Potent STAT3 Inhibitor Research Articles

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.

Repurposing of the anti-helminthic drug niclosamide to treat melanoma and pulmonary metastasis via the STAT3 signaling pathway

The incidence of melanoma is increasing rapidly worldwide. Additionally, new and effective candidates for treating melanoma are needed because of the increase in drug resistance and the high metastatic potential of this cancer. The STAT3 signaling pathway plays a pivotal role in pathogenesis of melanoma, making STAT3 a promising anticancer target for melanoma therapy. Niclosamide, an FDA-approved anti-helminthic drug, has been identified as a potent STAT3 inhibitor that suppresses STAT3 phosphorylation at Tyr705 and its transcript activity. In this study, we evaluated the biological activities of niclosamide in melanoma in vitro and in vivo. Niclosamide potently inhibited the growth of four melanoma cell lines and induced the apoptosis of melanoma cells via the mitochondrial apoptotic pathway. Further, western blot analysis indicated that cell apoptosis was correlated with activation of Bax and cleaved caspase-3 and decreased expression of Bcl-2. Moreover, niclosamide markedly impaired melanoma cell migration and invasion, reduced phosphorylated STAT3Tyr705 levels, and inhibited matrix metalloproteinase-2 and −9 expression. Additionally, in a xenograft model of A375, intraperitoneal administration of niclosamide inhibited tumor growth and tumor weight in a dose-dependent manner without obvious side effects. Histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells and p-STAT3Try705-positive cells and increase in cleaved caspase-3-positive cells. Notably, niclosamide significantly inhibited pulmonary metastasis in a B16-F10 melanoma lung metastasis model, including the number of lung metastatic nodules and lung/body coefficient. Importantly, a marked reduction in myeloid-derived suppressor cells (Gr1+CD11b+) infiltration in the pulmonary metastasis tissue was observed. Taken together, these results demonstrate that niclosamide is a promising candidate for treating melanoma.

Pharmacokinetic evaluation, molecular docking and in vitro biological evaluation of 1, 3, 4-oxadiazole derivatives as potent antioxidants and STAT3 inhibitors

1, 3, 4-Oxadiazole derivatives (4a–5f) were previously synthesized to investigate their anticancer properties. However, studies relating to their antioxidant potential and signal transducer and activator of transcription (STAT) inhibition have not been performed. We investigated previously synthesized 1, 3, 4-oxadiazole derivatives (4a–5f) for various radical scavenging properties using several in vitro antioxidant assays and also for direct inhibition of STAT3 through molecular docking. The data obtained from various antioxidant assays such as 2, 2,-diphenyl-1-picrylhydrazyl radical (DPPH), nitric oxide, hydrogen peroxide, and superoxide anion radical revealed that among all the derivatives, compound 5e displayed high antioxidant activities than the standard antioxidant L-ascorbic acid. Additionally, the total reduction assay and antioxidant capacity assay further confirmed the antioxidant potential of compound 5e. Furthermore, the molecular docking studies performed for all derivatives along with the standard inhibitor STX-0119 showed that binding energy released in direct binding with the SH2 domain of STAT3 was the highest for compound 5e (-9.91kcal/mol). Through virtual screening, compound 5e was found to exhibit optimum competency in inhibiting STAT3 activity. Compound 5e decreased the activation of STAT3 as observed with Western blot. In brief, compound 5e was identified as a potent antioxidant agent and STAT3 inhibitor and effective agent for cancer treatment.

L61H46 shows potent efficacy against human pancreatic cancer through inhibiting STAT3 pathway

BackgroundPancreatic cancer is the fourth leading cause of cancer-related death worldwide. The poor prognosis of this disease highlights the urgent need to develop more effective therapies. Activation of the STAT3 represents a potential drug target for pancreatic cancer therapy. Currently, clinically available small-molecule inhibitors targeting STAT3 are lacking.MethodsThrough bioassay screening and molecular docking, we identified a small molecule L61H46 that can potently target constitutive STAT3 signaling and kill human pancreatic cancer cells in vitro and in vivo.ResultsL61H46 effectively reduced colony formation and the viability of pancreatic cancer cells in a dose-dependent manner with half-maximal inhibitory concentration (IC50) values in the range between 0.86 and 2.83 µM. L61H46 significantly inhibited STAT3 phosphorylation (Tyr705) and the subsequent nucleus translocation but did not downregulate STAT1 phosphorylation. Moreover, L61H46 demonstrated a potent activity in suppressing pancreatic tumor growth in BXPC-3 xenograft model in vivo. Furthermore, L61H46 showed no signs of adverse effects on liver, heart, and kidney cells in vivo.ConclusionCollectively, our results suggest that L61H46 could be further optimized into a highly potent STAT3 inhibitor for the treatment of pancreatic cancer.

Apoptosis induced by methanol extract of Potentilladiscolor in human mucoepidermoid carcinoma cells through STAT3/PUMA signaling axis.

Potentilla discolor has been used in traditional Chinese medicine for the treatment of hyperglycemia. However, the potential role of Potentilla discolor against cancer and its mode of action remain to be fully elucidated. The present study explored the apoptotic effect of methanol extract of Potentilla discolor (MEPD) in human mucoepidermoid carcinoma (MEC) cell lines of salivary glands. MEPD markedly suppressed the growth and induced apoptotic cell death in MC3 and YD15 cells. MEPD treatment significantly upregulated the expression of PUMA and reduced STAT3 phosphorylation. Overexpression of STAT3 partially recovered the growth of MEC cells inhibited by MEPD. In addition, dephosphorylation of STAT3 by cryptotanshinone (a potent STAT3 inhibitor) was sufficient to inhibit the growth of MEC cells and induce apoptosis via affecting PUMA protein. These results suggest that MEPD has a potential anticancer property via the STAT3/PUMA signaling axis in human MEC cells of salivary gland.

Abstract B137: Novel potent salicylic, benzoic, and benzohydroxamic acid-based small-molecule STAT3 inhibitors induce antitumor effects in breast cancer xenografts

Abstract We investigated the molecular determinants for the activity of the previously reported salicylic BP-1-102 (IC50 6.8 ± 2.5 μM), benzoic SH4-54 (IC50 4.4 ± 0.3 μM), and benzohydroxamic acid SH5-07 (IC50 3.9 ± 0.6 μM) small-molecule STAT3 inhibitors in order to derive structurally optimum analogs with improved potency and pharmacokinetic parameters. All three leads are based on N-methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. The three functionalities were separately modified to generate various series of new analogs. The inhibitory activities of the new analogs against STAT3 DNA-binding in vitro were determined by electrophoretic motility shift assay. Some new analogs show dramatically improved potency compared to the corresponding lead compounds. Notably, analogs H098, H127, H142, H145, H152, and H155 showed more than 10-fold improved potency in inhibiting STAT3 DNA-binding activity in vitro, with IC50 of 300 - 600 nM. Using analogs H105 and H120, we show that the new analogs decreased STAT3 DNA-binding activity and phosphorylation at Tyr705 in MDA-MB-231 and MDA-MB-468 cells, two human triple-negative breast cancer lines harboring constitutively active STAT3, within as early as 30 min. Furthermore, the new analogs potently inhibited the growth and viability of the human cancer cells with persistently active STAT3, with IC50 of 1.2 - 2.0 µM. The potent analogs specifically disrupted STAT3 DNA-binding activity in vitro over that of STAT1 and STAT5, and they further showed minimum inhibitory effect on the proliferation of human cancer cells that do not harbor persistently active STAT3. Initial in vivo efficacy study in nude mouse model showed that oral administration of H105 and H120 suppressed the growth of tumor xenografts generated from human breast cancer line harboring constitutively active STAT3. Thus, we have made substantial progress and broken the nanomolar barrier in developing STAT3 inhibitors, and these compounds are the first nanomolar STAT3 inhibitors reported to date that are direct disruptors of STAT3 activation, which show in vivo activity. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Christine Brotherton-Pleiss, Marcus Tius, James Turkson. Novel potent salicylic, benzoic, and benzohydroxamic acid-based small-molecule STAT3 inhibitors induce antitumor effects in breast cancer xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B137.

Development of Erasin: a chromone-based STAT3 inhibitor which induces apoptosis in Erlotinib-resistant lung cancer cells

Inhibition of protein-protein interactions by small molecules offers tremendous opportunities for basic research and drug development. One of the fundamental challenges of this research field is the broad lack of available lead structures from nature. Here, we demonstrate that modifications of a chromone-based inhibitor of the Src homology 2 (SH2) domain of the transcription factor STAT5 confer inhibitory activity against STAT3. The binding mode of the most potent STAT3 inhibitor Erasin was analyzed by the investigation of structure-activity relationships, which was facilitated by chemical synthesis and biochemical activity analysis, in combination with molecular docking studies. Erasin inhibits tyrosine phosphorylation of STAT3 with selectivity over STAT5 and STAT1 in cell-based assays, and increases the apoptotic rate of cultured NSCLC cells in a STAT3-dependent manner. This ability of Erasin also extends to HCC-827 cells with acquired resistance against Erlotinib, a clinically used inhibitor of the EGF receptor. Our work validates chromone-based acylhydrazones as privileged structures for antagonizing STAT SH2 domains, and demonstrates that apoptosis can be induced in NSCLC cells with acquired Erlotinib resistance by direct inhibition of STAT3.

Amorfrutin A inhibits TNF-α induced JAK/STAT signaling, cell survival and proliferation of human cancer cells

Context: Amorfrutin A is a natural product isolated from the fruits of Amorpha fruticosa L. and has been shown to exhibit multiple bioeffector functions. In the present study, we investigated whether amorfrutin A exerts anticancer effects by inhibiting STAT3 activation in cervical cancer cells.Objective: To investigate the effectiveness of amorfrutin A as a treatment of cancer, and determine the underlying pharmacological mechanism of action.Materials and methods: HeLa, SK-Hep1, MDA-MB-231 and HCT116 cells were used in this study. Major assays were luciferase reporter assay, MTT, Western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, EdU labeling and immunofluorescence, xenografted assay.Results: Amorfrutin A significantly inhibited tumor necrosis factor-α (TNF-α)-induced phosphorylation and nuclear translocation of STAT3 in human cervical carcinoma cells. Amorfrutin A also inhibited activation of the upstream kinases Janus-activated kinase 1 (JAK1), JAK2 and Src signaling pathways. Furthermore, amorfrutin A increased the expression of p53, p21, p27, induced cell cycle arrest in the G1 phase as well as decreased levels of various oncogene protein products. In vivo studies further confirmed the inhibitory effect of amorfrutin A on the expression of STAT3 proteins, leading to a decrease growth of HeLa cells in a xenograft tumor model.Discussion and conclusions: The results indicated that amorfrutin A is a potent inhibitor of STAT3 and provide new perspectives into the mechanism of its anticancer activity.

Nitidine chloride acts as an apoptosis inducer in human oral cancer cells and a nude mouse xenograft model via inhibition of STAT3

Nitidine chloride (NC) is a natural alkaloid compound derived from the plant Zanthoxylum nitidum and is known for its therapeutic anticancer potential. In this study, we investigated the effects of NC on growth and signaling pathways in human oral cancer cell lines and a tumor xenograft model. The apoptotic effects and related molecular targets of NC on human oral cancer were investigated using trypan blue exclusion assay, DAPI staining, Live/Dead assay, Western blotting, Immunohistochemistry/Immunofluorescence and a nude mouse tumor xenograft. NC decreased cell viability in both HSC3 and HSC4 cell lines; further analysis demonstrated that cell viability was reduced via apoptosis. STAT3 was hyper-phosphorylated in human oral squamous cell carcinoma (OSCC) compared with normal oral mucosa (NOM) and dephosphorylation of STAT3 by the potent STAT3 inhibitor, cryptotanshinone or NC decreased cell viability and induced apoptosis. NC also suppressed cell viability and induced apoptosis accompanied by dephosphorylating STAT3 in four other oral cancer cell lines. In a tumor xenograft model bearing HSC3 cell tumors, NC suppressed tumor growth and induced apoptosis by regulating STAT3 signaling without liver or kidney toxicity. Our findings suggest that NC is a promising chemotherapeutic candidate against human oral cancer.

Abstract 3950: A novel combination therapy targeting BCL6 and phospho-STAT3 defeats intratumor heterogeneity in a subset of non-small cell lung cancers

Abstract Purpose: Lung cancer is a disease of great oncogenotype complexity (oncogenes and tumor suppressor gene alterations). These alterations can appear in different combinations even within histologically defined lung cancer subtypes. The success of targeted therapy has led to a search for oncogenotype-specific therapies. But, no one therapy fits all oncogenotypes. Here, we investigate whether characterization of oncogene-specific alterations in cellular signaling at single cell level indicate heterogeneity even within cells from the same patient with defined oncogenotype, and whether they can suggest new targets to deal with this heterogeneity. Methods: We compared signaling alterations in single cells for β-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1 and phospho-ERK1/2 among a collection of Human Bronchial Epithelial Cells (HBECs) that have been oncogenically transformed with combinations of TP53, K-RAS, and MYC, commonly found alterations in non-small cell lung cancer (NSCLC). We studied ~3000 cells/signaling marker/HBEC oncogenotype variant using immunofluorescence assays and single-cell image analysis (>1M data points). For downstream target identification and validation we utilized gene expression, Western blot and siRNA mediated knockdown assays. We utilized inhibitors to STAT3 and BCL6 in MTT drug sensitivity and colony formation assay in a panel of NSCLC lines. We used xenografted subcutaneous tumors for the in vivo validation of our results. Results: When all three oncogenic changes were present and the HBECs were tumorigenic, we observed STAT3 upregulation and SMAD2/3 downregulation. Interestingly, these STAT3 and SMAD2/3 signaling changes were found to be mutually exclusive in single cells within the transformed HBEC strain. We targeted the STAT3 upregulated subpopulation with the STAT3 inhibitor Stattic. But, Stattic treatment failed to eliminate the SMAD2/3 downregulated subpopulation. To target the SMAD2/3 down-regulated subpopulation, we identified BCL6, a downstream gene of SMAD2/3, as a novel target in transformed HBECs. Next, to test the generality of BCL6 as a target, we studied 5 NSCLC cell lines with various level of BCL6 expression: H1693, H1819, H1993, HCC827 and H2009. Our data suggests that BCL6 can also be a therapeutic target in a subset of NSCLC lines. Then we tested the response of these NSCLC lines to a combination of BBI608 (potent STAT3 inhibitor) and FX1 (BCL6 inhibitor). The combination treatment eliminated more cancer cells than the single treatments alone. Finally, we confirmed the benefit of the combination therapy in H1993 xenografted tumors. Conclusions: We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (Phospho-STAT3 and BCL6) downstream of common oncogenes and tumor suppressors (TP53, K-RAS, and MYC) may provide a potent way to defeat intra-tumor heterogeneity. Citation Format: Dhruba Deb, Satwik Rajaram, Jill E. Larsen, Patrick P. Dospoy, Rossella Marullo, Longshan Li, Kimberley Avila, Leandro Cerchietti, John D. Minna, Lani F. Wu, Steven J. Altschuler. A novel combination therapy targeting BCL6 and phospho-STAT3 defeats intratumor heterogeneity in a subset of non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3950. doi:10.1158/1538-7445.AM2017-3950

Mitochondrial dysfunction induced by a SH2 domain-targeting STAT3 inhibitor leads to metabolic synthetic lethality in cancer cells

In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using high-affinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.

623 - Characterisation of a novel and potent STAT3 inhibitor, VS-43

623 - Characterisation of a novel and potent STAT3 inhibitor, VS-43

Neuroactive and Anti-inflammatory Frankincense Cembranes: A Structure-Activity Study.

An expeditious isolation method for the cembrane diterpene alcohols incensol (1a) and serratol (2) has been developed from respectively African and Indian frankincense. The two native alcohols and a series of semisynthetic derivatives of incensol were evaluated for transient receptor potential vanilloid 3 (TRPV3) activation and the inhibition of NF-κB, the putative molecular targets underlying the psychotropic and anti-inflammatory activities of incensol acetate (IA, 1b). Serratol (2) was the most potent TRPV3 activator, outperforming by 2 orders of magnitude the reference agonist thymol and by 1 order of magnitude incensol acetate (1b). Acylation, epimerization, and oxidation did not significantly improve the affinity of incensol for TRPV3, while NF-κB inhibition, marginal for both natural alcohols, could be improved by esterification of incensol (1a) with lipophilic acids. Interestingly, incensol (1a) but not IA (1b) was a potent inhibitor of STAT3, raising the possibility that hydrolysis to incensol (1a) might be involved in the in vivo biological activity of IA (1b). Serratol was not amenable to chemical modification, but some marine cembranoids related to the frankincense diterpenoids showed a certain degree of TRPV3-activating properties, qualifying the aliphatic macrocyclic cembrane skeleton as a selective chemotype to explore the pharmacology of TRPV3, a thermo-TRP otherwise resistant to modulation by small molecules.

DHA is a newly defined STAT3 inhibitor that may be of multiple potential uses in cancer treatment

Accumulating evidence from epidemiological, pharmacological and case control studies has shown that dihydroartemisinin (DHA), one of first-line antimalarial drugs recommended by World Health Organization (WHO), possesses antineoplastic activity and selective cytotoxicity to a variety of human cancer cell model systems. Although some antitumor mechanisms of DHA have been confirmed, the findings from the previous studies are insufficient to fully reveal the whole picture of tumor suppression by DHA. In a recent investigation, we demonstrated that DHA exhibits antitumor properties against a variety of human HNSCC cells both in vitro and in vivo. The underlying mechanism involves selective inhibition of Jak2/STAT3 signaling. By specific inhibition of STAT3 activation, DHA exerted the chemotherapeutic efficacy, including reduction in cell viability and migratory capability, along with induction of G1 phase cell cycle arrest and apoptosis in HNSCC cells. All of data suggested that DHA may be a potent inhibitor of STAT3 that can be potentially used to treat patients with HNSCC, as well as other human cancers harboring STAT3 activation.

Poly-Electrophilic Sesquiterpene Lactones fromVernonia amygdalina: New Members and Differences in Their Mechanism of Thiol Trapping and in Bioactivity

In addition to known compounds, the leaves of Vernonia amygdalina afforded the new sesquiterpene lactones 14-O-methylvernolide (2), 3'-deoxyvernodalol (6), and vernomygdalin (8). These and related compounds were evaluated for modulation of a series of thiol trapping-sensitive transcription factors (NF-κB, STAT3, and Nrf2), involved in the maintenance of the chronic inflammatory condition typical of human degenerative diseases. Vernolide (1) emerged as a potent inhibitor of STAT3 and NF-κB and showed cytostatic activity toward the prostate cancer cell line DU45, arresting the cell cycle at the S phase. The exomethylene lactones are characterized by multiple Michael acceptor sites, as exemplified by vernolide (1) and vernodalol (5). By using the nuclear magnetic resonance-based cysteamine assay, the most reactive thiophilic site could be identified in both compounds, and competitive experiments qualified vernolide (1) as being more thiophilic than vernodalol (5), in agreement with the results of the pharmacological assays.

Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model

Breast carcinoma is the most common female cancer with considerable metastatic potential. Signal transducers and activators of the transcription 3 (Stat3) signaling pathway is constitutively activated in many cancers including breast cancer and has been validated as a novel potential anticancer target. Here, we reported our finding with nifuroxazide, an antidiarrheal agent identified as a potent inhibitor of Stat3. The potency of nifuroxazide on breast cancer was assessed in vitro and in vivo. In this investigation, we found that nifuroxazide decreased the viability of three breast cancer cell lines and induced apoptosis of cancer cells in a dose-dependent manner. In addition, western blot analysis demonstrated that the occurrence of its apoptosis was associated with activation of cleaved caspases-3 and Bax, downregulation of Bcl-2. Moreover, nifuroxazide markedly blocked cancer cell migration and invasion, and the reduction of phosphorylated-Stat3Tyr705, matrix metalloproteinase (MMP) MMP-2 and MMP-9 expression were also observed. Furthermore, in our animal experiments, intraperitoneal administration of 50 mg/kg/day nifuroxazide suppressed 4T1 tumor growth and blocked formation of pulmonary metastases without detectable toxicity. Meanwhile, histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells, MMP-9-positive cells and an increase in cleaved caspase-3-positive cells upon nifuroxazide. Notably, nifuroxazide reduced the number of myeloid-derived suppressor cell in the lung. Our data indicated that nifuroxazide may potentially be a therapeutic agent for growth and metastasis of breast cancer.

STAT3 as a mediator of BCR-ABL1-independent resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic cell disorder, with an annual incidence of 1–1.5 per 100 000 in the United States. The BCR-ABL1 oncogene is necessary and sufficient to cause the leukemic phenotype. CML is typically diagnosed in the chronic phase, where the myeloid cell compartment is expanded, but cellular differentiation maintained. Without effective therapy, there is inevitable transformation to the blastic phase, which resembles an acute leukemia of myeloid or lymphoid phenotype. Current CML therapy is based on tyrosine kinase inhibitors (TKIs), which block progression from the chronic to the blastic phase in the majority of patients. The first-generation TKI, imatinib mesylate (IM), is generally well tolerated, but has a failure rate of about 25% due to resistance or intolerance. Second-generation TKIs (dasatinib; nilotinib) provide effective salvage options, but some patients will eventually progress to blastic phase and have a poor prognosis. Additionally, even in patients with profound responses (complete molecular response), recurrence is common upon discontinuation of TKI therapy, a situation referred to as persistence. TKI resistance can result from two fundamentally different mechanisms: BCR-ABL1-dependent resistance, in which kinase domain mutations restore kinase activity by disrupting TKI binding,1, 2 and BCR-ABL1-independent resistance, which can be divided into (i) extrinsic resistance, in which contact between CML cells and bone marrow-derived factors protects CML cells from TKIs,3, 4 and (ii) cell-autonomous (intrinsic) resistance, in which CML cells activate alternative signaling pathways in the absence of bone marrow-derived factors.5, 6 Importantly, BCR-ABL1-independent resistance occurs despite continued suppression of BCR-ABL1 activity. It is thought that persistence of residual leukemia in patients responding to TKIs is due to the innate BCR-ABL1 independence of CML stem cells.7 Consistent with this, we have found that primitive CML progenitor cells, including quiescent Lin− CML cells, survive despite TKI inhibition of BCR-ABL1.8 Our recent studies have implicated STAT3 in BCR-ABL1-independent resistance. In line with previous reports, we have found upregulation of pSTAT3Y705 in TKI-naive CML progenitor cells grown in HS-5 bone marrow stromal cell-conditioned medium in the presence of IM, and that pSTAT3Y705 promotes CML cell survival, implicating STAT3 as a critical mediator of extrinsic BCR-ABL1-independent resistance. Interestingly, we have also found pSTAT3Y705 activation in CML CD34+ cells from patients with intrinsic TKI resistance (clinical resistance in the absence of BCR-ABL1 mutations). This suggests that extrinsic and intrinsic BCR-ABL1-independent resistance mechanisms converge on pSTAT3Y705, a notion that was validated with STAT3-specific small hairpin RNA and dominant-negative mutants. Our observations prompted us to evaluate the effects of small-molecule STAT3 inhibitors that block STAT3 dimerization through binding to the SH2 domain. Successive STAT3 inhibitor library screens identified BP-5-087 as a highly potent and selective STAT3 inhibitor. Computational modeling and physicochemical assays confirmed binding of BP-5-087 with STAT3. In biological assays using TKI-resistant progenitors, we found that BP-5-087 combined with imatinib reduces survival and colony formation by CML progenitor and stem cells from patients with BCR-ABL1-independent resistance, consistent with a synthetically lethal pharmacologic interaction. Taken together, our data indicate that activation of pSTAT3Y705 is central to BCR-ABL1 kinase-independent TKI resistance, and that genetic, functional or pharmacologic inhibition of STAT3 reduces TKI-resistant clonogenic potential in vitro. BP-5-087 is a highly potent and selective STAT3 inhibitor. Combinations of BP5-087 with imatinib greatly reduce the survival of primary TKI-resistant stem and progenitor cells, and as such, may be considered a promising combinatorial therapy to overcome BCR-ABL1-independent TKI resistance and eliminate residual leukemia.

STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma.

Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib ([Formula: see text] mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib.

Abstract LB-227: Preclinical pharmacology and clinical efficacy of AZD9150 (ISIS-STAT3Rx), a potent next-generation antisense oligonucleotide inhibitor of STAT3

Abstract Genomic technologies have greatly expanded our understanding of cancer biology, although they have not yet been effectively translated into improved cancer therapeutics, partly due to the inability of available therapeutic modalities to target the most promising cancer driver pathways. In contrast to other therapeutic approaches, antisense technology allows the rational design of potent, sequence-specific inhibitors based on genome sequencing information alone. Recent human clinical data have demonstrated the potent activity of antisense oligonucleotides (ASOs) targeted to genes expressed by the liver. Here we describe preclinical and clinical activity of a high affinity, new generation chemistry (constrained ethyl modified) ASO, AZD9150. AZD9150 targets the mRNA coding for the transcription factor STAT3, which is considered a difficult protein to target therapeutically. Systemic delivery of unformulated AZD9150 resulted in strong inhibition of STA3 RNA and protein levels in demonstrates in a broad range of preclinical cancer models in vivo including several patient-derived xenograft models. AZD9150 administration resulted in 2 partial responses highly treatment-refractory diffuse large B-cell lymphoma and in 2 mixed responses in Hodgkin's Lymphoma patients in the phase I dose escalation study. These findings suggest that this technology has the potential to help bridge the pharmacogenomic divide in cancer drug discovery. [D.H. and Y.K. contributed equally to this work.] Citation Format: David Hong, Youngsoo Kim, Anas Younes, John Nemunaitis, Nathan Fowler, Jeff Hsu, Tianyuan Zhou, Luis Fayad, Nancy Zhang, Sarina Piha-Paul, Richard Woessner, Murali VP Nadella, Deborah Lawson, Corinne Reimer, Minji Jo, Joanna Schmidt, Xiaokun Xiao, Sarah Greenlee, Gene Hung, Mason Yamashita, David C. Blakey, Brett P. Monia, A. Robert Macleod, Razelle Kurzrock. Preclinical pharmacology and clinical efficacy of AZD9150 (ISIS-STAT3Rx), a potent next-generation antisense oligonucleotide inhibitor of STAT3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-227. doi:10.1158/1538-7445.AM2014-LB-227

Disruption of STAT3 by Niclosamide Reverses Radioresistance of Human Lung Cancer

A major challenge affecting the outcomes of patients with lung cancer is the development of acquired radioresistance. However, the mechanisms underlying the development of resistance to therapy are not fully understood. Here, we discovered that ionizing radiation induces phosphorylation of Janus-associated kinase (JAK)-2 and STAT3 in association with increased levels of Bcl2/Bcl-XL in various human lung cancer cells. To uncover new mechanism(s) of radioresistance of lung cancer, we established lung cancer cell model systems with acquired radioresistance. As compared with radiosensitive parental lung cancer cells (i.e., A549, H358, and H157), the JAK2/STAT3/Bcl2/Bcl-XL survival pathway is significantly more activated in acquired radioresistant lung cancer cells (i.e., A549-IRR, H358-IRR, and H157-IRR). Higher levels of STAT3 were found to be accumulated in the nucleus of radioresistant lung cancer cells. Niclosamide, a potent STAT3 inhibitor, can reduce STAT3 nuclear localization in radioresistant lung cancer cells. Intriguingly, either inhibition of STAT3 activity by niclosamide or depletion of STAT3 by RNA interference reverses radioresistance in vitro. Niclosamide alone or in combination with radiation overcame radioresistance in lung cancer xenografts. These findings uncover a novel mechanism of radioresistance and provide a more effective approach to overcome radioresistance by blocking the STAT3/Bcl2/Bcl-XL survival signaling pathway, which may potentially improve lung cancer outcome, especially for those patients who have resistance to radiotherapy.