Spatial molecular and cellular determinants of STAT3 activation in liver fibrosis progression in non-alcoholic fatty liver disease.

Background & Aims: The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is increasing. Individuals with NASH often develop liver fibrosis and advanced liver fibrosis is the main determinant of mortality in individuals with NASH. We and others have reported that STAT3 contributes to liver fibrosis and hepatocellular carcinoma in mice. Methods: Here, we explored whether STAT3 activation in hepatocyte and non-hepatocyte areas, measured by phospho-STAT3 (pSTAT3), is associated with liver fibrosis progression in 133 patients with NAFLD. We further characterized the molecular and cellular determinants of STAT3 activation by integrating spatial distribution and transcriptomic changes in fibrotic NAFLD livers.Results: pSTAT3 scores in non-hepatocyte areas progressively increased with fibrosis severity (r = 0.53, p <0.001). Correlation analyses between pSTAT3 scores and expression of 1,540 immune- and cancer-associated genes revealed a large effect of STAT3 activation on gene expression changes in non-hepatocyte areas and confirmed a major role for STAT3 activation in fibrogenesis. Digital spatial transcriptomic profiling was also performed on 13 regions selected in hepatocyte and non-hepatocyte areas from four NAFLD liver biopsies with advanced fibrosis, using a customized panel of markers including pSTAT3, PanCK+CK8/18, and CD45. The regions were further segmented based on positive or negative pSTAT3 staining. Cell deconvolution analysis revealed that activated STAT3 was enriched in hepatic progenitor cells (HPCs) and sinusoidal endothelial cells. Regression of liver fibrosis upon STAT3 inhibition in mice with NASH resulted in a reduction of HPCs, demonstrating a direct role for STAT3 in HPC expansion. Conclusion: Increased understanding of the spatial dependence of STAT3 signaling in NASH and liver fibrosis progression could lead to novel targeted treatment approaches. Impact and implications: Advanced liver fibrosis is the main determinant of mortality in patients with NASH. This study showed using liver biopsies from 133 patients with NAFLD, that STAT3 activation in non-hepatocyte areas is strongly associated with fibrosis severity, inflammation, and progression to NASH. STAT3 activation was enriched in hepatic progenitor cells (HPCs) and sinusoidal endothelial cells (SECs), as determined by innovative technologies interrogating the spatial distribution of pSTAT3. Finally, STAT3 inhibition in mice resulted in reduced liver fibrosis and depletion of HPCs, suggesting that STAT3 activation in HPCs contributes to their expansion and fibrogenesis in NAFLD.

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

STAT3 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.

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, ß) which are expressed in most cells in a 4:1 ratio (α: ß). STAT3α has pro-inflammatory and anti-apoptotic functions, while STAT3ß 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 (∆ß/∆ß), 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 ∆ß/∆ß 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.

DRUGGING "UNDRUGGABLE" DISEASE-CAUSING PROTEINS: FOCUS ON SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION (STAT) 3.

Signal transducer and activator of transcription (STAT) 3 has been assigned to the group of "undruggable" disease-causing proteins, despite its containing a Src-homology (SH) 2 domain, a potential Achilles' heel that has eluded successful targeting by academic and pharmaceutical groups over the past 30 years. Based on mutational and modeling studies, our group developed a unique virtual ligand screening strategy targeting the STAT3 SH2 domain that was coupled to robust biochemical and cellular assays and structure-based medicinal chemistry and led to the identification of TTI-101. TTI-101 represents one of the most advanced, direct, small-molecule inhibitors of an SH2 domain-containing, disease-causing protein in clinical development. TTI-101 is currently being evaluated in a Phase 1 study to determine safety and tolerability in addition to pharmacodynamic effects and efficacy in patients with advanced solid tumors.

Signal Transducer and Activator of Transcription-3 Modulation of Cardiac Pathology in Chronic Chagasic Cardiomyopathy.

Chronic Chagasic cardiomyopathy (CCC) is a severe clinical manifestation that develops in 30%-40% of individuals chronically infected with the protozoal parasite Trypanosoma cruzi and is thus an important public health problem. Parasite persistence during chronic infection drives pathologic changes in the heart, including myocardial inflammation and progressive fibrosis, that contribute to clinical disease. Clinical manifestations of CCC span a range of symptoms, including cardiac arrhythmias, thromboembolic disease, dilated cardiomyopathy, and heart failure. This study aimed to investigate the role of signal transducer and activator of transcription-3 (STAT3) in cardiac pathology in a mouse model of CCC. STAT3 is a known cellular mediator of collagen deposition and fibrosis. Mice were infected with T. cruzi and then treated daily from 70 to 91 days post infection (DPI) with TTI-101, a small molecule inhibitor of STAT3; benznidazole; a combination of benznidazole and TTI-101; or vehicle alone. Cardiac function was evaluated at the beginning and end of treatment by echocardiography. By the end of treatment, STAT3 inhibition with TTI-101 eliminated cardiac fibrosis and fibrosis biomarkers but increased cardiac inflammation; serum levels of interleukin-6 (IL-6), and IFN-γ; cardiac gene expression of STAT1 and nuclear factor-κB (NF-κB); and upregulation of IL-6 and Type I and Type II IFN responses. Concurrently, decreased heart function was measured by echocardiography and myocardial strain. These results indicate that STAT3 plays a critical role in the cardiac inflammatory-fibrotic axis during CCC.

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).

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.

Pharmacokinetics and pharmacodynamics of TTI-101, a STAT3 inhibitor that blocks muscle proteolysis in rats with chronic kidney disease.

Loss of muscle proteins increases the morbidity and mortality of patients with chronic kidney disease (CKD), and there are no reliable preventive treatments. We uncovered a STAT3/CCAAT-enhancer-binding protein-δ to myostatin signaling pathway that activates muscle protein degradation in mice with CKD or cancer; we also identified a small-molecule inhibitor of STAT3 (TTI-101) that blocks this pathway. To evaluate TTI-101 as a treatment for CKD-induced cachexia, we measured TTI-101 pharmacokinetics and pharmacodynamics in control and CKD rats that were orally administered TTI-101or its diluent. The following two groups of gavage-fed rats were studied: sham-operated control rats and CKD rats. Plasma was collected serially (0, 0.25, 0.5, 1, 2, 4, 8, and 24 h) following TTI-101 administration (at oral doses of 0, 10, 30, or 100 mg/kg). Plasma levels of TTI-101 were measured by LC-MS/MS, and pharmacokinetic results were analyzed with the PKSolver program. Plasma TTI-101 levels increased linearly with doses; the maximum plasma concentrations and time to maximal plasma levels (~1 h) were similar in sham-operated control rats and CKD rats. Notably, gavage treatment of TTI-101 for 3 days produced TTI-101 muscle levels in sham control rats and CKD rats that were not significantly different. CKD rats that received TTI-101 for 7 days had suppression of activated STAT3 and improved muscle grip strength; there also was a trend for increasing body and muscle weights. TTI-101 was tolerated at doses of 100 mg·kg-1·day-1 for 7 days. These results with TTI-101 in rats warrant its development as a treatment for cachexia in humans.

Targeting STAT3 anti-apoptosis pathways with organic and hybrid organic-inorganic inhibitors.

Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (AML) that spur efforts to identify new clinical targets and active agents. STAT3 has emerged as a potential target in resistant AML, but inhibiting STAT3 function has proven challenging. This paper describes synthetic studies and biological assays for a naphthalene sulfonamide inhibitor class of molecules that inhibit G-CSF-induced STAT3 phosphorylation in cellulo and induce apoptosis in AML cells. We describe two different approaches to inhibitor design: first, variation of substituents on the naphthalene sulfonamide core allows improvements in anti-STAT activity and creates a more thorough understanding of anti-STAT SAR. Second, a novel approach involving hybrid sulfonamide-rhodium(ii) conjugates tests our ability to use cooperative organic-inorganic binding for drug development, and to use SAR studies to inform metal conjugate design. Both approaches have produced compounds with improved binding potency. In vivo and in cellulo experiments further demonstrate that these approaches can also lead to improved activity in living cells, and that compound 3aa slows disease progression in a xenograft model of AML.

Targeting Janus Kinases and Signal Transducer and Activator of Transcription 3 to Treat Inflammation, Fibrosis, and Cancer: Rationale, Progress, and Caution.

Before it was molecularly cloned in 1994, acute-phase response factor or signal transducer and activator of transcription (STAT)3 was the focus of intense research into understanding the mammalian response to injury, particularly the acute-phase response. Although known to be essential for liver production of acute-phase reactant proteins, many of which augment innate immune responses, molecular cloning of acute-phase response factor or STAT3 and the research this enabled helped establish the central function of Janus kinase (JAK) family members in cytokine signaling and identified a multitude of cytokines and peptide hormones, beyond interleukin-6 and its family members, that activate JAKs and STAT3, as well as numerous new programs that their activation drives. Many, like the acute-phase response, are adaptive, whereas several are maladaptive and lead to chronic inflammation and adverse consequences, such as cachexia, fibrosis, organ dysfunction, and cancer. Molecular cloning of STAT3 also enabled the identification of other noncanonical roles for STAT3 in normal physiology, including its contribution to the function of the electron transport chain and oxidative phosphorylation, its basal and stress-related adaptive functions in mitochondria, its function as a scaffold in inflammation-enhanced platelet activation, and its contributions to endothelial permeability and calcium efflux from endoplasmic reticulum. In this review, we will summarize the molecular and cellular biology of JAK/STAT3 signaling and its functions under basal and stress conditions, which are adaptive, and then review maladaptive JAK/STAT3 signaling in animals and humans that lead to disease, as well as recent attempts to modulate them to treat these diseases. In addition, we will discuss how consideration of the noncanonical and stress-related functions of STAT3 cannot be ignored in efforts to target the canonical functions of STAT3, if the goal is to develop drugs that are not only effective but safe. SIGNIFICANCE STATEMENT: Key biological functions of Janus kinase (JAK)/signal transducer and activator of transcription (STAT)3 signaling can be delineated into two broad categories: those essential for normal cell and organ development and those activated in response to stress that are adaptive. Persistent or dysregulated JAK/STAT3 signaling, however, is maladaptive and contributes to many diseases, including diseases characterized by chronic inflammation and fibrosis, and cancer. A comprehensive understanding of JAK/STAT3 signaling in normal development, and in adaptive and maladaptive responses to stress, is essential for the continued development of safe and effective therapies that target this signaling pathway.

Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy.

High dose interleukin-2 (IL-2) is active against metastatic melanoma and renal cell carcinoma, but treatment-associated toxicity and expansion of suppressive regulatory T cells (Tregs) limit its use in patients with cancer. Bempegaldesleukin (NKTR-214) is an engineered IL-2 cytokine prodrug that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rß). Here we assess the therapeutic impact and mechanism of action of NKTR-214 in combination with anti-PD-1 and anti-CTLA-4 checkpoint blockade therapy or peptide-based vaccination in mice. NKTR-214 shows superior anti-tumor activity over native IL-2 and systemically expands anti-tumor CD8+ T cells while inducing Treg depletion in tumor tissue but not in the periphery. Similar trends of intratumoral Treg dynamics are observed in a small cohort of patients treated with NKTR-214. Mechanistically, intratumoral Treg depletion is mediated by CD8+ Teff-associated cytokines IFN-γ and TNF-α. These findings demonstrate that NKTR-214 synergizes with T cell-mediated anti-cancer therapies.

Stat3 activation induces insulin resistance via a muscle-specific E3 ubiquitin ligase Fbxo40.

Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly understood. One potential mechanism is that CKD-induced inflammation activates the signal transducer and activator of transcription 3 (Stat3) in muscle. We uncovered increased p-Stat3 in muscles of mice with CKD or mice fed high-fat diet (HFD). Activated Stat3 stimulates the expression of Fbxo40, a muscle-specific E3 ubiquitin ligase that stimulates ubiquitin conjugation leading to degradation of insulin receptor substrate 1 (IRS1). Evidence that Stat3 activates Fbxo40 includes 1) potential Stat3 binding sites in Fbxo40 promoters; 2) Stat3 binding to the Fbxo40 promoter; and 3) constitutively active Stat3 stimulating both Fbxo40 expression and its promoter activity. We found that IL-6 activates Stat3 in myotubes, increasing Fbxo40 expression with reduced IRS1 and p-Akt. Knockdown Fbxo40 using siRNA from myotubes results in higher levels of IRS1 and p-Akt despite the presence of IL-6. We treated mice with a small-molecule inhibitor of Stat3 (TTI-101) and found improved glucose tolerance and insulin signaling in skeletal muscles of mice with CKD or fed an HFD. Finally, we uncovered improved glucose tolerance in mice with muscle-specific Stat3 KO versus results in Stat3f/f mice in response to the HFD. Thus Stat3 activation in muscle increases IR in mice. Inhibition of Stat3 by TTI-101 could be developed into clinical strategies to improve muscle insulin signaling in inflammation and other catabolic diseases.

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.].

Contribution of STAT3 to Inflammatory and Fibrotic Diseases and Prospects for its Targeting for Treatment.

Signal transducer and activator of transcription (STAT) 3 plays a central role in the host response to injury. It is activated rapidly within cells by many cytokines, most notably those in the IL-6 family, leading to pro-proliferative and pro-survival programs that assist the host in regaining homeostasis. With persistent activation, however, chronic inflammation and fibrosis ensue, leading to a number of debilitating diseases. This review summarizes advances in our understanding of the role of STAT3 and its targeting in diseases marked by chronic inflammation and/or fibrosis with a focus on those with the largest unmet medical need.

Intratumoral CD40 activation and checkpoint blockade induces T cell-mediated eradication of melanoma in the brain.

CD40 agonists bind the CD40 molecule on antigen-presenting cells and activate them to prime tumor-specific CD8+ T cell responses. Here, we study the antitumor activity and mechanism of action of a nonreplicating adenovirus encoding a chimeric, membrane-bound CD40 ligand (ISF35). Intratumoral administration of ISF35 in subcutaneous B16 melanomas generates tumor-specific, CD8+ T cells that express PD-1 and suppress tumor growth. Combination therapy of ISF35 with systemic anti-PD-1 generates greater antitumor activity than each respective monotherapy. Triple combination of ISF35, anti-PD-1, and anti-CTLA-4 results in complete eradication of injected and noninjected subcutaneous tumors, as well as melanoma tumors in the brain. Therapeutic efficacy is associated with increases in the systemic level of tumor-specific CD8+ T cells, and an increased ratio of intratumoral CD8+ T cells to CD4+ Tregs. These results provide a proof of concept of systemic antitumor activity after intratumoral CD40 triggering with ISF35 in combination with checkpoint blockade for multifocal cancer, including the brain.

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.

Protein stabilization improves STAT3 function in autosomal dominant hyper-IgE syndrome.

Autosomal dominant hyper-IgE syndrome (AD-HIES) is caused by dominant-negative mutations in STAT3; however, the molecular basis for mutant STAT3 allele dysfunction is unclear and treatment remains supportive. We hypothesized that AD-HIES mutations decrease STAT3 protein stability and that mutant STAT3 activity can be improved by agents that increase chaperone protein activity. We used computer modeling to characterize the effect of STAT3 mutations on protein stability. We measured STAT3 protein half-life (t1/2) and determined levels of STAT3 phosphorylated on tyrosine (Y) 705 (pY-STAT3) and mRNA levels of STAT3 gene targets in Epstein-Barr virus-transformed B (EBV) cells, human peripheral blood mononuclear cells (PBMCs), and mouse splenocytes incubated without or with chaperone protein modulators-HSF1A, a small-molecule TRiC modulator, or geranylgeranylacetone (GGA), a drug that upregulates heat shock protein (HSP) 70 and HSP90. Computer modeling predicted that 81% of AD-HIES mutations are destabilizing. STAT3 protein t1/2 in EBV cells from AD-HIES patients with destabilizing STAT3 mutations was markedly reduced. Treatment of EBV cells containing destabilizing STAT3 mutations with either HSF1A or GGA normalized STAT3 t1/2, increased pY-STAT3 levels, and increased mRNA levels of STAT3 target genes up to 79% of control. In addition, treatment of human PBMCs or mouse splenocytes containing destabilizing STAT3 mutations with either HSF1A or GGA increased levels of cytokine-activated pY-STAT3 within human CD4+ and CD8+ T cells and numbers of IL-17-producing CD4+ mouse splenocytes, respectively. Thus, most AD-HIES STAT3 mutations are destabilizing; agents that modulate chaperone protein function improve STAT3 stability and activity in T cells and may provide a specific treatment.

Chaperonin TRiC/CCT Recognizes Fusion Oncoprotein AML1-ETO through Subunit-Specific Interactions.

AML1-ETO is the translational product of a chimeric gene created by the stable chromosome translocation t (8;21)(q22;q22). It causes acute myeloid leukemia (AML) by dysregulating the expression of genes critical for myeloid cell development and differentiation and recently has been reported to bind multiple subunits of the mammalian cytosolic chaperonin TRiC (or CCT), primarily through its DNA binding domain (AML1-175). Through these interactions, TRiC plays an important role in the synthesis, folding, and activity of AML1-ETO. Using single-particle cryo-electron microscopy, we demonstrate here that a folding intermediate of AML1-ETO's DNA-binding domain (AML1-175) forms a stable complex with apo-TRiC. Our structure reveals that AML1-175 associates directly with a specific subset of TRiC subunits in the open conformation.