance of oligonucleotide sequences in STAT3 degradation. Moreover, POM-STAT3-induced STAT3 degradation was suppressed by the CRBN binder thalidomide, proteasome inhibitor bortezomib, E1 inhibitor MLN7243, and siRNA-mediated depletion of CRBN, indicating that STAT3 degradation is mediated by the ubiquitin-proteasome system, which involves CRBN as the responsible E3 ubiquitin ligase. Consistent with STAT3 degradation, NCI-H2087 cell viability was severely reduced following POM-STAT3 treatment. Thus, POM-STAT3 is a STAT3 degrader that potentially has cytocidal activity against cancer cells that are highly dependent on STAT3 signaling, which implies that inducing protein degradation by decoy oligonucleotide-warheaded PROTAC molecules could be harnessed to be therapeutic against oncogenic transcription factors.