Small Molecule STAT3 Inhibitor Research Articles

STAT3 Inhibitor OPB-51602 Is Cytotoxic to Tumor Cells Through Inhibition of Complex I and ROS Induction.

SummarySTAT3 is a transcription factor involved in several cellular activities including inflammation, proliferation, and survival, but it also plays a non-transcriptional role in modulating mitochondrial metabolism. Given its diverse functions in human cancers, it is an emerging therapeutic target. Here we show that OPB-51602, a small molecule inhibitor of STAT3, is highly toxic in a STAT3-dependent manner. Specifically, drug toxicity depends on mitochondrial STAT3 as tumor cells expressing only a mitochondrially restricted form of STAT3 are sensitive to the compound, whereas STAT3-null cells are protected. OPB-51602 inhibited complex I activity and led to increased ROS production, which in turn induced mitophagy, actin rearrangements, and cell death. Cells undergoing reduced oxidative phosphorylation or expressing NDI1 NADH dehydrogenase from Saccharomyces cerevisiae, which bypasses mammalian complex I, were resistant to OPB-51602 toxicity. These results show that targeting mitochondrial STAT3 function causes synthetic lethality through complex I inhibition that could be exploited for cancer chemotherapy.

A small molecule STAT3 inhibitor, LLL12, enhances cisplatin‑ and paclitaxel‑mediated inhibition of cell growth and migration in human ovarian cancer cells.

Ovarian cancer is one of the most lethal cancer types in American women. The platinum agent cisplatin, and/or paclitaxel, remains the first‑line chemotherapy for ovarian cancer, but the treatment success is severely limited by chemoresistance. As previously reported, persistent STAT3 signaling is associated with resistance to cisplatin and paclitaxel. To investigate whether the STAT3 small molecule inhibitor LLL12 can enhance the treatment effect of cisplatin and paclitaxel in ovarian cancer cells, A2780, SKOV3, CAOV‑3 and OVCAR5 cells were treated with LLL12, cisplatin and paclitaxel, alone or combination, and cell viability, cell migration, cell growth and protein expression levels were then evaluated. It was found that, for all four human ovarian cancer cell lines, STAT3 phosphorylation was significantly inhibited by LLL12. The combined treatment of LLL12 with paclitaxel or LLL12 with cisplatin exerted significantly greater inhibition of cell viability, cell migration and cell growth than did monotherapy. In addition, LLL12 and cisplatin in combination, or the three drugs in combination, also led to greater inhibition of cell viability and cell migration than combined cisplatin and paclitaxel treatment, a standard treatment for ovarian cancer. The present results demonstrated that the STAT3 small molecule inhibitor LLL12 is a potent inhibitor of STAT3 phosphorylation, cell viability and migration in human ovarian cancer cells. Combining LLL12 with cisplatin or paclitaxel may be a viable therapeutic approach in the treatment of patients with ovarian cancer exhibiting persistent STAT3 signaling.

Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition.

Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

Pharmacokinetics and pharmacodynamics of TTI-101, a STAT3 inhibitor that blocks muscle proteolysis in rats with chronic kidney disease.

Loss of muscle proteins increases the morbidity and mortality of patients with chronic kidney disease (CKD), and there are no reliable preventive treatments. We uncovered a STAT3/CCAAT-enhancer-binding protein-δ to myostatin signaling pathway that activates muscle protein degradation in mice with CKD or cancer; we also identified a small-molecule inhibitor of STAT3 (TTI-101) that blocks this pathway. To evaluate TTI-101 as a treatment for CKD-induced cachexia, we measured TTI-101 pharmacokinetics and pharmacodynamics in control and CKD rats that were orally administered TTI-101or its diluent. The following two groups of gavage-fed rats were studied: sham-operated control rats and CKD rats. Plasma was collected serially (0, 0.25, 0.5, 1, 2, 4, 8, and 24 h) following TTI-101 administration (at oral doses of 0, 10, 30, or 100 mg/kg). Plasma levels of TTI-101 were measured by LC-MS/MS, and pharmacokinetic results were analyzed with the PKSolver program. Plasma TTI-101 levels increased linearly with doses; the maximum plasma concentrations and time to maximal plasma levels (~1 h) were similar in sham-operated control rats and CKD rats. Notably, gavage treatment of TTI-101 for 3 days produced TTI-101 muscle levels in sham control rats and CKD rats that were not significantly different. CKD rats that received TTI-101 for 7 days had suppression of activated STAT3 and improved muscle grip strength; there also was a trend for increasing body and muscle weights. TTI-101 was tolerated at doses of 100 mg·kg-1·day-1 for 7 days. These results with TTI-101 in rats warrant its development as a treatment for cachexia in humans.

LLY17, a novel small molecule STAT3 inhibitor induces apoptosis and suppresses cell migration and tumor growth in triple-negative breast cancer.

Persistent STAT3 signaling is frequently detected in many cancer types including triple-negative breast cancer, and thus could potentially serve as a viable therapeutic target. We have designed a novel non-peptide compound LLY17 targeting STAT3 using Advanced Multiple Ligand Simultaneous Docking (AMLSD) methods. However, the efficacy of LLY17 has not been evaluated extensively in human and murine triple-negative breast cancer cells. In this study, we tested LLY17 in multiple human and murine triple-negative breast cancer cell lines. Human triple-negative breast cancer MDA-MB-468, MDA-MB-231, SUM159, and BT-549 cells, and murine triple-negative breast cancer 4T1 cells were used to study the inhibition effects of LLY17. The inhibition of STAT3 activation of LLY17 was investigated using western blot analysis. Cell viability, apoptosis and migration assays were carried out by MTT assay, Caspase-3/7 assay and wound healing assay, respectively. A mammary fat pad syngeneic mouse model was used to evaluate the antitumor effect of LLY17 in vivo. LLY17 inhibited IL-6-mediated induction of STAT3 phosphorylation but had no effect on IFN-γ-induced STAT1 phosphorylation or EGF-induced ERK phosphorylation. LLY17 inhibited STAT3 phosphorylation and induced apoptosis in human and murine triple-negative breast cancer cells but exhibited minimal toxicity toward Luminal A subtype breast cancer MCF-7 cells. RNAi attenuation experiments supported the requirement of STAT3 for LLY17-mediated inhibition of cell viability in triple-negative breast cancer cells. In addition, LLY17 inhibited cell migration of human and murine triple-negative breast cancer cells. Furthermore, LLY17 suppressed tumor growth and STAT3 phosphorylation of triple-negative breast cancer cells in a mammary fat pad syngeneic mouse model in vivo. Together, our findings suggest that targeting persistent STAT3 signaling by novel small molecule LLY17 may be a potential approach for the therapy of triple-negative breast cancer.

STAT3 Inhibitor ODZ10117 Suppresses Glioblastoma Malignancy and Prolongs Survival in a Glioblastoma Xenograft Model.

Constitutively activated STAT3 plays an essential role in the initiation, progression, maintenance, malignancy, and drug resistance of cancer, including glioblastoma, suggesting that STAT3 is a potential therapeutic target for cancer therapy. We recently identified ODZ10117 as a small molecule inhibitor of STAT3 and suggested that it may have an effective therapeutic utility for the STAT3-targeted cancer therapy. Here, we demonstrated the therapeutic efficacy of ODZ10117 in glioblastoma by targeting STAT3. ODZ10117 inhibited migration and invasion and induced apoptotic cell death by targeting STAT3 in glioblastoma cells and patient-derived primary glioblastoma cells. In addition, ODZ10117 suppressed stem cell properties in glioma stem cells (GSCs). Finally, the administration of ODZ10117 showed significant therapeutic efficacy in mouse xenograft models of GSCs and glioblastoma cells. Collectively, ODZ10117 is a promising therapeutic candidate for glioblastoma by targeting STAT3.

Stat3 activation induces insulin resistance via a muscle-specific E3 ubiquitin ligase Fbxo40.

Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly understood. One potential mechanism is that CKD-induced inflammation activates the signal transducer and activator of transcription 3 (Stat3) in muscle. We uncovered increased p-Stat3 in muscles of mice with CKD or mice fed high-fat diet (HFD). Activated Stat3 stimulates the expression of Fbxo40, a muscle-specific E3 ubiquitin ligase that stimulates ubiquitin conjugation leading to degradation of insulin receptor substrate 1 (IRS1). Evidence that Stat3 activates Fbxo40 includes 1) potential Stat3 binding sites in Fbxo40 promoters; 2) Stat3 binding to the Fbxo40 promoter; and 3) constitutively active Stat3 stimulating both Fbxo40 expression and its promoter activity. We found that IL-6 activates Stat3 in myotubes, increasing Fbxo40 expression with reduced IRS1 and p-Akt. Knockdown Fbxo40 using siRNA from myotubes results in higher levels of IRS1 and p-Akt despite the presence of IL-6. We treated mice with a small-molecule inhibitor of Stat3 (TTI-101) and found improved glucose tolerance and insulin signaling in skeletal muscles of mice with CKD or fed an HFD. Finally, we uncovered improved glucose tolerance in mice with muscle-specific Stat3 KO versus results in Stat3f/f mice in response to the HFD. Thus Stat3 activation in muscle increases IR in mice. Inhibition of Stat3 by TTI-101 could be developed into clinical strategies to improve muscle insulin signaling in inflammation and other catabolic diseases.

Abstract P5-05-11: Novel small molecule STAT3 inhibitors induce apoptosis and suppress cell migration and tumor growth in triple-negative breast cancer cells

Abstract Persistent STAT3 signaling is frequently detected in many cancer types and thus could potentially serve as a viable therapeutic target. In this study, we tested two novel small STAT3 inhibitors, LLY17 and LLL12B in triple-negative breast cancer cells, which frequently express persistent STAT3 phosphorylation. LLY17 and LLL12B selectively inhibited IL-6-mediated induction of STAT3 phosphorylation but no effect on IFN-γ mediated induction of STAT1 phosphorylation. LLY17 and LLL12B induced apoptosis in triple-negative breast cancer cells but exhibited minimal toxicity toward normal mammary epithelial cells. RNAi attenuation experiments confirmed the requirement of STAT3 for LLY17 and LLL12B-mediated inhibition of cell viability in triple-negative breast cancer cells. LLY17 and LLL12B inhibited STAT3 phosphorylation in human and murine triple-negative breast cancer cells. In addition, LLY17 and LLL12B inhibited cell migration of human and murine triple-negative breast cancer cells. Furthermore, LLY17 and LLL12B suppressed tumor growth and STAT3 phosphorylation of triple-negative breast cancer cells in vivo in a mammary fat pad murine syngeneic mouse model. Together, our findings suggest that targeting persistent STAT3 signaling pathway by novel oral bioavailable small molecules LLY17 and LLL12B may be a potential approach for the therapy of triple-negative breast cancer. Citation Format: Li Pan, Xiang Chen, Shengling Fu, Chenglong Li, Hui-Wen Lo, Jiayuh Lin. Novel small molecule STAT3 inhibitors induce apoptosis and suppress cell migration and tumor growth in triple-negative breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-11.

OPB-51602, a Direct Inhibitor of STAT3, Impairs Growth and Viability of Tumor Cells Through Inhibition of Respiratory Complex I

STAT3 is a transcription factor involved in numerous cellular activities, including inflammation, proliferation, and survival, but it also plays a non-transcriptional role in modulating mitochondrial metabolism. Given its diverse functions in human cancer, it is an emerging therapeutic target. Here we show that OPB-51602, a small molecule inhibitor of STAT3, is highly toxic in a STAT3- dependent manner. Specifically, drug toxicity depends on STAT3 mitochondrial functions as tumor cells expressing only a mitochondrially-restricted form of STAT3 are highly sensitive to the compound, while STAT3-null cells are protected. OPB-51602 treatment abrogated complex I activity and led to mitophagy, actin skeletal rearrangements, and apoptosis. Cells undergoing reduced oxidative phosphorylation or expressing the NDI1 NADH dehydrogenase enzyme from Saccharomyces cerevisiae that bypasses mammalian complex I, were resistant to OPB-51602 toxicity. These results show that targeting mitochondrial STAT3 function causes a synthetic lethality involving complex I inhibition that could be exploited for cancer chemotherapy.

MKL1 promotes endothelial-to-mesenchymal transition and liver fibrosis by activating TWIST1 transcription

Excessive fibrogenic response in the liver disrupts normal hepatic anatomy and function heralding such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. Sinusoidal endothelial cells contribute to myofibroblast activation and liver fibrosis by undergoing endothelial-mesenchymal transition (EndMT). The underlying mechanism remains poorly defined. Here we report that inhibition or endothelial-specific deletion of MKL1, a transcriptional modulator, attenuated liver fibrosis in mice. MKL1 inhibition or deletion suppressed EndMT induced by TGF-β. Mechanistically, MKL1 was recruited to the promoter region of TWIST1, a master regulator of EndMT, and activated TWIST1 transcription in a STAT3-dependent manner. A small-molecule STAT3 inhibitor (C188-9) alleviated EndMT in cultured cells and bile duct ligation (BDL) induced liver fibrosis in mice. Finally, direct inhibition of TWIST1 by a small-molecule compound harmine was paralleled by blockade of EndMT in cultured cells and liver fibrosis in mice. In conclusion, our data unveil a novel mechanism underlying EndMT and liver fibrosis and highlight the possibility of targeting the STAT3-MKL1-TWIST1 axis in the intervention of aberrant liver fibrogenesis.

Development of Oxadiazole-Based ODZ10117 as a Small-Molecule Inhibitor of STAT3 for Targeted Cancer Therapy.

Persistently activated STAT3 is a promising target for a new class of anticancer drug development and cancer therapy, as it is associated with tumor initiation, progression, malignancy, drug resistance, cancer stem cell properties, and recurrence. Here, we discovered 3-(2,4-dichloro-phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole (ODZ10117) as a small-molecule inhibitor of STAT3 to be used in STAT3-targeted cancer therapy. ODZ10117 targeted the SH2 domain of STAT3 regardless of other STAT family proteins and upstream regulators of STAT3, leading to inhibition of the tyrosine phosphorylation, dimerization, nuclear translocation, and transcriptional activity of STAT3. The inhibitory effect of ODZ10117 on STAT3 was stronger than the known STAT3 inhibitors such as S3I-201, STA-21, and nifuroxazide. ODZ10117 suppressed the migration and invasion, induced apoptosis, reduced tumor growth and lung metastasis, and extended the survival rate in both in vitro and in vivo models of breast cancer. Overall, we demonstrated that ODZ10117 is a novel STAT3 inhibitor and may be a promising agent for the development of anticancer drugs.

TWO HITS FOR LACTIC ACIDOSIS: A STUDY IN DRUG-DRUG INTERACTION

SESSION TITLE: Wednesday Fellows Case Report Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: Tumors with high mitotic activity undergo oxidative phosphorylation for ATP synthesis. In acute myelogenous leukemia (AML) new therapies that target energy metabolism and mitochondrial protein synthesis are being studied. When these new drugs are combined with medications that also effect mitochondrial activity serious complications may occur. Particularly when side-effects from new investigation therapies are not widely known, nor available to automated electronic medical record (EMR) drug interaction checks. We present a case of life-threatening lactic acidosis due to the use of a STAT3 (signal transduction activator of transcription) inhibitor - a drug currently in clinical trials -- in a patient also receiving linezolid, in whom the risk of a life-threatening drug interaction and toxicity was not fully appreciated. CASE PRESENTATION: A 61 year-old man with a history of refractory AML in a clinical trial with a STAT 3 inhibitor presented with sepsis, but despite appropriate antibiotics his clinical status declined requiring mechanical ventilation. Lab studies showed a rapidly worsening lactic acidosis (3.9 to 18 mmol/L) over a few hours. Later it was discovered that he had completed a prolonged course of linezolid for vancomycin-resistant enterococcus bacteremia the day prior to admission. Though inputted in the medical record as an investigation study drug, the drug-drug interaction with linezolid was not registered by the EMR. Plasmapheresis and dialysis were used to remove residual linezolid and the study drug. Subsequently his lactate decreased to 2.4 and he was extubated. However, given the lack of treatment options for refractory AML the family decided to pursue comfort measures. DISCUSSION: STAT3 is a member of the STAT family of proteins involved in intracellular and extracellular signals to the nucleus, controlling gene expression. STAT3 is involved in AML cells, antiapoptosis genes, cell cycle regulators and promotes OXPHOS (oxidative phosphorylation). A major concern for the use of OXPHOS inhibitors is lactic acidosis. Similarly, linezolid decreases mitochondrial respiratory chain enzyme activity, hence disrupting cellular energy production. In our patient, it is likely that the use of two OXPHOS inhibitors resulted in severe mitochondrial toxicity and lactic acidosis. With new medications being developed that inhibit mitochondrial oxidative phosphorylation, intensive care units may see patients who are suffering from their side effects. CONCLUSIONS: This case highlights the importance of reviewing mechanisms and adverse effects of study drugs. Our case also reminds us that study drugs may not be included in the EMR or not flagged by computerized drug interaction alerts. We believe the use of STAT3 inhibitor and other new OXPHOS inhibitors warrant careful monitoring, particularly when used simultaneously with other medications that increase the risk of mitochondrial toxicity. Reference #1: Redell MS, Ruiz MJ, Alonzo TA, Gerbing RB, Tweardy DJ. Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor. Blood. 2011;117(21):5701-9. DISCLOSURES: No relevant relationships by Alan Gandler, source=Web Response No relevant relationships by Alexys Monoson, source=Web Response No relevant relationships by David Oxman, source=Web Response No relevant relationships by MRINALINI VENKATA SUBRAMANI, source=Web Response

Myeloid maturation potentiates STAT3-mediated atypical IFN-\u03b3 signaling and upregulation of PD-1 ligands in AML and MDS

Interferon (IFN)-γ is the major mediator of anti-tumor immune responses; nevertheless, cancer cells use intrigue strategies to alter IFN-γ signaling and avoid elimination. Understanding the immune regulatory mechanisms employed by acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells upon exposure to IFN-γ is critical for development of immunotherapy and checkpoint blockade therapy approaches. This study aims to explore the influence of myeloid maturation on IFN-γ-induced PD-L1 and PD-L2 expression and on pro-leukemogenic transcription factor STAT3 signaling in AML and MDS. Stimulation of myeloid blasts’ maturation by all-trans retinoic acid (ATRA) or 1α,25-dihydroxyvitamin D3 (vitamin D) increased the CD11b+ fraction that expressed PD-1 ligands in response to IFN-γ. Intriguingly, STAT3 pathway was potently induced by IFN-γ and strengthened upon prolonged exposure. Nonetheless, STAT3-mediated atypical IFN-γ signaling appeared as a negligible factor for PD-L1 and PD-L2 expression. These negative influences of IFN-γ could be alleviated by a small-molecule inhibitor of STAT3, stattic, which also inhibited the upregulation of PD-L1. In conclusion, induction of myeloid maturation enhances the responsiveness of AML and MDS cells to IFN-γ. However, these malignant myeloid cells can exploit both STAT3 pathway and PD-1 ligands to survive IFN-γ-mediated immunity and maintain secondary immune resistance.

Napabucasin, a novel STAT3 inhibitor suppresses proliferation, invasion and stemness of glioblastoma cells

BackgroundGlioblastoma (GBM) cells with stem cell-like properties are called glioma stem cells (GSCs). GSCs display highly treatment resistance and are responsible for tumor recurrence. Napabucasin (BBI608), a novel small molecule inhibitor of STAT3, has been identified to eliminate stemness-like tumor cells in some cancers. However, the influence of Napabucasin on GBM cells, especially on GSCs, is currently unclear. In this study, we explored the influence and underlying mechanisms of Napabucasin on GBM cells.MethodsSTAT3 expression and its correlation with the glioma grade and patient survival were analyzed using CGGA and TCGA glioma databases. The influence of Napabucasin on proliferation, stemness, the cell cycle, apoptosis, and invasion of human GBM cell lines U87MG and LN229 was tested by CCK8, EdU incorporation, colony formation, Transwell invasion, and three-dimensional spheroid assays as well as flow cytometry, qPCR, and western blot analysis. The ability of Napabucasin to inhibit cell proliferation of U87MG tumor xenografts in mice was assessed using a live animal bioluminescence imaging system and immunohistochemistry.ResultsNapabucasin suppressed the proliferation, colony formation, and invasion of U87MG and LN229 cells. Furthermore, Napabucasin induced cell cycle arrest and apoptosis. More importantly, Napabucasin treatment obviously inhibited expression of stemness-associated genes including STAT3 and suppressed the spheroid formation of glioma cells in vitro. Napabucasin also disrupted the NF-κB signaling pathway via downregulation of RelA (p65). Finally, glioma growth was effectively impaired by Napabucasin in nude mice bearing intracranial glioma xenografts.ConclusionsNapabucasin treatment may be a novel approach for the treatment of GBM, particularly GSCs.

Abstract 17: Novel STAT3 inhibitors identified by Structure-Based Virtual Ligand Screening incorporating SH2 domain flexibility

Abstract Efforts to develop STAT3 inhibitors have focused on its SH2 domain, starting with short phosphotyrosylated peptides with STAT3 binding motifs, e.g. pY905LPQTV within gp130. Despite binding to STAT3 with high affinity, issues regarding stability, bioavailability, and membrane permeability of these peptides, as well as peptidomimetics such as CJ-887, have limited their further clinical development and led to increased interest in small-molecule inhibitors. Some small molecule STAT3 inhibitors have been identified using structure-based virtual ligand screening (SB-VLS). While having favorable drug-like properties, most suffer from weak binding affinities, possibly due to the high flexibility of the target domain, especially within the region involved in pY-peptide binding. We conducted molecular dynamic (MD) simulations of the SH2 domain in a complex with CJ-887, with a focus on ligand-induced protein conformation changes that increase binding affinity. We used an averaged structure from this MD trajectory as “induced-active site” receptor model for SB-VLS of 110,000 compounds in the SPEC database. Screening was followed by re-docking, re-scoring of the top 30% of hits and selection for compounds that directly interact with pY+0 binding pocket residues R609-S613, resulting in 110 initial hits, which were then tested for STAT3 targeting using a panel of cellular and biochemical assays. 24 out of 110 compounds could inhibit G-CSF-stimulated pY-STAT3 by > 50% at 10 µM with 9 compounds inhibiting by > 99%; 8/24 compounds (SPEC-29, 8, 93, 98, 106, 57, 101 and 85) inhibited STAT3-binding to EGFR pY-peptide (SPR) by 29-71% at 10 µM; 5 of these (SPEC-29, 8, 93, 98, and 106) had pY-STAT3 IC50s ranging from 2.7-19.0 µM; 3 compounds (SPEC-29, 8, and 93) potently inhibited growth of pSTAT3-high MDA-MB-468 (IC50 = 2.4-12.1 µM) and MDA-MB-231 (IC50s = 3.0-12.2 µM) cells and the growth-inhibitory abilities correlated to abilities to inhibit pY-STAT3. Thus, we used a dynamic ligand-STAT3, bound-fit docking model to screen libraries that successfully identified two hits with potent activity against STAT3 both of which comply with all of Lipinski’s “rules of five” for drug-likeness. Importantly, while most STAT3 inhibitors previously identified have negatively charged moieties, which mediate their binding to the pY+0 binding pocket but limit cell penetration, the two hits identified are uncharged and likely will serve as superior candidates for future hit-to-lead development leading to an effective direct small-molecule STAT3 inhibitor. Furthermore, the ability of the technique employed here for identifying neutral hits may also be useful in designing inhibitors targeting SH2 domains within other oncogenic targets. Citation Format: Ren Kong, Uddalak Bharadwaj, Thomas Kris Eckols, Moses Kasembeli, Mikhail Kolosov, Anh Tran, Oluwatomilona I. Ifelayo, Hong Zhao, Stephen T. Wong, David J. Tweardy. Novel STAT3 inhibitors identified by Structure-Based Virtual Ligand Screening incorporating SH2 domain flexibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 17.

Design, synthesis and activity of novel 2,6-disubstituted purine derivatives, potential small molecule inhibitors of signal transducer and activator of transcription 3

Sustained activation of STAT3 is closely related to the cancer development, but the inhibitors for STAT3 overexpression are still in the clinical research stage. In this study, a series of 2,6-disubstituted purine derivatives were designed and synthesized, and their biological activities, as small molecule inhibitors of STAT3, were assessed. Compound PD26-TL07 exhibited remarkable antiproliferative activity against three cancer cell lines (IC50 values for HCT-116, SW480 and MDA-MB-231 were 1.77 ± 0.35, 1.51 ± 0.19, and 1.25 ± 0.38 μM, respectively). Moreover, detailed biological assays revealed that PD26-TL07 could effectively inhibited STAT3 phosphorylation, and had little inhibition to others’. The newly discovered PD26-TL07 displayed an expecting anticancer effect both in vitro and in vivo. The molecular docking models revealed that PD26-TL07 could bind to the SH2 domain of STAT3. Three additional compounds (PD26-BZ01, PD26-TL03 and PD26-AS06) were also able to inhibit this phosphorylation. This study described novel 2,6-disubstituted purine derivatives as potent anticancer agents targeting STAT3.

Cryptotanshinone protects against pulmonary fibrosis through inhibiting Smad and STAT3 signaling pathways

Cryptotanshinone (CTS), a lipophilic compound extracted from root of Salvia miltiorrhiza (Danshen), has demonstrated multiple pharmacological activities, including anti-inflammation, anti-proliferation and anti-infection. However, the effect of CTS on pulmonary fibrosis is unknown. This study aims to investigate the effects of CTS treatment on pulmonary fibrosis and its underlying mechanism. The pulmonary fibrosis model was established by intratracheal instillation of bleomycin (5 mg/kg) in Sprague-Dawley rats (in vivo) and stimulating human fetal lung fibroblasts (HLFs) with transforming growth factor-beta 1 (TGF-β1) (in vitro). CTS (7.5, 15, 30, 60 mg/kg/day) and pirfenidone (150 mg/kg/day, positive control) were administered by oral gavage for 28 days. In this study, we found CTS treatment improved pulmonary function, relieved pathological changes and attenuated the accumulation of extracellular matrix in pulmonary fibrosis rat model induced by bleomycin. Mechanistically, CTS suppressed phosphorylation of Smad2/3 and STAT3 induced by TGF-β1 in HLFs. Stattic, a 1-benzothiophene based small-molecule STAT3 inhibitor, resulted in a significant down-regulation of fibrosis biomarkers including fibronectin, collagen type I and alpha smooth muscle actin (α-SMA). Overexpression of STAT3 promoted expression of fibrosis biomarkers in HLFs cell model induced by TGF-β1 and partially blocked the inhibitory effect of CTS on TGF-β1-induced fibrosis response. Taken together, these results suggested that CTS protects against pulmonary fibrosis via inhibition of Smad and STAT3 signaling pathways. Thus, CTS may represent a promising drug candidate for treating pulmonary fibrosis.

IL-6 Mediates Cross-Talk between Tumor Cells and Activated Fibroblasts in the Tumor Microenvironment.

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR.

STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions

STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided.

Abstract 4880: Novel nano-molar small-molecule STAT3 inhibitor series with antitumor activities against human breast cancer

Abstract Constitutively-active Signal transducer and activator of transcription (STAT) 3 has been well validated as a significant mechanistic underpinning of many human cancers and a target for anticancer therapy. To date, no small molecule STAT3 inhibitor has been successfully developed for clinical application as a therapeutic agent, despite the discovery of many molecular entities that inhibit the protein. To advance the therapeutic development of small molecule STAT3 inhibitors, we focused on the existing lead compounds, BP-1-102, SH4-54, and SH5-07 (all three showing IC50 of 4-7 μM). All three leads are based on an N-methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. Intensive medicinal chemistry campaign was directed towards understanding and optimizing the molecular determinants critical for enhanced STAT3-inhibitory potency and strong antitumor effects. We report the development of nanomolar potent inhibitors of STAT3 DNA-binding activity. Most notably, the analogs H127, H145, H171, H174, H181, and H182 preferentially inhibit STAT3 activity in DNA-binding assay in vitro, with IC50 of 300 - 800 nM, compared to little or no observed effect on STAT1 and STAT5 DNA-binding activity at concentrations up to 20 μM. Treatment of human breast cancer MDA-MB-231 and MDA-MB-468 cells with H171, H174 or H182 inhibited constitutive STAT3 DNA-binding activity and phosphorylation at Tyr705 in both time- and dose-dependent manner. H171, H174 or H182 further blocked the growth and viability, colony formation, and oncogenic transformation of the human breast cancer cells that harbor persistently active STAT3, with IC50 of 1.0 - 1.9 μM, compared to the IC50 of 3.8 - 8.1 μM against the viability of the immortalized breast epithelial cells that do not harbor persistently active STAT3. These compounds represent some of the first nanomolar potent small molecules observed to directly inhibit STAT3 activity, with potent antitumor cell effects. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Christine Brotherton-Pleiss, Casie Kubota, Jasmine Chen, Marcus Tius, James Turkson. Novel nano-molar small-molecule STAT3 inhibitor series with antitumor activities against human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4880.