Novel STAT3 small-molecule inhibitors identified by structure-based virtual ligand screening incorporating SH2 domain flexibility.

Efforts to develop STAT3 inhibitors have focused on its SH2 domain starting with short phosphotyrosylated peptides based on 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 interest in small-molecule inhibitors. Some small molecule STAT3 inhibitors, identified using structure-based virtual ligand screening (SB-VLS); while having favorable drug-like properties, suffer from weak binding affinities, possibly due to the high flexibility of the target domain. We conducted molecular dynamic (MD) simulations of the SH2 domain in complex with CJ-887, and used an averaged structure from this MD trajectory as an "induced-active site" receptor model for SB-VLS of 110,000 compounds within the SPEC database. Screening was followed by re-docking and re-scoring of the top 30% of hits, selection for hit compounds that directly interact with pY + 0 binding pocket residues R609 and S613, and testing for STAT3 targeting in vitro, which identified two lead hits with good activity and favorable drug-like properties. Unlike most small-molecule STAT3 inhibitors previously identified, which contain negatively-charged moieties that mediate binding to the pY + 0 binding pocket, these compounds are uncharged and likely will serve as better candidates for anti-STAT3 drug development. IMPLICATIONS: SB-VLS, using an averaged structure from molecular dynamics (MD) simulations of STAT3 SH2 domain in a complex with CJ-887, a known peptidomimetic binder, identify two highly potent, neutral, low-molecular weight STAT3-inhibitors with favorable drug-like properties.

Combined Inhibition of STAT3 and DNA Repair in Palbociclib-Resistant ER-Positive Breast Cancer.

PURPOSE: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are currently used in combination with endocrine therapy to treat advanced hormone receptor-positive, HER2-negative breast cancer. Although this treatment doubles time to progression compared with endocrine therapy alone, about 25%-35% of patients do not respond, and almost all patients eventually acquire resistance. Discerning the mechanisms of resistance to CDK4/6 inhibition is crucial in devising alternative treatment strategies. EXPERIMENTAL DESIGN: Palbociclib-resistant cells (MCF-7 and T47D) were generated in a step-wise dose-escalading fashion. Whole-exome sequencing, genome-wide expression analysis, and proteomic analysis were performed in both resistant and parental (sensitive) cells. Pathway alteration was assessed mechanistically and pharmacologically. Biomarkers of altered pathways were examined in tumor samples from patients with palbociclib-treated breast cancer whose disease progressed while on treatment. RESULTS: Palbociclib-resistant cells are cross-resistant to other CDK4/6 inhibitors and are also resistant to endocrine therapy (estrogen receptor downregulation). IL6/STAT3 pathway is induced, whereas DNA repair and estrogen receptor pathways are downregulated in the resistant cells. Combined inhibition of STAT3 and PARP significantly increased cell death in the resistant cells. Matched tumor samples from patients with breast cancer who progressed on palbociclib were examined for deregulation of estrogen receptor, DNA repair, and IL6/STAT3 signaling, and results revealed that these pathways are all altered as compared with the pretreatment tumor samples. CONCLUSIONS: Palbociclib resistance induces endocrine resistance, estrogen receptor downregulation, and alteration of IL6/STAT3 and DNA damage response pathways in cell lines and patient samples. Targeting IL6/STAT3 activity and DNA repair deficiency using a specific STAT3 inhibitor combined with a PARP inhibitor could effectively treat acquired resistance to palbociclib.

Correction: Small-molecule inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.8368.].

Multifunctional Effects of a Small-Molecule STAT3 Inhibitor on NASH and Hepatocellular Carcinoma in Mice.

Purpose: The incidence of hepatocellular carcinoma is increasing in the United States, and liver cancer is the second leading cause of cancer-related mortality worldwide. Nonalcoholic steatohepatitis (NASH) is becoming an important risk for hepatocellular carcinoma, and most patients with hepatocellular carcinoma have underlying liver cirrhosis and compromised liver function, which limit treatment options. Thus, novel therapeutic strategies to prevent or treat hepatocellular carcinoma in the context of NASH and cirrhosis are urgently needed.Experimental Design: Constitutive activation of STAT3 is frequently detected in hepatocellular carcinoma tumors. STAT3 signaling plays a pivotal role in hepatocellular carcinoma survival, growth, angiogenesis, and metastasis. We identified C188-9, a novel small-molecule STAT3 inhibitor using computer-aided rational drug design. In this study, we evaluated the therapeutic potential of C188-9 for hepatocellular carcinoma treatment and prevention.Results: C188-9 showed antitumor activity in vitro in three hepatocellular carcinoma cell lines. In mice with hepatocyte-specific deletion of Pten (HepPten- mice), C188-9 treatment blocked hepatocellular carcinoma tumor growth, reduced tumor development, and reduced liver steatosis, inflammation, and bile ductular reactions, resulting in improvement of the pathological lesions of NASH. Remarkably, C188-9 also greatly reduced liver injury in these mice as measured by serum aspartate aminotransferase and alanine transaminase levels. Analysis of gene expression showed that C188-9 treatment of HepPten- mice resulted in inhibition of signaling pathways downstream of STAT3, STAT1, TREM-1, and Toll-like receptors. In contrast, C188-9 treatment increased liver specification and differentiation gene pathways.Conclusions: Our results suggest that C188-9 should be evaluated further for the treatment and/or prevention of hepatocellular carcinoma. Clin Cancer Res; 23(18); 5537-46. ©2017 AACR.

Small-molecule inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma.

While STAT3 has been validated as a target for treatment of many cancers, including head and neck squamous cell carcinoma (HNSCC), a STAT3 inhibitor is yet to enter the clinic. We used the scaffold of C188, a small-molecule STAT3 inhibitor previously identified by us, in a hit-to-lead program to identify C188-9. C188-9 binds to STAT3 with high affinity and represents a substantial improvement over C188 in its ability to inhibit STAT3 binding to its pY-peptide ligand, to inhibit cytokine-stimulated pSTAT3, to reduce constitutive pSTAT3 activity in multiple HNSCC cell lines, and to inhibit anchorage dependent and independent growth of these cells. In addition, treatment of nude mice bearing xenografts of UM-SCC-17B, a radioresistant HNSCC line, with C188-9, but not C188, prevented tumor xenograft growth. C188-9 treatment modulated many STAT3-regulated genes involved in oncogenesis and radioresistance, as well as radioresistance genes regulated by STAT1, due to its potent activity against STAT1, in addition to STAT3. C188-9 was well tolerated in mice, showed good oral bioavailability, and was concentrated in tumors. Thus, C188-9, either alone or in combination with radiotherapy, has potential for use in treating HNSCC tumors that demonstrate increased STAT3 and/or STAT1 activation.

Small-molecule targeting of signal transducer and activator of transcription (STAT) 3 to treat non-small cell lung cancer.

OBJECTIVE: Lung cancer is the leading cause of cancer death in both men and women. Non-small cell lung cancer (NSCLC) has an overall 5-year survival rate of 15%. While aberrant STAT3 activation has previously been observed in NSCLC, the scope of its contribution is uncertain and agents that target STAT3 for treatment are not available clinically. METHODS: We determined levels of activated STAT3 (STAT3 phosphorylated on Y705, pSTAT3) and the two major isoforms of STAT3 (α and ß) in protein extracts of 8 NSCLC cell lines, as well as the effects of targeting STAT3 in vitro and in vivo in NSCLC cells using short hairpin (sh) RNA and two novel small-molecule STAT3 inhibitors, C188-9 and piperlongumine (PL). RESULTS: Levels of pSTAT3, STAT3α, and STATß were increased in 7 of 8 NSCLC cell lines. Of note, levels of pSTAT3 were tightly correlated with levels of STAT3ß, but not STAT3α. Targeting of STAT3 in A549 cells using shRNA decreased tSTAT3 by 75%; this was accompanied by a 47-78% reduction in anchorage-dependent and anchorage-independent growth and a 28-45% reduction in mRNA levels for anti-apoptotic STAT3 gene targets. C188-9 and PL (@30 µM) each reduced pSTAT3 levels in all NSCLC cell lines tested by ≥50%, reduced anti-apoptotic protein mRNA levels by 25-60%, and reduced both anchorage-dependent and anchorage-independent growth of NSCLC cell lines with IC50 values ranging from 3.06 to 52.44 µM and 0.86 to 11.66 µM, respectively. Treatment of nude mice bearing A549 tumor xenografts with C188-9 or PL blocked tumor growth and reduced levels of pSTAT3 and mRNA encoding anti-apoptotic proteins. CONCLUSION: STAT3 is essential for growth of NSCLC cell lines and tumors and its targeting using C188-9 or PL may be a useful strategy for treatment.

Monoclonal Antibodies Specific for STAT3ß Reveal Its Contribution to Constitutive STAT3 Phosphorylation in Breast Cancer.

Since its discovery in mice and humans 19 years ago, the contribution of alternatively spliced Stat3, Stat3ß, to the overall functions of Stat3 has been controversial. Tyrosine-phosphorylated (p) Stat3ß homodimers are more stable, bind DNA more avidly, are less susceptible to dephosphorylation, and exhibit distinct intracellular dynamics, most notably markedly prolonged nuclear retention, compared to pStat3α homodimers. Overexpression of one or the other isoform in cell lines demonstrated that Stat3ß acted as a dominant-negative of Stat3α in transformation assays; however, studies with mouse strains deficient in one or the other isoform indicated distinct contributions of Stat3 isoforms to inflammation. Current immunological reagents cannot differentiate Stat3ß proteins derived from alternative splicing vs. proteolytic cleavage of Stat3α. We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to Stat3ß (CT7) and do not cross-react with Stat3α. Immunoblotting studies revealed that levels of Stat3ß protein, but not Stat3α, in breast cancer cell lines positively correlated with overall pStat3 levels, suggesting that Stat3ß may contribute to constitutive Stat3 activation in this tumor system. The ability to unambiguously discriminate splice alternative Stat3ß from proteolytic Stat3ß and Stat3α will provide new insights into the contribution of Stat3ß vs. Stat3α to oncogenesis, as well as other biological and pathological processes.

Synthetic and biological studies of phaeosphaerides.

The signal transducer and activator of transcription 3 (STAT3) has been validated as a suitable target for cancer therapy. Recent evidence by our group and others has shown that phaeosphaerides act as inhibitors of the STAT3 pathway. An efficient synthetic sequence to phaeosphaeride 1a has been previously disclosed. In this work, the first total synthesis of (±)-phaeosphaeride B (1d) and the unnatural phaeosphaeride 1b is reported. Additionally, the biological activities of 1a and 1b were investigated. (6S,7S,8S)-1a and (6R,7S,8S)-1b inhibited granulocyte colony-stimulating factor (GCSF)-stimulated phosphorylation of STAT1, STAT3, and STAT5 and IL-6-stimulated nuclear translocation of STAT3 alpha. In an SPR-based assay, (6S,7S,8S)-1a and (6R,7S,8S)-1b showed minimal ability to inhibit binding of STAT3 to its immobilized phosphotyrosylpeptide ligand (IC50 > 100 µM). Thus, (6S,7S,8S)-1a and (6R,7S,8S)-1b are likely upstream inhibitors of a kinase in the STAT signaling pathway and do not act through the inhibition of STAT3 dimerization by the blocking of the SH2 binding domain.

Modulation of STAT3 folding and function by TRiC/CCT chaperonin.

Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the ß-strand rich, DNA-binding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC.

Substance P is required for the pathogenesis of EMCV infection in mice.

Myocarditis is an important cause of heart failure in adolescents and young adults and is caused, most commonly, by viral infections. Viral myocarditis is characterized by cardiac inflammation and cardiomyocyte necrosis. The molecular pathogenesis of viral myocarditis is incomplete and specific therapies are not available. Proinflammatory cytokines such as IL-1beta, TNF-alpha and IL-6 have been implicated in the pathogenesis of myocarditis caused by encephalomyocarditis virus (EMCV) infection, a model of viral myocarditis in mice. Substance P (SP), a neuropeptide and pain transmitter, stimulates the production of proinflammatory cytokines and has been demonstrated by us and others to contribute to the pathogenesis of several viral, protozoan and helminth infections in mouse and man. Receptors for SP are expressed on the surface of cardiomyocytes, neurons, endothelial cells and immunocytes, including lymphocytes and macrophages. The current studies were performed to evaluate the role of SP in the pathogenesis of EMCV-induce myocarditis. SP levels were increased 61 fold in EMCV infected wild-type mice. EMCV infection resulted in 51% mortality at 14 days and a 1.56 fold increase in heart-to-body weight ratio that was accompanied by cardiac inflammation and necrosis and along with cardiomyocyte apoptosis and hypertrophy of surviving cells. In contrast, SP precursor knockout mice were completely protected from EMCV-mortality, cardiomegaly, cardiac inflammation and necrosis as well as cardiomyocyte apoptosis and hypertrophy. These results indicate that SP is essential for the pathogenesis of EMCV myocarditis and suggest that targeting this signaling pathway may be beneficial in viral myocarditis in humans.