Therapeutics, Targets, and Chemical Biology HO-3867, a Safe STAT3 Inhibitor, Is Selectively Cytotoxic to Ovarian Cancer

STAT3 iswell corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (–NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecularmodeling studies of this class suggested direct interactionwith the STAT3DNAbinding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to bemultifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated frompatient ascites, HO-3867 inhibited cellmigration/invasion and survival. Our results offer preclinical proof-ofconcept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated. Cancer Res; 74(8); 2316–27. 2014 AACR. Introduction The toxic side effects that standard anticancer drugs exert on healthy tissues and normal cells present obstacles in cancer treatment. These effects lead to dose reductions, treatment delays, and even the discontinuation of therapy. "Targeted therapy" is a relativelymodern term commonly used to describe new agents, including small molecules and monoclonal antibodies, specifically designed to take advantage molecular pathways involved in the pathophysiology to be treated. A secondary goal of such developments is to limit the negative side effects these molecules exert on normal tissues. Unfortunately, even new therapeutics cause significant adverse effects on normal tissues, leading to toxicity. Thus, development of safe and targeted anticancer therapies that selectively kill cancer cells while sparing the surrounding healthy tissues is essential. We identified a novel class of bifunctional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (–NOH; a nitroxide precursor) group for their inhibitory property on STAT3 activation (1, 2). Previous work found that DAP compounds exhibit more toxicity toward cancer cells when compared with noncancerous cells. However, the mechanism behind this selectivity was not fully elucidated. We hypothesize that the addition of the –NOH moiety functions as a modulator of cytotoxicity, imparting antioxidant protection to noncancerous tissues, while allowing the compound to maintain toxicity toward cancer cells. Two compounds, HO-3867 (possessing the –NOH moiety) and H-4073 (lacking the –NOH moiety) with the same DAP backbone were used to expound upon this concept (1, 3–5). In this study, we confirm the selective toxicity of HO-3867 as compared with H-4073. The mechanism of this selectivity is a differential interaction of HO-3867 and H-4073 with STAT3 in normal and cancer cells, with no impact upon other STATs. In addition, both compounds exhibit greater uptake and bioavailability in cancer cells when compared with normal cells. HO-3867 demonstrated anticancer efficacy in both a Authors' Affiliations: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Comprehensive Cancer Center; Center for Childhood Cancer, Nationwide Children's Hospital; Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Wexner Medical Center; EPR Imaging Center, Geisel School of Medicine, Dartmouth, New Hampshire; and Institute of Organic and Medicinal Chemistry, University of P ecs, P ecs, Hungary Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). K.S. Rath, S.K. Naidu, and P. Lata contributed equally to this work. Corresponding Author: Karuppaiyah Selvendiran, Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210. Phone: 614-685-6574; Fax: 614-2928454; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-2433 2014 American Association for Cancer Research. Cancer Research Cancer Res; 74(8) April 15, 2014 2316 on April 15, 2017. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 3, 2014; DOI: 10.1158/0008-5472.CAN-13-2433