Abstract 1986: Effect of the PBD dimer SJG-136 on expression of STAT3 dependent genes
Authors: Julia Mantaj, David E. Thurston, Khondaker M. Rahman
Journal: Cancer Research (2015)

Abstract Signal Transducer and Activator of Transcription 3 (STAT3) regulates the transcription of genes involved in cell differentiation, proliferation, apoptosis, angiogenesis, metastasis, and immune responses. STAT3 plays a crucial role in cancer progression as it is known to be over-expressed in many human tumours, including leukaemia, breast cancer and ovarian carcinoma. The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents which form an aminal linkage with the C2-NH2 group of a guanine base within the DNA minor groove. The PBD dimer SJG-136 has completed a Phase II clinical trial in ovarian cancer, and is presently being evaluated in leukaemia. There is growing evidence that PBD monomers exert their pharmacological effects through transcription factor inhibition. For example, GWL-78, a C8-linked PBD-Py-Py conjugate, has been shown to block interaction of the transcription factor NF-Y, and KMR-28-39, a GC sequence selective C8-linked PBD-MPB conjugate, inhibits transcription factors including NFκB. It has been recently observed that the PBD dimer SJG-136 can form covalent adducts with STAT3 consensus sequences. The aim of this study was to investigate whether the interaction of SJG-136 with STAT3 binding sequences is biologically relevant, and to explore whether STAT3 inhibition is one of the key mechanisms in addition to previously described DNA strand-breakage, inhibition of endonuclease and RNA polymerases, and arrest of the replication fork. We have developed an ion pair reversed phase HPLC/MS analytical methodology, and have previously demonstrated for the first time the ability of SJG-136 to bind to specific DNA consensus sequences of the transcription factors NFκB, EGR-1, AP-1 and STAT3. After completing this biophysical study, the effect of SGJ-136 on the expression of STAT3-dependent genes and proteins utilizing the breast cancer cell line MDA-MB-231 was studied using the quantitative polymerase chain reaction (qPCR) and Western blot analyses. Briefly, cells were stimulated with LPS and incubated with various concentrations of SJG-136 for 24 hours. The cells were lysed, and gene expression profiles compared with control. The results demonstrate that SJG-136 appears to produce a significant dose-dependent down-regulation of STAT3-dependent genes including Bcl-2, cyclin D1, survivin, NNMT and fascin compared to the reference genes GAPDH, ß actin and α tubulin. These findings are in broad agreement with our bio-physical observations, have implications for understanding the mechanism of action of SJG-136, and may potentially explain the differences in activity of SJG-136 toward various tumour cell lines. Citation Format: Julia Mantaj, David E. Thurston, Khondaker M. Rahman. Effect of the PBD dimer SJG-136 on expression of STAT3 dependent genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1986. doi:10.1158/1538-7445.AM2015-1986