Direct STAT3 Inhibitor Research Articles

Corrigendum to: The direct STAT3 inhibitor 6-ethoxydihydrosanguinarine exhibits anticancer activity in gastric cancer

Corrigendum to: The direct STAT3 inhibitor 6-ethoxydihydrosanguinarine exhibits anticancer activity in gastric cancer

Discovery of new STAT3 inhibitors as anticancer agents using ligand-receptor contact fingerprints and docking-augmented machine learning.

STAT3 belongs to a family of seven vital transcription factors. High levels of STAT3 are detected in several types of cancer. Hence, STAT3 inhibition is considered a promising therapeutic anti-cancer strategy. In this work, we used multiple docked poses of STAT3 inhibitors to augment training data for machine learning QSAR modeling. Ligand-Receptor Contact Fingerprints and scoring values were implemented as descriptor variables. Escalating docking-scoring consensus levels were scanned against orthogonal machine learners, and the best learners (Random Forests and XGBoost) were coupled with genetic algorithm and Shapley additive explanations (SHAP) to identify critical descriptors that determine anti-STAT3 bioactivity to be translated into pharmacophore model(s). Two successful pharmacophores were deduced and subsequently used for in silico screening against the National Cancer Institute (NCI) database. A total of 26 hits were evaluated in vitro for their anti-STAT3 bioactivities. Out of which, three hits of novel chemotypes, showed cytotoxic IC50 values in the nanomolar range (35 nM to 6.7 μM). However, two are potent dihydrofolate reductase (DHFR) inhibitors and therefore should have significant indirect STAT3 inhibitory effects. The third hit (cytotoxic IC50 = 0.44 μM) is purely direct STAT3 inhibitor (devoid of DHFR activity) and caused, at its cytotoxic IC50, more than two-fold reduction in the expression of STAT3 downstream genes (c-Myc and Bcl-xL). The presented work indicates that the concept of data augmentation using multiple docked poses is a promising strategy for generating valid machine learning models capable of discriminating active from inactive compounds.

Inhibition of STAT3 Promotes Effector T Cell Infiltration But also Immunosuppression in the HCC Tumor Microenvironment.

STAT3 is involved in the progression of several cancers, and has been proposed as target for therapy. Indeed, the multitargeted tyrosine kinase inhibitor drug regorafenib, which indirectly inhibits STAT3, can significantly enhance the effects of anti-programmed death receptor (PD)-1 therapy in hepatocellular carcinoma (HCC) models. Here, we studied the impact of a direct STAT3 inhibitor on the tumor microenvironment and PD-1 blockade efficacy in HCC models. Orthotopic mouse models of HCC (RIL-175 and HCA-1 grafts in syngeneic mice) were used to test the efficacy of the selective STAT3 inhibitor STX-0119 alone or combined with anti-PD-1 antibodies. We evaluated the effects of therapy on tumor vasculature and the immune microenvironment using immunofluorescence (IF), cell viability assay and quantitative real-time (qRT)-PCR in tumor tissues. Combining anti-PD-1 antibodies with a STX-0119 failed to show a growth delay or survival benefit compared to each agent alone or control in any of the HCC models. Interestingly, evaluation of intratumoral CD8+ T cell infiltration by IF showed a significant increase after one-week treatment with STX-0119 (p=0.034). However, STX-0119 treatment simultaneously promoted increased immunosuppression in the tumor microenvironment by increasing the proportion of Tregs, tissue hypoxia and α-SMA activated cancer-associated fibroblasts (CAFs) measured by IF. Consistent with these findings, we found increased immature tumor vessels by IF and VEGF, Tgf-β and Vash2 expression by qPCR. Pharmacologic STAT3 inhibition could significantly enhance CD8+ T cell infiltration in HCC but also significantly alter the immunosuppression and vascular abnormalization in the tumor microenvironment.

Etanercept Ameliorates Cardiac Fibrosis in Rats with Diet-Induced Obesity.

Diet-induced obesity (DIO) is considered the main risk factor for cardiovascular diseases. Increases in the plasma levels of tumor necrosis factor alpha (TNF-α) is associated with DIO. Etanercept, a TNF-α inhibitor, has been shown to alleviate cardiac hypertrophy. To investigate the effect of etanercept on cardiac fibrosis in DIO model, rats on high fat diet (HFD) were subdivided into two groups: the etanercept group and vehicle group. Cardiac injury was identified by classic methods, while fibrosis was characterized by histological analysis of the hearts. Etanercept treatment at 0.8 mg/kg/week twice weekly by subcutaneous injection effectively alleviates the cardiac fibrosis in HFD-fed rats. STAT3 activation seems to be induced in parallel with fibrosis-related gene expression in the hearts of HFD-fed rats. Decreased STAT3 activation plays a role in the etanercept-treated animals. Moreover, fibrosis-related genes are activated by palmitate in parallel with STAT3 activation in H9c2 cells. Etanercept may inhibit the effects of palmitate, but it is less effective than a direct inhibitor of STAT3. Direct inhibition of STAT3 activation by etanercept seems unlikely. Etanercept has the ability to ameliorate cardiac fibrosis through reduction of STAT3 activation after the inhibition of TNF-α and/or its receptor.

Silibinin and SARS-CoV-2: Dual Targeting of Host Cytokine Storm and Virus Replication Machinery for Clinical Management of COVID-19 Patients.

COVID-19, the illness caused by infection with the novel coronavirus SARS-CoV-2, is a rapidly spreading global pandemic in urgent need of effective treatments. Here we present a comprehensive examination of the host- and virus-targeted functions of the flavonolignan silibinin, a potential drug candidate against COVID-19/SARS-CoV-2. As a direct inhibitor of STAT3—a master checkpoint regulator of inflammatory cytokine signaling and immune response—silibinin might be expected to phenotypically integrate the mechanisms of action of IL-6-targeted monoclonal antibodies and pan-JAK1/2 inhibitors to limit the cytokine storm and T-cell lymphopenia in the clinical setting of severe COVID-19. As a computationally predicted, remdesivir-like inhibitor of RNA-dependent RNA polymerase (RdRp)—the central component of the replication/transcription machinery of SARS-CoV-2—silibinin is expected to reduce viral load and impede delayed interferon responses. The dual ability of silibinin to target both the host cytokine storm and the virus replication machinery provides a strong rationale for the clinical testing of silibinin against the COVID-19 global public health emergency. A randomized, open-label, phase II multicentric clinical trial (SIL-COVID19) will evaluate the therapeutic efficacy of silibinin in the prevention of acute respiratory distress syndrome in moderate-to-severe COVID-19-positive onco-hematological patients at the Catalan Institute of Oncology in Catalonia, Spain.

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.

Berbamine Enhances the Efficacy of Gefitinib by Suppressing STAT3 Signaling in Pancreatic Cancer Cells.

BackgroundSmall molecular inhibitors such as gefitinib (Gefi), which target EGF receptor (EGFR), are considered to be a viable pathway for the selective inhibition of pancreatic cancer (PC) development. However, the large difference in Gefi response between PC patient individuals and PC cell lines severely limits the clinical efficacy of Gefi. Berbamine (BBM) is a well-known natural-derived antitumor agent. However, no study yet exists on whether BBM can enhance the sensitivity of PC cells to Gefi or its underlying mechanisms.MethodsMTS assay and clonogenic assay were used to determine whether BBM could enhance the anti-PC activity of Gefi by. Flow cytometric analysis was performed to study the cell cycle progression and rate of apoptosis after combined treatment with BBM and Gefi. Surface plasmon resonance (SPR) and Western blot experiments were carried out to detect the STAT3 binding affinity and the STAT3 inhibitory effect of BBM. Molecular docking and Molecular dynamic simulation were used to predicting the dominant interaction between BBM and STAT3.ResultsThis study found that BBM synergizes with Gefi to inhibit cell growth and induce cell cycle arrest and PC cell apoptosis. Mechanistically, our results showed that BBM and Gefi have synergistic inhibitory effects on STAT3 phosphorylation, but have little effect on other EGFR downstream pathways, suggesting that BBM may exert sensitization through the inhibition of STAT3. Besides, BBM has a high affinity for STAT3 and a good inhibitory effect on STAT3 activation, further indicating that BBM was a potent direct STAT3 inhibitor. Molecular modeling between STAT3 and BBM suggested that BBM formed several key hydrophilic interactions with STAT3.ConclusionOur findings suggest that the combination of BBM and Gefi could be further developed as a potential PC therapy.

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.

Silibinin is a direct inhibitor of STAT3

We herein combined experimental and computational efforts to delineate the mechanism of action through which the flavonolignan silibinin targets STAT3. Silibinin reduced IL-6 inducible, constitutive, and acquired feedback activation of STAT3 at tyrosine 705 (Y705). Silibinin attenuated the inducible phospho-activation of Y705 in GFP-STAT3 genetic fusions without drastically altering the kinase activity of the STAT3 upstream kinases JAK1 and JAK2. A comparative computational study based on docking and molecular dynamics simulation over 14 different STAT3 inhibitors (STAT3i) predicted that silibinin could directly bind with high affinity to both the Src homology-2 (SH2) domain and the DNA-binding domain (DBD) of STAT3. Silibinin partially overlapped with the cavity occupied by other STAT3i in the SH2 domain to indirectly prevent Y705 phosphorylation, yet showing a unique binding mode. Moreover, silibinin was the only STAT3i predicted to establish direct interactions with DNA in its targeting to the STAT3 DBD. The prevention of STAT3 nuclear translocation, the blockade of the binding of activated STAT3 to its consensus DNA sequence, and the suppression of STAT3-directed transcriptional activity confirmed silibinin as a direct STAT3i. The unique characteristics of silibinin as a bimodal SH2- and DBD-targeting STAT3i make silibinin a promising lead for designing new, more effective STAT3i.

Preclinical Characterization of 3β-(N-Acetyl l-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (GPA512), a Prodrug of a Direct STAT3 Inhibitor for the Treatment of Prostate Cancer.

The transcription factor STAT3 is a potential target for the treatment of castration-resistant prostate cancer. Galiellalactone (1), a direct inhibitor of STAT3, prevents the transcription of STAT3 regulated genes. In this study we characterized 6 (GPA512, Johansson , M. ; Sterner , O. Patent WO 2015/132396 A1, 2015 ), a prodrug of 1. In vitro studies showed that 6 is rapidly converted to 1 in plasma and is stable in a buffer solution. The pharmacokinetics of 6 following a single oral dose indicated that the prodrug was rapidly absorbed and converted to 1 with a tmax of 15 min. Oral administration of 6 in mice increased the plasma exposure of the active parent compound 20-fold compared to when 1 was dosed orally. 6 treated mice bearing DU145 xenograft tumors had significantly reduced tumor growth compared to untreated mice. The favorable druglike properties and safety profile of 6 warrant further studies of 6 for the treatment of castration-resistant prostate cancer.

Abstract C179: Preclinical characterization of GPA512: A prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer

Abstract Background: The transcription factor STAT3 is a promising target for the treatment of castration-resistant prostate cancer (CRPC) as STAT3 is implicated in drug resistance and castration resistance as well as metastatic spread, tumor growth and immune escape. Galiellalactone (GL) is a direct inhibitor of STAT3 that prevents DNA binding, inhibits proliferation of prostate cancer cells expressing active STAT3 and induces apoptosis by down-regulation of STAT3 activated genes. In this study we aimed to characterize a prodrug of GL, GPA512, with improved drug-like properties and to demonstrate its effect on tumor growth in a xenograft model of prostate cancer following oral administration. Methods: Stability studies of prodrugs based on GL were performed in 0.1 M PBS buffer (pH 7.4) and in plasma at 37°C. In vitro efficacy of prodrugs was studied by WST-1 proliferation assay in DU145 prostate cancer cells expressing active STAT3. The systemic exposure of GL in mice was studied following a single oral dose of GPA512 or GL (both 10 mg/kg). The plasma concentrations of GL were determined by LC-MS/MS. For the xenograft study NMRI-nude male mice were inoculated subcutaneously with DU145 cells and once tumors were established the mice were divided in two groups with ten mice in each. Mice were treated orally with 40 mg/kg GPA512 daily five times/week for four weeks. Tumor growth was measured by caliper and at the end of the study tumors were harvested for subsequent analyses using immunohistochemistry and mRNA expression analysis. Results: In vitro studies showed that the prodrug GPA512 is rapidly converted to GL in plasma and that GPA512 is stable in buffer solution and has similar inhibitory effects on proliferation as GL on DU145 cells. The pharmacokinetics of GPA512 following a single oral dose indicated that the compound was rapidly absorbed and converted to GL with a tmax of 15 min. Oral administration of GPA512 in mice increased the plasma exposure (AUC) of the active parent compound 20-fold compared to when GL was dosed orally. GPA512 treated mice bearing subcutaneous DU145 tumors had significantly smaller tumors compared to mice treated with vehicle. No adverse effects or weight loss were observed. Analysis of tumors showed decreased cell proliferation and increased amount of apoptotic cells in GPA512 treated mice compared to control. The mRNA expression of STAT3 regulated anti-apoptotic gene MCl-1 was significantly reduced by GPA512 treatment. Conclusions: The drug-like properties and safety profile of the prodrug GPA512 and galiellalactone's direct inhibition of STAT3, warrant further studies of GPA512 as a drug candidate for treatment of patients with CRPC. Citation Format: Rebecka Hellsten, Zilma Escobar, Anders Bjartell, Giacomo Canesin, Susan Evans Axelsson, Olov Sterner, Martin Johansson. Preclinical characterization of GPA512: A prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C179.

STAT3 the oncogene - still eluding therapy?

The STAT family of transcription factors (signal transducers and activators of transcription) transduce signals from cytokine receptors to the nucleus, where STAT dimers bind to DNA and regulate transcription. STAT3 is the most ubiquitous of the STATs, being activated by a wide variety of cytokines and growth factors. STAT3 has many roles in physiological processes such as inflammatory signalling, aerobic glycolysis and immune suppression, and was also the first family member shown to be aberrantly activated in a wide range of both solid and liquid tumours. STAT3 promotes tumorigenesis by regulating the expression of various target genes, including cell-cycle regulators, angiogenic factors and anti-apoptosis genes. Paradoxically, in some circumstances, STAT3 signalling induces cell death. The best known example is the involuting mammary gland, where STAT3 is essential for induction of a lysosomal pathway of cell death. Nevertheless, direct silencing or inhibition of STAT3 diminishes tumour growth and survival in both animal and human studies. This suggests that abolishing STAT3 activity may be an effective cancer therapeutic strategy. However, despite this potential as a therapeutic target, and the extensive attempts by many laboratories and pharmaceutical companies to develop an effective STAT3 inhibitor for use in the clinic, no direct STAT3 inhibitor has been approved for clinical use. In this review, we focus on the role of STAT3 in tumorigenesis, and discuss its potential as a therapeutic target for cancer treatment.

Galiellalactone Is a Direct Inhibitor of the Transcription Factor STAT3 in Prostate Cancer Cells

The transcription factor STAT3 is constitutively active in several malignancies including castration-resistant prostate cancer and has been identified as a promising therapeutic target. The fungal metabolite galiellalactone, a STAT3 signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing active STAT3 and induces apoptosis of prostate cancer stem cell-like cells expressing phosphorylated STAT3 (pSTAT3). However, the molecular mechanism of this STAT3-inhibiting effect by galiellalactone has not been clarified. A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of galiellalactone target proteins. By adding streptavidin-Sepharose beads to GL-biot-treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Confocal microscopy revealed GL-biot in both the cytoplasm and the nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. Galiellalactone inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. Mass spectrometry analysis of recombinant STAT3 protein pretreated with galiellalactone revealed three modified cysteines (Cys-367, Cys-468, and Cys-542). Here we demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer.

Targeting STAT3 in castration-resistant prostate cancer: Galiellalactone as a direct inhibitor of STAT3 in prostate cancer cells.

e16065 Background: The transcription factor STAT3 is constitutively active in castration-resistant prostate cancer (CRPC) and constitutes a promising therapeutic target. The reactive fungal metabolite galiellalactone (GL), a STAT3-signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing activated STAT3 and inhibits growth and induces apoptosis of prostate cancer stem cell-like cells expressing pSTAT3. We aim in this study to identify the STAT3 signaling inhibiting mechanism of GL. Methods: A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of GL target proteins. The STAT3 inhibitory activity of GL-biot was confirmed with STAT3 luciferase reporter gene assay. The pSTAT3 expressing human prostate cancer cell line DU145 was incubated with GL-biot, followed by streptavidin beads to isolate proteins bound to GL-biot. Localization of GL-biot in DU145 cells was assessed using confocal microscopy. EMSA was performed on...

Preclinical characterization of GPA512: A prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer.

e22186 Background: The transcription factor STAT3 is a promising target for the treatment of castration-resistant prostate cancer (CRPC) as STAT3 is implicated in drug resistance, the progression o...

Drug-repositioning screening identified piperlongumine as a direct STAT3 inhibitor with potent activity against breast cancer.

Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect, and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been employed to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. Piperlongumine inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that piperlongumine directly inhibited binding of Stat3 to its phosphotyrosyl (pY) peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as JAKs, Src kinase family members, or RTKs. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient derived xenografts (PDX). PL’s anti-tumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/Kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 plays a role.

TPCA-1 Is a Direct Dual Inhibitor of STAT3 and NF-κB and Regresses Mutant EGFR-Associated Human Non–Small Cell Lung Cancers

Epidermal growth factor receptor (EGFR) is a clinical therapeutic target to treat a subset of non-small cell lung cancer (NSCLC) harboring EGFR mutants. However, some patients with a similar kind of EGFR mutation show intrinsic resistance to tyrosine kinase inhibitors (TKI). It indicates that other key molecules are involved in the survival of these cancer cells. We showed here that 2-[(aminocarbonyl)amino]-5 -(4-fluorophenyl)-3- thiophenecarboxamide (TPCA-1), a previously reported inhibitor of IκB kinases (IKK), blocked STAT3 recruitment to upstream kinases by docking into SH2 domain of STAT3 and attenuated STAT3 activity induced by cytokines and cytoplasmic tyrosine kinases. TPCA-1 is an effective inhibitor of STAT3 phosphorylation, DNA binding, and transactivation in vivo. It selectively repressed proliferation of NSCLC cells with constitutive STAT3 activation. In addition, using pharmacologic and genetic approaches, we found that both NF-κB and STAT3 could regulate the transcripts of interleukin (IL)-6 and COX-2 in NSCLC harboring EGFR mutations. Moreover, gefitinib treatment only did not efficiently suppress NF-κB and STAT3 activity. In contrast, we found that treatment with TKIs increased phosho-STAT3 level in target cells. Inhibiting EGFR, STAT3, and NF-κB by combination of TKIs with TPCA-1 showed increased sensitivity and enhanced apoptosis induced by gefitinib. Collectively, in this work, we identified TPCA-1 as a direct dual inhibitor for both IKKs and STAT3, whereas treatment targeting EGFR only could not sufficiently repress NF-κB and STAT3 pathways for lung cancers harboring mutant EGFR. Therefore, synergistic treatment of TPCA-1 with TKIs has potential to be a more effective strategy for cancers.

BP5-087, a Novel STAT3 Inhibitor, Combines With BCR-ABL1 Inhibition To Overcome Kinase-Independent Resistance In Chronic Myeloid Leukemia

Mutations in the BCR-ABL1 kinase domain are a well-established mechanism of tyrosine kinase inhibitor (TKI) resistance, but fail to explain many cases of clinical TKI failure. In the remaining patients, resistance occurs via activation of alternative signaling pathways that maintain survival despite BCR-ABL1 inhibition (BCR-ABL1-independent resistance). STAT3 mediates TKI resistance in chronic myeloid leukemia (CML) cells cultured in the presence of bone marrow-derived factors (Bewry et al., 2008; Traer et al., 2012; Nair et al., 2012), and also plays a critical role in survival of CML cells with BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). While targeting transcription factors is notoriously difficult, our combination of synthetic chemistry, in vitro reporter assays, and computational modeling has led to a low micromolar mechanism-based STAT3 inhibitor, which, in combination with TKIs, shows promise as a treatment for CML patients with BCR-ABL1-independent resistance.The original compound of the series, SF1-066 (10 µM; Fletcher et al., 2009), combines with TKIs to reduce survival of CML CD34+ cells exhibiting BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). To improve the potency and selectivity of SF1-066, we synthesized successive STAT3 inhibitor libraries and ranked candidates by structure-activity relationship using a luciferase-based reporter screen (Kraft et al. #2445, ASH 2012). This reporter assay quantifies STAT3 transcriptional activity in TKI-resistant AR230R cells, which grow in the continuous presence of imatinib (1.0 µM), lack BCR-ABL1 kinase domain mutations, and exhibit high levels of pSTAT3Y705, thereby enabling convenient, high-throughput screening for potency and selectivity in the context of endogenous STAT3 activation. Among three sequential STAT3 inhibitor libraries, BP5-087 emerged as the new lead compound. Fluorescence polarization assays verified that BP5-087 was 5-fold more effective than SF1-066 in outcompeting an SH2 peptide probe, and computational simulations predicted better overall binding of BP5-087 (-9.6 kcal/mol) versus SF1-066 (-7.6 kcal/mol) to the STAT3 SH2 interface. In AR230R cell growth assays, BP5-087 was effective at a 5-fold lower dose compared to SF1-066, with minimal effects on TKI-sensitive parental controls. Therefore, we tested BP5-087 in the context of primary TKI resistance.BP5-087 (1 µM) in combination with imatinib (2.5 µM) reduced colony formation and increased apoptosis of CD34+ cells from CML patients with BCR-ABL1-independent resistance. These cells have no BCR-ABL1 kinase domain mutations and undergo BCR-ABL1 kinase inhibition as detected by immunoblot analyses. In contrast, BP5-087 had no effect on CD34+ cells from newly diagnosed CML patients or normal individuals. Immunofluorescence demonstrated that dual treatment of TKI-resistant CML CD34+ cells resulted in reduced levels of nuclear pSTAT3Y705, consistent with an inhibitor of STAT3 dimerization. In more primitive CML stem cells, long term culture-initiating cell (LTC-IC) assays revealed that neither inhibitor alone had any effect on colony formation of primitive LTC-IC progenitors, whereas imatinib (2.5 µM) in combination with BP5-087 (1.0 µM) reduced LTC-IC colony formation by 66%. Consistent with this observation, immunofluorescence showed high levels of pSTAT3Y705 in primitive TKI-resistant CD34+CD38- cells when cultured in the presence but not absence of TKIs. To test the feasibility of BP5-087 for in vivo use, we treated mice orally with BP5-087 (25 mg/kg/day) for 4 weeks and observed no changes in body weight, peripheral blood cellularity, or bone marrow colony forming ability. Mass spectrometry confirmed that BP5-087 is orally bioavailable.In summary, BP5-087 is a systematically-derived, direct inhibitor of STAT3 that, in combination with TKIs, reduces survival of CML cells with BCR-ABL1-independent resistance. Further rounds of structure-activity optimization may reveal an inhibitor with a clinically-relevant effective concentration. Disclosures:Deininger:Bristol Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

A Novel Direct STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition In a Wide Range of Hematopoietic Malignancies and Effectively Suppresses the Chemotherapy Resistant Quiescent Cells In Vivo

AbstractAbstract 3277Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like Janus kinase (JAK) family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis-resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials.Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases. OPB-31121 treatment of HEL92.1.7 cells that had constitutive active mutation of JAK2 inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). STAT3 phosphorylation by JAK2 in vitro was also inhibited by OPB-31121 under constant JAK2 autophosphorylation. On the other hand, it did not inhibit dimerization and nuclear translocation of STAT3 once STAT3 was phosphorylated. Also, direct association between OPB-31121 and STAT3 was suggested in vitro. These data implies that one of the mechanisms of OPB-31121 action was the direct inhibition of STAT3 phosphorylation without JAK kinase inhibition. [Display omitted] OPB-31121 demonstrated strong growth suppressive effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including acute myeloid leukemia (AML) with JAK2 mutation or fms-related tyrosine kinase 3 (FLT3) mutation, chronic myeloid leukemia (CML), and myeloma. It is revealed that STAT3 is constitutively activated by tyrosine kinase signal from oncoprotein or oncogenic autocrine of IL-6 pathway in these cell lines. Of note, OPB-31121 had little growth inhibitory effect on normal hematopoietic cells and hardly affected colony formation of human cord blood cells at 100 nM. We also demonstrated growth suppression or regression of cell lines including HEL92.1.7, KU812 (CML), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation) in NOD/SCID mice (T/C: 1.8 to 39.5 %). For further analyses, we used human leukemia model mouse where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth suppression of leukemia cells of BCR-ABL-positive acute lymphoblastic leukemia (ALL), CML-blast crisis (BC), CML-BC with T315I mutation in BCR-ABL, and AML with FLT3/ITD (T/C: 4 to 58 %, Figure B). Furthermore, treatment with cytarabine induced accumulation of quiescent cells that were thought to be relatively resistant to chemotherapy, whereas OPB-31121 did not cause such accumulation, suggesting its effectiveness on quiescent cells (Figure C). We are now investigating the effect of OPB-31121 on leukemia-initiating cells and the results will be shown at the meeting. Taken together, we conclude that OPB-31121 holds promise as a therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase 1 trial in Hong-Kong, Korea, and the USA. Disclosures:Hayakawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Matsuyama:Otsuka Pharmaceutical Co., Ltd.: Employment. Harada:Otsuka Pharmaceutical Co., Ltd.: Employment. Hashimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Ohi:Otsuka Pharmaceutical Co., Ltd.: Employment. Kodama:Otsuka Pharmaceutical Co., Ltd.: Employment. Sumida:Otsuka Pharmaceutical Co., Ltd.: Employment. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.

Galiellalactone is a novel therapeutic candidate against hormone‐refractory prostate cancer expressing activated Stat3

Signal transducer and activator of transcription 3 (Stat3) is constitutively active (phosphorylated) in several forms of cancer, including prostate cancer (PCa). Stat3 signaling may be an interesting target for cancer therapy since inhibition of this pathway mediates growth inhibition and apoptosis of these cells. In this study we investigated the in vitro and in vivo effects of the fungal metabolite galiellalactone, a direct inhibitor of Stat3, on PCa cells. The human PCa cell lines DU145, PC-3, and LNCaP were used. Nude mice with subcutaneous PCa cell xenografts were subjected to daily intraperitoneal injections of galiellalactone for 3 weeks. The effect of galiellalactone on the induction of apoptosis of cultured PCa cells was investigated by Western blot analysis, immunocytochemistry, and annexin V staining. Effects of galiellalactone on Stat3 signaling were investigated by a luciferase reporter gene assay. Expression of Stat3 associated proteins and mRNA was investigated by Western blot and real-time quantitative PCR analysis. Galiellalactone induced apoptosis of p-Stat3 positive PCa cells (androgen-insensitive DU145 and PC-3) but not in cells lacking p-Stat3 (androgen-sensitive LNCaP). Galiellalactone inhibited Stat3-mediated luciferase activity (IC(50) approximately 5 microM) and reduced the expression of Bcl-2, Bcl-x(L), c-myc, and cyclin D1. Furthermore, galiellalactone significantly suppressed DU145 xenograft growth in vivo (42% growth reduction; P<0.002) and reduced the relative mRNA expression of Bcl-x(L) and Mcl-1. Galiellalactone induced growth inhibition and apoptosis in androgen-insensitive PCa cells expressing p-Stat3. We suggest that galiellalactone is a potential anti-tumor lead against hormone-refractory PCa with constitutively active Stat3.