Abstract 1230: High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype
Authors: Yue Peibin , Lopez-Tapia Francisco , Zhu Yinsong , Brotherton-Pleiss Christine , Fu Wenzhen , Alonso-Valenteen Felix , Mikhael Simoun , Medina-Kauwe Lali , Tius Marcus , Turkson James 
Published: July 2021

Signal Transducer and Activator of Transcription (STAT)3 is a member of the STAT family of transcription factors that is frequently aberrantly-activated in breast cancer and many other cancers. STAT3 represents a valid target for the development of novel anticancer therapeutics. However, it has posed significant challenges to the discovery efforts to identify potent and suitable small molecule inhibitors. As a result, there is currently no anti-STAT3 drugs in clinical application to realize the potential clinical benefits of inhibiting abnormal STAT3 function. Herein is a new series of (R)-azetidine-2-carboxamide-based small molecules, exemplified by H098, H127, H152, H169, H174, H182, and H203, which selectively inhibit STAT3 DNA-binding activity, with in vitro potency (IC50) in the range of 0.28-0.66 μM, compared to potencies greater than 18 µM against the activities of the family members, STAT1 and STAT5. Equally notable is the high affinity binding to STAT3, as shown by KD of 1 pM and 3.7 nM for H169 and H174, respectively. Treatment of human triple-negative breast cancer (TNBC) cells with H182 or H169 inhibited constitutive STAT3 phosphorylation (pY705STAT3) and DNA-binding activity in both time- and dose-dependent manner. Furthermore, both inhibitors potently inhibited anchorage-dependent and independent growth, colony survival, and migration of TNBC cells in vitro, with potencies, EC50 of 1.0-1.9 μM, and the compounds also induced apoptosis of same cells. Additionally, the expression of STAT3 target genes, including c-Myc, VEGF, and survivin, were suppressed in the compound-treated TNBC cells subsequent to the inhibition of constitutive STAT3 activation, thereby validating the inhibition of constitutively-active STAT3 and its downstream targets as being responsible for the antitumor cell responses. Moreover, H169 and docetaxel combinatorial treatment led to enhanced suppression of TNBC cell viability. By contrast, treatments with the new compounds had minimal effects on the induction of STAT1, Janus kinase 2, epidermal growth factor receptor, extracellular signal-regulated kinases (Erk1/2), Src, Shc phosphatase, or Akt in TNBC cells, and they were relatively weak on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively-active STAT3. Significantly, H182 delivered at 5-20 mg/kg via oral gavage or intra-peritoneal route inhibited growth of TNBC tumor xenograft in mice, with no evidence of toxicity. These studies identify novel, sub-micromolar potent azetidine-based small molecule STAT3 inhibitors that have shown antitumor efficacy against human TNBC in xenograft models. H182 and H169 represent suitable novel chemical entities for developing new therapeutics against TNBC and other cancers that harbor constitutively-active STAT3.

Citation Format: Peibin Yue, Francisco Lopez-Tapia, Yinsong Zhu, Christine Brotherton-Pleiss, Wenzhen Fu, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe, Marcus Tius, James Turkson. High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1230.