Strategies and Approaches of Targeting STAT3 for Cancer Treatment

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Strategies and Approaches of Targeting STAT3 for Cancer Treatment Steffanie L. Furtek, Donald S. Backos, Christopher J. Matheson, and Philip Reigan* Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 East Montview Boulevard, Aurora, Colorado 80045, United States ABSTRACT: Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates the expression of genes related to cell cycle, cell survival, and immune response associated with cancer progression and malignancy in a number of cancer types. Once activated, STAT3 forms a homodimer and translocates to the nucleus where it binds DNA promoting the translation of target genes associated with antiapoptosis, angiogenesis, and invasion/migration. In normal cells, levels of activated STAT3 remain transient; however, STAT3 remains constitutively active in approximately 70% of human solid tumors. The pivotal role of STAT3 in tumor progression has promoted a campaign in drug discovery to identify small molecules that disrupt the function of STAT3. A range of approaches have been used to identify novel small molecule inhibitors of STAT3, including high-throughput screening of chemical libraries, computational-based virtual screening, and fragment-based design strategies. The most common approaches in targeting STAT3 activity are either via the inhibition of tyrosine kinases capable of phosphorylating and thereby activating STAT3 or by preventing the formation of functional STAT3 dimers through disruption of the SH2 domains. However, the targeting of the STAT3 DNAbinding domain and disruption of binding of STAT3 to its DNA promoter have not been thoroughly examined, mainly due to the lack of adequate assay systems. This review summarizes the development of STAT3 inhibitors organized by the approach used to inhibit STAT3, the current inhibitors of each class, and the assay systems used to evaluate STAT3 inhibition and offers an insight into future approaches for small molecule STAT3 inhibitor development.

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sponding receptors, the receptors form a dimer complex. Activation of glycoprotein 130 (gp130) is also initiated, inducing dimerization of gp130 and the α-receptor subunit of the receptor.9 Together the ligand receptor and gp130 complex recruit Janus kinases (JAKs). The aggregation of JAKs leads to their activation via phosphorylation which in turn phosphorylates the cytoplasmic tyrosine residues on the receptors that serve as a dock for the SH2 domain of STAT3.10 STAT3 becomes activated (p-STAT3) through the phosphorylation of its Tyr705 residue located within its SH2 domain.11 Activation of STAT3 triggers p-STAT3 to form a homodimer via the interaction of the p-Tyr705 of one monomer and the SH2 domain of another.2,12 The activated dimer dissociates from the receptor and subsequently translocates from the cytoplasm to the nucleus where it binds specific DNA sequences and induces transcription (Figure 2).12 Transcriptional activity of STAT3 also relies on the binding of coactivators such as APE/ref-1, CBP/p300, and NCOA/SRC1a.8 STAT3 regulates gene expression involved in cell cycle (c-Myc and cyclin D1), antiapoptosis (Bcl-xL, Bcl-2, and survivin), angiogenesis (VEGF and IL-8), and invasion/migration (MMP-2 and MMP-9).2,13 In addition to JAKs, STAT3 can be activated by nonreceptor tyrosine kinases such as Src and ABL.14 The expression and activation levels of STAT3 vary in normal tissues and cells, and STAT3 is highly expressed in the

he signal transducer and activator of transcription (STAT) family of proteins is a group of transcription factors that regulate gene expression related to cell cycle, cell survival, and immune response.1,2 The STAT family of proteins is comprised of seven structurally and functionally related proteins STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6.1 Each of the STAT proteins is encoded by a separate gene, although all six members are structurally similar (Figure 1A).3 The aminoterminal domain (NH2), the coiled−coiled domain (CCD), the DNA-binding domain (DBD), the linker domain, and the Src homology 2 domain (SH2) remain highly conserved between the STAT family members, while STAT specificity is established by the divergence in the carboxy-terminal transcriptional activation domain (TAD).4 Alternate mRNA splicing or proteolytic processes can give rise to multiple isoforms, which have been isolated in the cases of STAT1, STAT3, and STAT5 and have demonstrated distinct functional relevancies.5 Akira and colleagues and Zhong and colleagues independently discovered STAT3 in 1994, classifying STAT3 as an acute-phase response factor that selectively bound to the IL-6 response element on DNA within the acute-phase gene promoter,6 and determined that STAT3 bound to DNA in response to epidermal growth factor (EGF),7 respectively. STAT3 is activated through the binding of cytokines or growth factors to cell surface receptors.4 Cytokines such as the interleukins IL-6, IL-10, and IL-11, as well as growth factors such as EGF, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) can activate the tyrosine phosphorylation cascade.8 Once ligands bind to their corre© 2016 American Chemical Society

Received: November 16, 2015 Accepted: January 5, 2016 Published: January 5, 2016 308

DOI: 10.1021/acschembio.5b00945 ACS Chem. Biol. 2016, 11, 308−318

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Figure 1. STAT3 protein structure. A) Global view of the STAT3 homodimer crystal structure containing DNA (PDB ID: 4E68) with the individual domains indicated (magenta = coiled-coil, orange = DNA binding, blue = linker, yellow = SH2, green = transactivation). Middle inset close up of the DNA binding interface with residues involved in direct DNA binding highlighted in yellow and those involved in drug binding of previously reported DNA binding site inhibitors highlighted in cyan. Bottom inset close up of the two interacting SH2 domains (bottom) with the previously reported SH2 inhibitor binding pockets highlighted. B) An individual STAT3 monomer from the same crystal structure. Top inset location of the residues comprising the SH2 domain inhibitor binding pockets (pY = red, pY-X = magenta, pY+1 = purple). Middle inset location of the redox-sensitive C687 residue and Y705 the target of activating phosphorylation. Bottom inset residues involved in direct DNA interaction (yellow) or those reported as the DNA binding domain inhibitor interface (cyan).

(PIAS), which prevents active STAT3 from binding DNA,22 and dephosphorylation of STAT3 at Tyr705 by protein tyrosine phosphatase receptor T (PTPRT).23 Approximately 70% of human solid and hematological tumors display overexpression or constitutively active STAT3 compared with normal cells.4 It has been demonstrated that the induction of oncogenesis in both cultured cells and nude mice can be mediated by the expression of mutated STAT3 (STAT3-C) that remains constitutively dimerizeable.24 By comparison, levels of pSTAT3 expression have greater impact on tumorigenesis than total STAT3. Constitutively active STAT3 has been shown to promote the induction and survival of cancer.11 It has been proposed that elevated levels of p-STAT3 promote resistance to apoptotic cues and facilitates rapid proliferation and tumorigenesis.25 Recently, it has been demonstrated that activated STAT3 can be induced by hypoxia26 and that the mechanism of action contributing to hypoxia-induced increases in p-STAT3 levels is a reduction in SOCS3.27 Constitutively active STAT3 have also been shown to play a role in impairing both the innate and adaptive immune responses,28 and STAT3 is constitutively active in both immune cells and tumor microglia.29,30 Upon recruitment into the tumor microenvironment, M1 macrophages and microglia become designated as the M2 phenotype following exposure to hypoxic conditions.31,32 Polarization of tumor-

peritoneum, leukocytes of the peripheral blood, and neutrophils of the bone marrow. Certain tissues and organs also display higher basal levels of STAT3 expression including the peripheral nervous system, digestive tract, and bone marrow, suggesting a physiological role of STAT3 that is necessary for the function of these systems.8 The activation of STAT3 signaling is a controlled and transient process in normal cells that can last from half an hour to several hours.15 Inactivation of STAT3 occurs through dephosphorylation of Tyr705 by nuclear protein-Tyr phosphatases such as TC-PTP and TC45, resulting in the shuttling of STAT3 back into the cytoplasm.16,17 Additional regulatory mechanisms exist, such as the immediate degradation or recycling of cytokine/growth factor receptors following the binding of their respective ligands, and the inactivation of JAK, STAT3, and the receptor via dephosphorylation through the interaction of the Src homology domain-containing tyrosine phosphatases 1/2 (SHP-1/2) and the intracellular domain of the cell surface receptor.18,19 Suppressors of cytokine signaling (SOCS) are transcribed as a negative feedback loop as part of STAT3 signaling and interact with the JAK domains or intracellular portions of the receptors to reduce STAT3 activation;20 however, Src-mediated STAT3 activation is not impacted by SOCS.21 STAT3 transcriptional activity is also regulated through a protein inhibitor of activated STAT3 309


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and atixinib and have been shown to reduce activation of STAT3.37 Recently, the novel VEGFR-2 small molecule inhibitor LCB03-0110 (Figure 3) was reported by Kim et al. and demonstrated blockage of angiogenesis mediated by VEGF/ JAK/STAT signaling.38 The widespread prevalence and importance of the IL-6/JAK/ STAT signaling pathway in a variety of human malignancies has spurred the development of several IL-6 and IL-6 receptor inhibitors. Inhibition of IL-6 signaling has been accomplished using IL-6 monoclonal antibodies, such as the FDA approved tocilizumab, which has shown promising results in rheumatoid arthritis and is currently being evaluated preclinically in cancer.39−41 In contrast, orally available small molecule inhibitors of IL-6 have not been extensively explored, likely due in part to the development of bacterial infections associated with prolonged blockade of IL-6 due to the cytokine’s role in suppressing the immune response.42 Recently, LMT-28 (Figure 3), which was discovered through a library screen by Hong et al., was demonstrated to bind to gp130, inhibit IL-6 activation, and downregulate levels of p-STAT3.43 Another gp130 small molecule inhibitor, SC144 (Figure 3), demonstrates inhibition of STAT3 activation, reduces expression of downstream targets, and exhibits oral activity in ovarian cancer.44 Although targeting cell surface receptors has demonstrated an ability to abrogate STAT3 signaling in response to growth factor or cytokine stimulation, a more widely explored strategy has been through the development of kinase small molecule inhibitors that target STAT3 phosphorylation. Kinase Inhibitors. As the majority of STAT3 biological activity is reliant on its activation, a rational approach to inhibiting p-STAT3 is through the inhibition of activating kinases upstream of STAT3. While there are many protein tyrosine kinases that are capable of phosphorylating STAT3, a major area of small molecule development has focused on targeting JAK and Src kinases. Over a dozen small molecule JAK inhibitors have been developed, many of which are currently in various stages of clinical trial (Figure 3). Design of JAK inhibitors has centered around two major indications for JAK/STAT signaling, namely oncology/hematology and inflammatory syndromes. In both areas, there have been clinical trials resulting in approved drugs, specifically ruxolitinib and tofacitinib, respectively.45 Other JAK inhibitors such as WP-1066, LS-104, CEP-701, LY2784544, and CYT387 (momelotinib) continue into phase I/II clinical trials with each compound demonstrating varying levels in reduction of p-STAT3, as well as assorted downstream implications such as increased apoptosis and decreased tumor growth. 10,15,45 Recently, however, a clinical trial of the JAK1/2 inhibitor AZD1480 in patients with solid tumors was terminated due to off-target neurotoxicity.46 Other limitations observed in clinical trials for JAK2 inhibitors include rate-limiting toxicities associated with anemia and thrombocytopenia.45 Similar to IL6 inhibition, inhibitors of JAK1 and JAK3 also have demonstrated on-target toxicities related to increased rates of infection in inflammatory conditions.47 Src was first identified as an onco-protein and is believed to play a critical role in cancer progression.48 Src inhibitors saracatinib (AZD0530), bosutinib (SKI-606), dasatinib, and KX2-391 are all in different points of phase II/III clinical trials for multiple cancer indications (Figure 3). PP2, another Src inhibitor, inhibits phosphorylation of both Src and STAT3 as well as decreases tumor growth through selective inhibition.49,50 Pan-JAK/Src inhibitors have also been explored for more complete targeting of STAT3 signaling. E738, a derivative of the

Figure 2. Signaling cascade for STAT3 activity. Activation of cell surface receptors through the binding of cytokines or growth factors recruits, phosphorylates, and activates STAT3. Nonreceptor Tyr kinases Src and ABL also activate STAT3. Activated STAT3 forms a homo- or heterodimer and translocates from the cytoplasm to the nucleus where it binds DNA and coactivators and induces gene transcription. STAT3 activation is negatively regulated by PIAS, SHP-1/2, SOCS, and PTPRT.

associated macrophages (TAMs) toward the M2 phenotype is mediated by the p-STAT3 signaling pathway and ultimately contributes to immunosuppression and tumor invasion.32 The promotion of cancer by TAMs is driven through the secretion of proangiogenic factors such as VEGF and MMP-9, which are downstream targets of STAT3 signaling.33 Given its critical role in both tumor onset and progression, STAT3 has emerged as an attractive target for small molecule therapeutics.

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UPSTREAM INHIBITION OF STAT3 Since the discovery of STAT3, small molecule inhibitors targeting various members of the STAT3 signaling pathway have been employed to disrupt STAT3 signaling and activity. In this section, we will briefly discuss selected inhibitors that target upstream of STAT3. Cell Surface Receptor Inhibitors. Several growth factors and cytokines have been implicated in the activation of STAT3, each involving a specific cell surface receptor. Growth factors known to induce the activation of STAT3 include EGF, human epidermal growth factor (HER2), FGF, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), VEGF, and insulin-like growth factor (IGF).8 Cytokines capable of stimulating STAT3 activation include the interleukins IL-6, IL10, IL-11, as well as IL-6 family members leukemia inhibitory factor (LIF) and leptin.13,34 Targeting aberrant STAT3 via inhibition of cytokine/growth factor binding has been shown to be an effective strategy in a variety of systems. EGF receptor (EGFR) inhibitors such as the peptide aptamer KDI1 and small molecule PD153035 (Figure 3) directly interact with EGFR and inhibit phosphorylation of STAT3 at Tyr-705, thus preventing its activation and dimerization. In multiple cancer cell lines PD153035 has an IC50 < 1 μM and demonstrates specificity for EGFR over other growth factor receptors.10,35 FGF receptor (FGFR) inhibition with ponatinib (Figure 3) also decreases both STAT3 phosphorylation and tumor growth in vivo.36 Approved VEGF receptor (VEGFR) inhibitors include sorafenib, sunitinib, 310


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Figure 3. Chemical structures of upstream STAT3 inhibitors.

thermore, the ability of kinases to activate more than one member of the STAT family can also lead to STAT1 and STAT5 inhibition and additional off-target effects. For these reasons, a larger strategy for STAT3 inhibition is through the direct targeting of functional p-STAT3.

natural product indirubin, has IC50 values against recombinant JAKs and Src family kinases ranging from 0.7−74.1 nM and 10.7−263.9 nM, respectively.51 While indirubin can be obtained from natural sources, synthetic bromoindirubins have been developed to enhance their binding affinity for kinases such as CDKs, JAKs, and Src family kinases.52 MLS-2384 is a 6bromoindirubin derivative that displays dual JAK/Src inhibition, a dose-dependent decrease in STAT3 phosphorylation, and anticancer activity in multiple tumor types.52 Although targeting the kinase activity responsible for activation of STAT3 can successfully reduce STAT3 activity, off-target toxicities are still a concern related to this therapeutic approach. Among the ∼500 distinct human kinases, ATP-binding sites remain highly homologous, resulting in off-target kinase inhibition.45 Fur-

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DIRECT STAT3 INHIBITORS

A number of small molecule compounds directly inhibit the activity and function of STAT3 and have been developed for use in cancer treatment and prevention.53,54 Although the functional outcome is similar, the mechanisms of action of the various direct STAT3 inhibitors vary considerably and include disrupting phosphorylation, dimerization, nuclear translocation, and/or 311


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Figure 4. Chemical structures of direct STAT3 inhibitors.

DNA-binding of STAT3.55−57 Several prominent examples of these small molecules are discussed in the following sections. STAT3 SH2 Domain Inhibitors. STAT3 homodimerization is mediated by the protein−protein interactions between the SH2 domains of individual monomers, specifically via the phosphorylation of Tyr705. This critical molecular interaction has been exploited as a molecular target for small molecule inhibitors that directly inhibit STAT3.58 Within the SH2 domain, STAT3 has three proximal binding subpockets that prove suitable for small molecule targeting (Figure 1A).59 These subpockets are comprised of the pY binding pocket, containing primarily polar residues responsible for H-bonding and electrostatic interactions, and two subpockets pY + 1 and pY − X comprised of hydrophobic residues, with pY − X being unique to STAT3 (Figure 1). The majority of developed STAT3 inhibitors that target the SH2 domain bind at least two of these three subdomains.60 One of the first small molecule inhibitors of STAT3 was discovered through a high-throughput screen (HTS) of diverse chemical libraries containing a little more than 17 000 compounds.12 Stattic (Stat three inhibitory compound) selectively inhibited STAT3 dimerization (IC50 = 5.1 μM), was selective over STAT1 and STAT5, and prevented STAT3 translocation to the nucleus in vitro.59 The inhibitory activity of

stattic was also determined to be time-, temperature-, and dithiothreitol-dependent, suggesting interaction with cysteine residues such as Cys687 located near the phosphopeptidebinding area of the SH2 domain (Figure 1B).61 More recently Zhang et al., demonstrated both in vitro and in vivo the treatment of esophageal squamous cell carcinoma (ESCC) with stattic radiosensitized cells and xenografts, suggesting a potential use as an adjuvant therapy in radioresistant ESCC.62 Similarly, stattic circumvents cisplatin resistance in ovarian cancer,63 suggesting a renewed use of this compound in recurrent cancers displaying constitutively active STAT3. Song et al., using a structure-based virtual screen of nearly 430 000 molecules, identified another STAT3 SH2 domain inhibitor.64 Their hit compound was identified as STA-21 (Figure 4) and demonstrated inhibition of active STAT3 binding to DNA, suppression of downstream target transcription, and induction of apoptosis in cancer cell lines with constitutively active STAT3 expression.64,65 This compound was subsequently derivatized to form a less complex anthraquinone-based compound (LLL-3) that possessed comparable antiproliferative activity as STA-21 and exhibited improved cell permeability compared with the parent compound.66 In K562 leukemic cells, Mencalha et al. demonstrated a decrease in tumor cell survival following treatment with LLL-3, as well as a synergistic effect 312


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ACS Chemical Biology with combination therapy of LLL-3 and Imatinib.67 Lin et al. further optimized LLL-3, replacing its acetyl group with a sulfonamide and producing another STAT3 inhibitor LLL-12.11 This compound was determined to have an IC50 against STAT3 ranging from 0.16 to 3.09 μM in breast, pancreatic, and glioblastoma cell lines.11,68−70 An in silico screen of the National Cancer Institute (NCI) chemical libraries identified another STAT3 SH2 domain inhibitor, S3I-201 (Figure 4). This compound was 3-fold more selective for STAT3 over STAT1, blocked the formation of STAT3 homodimers (IC50 = 86 μM), and inhibited proliferation of breast and hepatocellular cancer cells in mice.56 Additional computational-based docking studies suggested a less than optimal interaction of S3I-201 with the SH2 domain of STAT3. These results, taken together with high micromolar IC50 values for this initial hit, prompted the Gunning group to rationally design several analogs of S3I-201 in an effort to improve its in vitro activity.71 Several analogs of S3I-201 that showed promise from the SAR studies were SF-1-066, SF-1-087, and SF-1-121 (Figure 4) which had IC50 values of 37 μM, 24 μM, and 52 μM in DU145 cells, respectively.2,71 SF-1-066 was determined to bind the SH2 domain of STAT3 with a Kd= 2.74 μM, blocked the association of STAT3 with EGFR, inhibited growth of malignant cell lines with constitutively active STAT3, and suppressed the expression of downstream targets of STAT3-regulated genes.2 Additional SAR studies derivatized the sulfonamide moiety of SF-1-066 and an additional 15 analogs were developed. This study led to the identification of BP-1-102 (compound 17o), a compound that disrupted STAT3-DNA complex formation with an IC50 of 6.8 μM, a 5-fold improvement from SF-1-066.25,72 The highest potency of this compound was determined to be 10 μM in MDA-468 breast cancer cells, inhibiting growth of xenografts at 3 mg/kg, and displayed oral bioavailability, a first for designed STAT3 inhibitors.25 More recently S3I-1757, another S3I-201 analog, was developed by Zhang and colleagues (Figure 4).73 S3I-1757 was tested against the STAT3 SH2 domain and was found to have an IC50 of 13 μM.74 In both breast and lung cancer cell lines, S3I1757 decreased nuclear levels of active STAT3 as well as decreased the ability of activated STAT3 to bind DNA in a dosedependent manner beginning at 50 μM.74 MDA-MB-468 cells transfected with STAT3-C, a genetically engineered mutant capable of forming constitutively active STAT3 dimers via disulfide bonding in the absence of tyrosine phosphorylation, were able to rescue the inhibition of STAT3 transcriptional activity caused by S3I-1757. These data provided further support that S3I-1757 targets specifically at the SH2 domain of STAT3. From the inception of designing STAT3 SH2 domain inhibitors, there was a need to evaluate the effectiveness of these small molecules against STAT3 activity. Unfortunately, the lack of uniformity of assays used to determine potency varies among publications, making assessments across different classes of STAT3 inhibitors difficult. Schust and Burg developed the fluorescence polarization assay critical for evaluating small molecules as SH2 domain inhibitors against recombinant STAT3 protein. This assay uses competitive binding of a small fluorescent peptide that corresponds to the gp130 subunit of the IL-6 receptor and has been demonstrated to have a high affinity for the SH2 domain of recombinant STAT3.75 Interruption of the fluorescence-labeled SH2 peptide on the STAT3 protein is indicative of a compound binding to the SH2 domain of STAT3. This method allows for moderate-throughput screening of chemical libraries to identify potentially novel SH2 domain

inhibitors of STAT3. Alternatively, several groups have utilized time-resolved electrospray ionization mass spectrometry (TRESI-MS) and hydrogen−deuterium exchange (HDX) techniques to assess binding of an inhibitor to recombinant STAT3 as well as to evaluate its binding location and affinity for STAT3.76−79 The inhibition of cellular STAT3 activity by SH2 domain inhibition can be determined by STAT3 luciferase activity assay and electromobility shift assay (EMSA). These assays determine the overall abatement of STAT3 transcriptional activity and binding of STAT3 to DNA, respectively. STAT3 enzyme-linked immunosorbent assay (ELISA) kits are also available to evaluate the effect of the STAT3 inhibitors on the ability of STAT3 to bind DNA. However, methods surrounding dosing with compounds remain unclear, and there appears to be disparity on whether dosing previously prepared nuclear extracts has the capacity to disrupt functional dimers of STAT3. While useful in evaluating an inhibitor’s impact on STAT3, cell-based assays do not definitively display SH2 domain targeting or determine off target effects within the STAT3 signaling cascade. Less than 6 years after the initial discovery of the role of STAT3 in malignant transformation, peptides and peptide mimics of pTyr peptide PpYLKTK that bind the SH2 domain and prevent dimerization and STAT3 activity were developed.80 Due to their lack of membrane permeability and stability,58 the attractiveness of nonpeptidic small molecule STAT3 inhibitors became the forefront of drug discovery. Yet, most small molecule SH2 domain inhibitors remain in preclinical development,81 with the exception of STA-21 which has completed phase I/II trial for psoriasis.82 One of the major reasons for this is that STAT3:STAT3 dimerization is a protein−protein interaction involving a large surface area that is difficult to impact with small molecules.74 Furthermore, selectivity and mechanisms of effects still remain to be clearly defined.58 Morlacchi et al. recently examined STA-21 and its analogs in a fluorescence polarization assay and determined that STA-21 showed no affinity for the SH2 domain of STAT3 up to 100 μM.83 Morlacchi and colleagues demonstrated the ambiguity in the proclamations of an SH2 domain inhibitor without crucial assays that evaluate SH2 domain binding and affinity. Although promising, many of the mentioned compounds still display medium to high micromolar activities, suggesting the need for additional optimization before consideration for in vivo efficacy and transition into clinical trials. Taken together, small molecules that target the SH2 domain have been slow to progress to clinical development due to the difficulties in targeting a protein−protein interaction and the high concentrations required for STAT3 inhibition which increases the likelihood for off-target toxicities. STAT3 DNA-binding Domain Inhibitors. While STAT3 SH2 domain small molecule inhibitors remain the leading focus for drug discovery, there has been a recent interest in targeting the DBD.58,84,85 As a transcription factor, STAT3 activity relies on the physical interaction of an active STAT3 dimer with its corresponding DNA-binding consensus sequence (Figure 1A). Each monomer within an active STAT3 dimer has a total of four loops, three from the DBD and one from the linker domain that form interactions with the double helix of DNA.8 Platinum compounds are known to form DNA adducts and therefore can disrupt the ability of STAT3 to bind DNA, leading to apoptosis in STAT3 dependent human cancer cell lines.86,87 Platinum compounds proposed as STAT3 inhibitors are classified as platinum(IV) complexes and differ from chemotherapeutics such as cisplatin, a platinum(II) complex, which displays no inhibitory 313


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ACS Chemical Biology effect on STAT3.86,87 The most well-known platinum(IV) DBD inhibitors are CPA-1, CPA-7, and platinum(IV) tetrachloride. More recently another platinum(IV) compound, IS3−295, was determined to inhibit the DNA-binding capacity of STAT3 although its mechanism of action still requires elucidation.10 A more thorough review on this class of compounds can be found in a manuscript by Yue and Turkson.58 Similar to targeting the SH2 domain of STAT3, several research groups have identified peptides capable of disrupting the DBD of STAT3. DBD-1, a small peptide aptamer, disrupted STAT3-DNA binding but did not interfere with STAT3 phosphorylation. Significant levels of apoptosis were measured in B16 murine melanoma cells; however, in vivo findings showed only weak interactions of DBD-1 and the DBD of STAT3.88 HIC1 (hypermethylated in cancer 1), a tumor suppressor gene, was found to endogenously form a complex with the DBD of STAT3 and ultimately prevent the binding of STAT3 to its promoter sequence and induce transcription of downstream targets. The interaction of HIC1 and STAT3 was identified via mass spectrometry and confirmed using immunoprecipitation techniques using Flag-tagged HIC1 and HA-tagged STAT3 proteins in HeLa cells.89 More recently, Huang et al. identified InS3−54 through a virtual screening of the ChemDiv chemical libraries (Figure 4). InS3−54 exhibited a dose-dependent decrease in STAT3 activity as determined by luciferase assay (IC50 = 13.8 μM). It was also determined that InS3−54 displayed selectivity for STAT3 over STAT185 as observed in the decrease of downstream STAT3 target transcrition and the induction of apoptosis in both breast and lung cancer cell lines. This prompted the optimization of InS3−54 as a DBD inhibitor of STAT3. Through an additional structural comparison of the ChemDiv database, Huang et al. identified 79 commercially available compounds that showed at least 80% structural similarity to InS3−54. Of these compounds, several had better anti-STAT3 activity compared to the parent compound and further testing isolated compounds InS3−54A18 and InS3−54A26 (Figure 4) as lead candidates.84 Both InS3− 54A18 and InS3−54A26 displayed cytotoxicity IC50 values of ∼4 μM; however InS3−54A26 was disqualified from further study due to poor solubility properties and also having an IC50 of 4 μM in noncancerous lung fibroblast cells giving this compound too narrow of a therapeutic window.84 In contrast, InS3−54A18 had an IC50 in noncancerous cells ranging from 7.0 to 10.5 μM and an IC50 of 11 μM in a STAT3 luciferase assay, associated directly with STAT3, and was tolerated by mice up to 200 mg/kg with a multiple dosing regimen.84 InS3−54A18 warrants further study and optimization as a DBD inhibitor of STAT3. While there are several means of evaluating STAT3 inhibitors that bind to the SH2 domain, the breadth of assays available to confirm binding to the DBD of STAT3 remains quite narrow. An inhibitor of the STAT3 DBD will demonstrate effects in both a luciferase assay and an EMSA, similar to an SH2 domain inhibitor. While the FP assay for the SH2 domain has been well established, a similar recombinant protein assay evaluating the DBD has not yet been developed. This may be in part due to the only recent interest in targeting what many suggest is an “undruggable” domain.84 A novel approach to this issue was presented by Zhang and colleagues where the use of CNBractivated Sepharose beads conjugated with their compound InS3−54A26 and recombinant STAT3 protein identified the domain of binding in a pull-down assay. By incubating the conjugated beads with recombinant STAT3 containing only specific domains, Zhang and colleagues were able to demonstrate

that at the removal of the DBD, the conjugated beads were no longer successful in pulling down the recombinant protein.84 A major limitation of this assay is the requirement that the compound is capable of conjugation to the beads in such a way as to maintain interaction with STAT3. Further development of assay systems to quantitatively evaluate inhibitors of the DBD of STAT3 is needed before HTS methods can be employed effectively to identify novel STAT3 DBD inhibitors. While the SH2 domain has been widely explored, inhibitors targeting the DBD and more recently the N-terminus have had minor development,58 and exploration of these domains is warranted for further STAT3 inhibitor development.

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APPLICATIONS/DISCUSSION STAT3 is constitutively active in a large percentage of human solid and hematological tumors. These include ovarian, cervical, endometrial, breast, colon, pancreatic, lung, brain, glioma, head and neck squamous cell carcinoma, renal, prostate, melanoma, lymphoma, and leukemias. Most cultured human cancer cell lines also maintain constitutively active STAT3. Though the role of STAT3 in tumorigenesis continues to be elucidated, STAT3 continues to be a favorable drug target for cancer therapies. Likewise, there is significant evidence implicating the activation or suppression of STAT3 signaling in several noncancerous disease states,8 contributing to the development of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis, and both renal and pulmonary fibrosis. Both RA patients and RA mouse models exhibit hyperactivation of STAT3.90 In mice, enhanced STAT3 activation promoting RA development has been reported in conjunction with the amino acid substitution Y759F in gp130.91 This substitution has also been demonstrated to be linked to IL-6 stimulation and loss of the negative feedback loop mediated by SOCS3.91 In humans, p-STAT3 expression is significantly increased in peripheral blood fibrocytes, and the abnormal growth and survival of RA synoviocytes is linked to STAT3 activity.8 Abnormal STAT3 and/or p-STAT3 expression is observable in IBD with patients displaying elevated STAT3 activity in their intestinal epithelial cells.92 Peripheral blood granulocytes also display a 2-fold increase of p-STAT3 expression levels in active versus remissive patients.93 Crohn’s disease is characteristic of having increased expression and nuclear localization of STAT3 and treatment with antisense STAT3 oligonucleotides decreased in proinflammatory cytokines and reduced colonic tissue damage in a mouse colitis model.94 Taken together, these results indicate that STAT3 is also a viable drug target in autoimmune and inflammatory diseases and suggest a greater clinical scope for small molecule STAT3 inhibitors beyond cancer chemotherapy. Disruption of STAT3 signaling delays wound healing in normal mice, while constitutively active STAT3 in keratinocytes can induce spontaneous development of psoriatic-like skin lesions that can be reversed by STAT3 disruption.95,96 Similarly, inhibition of JAK/STAT signaling with compound WP1066 suppressed the onset of psoriasis in PPARβ/δ transgenic mice.97 In human patients, a completed clinical trial (ClinicalTrials.gov identifier: NCT01047943) of topical treatment of psoriatic lesions with small molecule STAT3 inhibitor STA-21 displayed improvement after 2 weeks of treatment.82 STAT3 has also been implicated in the development of renal and pulmonary fibrosis. In renal fibrosis, p-STAT3 is overexpressed in tubular epithelial cells, myofibroblasts, and interstitial cells and inhibition of STAT3 has been demonstrated to induce apoptosis in renal 314



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fibroblasts of obstructed kidney.8,98 Silica-exposed mice and human COPD patients exhibit overexpression of STAT3 in several airway tissues and downstream gene targets of STAT3 have been employed as biomarkers of COPD.99 It is important to note that the role of STAT3 is not entirely limited to disease formation and progression but is also involved with cardioprotection, liver protection, and obesity. In transgenic mice, cardiac-specific, constitutively active STAT3 provides protection from myocardial ischemia/reperfusion injury, while attenuation of fibrosis after myocardial infarction could be achieved through IL-11-mediated activation of cardiac STAT3.100,101 STAT3 also displays a protective role in liver injury. Inactivation of STAT3 in hepatic cells of mice that are multidrug resistance 2 knockouts (mdr2−/−) displayed bile acid induced hepatic injury and fibrosis.102 In obesity, neuronal STAT3 activation in response to leptin is necessary for maintaining energy homeostasis. Deletion of neuronal STAT3 or disruption of STAT3 activation by the leptin receptor (LEPRb) causes severe hyperphagia and morbid obesity. Activation of STAT3 via the binding of leptin downregulated the expression of both the obesity-associated and fat-mass genes in vitro and in vivo.103 The disruption of STAT3 signaling may therefore have long-term use implications related to cardioprotection, liver protection, and reduction of obesity. The future of STAT3 small molecule inhibitor development is reliant on a standardization for the evaluation of efficacy. The disparity between the assays and assay conditions reported in publications for assessing STAT3 inhibitors makes it difficult to compare one compound to another and to determine a ranking of effective inhibitors. While cell-based standards such as ELISA, luciferase, and EMSA evaluate the ability of an inhibitor to impact STAT3 signaling and activity, they provide little to no information on the direct binding to STAT3, and the results can vary depending on the cell type used. The recombinant protein pull-down method proposed by Zhang et al.84 lends a suggestion on the location of binding yet is limited by its inability to increase the throughput of the assay to accommodate larger chemical libraries. The development of additional recombinant STAT3 assays to evaluate direct STAT3 binding and inhibition is essential for the advancement of the field. While the preclinical evidence of direct STAT3 inhibition justifies further development of novel small molecule therapeutics, there currently are no direct STAT3 inhibitors in clinical trial for cancer therapy. Many of the upstream kinase inhibitors have demonstrated the effects of STAT3 signaling inhibition in cancer, suggesting a therapeutic relevancy for STAT3 inhibitors. Furthermore, the aberrant activity of STAT3 in cancer provides potential therapeutic strategies for increasing tumor selectivity of STAT3 inhibitors, as the signaling of STAT3 is primarily a transient process in normal tissues. With several upstream inhibitors progressing into the final stages of clinical trials for various cancer indications, the novelty of STAT3 inhibition is in the direct targeting of STAT3. In conclusion, the development of small molecule inhibitors against STAT3 is still an evolving field with the potential to exploit constitutively active STAT3 in cancer and other diseases; therefore, the continued development of effective and targeted STAT3 inhibitors is necessary to elucidate clinical candidates and further illuminate the therapeutic significance of STAT3 inhibition.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +1(303)724-6431. E-mail: philip.reigan@ ucdenver.edu. Notes

The authors declare no competing financial interest.

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KEYWORDS Transcription factors: A transcription factor is a protein that binds to specific DNA sequences in order to induce transcription of genetic material from DNA to mRNA. Small molecule inhibitors: A small molecule inhibitor is a low molecular weight organic compound used to regulate a biological process, through interaction with the active or allosteric site of a protein. Many drugs are considered small molecule inhibitors. STAT3: Signal transducer and activator of transcription 3 is a cytoplasmic transcription factor that plays a key role in many cellular processes related to cell growth and apoptosis. STAT3 inhibitors: STAT3 inhibitors are peptide or drug-like molecules that decrease the signaling or transcriptional activity of STAT3 through indirect or direct targeting of the STAT3 protein. DNA-binding domain: The DNA-binding domain is an independently folded protein domain that contains at least one motif that recognizes and binds single- and/or doublestranded DNA. SH2 domain: The Src homology 2 (SH2) domain is a structurally conserved protein domain which functions in recognizing phosphorylated tyrosine residues and facilitates protein−protein interactions with these phosphorylated sites. Tumorigenesis: The process related to the transformation of normal cells into cancerous cells producing a new tumor or tumors. It is also commonly known as carcinogenesis. Cancer therapy: Cancer therapy is the use of drugs or other substances to identify and attack cancer cells in order to reduce and/or eradicate tumors. REFERENCES

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(97) Romanowska, M., Reilly, L., Palmer, C. N., Gustafsson, M. C., and Foerster, J. (2010) Activation of PPARbeta/delta causes a psoriasis-like skin disease in vivo. PLoS One 5, e9701. (98) Pang, M., Ma, L., Gong, R., Tolbert, E., Mao, H., Ponnusamy, M., Chin, Y. E., Yan, H., Dworkin, L. D., and Zhuang, S. (2010) A novel STAT3 inhibitor, S3I-201, attenuates renal interstitial fibroblast activation and interstitial fibrosis in obstructive nephropathy. Kidney Int. 78, 257−268. (99) Qu, P., Roberts, J., Li, Y., Albrecht, M., Cummings, O. W., Eble, J. N., Du, H., and Yan, C. (2009) Stat3 downstream genes serve as biomarkers in human lung carcinomas and chronic obstructive pulmonary disease. Lung Cancer 63, 341−347. (100) Boengler, K., Hilfiker-Kleiner, D., Drexler, H., Heusch, G., and Schulz, R. (2008) The myocardial JAK/STAT pathway: from protection to failure. Pharmacol. Ther. 120, 172−185. (101) Obana, M., Maeda, M., Takeda, K., Hayama, A., Mohri, T., Yamashita, T., Nakaoka, Y., Komuro, I., Takeda, K., Matsumiya, G., Azuma, J., and Fujio, Y. (2010) Therapeutic activation of signal transducer and activator of transcription 3 by interleukin-11 ameliorates cardiac fibrosis after myocardial infarction. Circulation 121, 684−691. (102) Mair, M., Zollner, G., Schneller, D., Musteanu, M., Fickert, P., Gumhold, J., Schuster, C., Fuchsbichler, A., Bilban, M., Tauber, S., Esterbauer, H., Kenner, L., Poli, V., Blaas, L., Kornfeld, J. W., Casanova, E., Mikulits, W., Trauner, M., and Eferl, R. (2010) Signal transducer and activator of transcription 3 protects from liver injury and fibrosis in a mouse model of sclerosing cholangitis. Gastroenterology 138, 2499− 2508. (103) Wang, P., Yang, F. J., Du, H., Guan, Y. F., Xu, T. Y., Xu, X. W., Su, D. F., and Miao, C. Y. (2011) Involvement of leptin receptor long isoform (LepRb)-STAT3 signaling pathway in brain fat mass- and obesity-associated (FTO) downregulation during energy restriction. Mol. Med. 17, 523−532.

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Strategies and Approaches of Targeting STAT3 for Cancer Treatment

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