Small Molecule STAT3 Inhibitor Research Articles

Impacts of small-molecule STAT3 inhibitor SC-43 on toxicity, global proteomics and metabolomics of HepG2 cells

ObjectiveIn this study, we aimed to investigate the cytotoxicity and potential mechanisms of SC-43 by analyzing the global proteomics and metabolomics of HepG2 cells exposed to SC-43. MethodsThe effect of SC-43 on cell viability was evaluated through CCK-8 assay. Proteomics and metabolomics studies were performed on HepG2 cells exposed to SC-43, and the functions of differentially expressed proteins and metabolites were categorized. Drug affinity responsive target stability (DARTS) was utilized to identify the potential binding proteins of SC-43 in HepG2 cells. Finally, based on the KEGG pathway database, the co-regulatory mechanism of SC-43 on HepG2 cells was elucidated by conducting a joint pathway analysis on the differentially expressed proteins and metabolites using the MetaboAnalyst 5.0 platform. ResultsLiver cell viability is significantly impaired by continuous exposure to high concentrations of SC-43. Forty-eight dysregulated proteins (27 upregulated, 21 downregulated) were identified by proteomics analysis, and 184 dysregulated metabolites (65 upregulated, 119 downregulated) were determined by metabolomics in HepG2 cells exposed to SC-43 exposure compared with the control. A joint pathway analysis of proteomics and metabolomics data using the MetaboAnalyst 5.0 platform supported the close correlation between SC-43 toxicity toward HepG2 and the disturbances in pyrimidine metabolism, ferroptosis, mismatch repair, and ABC transporters. Specifically, SC-43 significantly affected the expression of several proteins and metabolites correlated with the above-mentioned functional pathways, such as uridine 5'-monophosphate, uridine, 3'-CMP, glutathione, γ-Glutamylcysteine, TF, MSH2, RPA1, RFC3, TAP1, and glycerol. The differential proteins suggested by the joint analysis were further selected for ELISA validation. The data showed that the RPA1 and TAP1 protein levels significantly increased in HepG2 cells exposed to SC-43 compared to the control group. The results of ELISA and joint analysis were basically in agreement. Notably, DARTS and biochemical analysis indicated that SART3 might be a potential target for SC-43 toxicity in HepG2 cells. ConclusionIn summary, prolonged exposure of liver cells to high concentrations of SC-43 can result in significant damage. Based on a multi-omics analysis, we identified proteins and metabolites associated with SC-43-induced hepatocellular injury and clarified the underlying mechanism, providing new insights into the toxic mechanism of SC-43.

WP1066, a small molecule inhibitor of STAT3, chemosensitizes paclitaxel-resistant ovarian cancer cells to paclitaxel by simultaneously inhibiting the activity of STAT3 and the interaction of STAT3 with Stathmin

Paclitaxel is widely used to treat cancer, however, drug resistance limits its clinical utility. STAT3 is constitutively activated in some cancers, and contributes to chemotherapy resistance. Currently, several STAT3 inhibitors including WP1066 are used in cancer clinical trials. However, whether WP1066 reverses paclitaxel resistance and the mechanismremains unknown. Here, we report that in contrast to paclitaxel-sensitive parental cells, the expressions of several pro-survival BCL2 family members such as BCL-2, BCL-XL and MCL-1 are higher in paclitaxel-resistant ovarian cancer cells. Meanwhile, STAT3 is constitutively activated while stathmin loses its activity in paclitaxel-resistant cells. Importantly, WP1066 amplifies the inhibition of cell proliferation, colony-forming ability and apoptosis of ovarian cancer cells induced by paclitaxel. Mechanistically, WP1066, on the one hand, interferes the STAT3/Stathmin interaction, causing unleash of STAT3/Stathmin from microtubule, thus destroying microtubule stability. This process results in reduction of Ac-α-tubulin, further causing MCL-1 reduction. On the other hand, WP1066 inhibits phosphorylation of STAT3 by JAK2, and blocks its nuclear translocation, therefore repressing the transcription of pro-survival targets such as BCL-2, BCL-XL and MCL-1. Finally, the two pathways jointly promote cell death. Our findings reveal a new mechanism wherein WP1066 reverses paclitaxel-resistance of ovarian cancer cells by dually inhibiting STAT3 activity and STAT3/Stathmin interaction, which may layfoundation for WP1066 combined with paclitaxel in treating paclitaxel-resistant ovarian cancer.

Relevance and mechanism of STAT3/miR-221-3p/Fascin-1 axis in EGFR TKI resistance of triple-negative breast cancer.

The epidermal growth factor receptor 1 (EGFR) plays a crucial role in the progression of various malignant tumors and is considered a potential target for treating triple-negative breast cancer (TNBC). However, the effectiveness of representative tyrosine kinase inhibitors (TKIs) used in EGFR-targeted therapy is limited in TNBC patients. In our study, we observed that the TNBC cell lines MDA-MB-231 and MDA-MB-468 exhibited resistance to Gefitinib. Treatment with Gefitinib caused an upregulation of Fascin-1 (FSCN1) protein expression and a downregulation of miR-221-3p in these cell lines. However, sensitivity to Gefitinib was significantly improved in both cell lines with either inhibition of FSCN1 expression or overexpression of miR-221-3p. Our luciferase reporter assay confirmed that FSCN1 is a target of miR-221-3p. Moreover, Gefitinib treatment resulted in an upregulation of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in MDA-MB-231 cells. Using Stattic, a small-molecule inhibitor of STAT3, we observed a significant enhancement in the inhibitory effect of Gefitinib on the growth, migration, and invasion of MDA-MB-231 cells. Additionally, Stattic treatment upregulated miR-221-3p expression and downregulated FSCN1 mRNA and protein expression. A strong positive correlation was noted between the expression of STAT3 and FSCN1 in breast cancer tissues. Furthermore, patients with high expression levels of both STAT3 and FSCN1 had a worse prognosis. Our findings suggest that elevated FSCN1 expression is linked to primary resistance to EGFR TKIs in TNBC. Moreover, we propose that STAT3 regulates the expression of miR-221-3p/FSCN1 and therefore modulates resistance to EGFR TKI therapy in TNBC. Combining EGFR TKI therapy with inhibition of FSCN1 or STAT3 may offer a promising new therapeutic option for TNBC.

Increased STAT3 Activation in Periodontitis Drives Inflammatory Bone Loss.

Periodontitis is one of the most prevalent human inflammatory diseases. It is characterized by periodontal tissue destruction, progressively driven by the host response. In this regard, cytokines associated with tissue destruction, such as interleukin (IL)-6 and IL-23, use a common signaling pathway mediated by STAT3. This transcription factor is also needed for IL-17A production, a key mediator in periodontitis pathogenesis. Although several studies have reported increased activation of STAT3 in experimental periodontitis, a detailed characterization of STAT3 activation in human gingival tissues and its involvement in alveolar bone loss has yet to be explored. Using a cross-sectional study design, we detected increased proportions of pSTAT3-positive cells during periodontitis compared with health, particularly in epithelial cells and T cells. Other cell types of hematopoietic and nonhematopoietic origin also display STAT3 activation in gingival tissues. We detected increased STAT3 phosphorylation and expression of STAT3-related genes during experimental periodontitis. Next, we evaluated the role of STAT3 in alveolar bone destruction using a mouse model of STAT3 loss of function (mut-Stat3 mice). Compared with controls, mut-Stat3 mice had reduced alveolar bone loss following ligature-induced periodontitis. We also evaluated pharmacologic inhibition of STAT3 in ligature-induced periodontitis. Like mut-Stat3 mice, mice treated with STAT3 small-molecule inhibitor had reduced bone loss compared with controls. Our results demonstrate that STAT3 activation is increased in epithelial and T cells during periodontitis and indicate a pathogenic role of STAT3 in inflammatory alveolar bone loss.

STA-21, a small molecule STAT3 inhibitor, ameliorates experimental autoimmune encephalomyelitis by altering Th-17/Treg balance.

Numerous studies have demonstrated the role of T helper (Th) 17 and T regulatory (reg) cells and pro-inflammatory and anti-inflammatory cytokines related to these cells in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). STAT3 is one of the downstream signaling proteins of IL-23, IL-6, and IL-21 that are required for Th17 cells differentiation. STA-21 is a STAT3 inhibitor that functions by inhibiting STAT3 dimerization and binding to DNA impairing the expression of STAT3 target genes including, RORγt, IL-21 and IL-23R that are also required for Th17 cell differentiation. In this study, we evaluated the effect of STA-21 on EAE Model and investigated how this small molecule can change Th17/Treg balance leading to amelioration of disease. After EAE induction and treatment with STA-21, its effects were assessed. Major assays were H&E and LFB staining, Flow cytometric analysis, Reverse transcription-PCR (RT-PCR), and ELISA. STA-21 ameliorated the EAE severity and decreased the EAE inflammation and demyelination. It also decreased STAT3 phosphorylation, the proportion of Th17 cells and the protein level of IL-17. In contrast, the balance of Tregs and the level of anti-inflammatory cytokine, IL-10 increased in STA-21-treated mice. Moreover, STA-21 significantly decreased the expression of Th17 related transcription factors, RORɣt and IL-23R while FOXP3 expression associated with Treg differentiation was increased. This study showed that STA-21 has therapeutic effects in EAE by reducing inflammation and shifting inflammatory immune responses to anti-inflammatory and can be used as a suitable treatment strategy for the treatment of EAE. The effectiveness of inhibiting or strengthening the functional cells of the immune system by these small molecules in terms of easy to access, simple construction and inexpensive expansion make them as a suitable tool for the treatment of inflammatory and autoimmune diseases.

Genetic and Small-Molecule Modulation of Stat3 in a Mouse Model of Crohn's Disease.

Crohn's disease (CD), is an inflammatory bowel disease that can affect any part of the gastro-intestinal tract (GI) and is associated with an increased risk of gastro-intestinal cancer. In the current study, we determined the role of genetic and small-molecule modulation of STAT3 in a mouse model of CD. STAT3 has 2 isoforms (α, β) which are expressed in most cells in a 4:1 ratio (α: β). STAT3α has pro-inflammatory and anti-apoptotic functions, while STAT3β has contrasting roles. We used an animal model of CD consisting of intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid and examined the severity of CD in transgenic-mice that express only STAT3α (∆β/∆β), as well as in wild-type (WT) mice administered TTI-101 (formerly C188-9), a small molecule STAT3 inhibitor. We determined that clinical manifestations of CD, such as mortality, rectal-bleeding, colonic bleeding, diarrhea, and colon shortening, were exacerbated in ∆β/∆β transgenic versus cage-control WT mice, while they were markedly decreased by TTI-101 treatment of WT mice. TTI-101 treatment also increased apoptosis of pathogenic CD4+ T cells and reduced colon levels of IL-17-positive cells. Our results indicate that STAT3 contributes to CD and that targeting of STAT3 with TTI-101 may be a useful approach to treating CD.

Aberrant function of pathogenic STAT3 mutant proteins is linked to altered stability of monomers and homodimers

AbstractSTAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide–binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

LLL12B, a Novel Small-Molecule STAT3 Inhibitor, Induces Apoptosis and Suppresses Cell Migration and Tumor Growth in Triple-Negative Breast Cancer Cells.

Persistent STAT3 signaling plays a pivotal role in human tumor malignancy, including triple-negative breast cancer (TNBC). There are few treatment options currently available for TNBC; thus, given its importance to cancer, STAT3 is a potential cancer therapeutic target and is the focus of drug discovery efforts. In this study, we tested a novel orally bioavailable small-molecule STAT3 inhibitor, LLL12B, in human MDA-MB-231, SUM159, and murine 4T1 TNBC cell lines. TNBC cells frequently expressed persistent STAT3 phosphorylation and their cell viability was sensitive to STAT3 knockdown by siRNA. LLL12B selectively inhibited the IL-6-mediated induction of STAT3 phosphorylation, but had little effect on the IFN-γ-mediated induction of STAT1 phosphorylation nor the EGF-mediated induction of ERK phosphorylation. In addition, targeting STAT3 with LLL12B induced apoptosis, reduced colony formation ability, and inhibited cell migration in TNBC cells. Furthermore, LLL12B suppressed the tumor growth of the MDA-MB-231 TNBC cells in a mammary fat pad mouse tumor model in vivo. Together, our findings support the concept that targeting persistent STAT3 signaling using the novel small-molecule LLL12B is a potential approach for TNBC therapy.

STAT3 is a potential therapeutic target in cervical cancer (257)

<b>Objectives:</b> STAT3 is known to be hyperactivated in most human cancers and is associated with poor prognoses. The STAT3 activation pathway within human papillomavirus (HPV)-positive cervical cancer cells is known to be essential for cervical cancer cell proliferation and survival. However, whether STAT3 activity is elevated in cervical cancer patient tumors remains to be determined. We sought to assess whether activated STAT3 (p-STAT3) was more highly expressed in cervical squamous cell carcinoma tissue microarrays compared to benign adjacent cervix tissue. We also assessed the ability of a small molecule inhibitor of STAT3 that has been used in the clinic to attenuate the STAT3 activation pathway and induce tumor cell death of HPV-associated human squamous cell carcinoma cells <i>in vitro</i>. <b>Methods:</b> Tumor microarrays from 12 patients with cervical squamous cell carcinoma with adjacent, benign cervix tissue were first probed with an anti-p-STAT3 antibody and detected with a fluorophore-conjugated secondary antibody for p-STAT3. Confocal imaging of immunofluorescence was performed, and fluorescence quantification was performed by ZEN 2.3 lite software and plotted in GraphPad Prism 8 software. The CaSki HPV16 infected cervical squamous cell carcinoma cell line was used for <i>in vitro</i> experiments. CaSki cells were treated with BBI608 (a small molecule inhibitor of STAT3) and assessed for changes in the STAT3 activation axis using Western Blot. Cell viability was analyzed using CellTiter-Glo® Luminescent Cell Viability Assay. <b>Results:</b> Quantification of p-STAT3 immunofluorescence from confocal imaging showed significantly higher expression of p-STAT3 in cervical squamous cell carcinoma tissue microarrays compared to adjacent, benign cervix tissue with mean expression levels of 37.7 relative units (RU) vs 29.1 RU, respectively (p=0.006). On Western Blot, BBI608 (3uM and 4uM) was found to attenuate the STAT3 activation axis in a dose-dependent fashion, including a significant decrease in p-JAK1 and p-STAT3 protein expressions. Furthermore, the protein expression of <i>HIF1A</i>, a known downstream STAT3 target gene and associated with poor survival and treatment resistance in cervical cancer, was also decreased by BBI608 (3uM and 4uM) in a similar fashion. On analysis of cell viability, BBI608 was found capable of inducing cell death in CaSki cells with an IC50 of 2.6uM after a 48-hour incubation period. <b>Conclusions:</b> The STAT3 activation pathway is a logical and promising target for therapeutic intervention in cervical cancer. On review of HPV-associated cervical cancer tumor arrays, we found a significantly higher expression of pSTAT3 in cervical tumor cells and tumor-infiltrating lymphocytes compared to adjacent, benign tissue. Furthermore, we found that BBI608 was able to attenuate the STAT3 activation axis and induce cell death in the CaSki cervical squamous cell carcinoma cell line.Fig. 1

Abstract 2678: LLL12B, a novel small molecule STAT3 inhibitor induces apoptosis and suppresses cell migration and tumor growth in triple-negative breast cancer cells

Abstract Persistent STAT3 signaling has been recognized as a cancer therapeutic target in some cancer types including triple-negative breast cancer, and the development of effective small molecule STAT3 inhibitors for potential cancer therapy is desirable. In this study, we tested a novel oral bioavailable small molecule STAT3 inhibitor, LLL12B in MDA-MB-231, SUM159, and 4T1 triple-negative breast cancer cells. Triple-negative breast cancer cells frequently express persistent STAT3 phosphorylation and its cell viability is sensitive to STAT3 knockdown by siRNA. LLL12B selectively inhibited IL-6 mediated induction of STAT3 phosphorylation but had little effect on IFN-γ mediated induction of STAT1 phosphorylation or EGF mediated induction of ERK phosphorylation. LLL12B inhibited STAT3 phosphorylation, induced cell apoptosis, and reduced cell colony formation in triple-negative breast cancer cells. In addition, LLL12B inhibited cell migration of triple-negative breast cancer cells. Furthermore, LLL12B suppressed tumor growth of MDA-MB-231 triple-negative breast cancer cells in a mammary fat pad syngeneic mouse model. Together, our findings suggest that targeting persistent STAT3 signaling pathway by novel small molecule LLL12B may be a potential approach for triple-negative breast cancer therapy. Citation Format: Li Pan, Xiang Chen, Feyruz V. Rassool, Chenglong Li, Jiayuh Lin. LLL12B, a novel small molecule STAT3 inhibitor induces apoptosis and suppresses cell migration and tumor growth in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2678.

Abstract 1774: Circulating IL-6, an early biomarker in HR-positive, HER2-negative metastatic breast cancer patients progressing on CDK4/6 inhibitors

Abstract Background: CDK4/6 inhibitors (CDK4/6i, e.g. palbociclib) in combination with endocrine therapy (ET) have proven successful in delaying progression in hormone receptor-positive (HR+), HER2-negative (HER2-) metastatic breast cancer patients; however, 60-70% will progress within 1-2 years of treatment. Thus, understanding the mechanisms of resistance and identifying novel treatment strategies are needed to improve survival. Our previously published data shows that palbociclib resistance is marked by significant upregulation of the IL6-STAT3 signaling pathway in HR+, HER2- breast cancer cells and treatment with TTI-101, a small-molecule STAT3 inhibitor completing Phase I studies in solid tumor patients, significantly decreased cell viability. Additionally, matched biopsies from advanced HR+, HER2- breast cancer patients who progressed on palbociclib plus ET had an upregulation in activated STAT3 (STAT3 phosphorylated on Y705, pY-STAT3) as compared to their pre-treatment biopsy. Collectively, these data suggest that targeting the IL-6/STAT3 pathway is a viable therapeutic strategy. Our ongoing studies are directed at delineating the role of IL-6 and pY-STAT3 as predictive biomarkers to provide preclinical rationale for targeting STAT3 in patients who develop CDK4/6i resistance. Methods: We downregulated IL-6 using shRNA in MCF7 and T47D palbociclib sensitive and resistant cells. We tested the efficacy of TTI-101 in patient-derived xenograft (PDX) models from patients with metastatic HR+, HER2- breast cancer who progressed on treatment with palbociclib + ET. We also collected plasma samples from patients with metastatic HR+, HER2- breast cancer just prior to CDK4/6i treatment and at disease progression. Results: Downregulation of IL-6 in palbociclib resistant cells reduced pY-STAT3 levels and re-sensitized cells to ET and palbociclib. Induction of IL-6 is an early event by treating sensitive cells with 1uM palbociclib for 21 days during which the cells adapted and eventually developed resistance. Suppression of IL-6 in sensitive cells revealed that it is needed for cells to overcome growth inhibition by palbociclib. Targeting STAT3 in palbociclib resistant PDX mouse models using TTI-101 (50mg/kg twice daily by oral gavage), resulted in significantly reduced tumor volume; a synergistic effect was observed when TTI-101 was combined with palbociclib. Lastly, IL-6 levels in plasma samples from metastatic HR+, HER2- breast cancer patients were significantly higher at disease progression compared to pre-treatment with CDK4/6i. Conclusion: Collectively, these studies suggest that IL-6 signaling through STAT3 is a key mediator of resistance to palbociclib. We provide the rationale for using circulating IL-6 as a biomarker of disease progression and tumor pY-STAT3 as a selection criterion for treatment with TTI-101 to prevent or reverse resistance. Citation Format: Nicole M. Kettner, Tuyen N. Bui, Juliana Navarro-Yepes, T Kris Eckols, Akshara S. Raghavendra, David J. Tweardy, Kelly K. Hunt, Debu Tripathy, Khandan Keyomarsi. Circulating IL-6, an early biomarker in HR-positive, HER2-negative metastatic breast cancer patients progressing on CDK4/6 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1774.

Pancreatic cancer growth promoted by bone marrow mesenchymal stromal cell–derived IL-6 is reversed predominantly by IL-6 blockade

Pancreatic cancer is a highly lethal cancer characterized by local invasiveness, early metastasis, recurrence and high resistance to current therapies. Extensive stroma or desmoplasia is a key histological feature of the disease, and interactions between cancer and stromal cells are critical for pancreatic cancer development and progression. Mesenchymal stromal cells [MSCs] exhibit preferential tropism to primary and metastatic tumor sites and may either suppress or support tumor growth. Although MSCs represent a potential source of pancreatic cancer stroma, their contribution to pancreatic tumor growth remains poorly known. Here, we show that bone marrow MSCs significantly contribute to pancreatic cancer growth in vitro and in vivo. Furthermore, MSCs create a pro-carcinogenic microenvironment through the release of key factors mediating growth and angiogenesis, including interleukin (IL)-6, IL-8, vascular endothelial growth factor and activation of STAT3 signaling in tumor cells. IL-6 released by MSCs was largely responsible for the pro-tumorigenic effects of MSCs. Knockdown of IL-6 expression in MSCs by small interfering RNA (siRNA) abolished the MSC growth-promoting effect in vitro, reducing tumor cell proliferation and clonogenic potential. In addition, in a heterotopic nude mouse model of human pancreatic tumor xenografts, blockade of IL-6 with the anti-IL-6 receptor antibody, tocilizumab, or of its downstream effector STAT3 with the small molecule STAT3 inhibitor S3I-201, abrogated MSC-mediated tumor promotion and delayed tumor formation significantly. Our data demonstrate that MSCs promote pancreatic cancer growth, with IL-6 produced by MSCs playing a pivotal role.

Mitochondrial Plasticity Promotes Resistance to Sorafenib and Vulnerability to STAT3 Inhibition in Human Hepatocellular Carcinoma.

Simple SummaryEnhanced expression of mitochondrial ribosomal proteins and marked reprogramming of the mitochondrial network are associated with sorafenib resistance in human cell lines and hepatocarcinoma patients, providing novel actionable targets for increasing therapeutic efficacy.The multi-kinase inhibitor sorafenib is a primary treatment modality for advanced-stage hepatocellular carcinoma (HCC). However, the therapeutic benefits are short-lived due to innate and acquired resistance. Here, we examined how HCC cells respond to sorafenib and adapt to continuous and prolonged exposure to the drug. Sorafenib-adapted HCC cells show a profound reprogramming of mitochondria function and marked activation of genes required for mitochondrial protein translation and biogenesis. Mitochondrial ribosomal proteins and components of translation and import machinery are increased in sorafenib-resistant cells and sorafenib-refractory HCC patients show similar alterations. Sorafenib-adapted cells also exhibited increased serine 727 phosphorylated (pSer727) STAT3, the prevalent form in mitochondria, suggesting that STAT3 might be an actionable target to counteract resistance. Consistently, a small-molecule STAT3 inhibitor reduces pSer727, reverts mitochondrial alterations, and enhances the response to sorafenib in resistant cells. These results sustain the importance of mitochondria plasticity in response to sorafenib and identify a clinically actionable strategy for improving the treatment efficacy in HCC patients.

Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. DisclosuresShastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

TTI-101: A competitive inhibitor of STAT3 that spares oxidative phosphorylation and reverses mechanical allodynia in mouse models of neuropathic pain

Signal Transducer and Activator of Transcription (STAT) 3 emerged rapidly as a high-value target for treatment of cancer. However, small-molecule STAT3 inhibitors have been slow to enter the clinic due, in part, to serious adverse events (SAE), including lactic acidosis and peripheral neuropathy, which have been attributed to inhibition of STAT3′s mitochondrial function. Our group developed TTI-101, a competitive inhibitor of STAT3 that targets the receptor pY705-peptide binding site within the Src homology 2 (SH2) domain to block its recruitment and activation. TTI-101 has shown target engagement, no toxicity, and evidence of clinical benefit in a Phase I study in patients with solid tumors. Here we report that TTI-101 did not affect mitochondrial function, nor did it cause STAT3 aggregation, chemically modify STAT3 or cause neuropathic pain. Instead, TTI-101 unexpectedly suppressed neuropathic pain induced by chemotherapy or in a spared nerve injury model. Thus, in addition to its direct anti-tumor effect, TTI-101 may be of benefit when administered to cancer patients at risk of developing chemotherapy-induced peripheral neuropathy (CIPN).

Abstract 1230: High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype

Abstract Signal Transducer and Activator of Transcription (STAT)3 is a member of the STAT family of transcription factors that is frequently aberrantly-activated in breast cancer and many other cancers. STAT3 represents a valid target for the development of novel anticancer therapeutics. However, it has posed significant challenges to the discovery efforts to identify potent and suitable small molecule inhibitors. As a result, there is currently no anti-STAT3 drugs in clinical application to realize the potential clinical benefits of inhibiting abnormal STAT3 function. Herein is a new series of (R)-azetidine-2-carboxamide-based small molecules, exemplified by H098, H127, H152, H169, H174, H182, and H203, which selectively inhibit STAT3 DNA-binding activity, with in vitro potency (IC50) in the range of 0.28-0.66 μM, compared to potencies greater than 18 µM against the activities of the family members, STAT1 and STAT5. Equally notable is the high affinity binding to STAT3, as shown by KD of 1 pM and 3.7 nM for H169 and H174, respectively. Treatment of human triple-negative breast cancer (TNBC) cells with H182 or H169 inhibited constitutive STAT3 phosphorylation (pY705STAT3) and DNA-binding activity in both time- and dose-dependent manner. Furthermore, both inhibitors potently inhibited anchorage-dependent and independent growth, colony survival, and migration of TNBC cells in vitro, with potencies, EC50 of 1.0-1.9 μM, and the compounds also induced apoptosis of same cells. Additionally, the expression of STAT3 target genes, including c-Myc, VEGF, and survivin, were suppressed in the compound-treated TNBC cells subsequent to the inhibition of constitutive STAT3 activation, thereby validating the inhibition of constitutively-active STAT3 and its downstream targets as being responsible for the antitumor cell responses. Moreover, H169 and docetaxel combinatorial treatment led to enhanced suppression of TNBC cell viability. By contrast, treatments with the new compounds had minimal effects on the induction of STAT1, Janus kinase 2, epidermal growth factor receptor, extracellular signal-regulated kinases (Erk1/2), Src, Shc phosphatase, or Akt in TNBC cells, and they were relatively weak on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively-active STAT3. Significantly, H182 delivered at 5-20 mg/kg via oral gavage or intra-peritoneal route inhibited growth of TNBC tumor xenograft in mice, with no evidence of toxicity. These studies identify novel, sub-micromolar potent azetidine-based small molecule STAT3 inhibitors that have shown antitumor efficacy against human TNBC in xenograft models. H182 and H169 represent suitable novel chemical entities for developing new therapeutics against TNBC and other cancers that harbor constitutively-active STAT3. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Yinsong Zhu, Christine Brotherton-Pleiss, Wenzhen Fu, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe, Marcus Tius, James Turkson. High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1230.

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. ImplicationsSB-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.

Inhibition of STAT3 augments antitumor efficacy of anti-CTLA-4 treatment against prostate cancer

There is an urgent need for new treatment options in metastatic drug-resistant prostate cancer. Combining immunotherapy with other targeted therapies may be an effective strategy for advanced prostate cancer. In the present study, we sought to investigate to enhance the efficacy of anti-CTLA-4 therapy against prostate cancer by the combination with STAT3 inhibition.Male C57BL6 mice were subcutaneously inoculated with the murine prostate cancer cell line RM-1. Tumor progression was monitored following treatment with vehicle, the small molecule STAT3 inhibitor GPB730, anti-CTLA-4 or GPB730 + anti-CTLA-4. Treatment with anti-CTLA-4 or anti-CTLA-4 + GPB730 significantly inhibited tumor growth and enhanced survival compared to vehicle. Combining anti-CTLA-4 treatment with GPB730 resulted in a significantly prolonged survival compared to anti-CTLA-4 alone. GPB730 significantly increased infiltration of CD45 + cells in tumors of anti-CTLA-4-treated mice compared to anti-CTLA-4 alone. The levels of tumor-infiltrating Tregs were significantly decreased and the CD8:Treg ratio significantly increased by GPB730 treatment in combination with anti-CTLA-4 compared to anti-CTLA-4 alone. Immunohistochemical analysis showed a significant increase in CD45-positive cells in anti-CTLA-4 and anti-CTLA-4 + GPB730-treated tumors compared to vehicle or GPB730 monotherapy. Plasma levels of IL10 were significantly increased by anti-CTLA-4 compared to vehicle but no increase was observed when combining anti-CTLA-4 with GPB730.In conclusion, STAT3 inhibition by GPB730 enhances the antitumoral activity of anti-CTLA-4 and decreases the intratumoral Treg frequency in a prostate cancer mouse model. These results support the combination of STAT3 inhibition with anti-CTLA-4 therapy to increase clinical responses in patients with prostate cancer.

Discovery of Novel Azetidine Amides as Potent Small-Molecule STAT3 Inhibitors.

We optimized our previously reported proline-based STAT3 inhibitors into an exciting new series of (R)-azetidine-2-carboxamide analogues that have sub-micromolar potencies. 5a, 5o, and 8i have STAT3-inhibitory potencies (IC50) of 0.55, 0.38, and 0.34 μM, respectively, compared to potencies greater than 18 μM against STAT1 or STAT5 activity. Further modifications derived analogues, including 7e, 7f, 7g, and 9k, that addressed cell membrane permeability and other physicochemical issues. Isothermal titration calorimetry analysis confirmed high-affinity binding to STAT3, with KD of 880 nM (7g) and 960 nM (9k). 7g and 9k inhibited constitutive STAT3 phosphorylation and DNA-binding activity in human breast cancer, MDA-MB-231 or MDA-MB-468 cells. Furthermore, treatment of breast cancer cells with 7e, 7f, 7g, or 9k inhibited viable cells, with an EC50 of 0.9–1.9 μM, cell growth, and colony survival, and induced apoptosis while having relatively weaker effects on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively active STAT3.

A novel circular RNA circ-LRIG3 facilitates the malignant progression of hepatocellular carcinoma by modulating the EZH2/STAT3 signaling

BackgroundCircular RNA (circRNA) is emerging as an important player in human diseases, especially cancer. In our previous study, we identified a series of deregulated circRNAs in hepatocellular carcinoma (HCC) by performing circRNA microarray expression profile. Here, we aimed to explore the role of circ-LRIG3 (hsa_circ_0027345) in HCC.MethodsqRT-PCR and western blot were used to asses gene and protein expression, respectively. CCK-8, EdU and Transwell assays were used to detect cell proliferation, migration and invasion. GSEA software was applied to analyze the pathway related to circ-LRIG3. Co-IP, RIP and ChIP assays were used to identify the positive feedback axis of circ-LRIG3/EZH2/STAT3. Animal study was carried to test the role of circ-LRIG3 in vivo.ResultsCirc-LRIG3 was notably upregulated in HCC and promoted HCC cell proliferation, migration, invasion and reduced apoptosis. Circ-LRIG3 formed a ternary complex with EZH2 and STAT3, facilitating EZH2-induced STAT3 methylation and subsequent phosphorylation, resulting in the activation of STAT3 signaling. In turn, activated STAT3 could directly bind to circ-LRIG3 promoter to increase circ-LRIG3 transcription activity, thus forming a positive feedback loop. The animal models showed that exogenous expression of circ-LRIG3 enhanced tumorigenicity and metastasis in vivo, whereas these effects were blocked after treatment with C188–9, a specific STAT3 small-molecule inhibitor. Clinically, high circ-LRIG3 was closely linked with aggressive clinicopathological features and was identified as an independent risk prognostic factor of overall survival. Importantly, plasma circ-LRIG3 was found to be a highly sensitive and specific non-invasive diagnostic indicator for HCC.ConclusionsOur study reveals the carcinogenic role of circ-LRIG3 in HCC, which may provide a new therapeutic target for HCC patients.