Transcription Factor Inhibition as a Potential Additional Mechanism of Action of Pyrrolobenzodiazepine (PBD) Dimers

Background: The pyrrolobenzodiazepine (PBD) dimer SJG-136 reached Phase II clinical trials in ovarian cancer and leukaemia in the UK and USA in the 2000s. Several structural analogues of SJG-136 are currently in clinical development as payloads for Antibody-Drug Conjugates (ADCs). There is growing evidence that PBDs exert their pharmacological effects through inhibition of transcription factors (TFs) in addition to arrest at the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. Hence, PBDs can be used to target specific DNA sequences to inhibit TFs as a novel anticancer therapy. Objective: To explore the ability of SJG-136 to bind to the cognate sequences of transcription factors using a previously described HPLC/MS method, to obtain preliminary mechanistic evidence of its ability to inhibit transcription factors (TF), and to determine its effect on TF-dependent gene expression. Methods: An HPLC/MS method was used to assess the kinetics and thermodynamics of adduct formation between the PBD dimer SJG-136 and the cognate recognition sequence of the TFs NF-κB, EGR-1, AP-1, and STAT3. CD spectroscopy, molecular dynamics simulations, and gene expression analyses were used to rationalize the findings of the HPLC/MS study. Results: Notable differences in the rate and extent of adduct formation were observed with different DNA sequences, which might explain the variations in cytotoxicity of SJG-136 observed across different tumour cell lines. The differences in adduct formation result in variable downregulation of several STAT3-dependent genes in the human colon carcinoma cell line HT-29 and the human breast cancer cell line MDA-MB-231. Conclusions: SJG-136 can disrupt transcription factor-mediated gene expression, which contributes to its exceptional cytotoxicity in addition to the DNA-strand cleavage initiated by its ability to crosslink DNA.