DNA-binding Domain Of STAT3 Research Articles

A New Renieramycin T Right-Half Analog as a Small Molecule Degrader of STAT3.

Constitutive activation of STAT3 contributes to tumor development and metastasis, making it a promising target for cancer therapy. (1R,4R,5S)-10-hydroxy-9-methoxy-8,11-dimethyl-3-(naphthalen-2-ylmethyl)-1,2,3,4,5,6-hexahydro-1,5-epiminobenzo[d]azocine-4-carbonitrile, DH_31, a new derivative of the marine natural product Renieramycin T, showed potent activity against H292 and H460 cells, with IC50 values of 5.54 ± 1.04 µM and 2.9 ± 0.58 µM, respectively. Structure-activity relationship (SAR) analysis suggests that adding a naphthalene ring with methyl linkers to ring C and a hydroxyl group to ring E enhances the cytotoxic effect of DH_31. At 1-2.5 µM, DH_31 significantly inhibited EMT phenotypes such as migration, and sensitized cells to anoikis. Consistent with the upregulation of ZO1 and the downregulation of Snail, Slug, N-cadherin, and Vimentin at both mRNA and protein levels, in silico prediction identified STAT3 as a target, validated by protein analysis showing that DH_31 significantly decreases STAT3 levels through ubiquitin-proteasomal degradation. Immunofluorescence and Western blot analysis confirmed that DH_31 significantly decreased STAT3 and EMT markers. Additionally, molecular docking suggests a covalent interaction between the cyano group of DH_31 and Cys-468 in the DNA-binding domain of STAT3 (binding affinity = -7.630 kcal/mol), leading to destabilization thereafter. In conclusion, DH_31, a novel RT derivative, demonstrates potential as a STAT3-targeting drug that significantly contribute to understanding of the development of new targeted therapy.

Abstract 663: Irreversible covalent azetidine-based small molecule inhibitors of Stat3 activity block growth of human pancreatic tumors

Abstract The incidence and death rates for pancreatic adenocarcinoma in the US have been increasing by about 1% per year and the death rate has increased by approximately 0.2% yearly. The five-year survival rate has remained below 10% for many years. With surgery, radiation, and chemotherapy being the main treatment options to extend survival or for symptom relief, there is an urgent unmet need for effective new treatment options. Signal Transducer and Activator of Transcription (Stat) 3, one of the Stat family members, is aberrantly activated in human pancreatic and many other cancers. Aberrantly-active Stat3 promotes abnormal tumor-cell intrinsic and extrinsic mechanisms, including dysregulation of gene expression and mitochondria energy metabolism in the tumor cells, and suppression of immune cell functions in the tumor microenvironment. Stat3 is therefore an important target for therapeutic development. Herein we present two small molecules, H182 and H333, which are covalent inhibitors of Stat3 and that potently block Stat3 DNA-binding activity in vitro, with IC50 0.66-0.98 µM. H182 and its structural analog, H333 bind Stat3 via irreversible covalent interactions with key cysteine residues in the Stat3 DNA-binding domain to induce a time-dependent inhibition of Stat3 activity. Both compounds inhibit intracellular constitutive Stat3 tyrosine phosphorylation and induce loss of viable cells and apoptosis in vitro of human pancreatic Panc-1 and Mia-Paca2 cells. H333 is 13-fold more specific and H182 is 4-fold more specific against tumor cells over normal cells. Furthermore, MiaPaca-2 cells treated with H182 and analyzed by Seahorse showed dramatically decreased mitochondria respiration. Notably, xenograft models of MiaPaca2 treated with H182 or H333 via i.p. every other day for 27 days showed dramatic growth inhibition. Collectively, our results identify H182 and H333 as therapeutically-viable small molecules with unique irreversible mechanism of Stat3 inhibition which accounts for their strong antitumor effects against human pancreatic tumor xenografts. Citation Format: Yue Chen, Ning Zhai, Peibin Yue, Christine Brotherton-Pleiss, Wenzhen Fu, Kayo Nakamura, Weiliang Chen, Marcus Tius, Francisco Lopez-Tapia, James Turkson. Irreversible covalent azetidine-based small molecule inhibitors of Stat3 activity block growth of human pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 663.

Mechanism of XiJiaQi in the treatment of chronic heart failure: Integrated analysis by pharmacoinformatics, molecular dynamics simulation, and SPR validation

ObjectiveChronic heart failure (CHF) is a complicated clinical syndrome with a high mortality rate. XiJiaQi (XJQ) is a traditional Chinese medicine used in the clinical treatment of CHF, but its bioactive components and their modes of action remain unknown. This study was designed to unravel the molecular mechanism of XJQ in the treatment of CHF using multiple computer-assisted and experimental methods. MethodsPharmacoinformatics-based methods were used to explore the active components and targets of XJQ in the treatment of CHF. ADMETlab was then utilized to evaluate the pharmacokinetic and toxicological properties of core components. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were to explore the underlying mechanism of XJQ treatment. Molecular docking, surface plasmon resonance (SPR), and molecular dynamics (MD) were employed to evaluate the binding of active components to putative targets. ResultsAstragaloside IV, formononetin, kirenol, darutoside, periplocin and periplocymarin were identified as core XJQ-related components, and IL6 and STAT3 were identified as core XJQ targets. ADME/T results indicated that periplocin and periplocymarin may have potential toxicity. GO and KEGG pathway analyses revealed that XJQ mainly intervenes in inflammation, apoptosis, diabetes, and atherosclerosis-related biological pathways. Molecular docking and SPR revealed that formononetin had a high affinity with IL6 and STAT3. Furthermore, MD simulation confirmed that formononetin could firmly bind to the site 2 region of IL6 and the DNA binding domain of STAT3. ConclusionThis study provides a mechanistic rationale for the clinical application of XJQ. Modulation of STAT3 and IL-6 by XJQ can impact CHF, further guiding research efforts into the molecular underpinnings of CHF.

Progressive multifocal leukoencephalopathy (PML) treated with pembrolizumab in a patient with mosaic STAT3 gain-of-function mutation

Progressive multifocal leukoencephalopathy (PML) is a rare and often fatal disease of the central nervous system caused by JC virus. It occurs exclusively in immunocompromised hosts and is strongly associated with HIV infection and iatrogenic immunosuppression. Monogenic defects underlying the pathogenesis and reactivation of JC virus are not well understood, although some reports of primary immunodeficiencies (including four cases of STAT1 gain-of-function) have been previously described.A 77 year old male with history of prostate cancer (in remission; treated with radiation and hormonal therapy >10 years ago) presented with dysarthria, ataxia, left-sided weakness, diagnosed with CSF and biopsy-confirmed PML. He underwent an extensive workup upon which no other chronic infectious or oncologic processes were identified. He has never received any form of immunomodulation (including chemotherapy) in the past. Workup revealed low CD4 (347/mm3) and CD8 (282/mm3) T cell counts with normal proportions of naive vs. memory populations. Mitogen stimulation demonstrated poor CD8T cell proliferation. Targeted sequencing revealed a heterozygous mosaic mutation in the DNA-binding domain of STAT3 (c.1229 A > G p.H410R, 14% in 96 reads). This variant had been previously identified in two individuals with large granular lymphocyte (LGL) leukemia and validated as a gain-of-function via downstream signaling assays.Our patient demonstrated a favorable response to PD-1 blockade with pembrolizumab (2 mg/kg) and was discharged to a rehabilitation facility. His MRI brain showed no further progression of cerebellar white matter lesions following three doses of pembrolizumab.To our knowledge, this is the first case to implicate that STAT3 gain-of-function may confer a predisposition to JC virus reactivation and/or control in the pathogenesis of PML. Furthermore, we propose that this patient’s response to pembrolizumab may be partially attributed to STAT3-mediated regulation of the PD-1/PD-L1 axis. Further studies are currently underway to characterize this patient’s mosaic variant in flow cytometry-sorted subsets (including CD8T cells). Additional studies may include measurement of PD-1/PD-L1 expression and basal vs. induced STAT3 phosphorylation in sorted cell populations.

Abstract 3738: A novel mechanism of STAT3 activation by genotoxic chemotherapy

Abstract CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling induced by genotoxic drugs. We have previously found that apoptosis induced by genotoxic drugs involved elevated levels of CARP-1. Exposure to genotoxic drugs also induced CARP-1 interaction with NF-κB kinase subunit γ (aka, NEMO) to promote NF-κB activation and cell survival. To further elucidate chemotherapy-activated, CARP-1-dependent cell survival mechanisms, we UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed STAT3 interaction with CARP-1 (614-638) peptide. Our mutagenesis and co-IP-WB experiments revealed CARP-1 (550-650) directly interacts with a 40 amino acid peptide located in the STAT3 DNA binding domain. Overexpression of mutant STAT3 with in-frame-deletion of CARP-1/CCAR1-binding epitope (CE) peptide (Gst-STAT3 (ΔCE) mutant), but not Gst-STAT3 (WT), failed to translocate to nucleus in Adriamycin-treated cells. Disruption of STAT3-CARP-1 interaction or expression of STAT3 (ΔCE) mutant abolished Adriamycin-induced STAT3 Y705 and S727 phosphorylation. Collectively our data demonstrate that CARP-1 interaction with STAT3 regulates genotoxic chemotherapy-activated STAT3 and its nuclear translocation to promote cancer cell survival and growth. Citation Format: Jaganathan Venkatesh, Magesh Muthu, Nuwan C. Acharige, Mary K. Pflum, Arun K. Rishi. A novel mechanism of STAT3 activation by genotoxic chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3738.

Abstract 514: Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice

Abstract Unlike classical Signal transducer and activator of transcription (Stat) signaling, which is transient in non-transformed cells, the aberrant activation of the family member, Stat3, occurs in malignant transformation and is implicated in breast, ovarian and many other human cancers. Thus, Stat3 remains a validated and important target for the discovery of novel anticancer drugs. Despite this, the discovery and development of potent Stat3 small molecule inhibitors has proven to be a significant challenge, and no drug is available yet in the market. We have discovered novel Stat3 irreversible azetidine inhibitors with unprecedent submicromolar potency through systematic medicinal chemistry structure activity relationship (SAR). Mechanism of action of most potent azetidine inhibitors depend on whether they are salicylic acids or not, i.e., salicylic acids preferably bind irreversibly to Stat3 Cys426 site in the DNA-binding domain (DBD), and non-salicylic acids, e.g., current lead H182, bind to Stat3 Cys468 DBD site. As expected, the inhibition of Stat3 DNA-binding activity was time dependent, with IC50 in the range of 0.27-0.87 µM at one hour incubation with active Stat3. On the other hand, azetidine salicylic acids also bind to the SH2 domain, although reversibly and at much weaker affinity, as determined in fluorescent polarization (FP) assay, with IC50 of 10-16 µM at one hour incubation, while non-salicylic acids, e.g., lead H182, present no binding affinity up to 600 µM for the SH2 domain. Despite that H182 does not bind to SH2 domain, it still inhibits its phosphorylation. Though lead compound H182 presents significant mouse in vivo efficacy, mouse in vivo pharmacokinetics shows very low plasma AUC, which correlates with quite high mouse in vitro hepatocyte CLint of 138 µL/min/106 cells; however, human in vitro hepatocyte assay gives much better results and is in the middle stability range (CLint of 14.6 µL/min/106 cells). The in vivo PK in other species, e.g., rat, is being determined. H182 represents a plausible molecule for further development. Citation Format: Yue Chen, Ning Zhai, Yinsong Zhu, Peibin Yue, Nagendra Verma, Christine Brotherton-Pleiss, Wenzhen Fu, Kayo Nakamura, Weiliang Chen, Marcus Tius, Francisco J. Lopez-Tapia, James Turkson. Novel potent azetidine-based inhibitors bind irreversibly to Stat3 DNA-binding domain (DBD) and are efficacious against tumor growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 514.

A novel STAT3 splice-site variant in a kindred with autosomal dominant hyper IgE syndrome

Background: Dominant negative STAT3 loss-of-function is the most common genetic cause of hyper-IgE syndrome (HIES). Patients may present with a host of both immune and non-immune manifestations, including connective tissue abnormalities, recurrent infections, malignant predisposition, and biochemical evidence of elevated serum IgE or eosinophilia. Aim: To describe a novel splice-site variant in STAT3 resulting in HIES. Methods: Case report of two family members with HIES. Results: A proband and his son presented with neonatal-onset pustular rash, recurrent skin and sinopulmonary infections and elevated serum IgE and were diagnosed with AD-HIES. They were identified to harbor a novel splice-site variant in the DNA-binding domain (DBD) of STAT3: c.1110-3C>G, predicted to result in defective splicing in exon 12. Interestingly, a number of other patients with AD-HIES have mutations affecting the same splice-site, suggesting this may be a hot-spot for mutagenesis. Conclusion: Splice-site mutations in the DBD of STAT3 are increasingly identified as a cause of AD-HIES. Future work is required to delineate whether patients with splice-site mutations have unique clinical characteristics, supporting efforts for genotype-phenotype correlation in this disease. Statement of Novelty: We present a novel splice-site mutation in the DNA-binding domain of STAT3 leading to autosomal dominant hyper-IgE syndrome.

Evaluation of the putative lymphoma-associated point mutation D427H in the STAT3 transcription factor

BackgroundSignal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that promotes cell proliferation and immunomodulation in untransformed cells and maintains stemness of transformed cells, facilitating invasion and metastasis. Numerous point mutations in the STAT3 protein have been identified that drive malignancy in various tumor entities. The missense mutation D427H localized in the STAT3 DNA-binding domain has been previously reported in patients with NK/T cell lymphomas. To assess the biological activity of this missense mutation, we compared the STAT3-D427H mutant to wild-type (WT) protein as well as the known hyper-active mutant F174A.ResultsAlthough previously reported as an activating mutation, the STAT3-D427H mutant neither showed elevated cytokine-induced tyrosine phosphorylation nor altered nuclear accumulation, as compared to the WT protein. However, the D427H mutant displayed enhanced binding to STAT-specific DNA-binding sites but a reduced sequence specificity and dissociation rate from DNA, which was demonstrated by electrophoretic mobility shift assays. This observation is consistent with the phenotype of the homologous E421K mutation in the STAT1 protein, which also displayed enhanced binding to DNA but lacked a corresponding increase in transcriptional activity.ConclusionsBased on our data, it is unlikely that the D427H missense mutation in the STAT3 protein possesses an oncogenic potential beyond the WT molecule.

Discovery of novel STAT3 DNA binding domain inhibitors.

Background: STAT3 is a pro-oncogenic transcription factor. Pyrimethamine (PYM) is a STAT3 inhibitor that suppresses the proliferation of some cancer cells through downregulation of STAT3 target proteins. Methodology& Results: We have used structure-based tools to design novel PYM-based compounds. Intracellular target validation studies revealed that representative compounds 11b-d and 15a downregulate STAT3 downstream proteins and inhibit STAT3 DNA binding domain (DBD). Relative to PYM, a cohort of these compounds are >100-fold more cytotoxic to cancer cells with constitutively active (high pSTAT3) and basal (low pSTAT3) STAT3 signaling, suggesting that STAT3 DBD inhibition is deleterious to the proliferation of cancer cells with low and high pSTAT3 levels. Conclusion: These are promising leads for further preclinical evaluation as therapeutic agents for STAT3-dependent cancers.

Synthesis, In Silico and In Vivo Evaluation of Novel 1, 3, 4-Thiadiazole Analogues as Novel Anticancer Agents

Background: 1,3,4-thiadiazole is a lead molécule which is versatile for a wide variety of biological activities and in continuation of our interest in establishing some novel heterocyclic compounds for antitumor activity. Objective: The objective of the study was to synthesize series of 5-(1,3-benzodioxol-5-yl)-1,3,4- thiadiazol-2-amine derivative and evaluated for their possible in vitro and in vivo anticancer activity. Methods: The synthesis of 2-aminonaphthoxy-1,3,4-thiadiazole and 5-(1,3-benzodioxol-5-yl)-1,3,4- thiadiazol-2-amine as intermediates were carried out by cyclization method. A mixture of thiosemicarbazide and naphthoxyacetic acid/piperonylic acid and phosphoryl chloride were subjected to cyclization with phosphorous oxychloride to obtain compound 3. Further compounds 1 and 3 were reacted with different aromatic aldehydes in methanol to form compounds 2a-e and 4a-e. The compounds 2a-e and 4a-e were characterized for the melting points, IR, Proton NMR and Mass spectra. The compounds were further evaluated for their anticancer activity. The docking study was performed using Discovery studio 4.1 (Accelrys) software against DNA-binding domain of STAT3. The compounds were analyzed for the ligand-protein binding interaction(s) by molecular docking into the active site of STAT3β using the CDOCKER protocol of Discovery studio (v4.1). Results: The title compounds were screened for in vitro anticancer on human breastadenocarcinoma cells (MCF-7 and Vero). Compounds 4c, 4d and 2d against MCF 7 and 4d against Vero cell lines were found to be the most active dérivatives with IC50 values of 1.03, 2.81 and 3.45 µg/ml against MCF 7 and 31.81 µg/ml against Vero cell lines, respectively. Conclusion: From the in vivo anticancer studies, it was concluded that the synthesized compounds 4c and 4d displayed anticancer activity comparable to the standard drug, while the rest of the compounds demonstrated mild potency for anticancer studies.

Silibinin is a direct inhibitor of STAT3

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

Cytostatic hydroxycoumarin OT52 induces ER/Golgi stress and STAT3 inhibition triggering non-canonical cell death and synergy with BH3 mimetics in lung cancer

Coumarins are natural compounds with antioxidant, anti-inflammatory and anti-cancer potential known to modulate inflammatory pathways. Here, non-toxic biscoumarin OT52 strongly inhibited proliferation of non-small cell lung cancer cells with KRAS mutations, inhibited stem-like characteristics by reducing aldehyde dehydrogenase expression and abrogated spheroid formation capacity. This cytostatic effect was characterized by cell cycle arrest and onset of senescence concomitant with endoplasmic reticulum and Golgi stress, leading to metabolic alterations. Mechanistically, this cellular response was associated with the novel capacity of biscoumarin OT52 to inhibit STAT3 transactivation and expression of its target genes linked to proliferation. These results were validated by computational docking of OT52 to the STAT3 DNA-binding domain. Combination treatments of OT52 with subtoxic concentrations of Bcl-xL and Mcl-1-targeting BH3 protein inhibitors triggered synergistic immunogenic cell death validated in colony formation assays as well as in vivo by zebrafish xenografts.

Inhibitory Effect of Carnosol on Phthalic Anhydride-Induced Atopic Dermatitis via Inhibition of STAT3.

Carnosol is a phenolic antioxidant present in rosemary (Rosmarinus officinalis). It is known for anti-inflammatory effects, analgesic activity and anti-cancer effects. However, no study has been dedicated yet to its effect on atopic dermatitis (AD). Here, we show that carnosol effectively inhibited LPS-induced nitric oxide (NO) generation and expression of inflammatory marker proteins (iNOS and COX-2) in RAW 264.7 cells. In addition, carnosol effectively inhibits the phosphorylation of STAT3 and DNA binding activity in RAW 264.7 cells. Pull down assay and docking model analysis showed that carnosol directly binds to the DNA binding domain (DBD) of STAT3. We next examined the anti-atopic activity of carnosol (0.05 μg/cm2) using 5% Phthalic anhydride (PA)-induced AD model in HR1 mice. Carnosol treatment significantly reduced 5% PA-induced AD like skin inflammation in skin tissues compared with control mice. Moreover, carnosol treatment inhibits the expression of iNOS and COX-2 in skin tissue. In addition, the levels of TNF-α, IL-1β, and Immunoglobulin-E in blood serum was significantly decreased in carnosol treated mice compared with those of 5% PA treated group. Furthermore, the activation of STAT3 in skin tissue was decreased in carnosol treated mice compared with control mice. In conclusion, these findings suggest that carnosol exhibited a potential anti-AD activity by inhibiting pro-inflammatory mediators through suppression of STAT3 activation via direct binding to DBD of STAT3.

The STAT3 HIES mutation is a gain-of-function mutation that activates genes via AGG-element carrying promoters.

Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, ‘TTC(N3)GAA’)-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320–494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence ‘AGG(N3)AGG’. Surprisingly, the helical N-terminal region (1–355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity.

Abstract 606: Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia

Abstract Introduction T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of CD8+ cytotoxic T-cells. We recently discovered that 40% of T-LGL leukemia patients have somatic mutations in the SH2-domain of the STAT3 gene (Koskela et al. NEJM 2012). As aberrant STAT3 activation can be observed in all patients with LGL-leukemia, we now aimed to discover whether patients without mutations in the STAT3 hotspot area harbor mutations in the other parts of the STAT3 gene. Methods Targeted STAT3 sequencing covering all 23 coding exons was done with in-house developed deep amplicon sequencing panel using the Illumina Miseq platform. The data was analyzed with a bioinformatics pipeline, which is based on calling of variants with specific counts/frequencies and filtering out false positives using the estimated error rate and quality data of amplicon reads. All samples with a frequency ratio ≥0.9 were considered to be true mutations after filtering of SNPs and low coverage variants. 111 LGL-leukemia patients with no known STAT3-mutations in the SH2 domain were analyzed. To explore the functional effects of mutations, expression constructs were generated with the identified variants and wild-type STAT3. The variants were expressed in HEK-293 cells carrying a STAT3-responsive SIE-reporter driven luciferase expression sequence to establish increased basal and IL6-stimulated STAT3 activity. Results With targeted amplicon sequencing, 3 patients were discovered to have STAT3 missense mutations in the DNA-binding domain. Two patients presented with the same H410R mutation with a variant allele frequency (VAF) of 49% and 8.8% respectively while another patient had a S381Y mutation (VAF 7%). The mutation H410R occurs in the DNA-binding domain in a highly conserved position, and results in conversion of histidine to arginine, which would predict for a slight increase in hydrophilicity. In addition to STAT3 DNA-binding domain mutations, one T-LGL patient had a novel F174S mutation in the coiled-coil domain of STAT3 (VAF 43%). The coiled-coil domain of STAT3 has previously been shown to be essential in SH2-domain mediated receptor binding and subsequent activation. Luciferase measurements of SIE-reporter HEK-293 cells transfected with constructs expressing either wild-type, variant F174S, H410R or Y640F STAT3 (the most common activating mutation in LGL leukemia) revealed the F174S and H410R variants to be as activating as the Y640F mutation in both unstimulated and IL-6 stimulated conditions. Conclusions T-LGL leukemia patients without STAT3 SH2-domain mutations harbor novel activating mutations in the DNA-binding and coiled-coil domain of STAT3. The frequency of mutations was 3.6% (4 of 111 patients). These findings further highlight the importance of screening the whole STAT3 gene in the diagnostic workup of LGL-leukemia and the central role of STAT3 in the pathogenesis of the disease. Citation Format: Emma I. Andersson, Hanna Rajala, Heikki Kuusanmäki, Arjan van Adrichem, Samuli Eldfors, Sonja Lagström, Thomas Olson, Michael Clemente, Pekka Ellonen, Caroline Heckman, Thomas P. Loughran, Jaroslaw P. Maciejewski, Satu Mustjoki. Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2015-606

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

Author SummaryStat3 is a multidomain transcription factor that contributes to many cellular functions by transmitting signals for over 40 peptide hormones from the cell surface to the nucleus. Understanding how multidomain proteins achieve their fully folded and functional state is of substantial biological interest. As Stat3 signaling is up-regulated in many pathological conditions, including cancer and inflammatory diseases, insight into what controls its folding may be useful for the identification of vulnerabilities that can be therapeutically exploited. We demonstrate that the major protein-folding machine or chaperonin within eukaryotic cells, TRiC/CCT, is required for Stat3 to fold during its synthesis and for Stat3 to be fully functional within the cell. We also find that TRiC can refold chemically denatured Stat3 and provide evidence that the CCT3 subunit of TRiC binds to the DNA-binding domain of Stat3. We also show that Stat3 activity is decreased by down-modulating levels of TRiC and can be increased by increasing Stat3's interaction with TRiC. TRiC therefore regulates both Stat3 protein levels and its function, making Stat3 modulation by manipulation of its interaction with TRiC a potential approach for the treatment of cancer and inflammatory diseases.

HO-3867, a Safe STAT3 Inhibitor, Is Selectively Cytotoxic to Ovarian Cancer

STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (-NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867-mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated.

Arginine residues within the DNA binding domain of STAT3 promote intracellular shuttling and phosphorylation of STAT3

Acetylation-dependent inactivation of STAT1 can be mimicked by the exchange of its lysine residues K410 and K413 to glutamine residues. STAT3 harbors non-acetylatable arginine moieties at the corresponding sites R414 and R417. It is unclear whether the mutation of these sites to glutamine residues antagonizes STAT3 activation. Here, we show that an arginine–glutamine-exchange at the STAT3 moieties R414 and R417 (R414Q and R417Q) reduces cytokine-dependent tyrosine phosphorylation of STAT3. This inhibitory effect can be partially rescued by phosphatase inhibition. In addition, the R414Q and R417Q mutations enhance the nuclear accumulation of unphosphorylated STAT3. STAT3 R414Q and STAT3 R417Q show a reduced response to cytokine stimulation emanating from the plasma membrane. Moreover, these STAT3 mutants have no direct inhibitory effect on the cytokine-induced activation of STAT1/STAT3-mediated gene expression. Since the mutations R414Q and R417Q reside within the STAT3 DNA binding domain (DBD), the STAT3 R414Q and R417Q mutants also lack intrinsic activity as transcription factors. Furthermore, in contrast to wild-type STAT3 they cannot compensate for a loss of STAT1 and they cannot promote STAT1/STAT3-dependent transcriptional activation. We further analyzed a STAT3 arginine–lysine-exchange mutant (R414K/R417K). This molecule mimics corresponding lysine residues found within the DBD of STAT1. Compared to wild-type STAT3, the STAT3 R414K/R417K mutant shows attenuated tyrosine phosphorylation and it is a less active transcription factor. In addition, STAT3 R414K/R417K is not activated by deacetylase inhibition. On the other hand, C-terminal acetylation of STAT3 is intact in STAT3 R414K/R417K. Our results suggest that the exchange of amino acid residues within the DBDs of STAT1/STAT3 affects their phosphorylation as well as their intracellular shuttling.

Mitochondrial Localized Stat3 Promotes Breast Cancer Growth via Phosphorylation of Serine 727

Signal transducer and activator of transcription 3 (Stat3) is a key mediator in the development of many cancers. For 20 years, it has been assumed that Stat3 mediates its biological activities as a nuclear localized transcription factor activated by many cytokines. However, recent studies from this laboratory and others indicate that Stat3 has an independent function in the mitochondria (mitoStat3) where it controls the activity of the electron transport chain (ETC) and mediates Ras-induced transformation of mouse embryo fibroblasts. The actions of mitoStat3 in controlling respiration and Ras transformation are mediated by the phosphorylation state of serine 727. To address the role of mitoStat3 in the pathogenesis of cells that are transformed, we used 4T1 breast cancer cells, which form tumors that metastasize in immunocompetent mice. Substitution of Ser-727 for an alanine or aspartate in Stat3 that has a mitochondrial localization sequence, MLS-Stat3, has profound effects on tumor growth, complex I activity of the ETC, and accumulation of reactive oxygen species (ROS). Cells expressing MLS-Stat3(S727A) display slower tumor growth, decreased complex I activity of the ETC, and increased ROS accumulation under hypoxia compared with cells expressing MLS-Stat3. In contrast, cells expressing MLS-Stat3(S727D) show enhanced tumor growth and complex I activity and decreased production of ROS. These results highlight the importance of serine 727 of mitoStat3 in breast cancer and suggest a novel role for mitoStat3 in regulation of ROS concentrations through its action on the ETC.

Abstract 5619: Drugging the “undrugable” DNA-binding domain of STAT3 for inhibition of cancer cell migration and invasion

Abstract Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors, and its activation is associated with high histological grade and advanced cancer stage. STAT3 has been shown to play important roles in multiple aspects of cancer aggressiveness including migration, invasion, survival, self-renewal, angiogenesis, and tumor-cell immune evasion by regulating the expression of multiple downstream target genes. Thus, inhibiting STAT3 promises an attracting strategy for treatment of advanced tumors with metastatic potential. Recently, using in-silico screening of a virtual compound database by docking onto the DNA-binding site of human STAT3, our laboratory identified specific STAT3 inhibitors that can disrupt DNA binding activity of STAT3 and inhibit STAT3-dependent cancer cell proliferation. In this study, we further characterized these STAT3 inhibitors and demonstrated that these inhibitors suppressed migration and invasion of both breast and lung cancer cells by inhibiting STAT3-dependent expression of metalloproteinase-2, -9 and vascular endothelial growth factor which are known to promote and enhance tumor metastasis. Taken together, we conclude that it is possible to inhibit STAT3 by targeting its DNA binding domain for discovery of anticancer therapeutics and for treatment of metastatic cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5619. doi:1538-7445.AM2012-5619