Constitutive pSTAT3 Research Articles

Recurrent PDL1 expression and PDL1 (CD274) copy number alterations in breast implant–associated anaplastic large cell lymphomas

Breast implant-associated anaplastic large cell lymphoma (BI-ALCL) is a variant of anaplastic large cell lymphoma arising within seroma effusion associated with breast implants. BI-ALCL is a rare disease, recently recognized as a new provisional entity by the 2017 revised World Health Organization classification. All BI-ALCLs tested so far showed a "triple-negative" genetic profile-negative for ALK, DUSP22, and TP63 rearrangements-and were characterized by mutational and gene expression profiles consistent with aberrant activation of the JAK/STAT pathway. The active form of STAT3 (pSTAT3) is constantly expressed in BI-ALCLs and may favor tumor immune escape by triggering the transcription of PDL1 (CD274), a gene encoding the immune-checkpoint molecule programmed cell death ligand 1 (PDL1); immunohistochemical positivity for PDL1 has been recently described in 3 BI-ALCL cases, and one of them also harbored PDL1 gene amplification. We evaluated PDL1 and pSTAT expression by immunohistochemistry and PDL1 copy number alterations (CNAs) at chromosome 9p24.1 by fluorescent in situ hybridization in a cohort of 9 BI-ALCL cases; we also investigated the presence of tumor-infiltrating programmed cell death 1 (PD1)+ T cells (tumor-infiltrating lymphocytes, or TILs) and PDL1+ tumor-associated macrophages (TAMs) in BI-ALCL microenvironment. Tumor cells expressed PDL1 in 5 (56%) of 9 cases and harbored PDL1 CNAs in 3 (33%) of 9 cases; immunohistochemistry for pSTAT3 was positive in all 6 cases tested (100%), indicative of active JAK/STAT signaling. We observed PDL1 CNAs only among PDL1-positive cases, whereas PD1+ TILs and PDL1+ TAMs were present at variable levels in both PDL1-positive and PDL1-negative BI-ALCLs. We report frequent PDL1 expression and recurrent PDL1 CNAs in BI-ALCLs: our data suggest that 9p24.1 alterations represent a common mechanism of PDL1 overexpression in this disease, likely acting in synergy with constitutive pSTAT3 signaling. In PDL1-positive cases without chromosomal aberration, PDL1 expression may be induced by JAK/STAT signaling alone and/or others alternative pathways. BI-ALCL microenvironment hosts variable amounts of PD1+ TILs and PDL1+ TAMs, suggesting the presence of an active PD1/PDL1 axis. These findings may be of therapeutic value in advanced-stage patients who may benefit from a PD1/PDL1 blocking treatment.

Constitutive STAT3 phosphorylation and IL-6/IL-10 co-expression are associated with impaired T-cell function in tuberculosis patients.

T-cells critically contribute to protection against Mycobacterium tuberculosis infection, and impaired T-cell responses can lead to disease progression. Pro-inflammatory and immunosuppressive cytokines affect T-cells, and fine-tuned regulation of cytokine signaling via the Jak/STAT signaling pathways is crucial for appropriate T-cell function. Constitutive STAT3 phosphorylation as a consequence of aberrant cytokine signaling has been described to occur in pathognomonic T-cell responses in inflammatory and autoimmune diseases. We characterized blood samples from tuberculosis patients (n=28) and healthy contacts (n=28) from Ghana for M. tuberculosis-specific T-cell responses, constitutive cytokine production, and SOCS3 and pSTAT3 expression. Lentiviral modulation of primary CD4+ T-cells was performed to determine the effects of SOCS3 on T-cell functions. T-cells from tuberculosis patients expressed higher levels of IL-10 and IL-6 and lower levels of T helper type (TH)17 cytokines after M. tuberculosis-specific stimulation compared to healthy contacts. In addition, tuberculosis patients had higher IL-10 and IL-6 levels in the supernatants of non-stimulated immune cells and plasma samples compared to healthy contacts. Notably, aberrant cytokine expression was accompanied by high constitutive pSTAT3 levels and SOCS3 expression in T-cells. Multivariate analysis identified an IL-6/IL-10 co-expression-based principal component in tuberculosis patients that correlated with high pSTAT3 levels. SOCS3 contributed to a regulatory component, and tuberculosis patients with high SOCS3 expression showed decreased TH1 cytokine expression and impaired IL-2-induced STAT5 phosphorylation. SOCS3 over-expression in primary CD4+ T-cells confirmed the SOCS3 inhibitory function on IL-2-induced STAT5 phosphorylation. We conclude that constitutive pSTAT3 and high SOCS3 expression are influential factors that indicate impaired T-cell functions in tuberculosis patients.

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

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

Phospho-Flow Cytometry Quantitation of STAT Signalling Checkpoints in Malignant and Non-Malignant Cells Identifies Patients Suitable for pSTAT3 and 5 Targeted Inhibitors

BackgroundIL-6 receptor/JAK/STAT is a major signalling pathway associated with pleiotropic functions including cell transformation and proliferation. STAT3, one of a family of 7 STAT members, sits at the apex of the signalling cascade and is a potential target for cancer therapy. Small molecular weight inhibitors either prevent STAT3 phosphorylation or target high constitutive levels of phosphorylated pSTAT3 (pSTAT3). Increased constitutive expression of pSTAT3 is a hallmark of solid tumors and is associated with increased morbidity. However recent studies suggest that this may not apply to hematological malignancies. Phospho-flow cytometry offers a novel approach for the quantitation of constitutive and cytokine induced pSTAT3 expression in subsets of hemopoietic cells. Constitutive pSTAT3 expression is not a prognostic factor for patients with either multiple myeloma (MM) or acute myeloid leukemia (AML), though we recently demonstrated that IL-6 induced pSTAT3 was associated with better prognosis in MM (χ2 =13.06; p<0.0003) (Leukemia 2015; 29:483) . There is considerable heterogeneity of pSTAT3 expression between patients which suggests that quantitation of these new biomarkers may help to identify those patients most likely to respond to STAT-targeted therapy. MethodsWe have determined constitutive and IL-6 induced pSTAT3 and 5 in malignant plasma cells of patients with MM (n=25) and for comparison, the blast cells in AML (n=8) and CD5+ B cells in chronic lymphocytic leukemia (CLL) (n=12) as well as the non-malignant B and T cells in each sample. Normal BM cells (n=9) served as a control. We have also determined pSTAT3 and 5 expression in "side population" cells (SP), the putative myeloma stem cells, and determined whether MM and AML tumor cells induce pSTAT3 on normal lymphocytes in vitro by co-culture experiments. IL-6 receptor expression was also determined on all cells.ResultsConstitutive pSTAT3 expression was increased (>5% of normal lymphocytes) in the malignant plasma cells of 44% of patients with MM but by comparison was not increased in AML or B-CLL. Constitutive pSTAT5 was increased (>5%) in 68% of MM plasma cells and 75% of AML cells and was either normal or decreased in CLL cells. Patients with increased pSTAT3 or 5 in MM cells also had high expression in autologous normal T cells. The level of constitutive pSTAT3 and 5 was not related to the disease status (relapse vs remission). In patients with MM, IL-6 induced an increased pSTAT3 expression (induced/constitutive ratio >1.5) in both MM cells and T cells in 88% of samples. IL-6 induced pSTAT3 expression in blasts of 89% of AML patients (median ratio = 79). There was no correlation between the level of pSTAT3 induced by IL-6 and IL-6 receptor expression. IL-6 did not increase pSTAT5 levels in MM, AML or CLL. SP cells in the MM cell line RPMI 8226 had lower constitutive pSTAT3 than the non-SP population and a lower IL-6 receptor expression, but had a greater response to cytokine stimulation, suggesting that SP cells may be more sensitive than non-SP cells to inhibitors of STAT phosphorylation. Addition of RPMI 8226 cells or supernatant to normal T cells caused an increase in pSTAT3, suggesting that the upregulation of pSTAT3 is at least partly due to a tumor-derived soluble agent in the microenvironment.ConclusionsThese results demonstrate the heterogeneity of constitutive and cytokine induced pSTAT3 and 5 expression in MM, AML and CLL. Elevated levels of constitutive pSTAT3 (approx 44% of MM) and pSTAT5 (88% of MM and AML) in the malignant clone were associated with high levels in autologous non-malignant T cells and are not of prognostic significance. Therefore elevated pSTAT3 and 5 may not be an exclusive hallmark of the malignant clone, as reported in solid tumors, but rather a result of the microenvironment and a high IL6-R expression. Phospho-flow cytometry enables precise quantitation of constitutive and cytokine-induced pSTAT3 and 5, and thus can identify the patients most likely to respond to direct STAT-3 or 5 inhibitor therapies. SP cells (putative MM stem cells) appear to be potentially targetable. Inhibitors targeting signalling pathways may be more efficacious if therapy is directed to patients with targetable checkpoints. DisclosuresHart:DendroCyte BioTech Pty Ltd: Equity Ownership.

IL-6-driven STAT signalling in circulating CD4+ lymphocytes is a marker for early anticitrullinated peptide antibody-negative rheumatoid arthritis

ObjectivesA previously identified signal transduction and activator of transcription-3 (STAT3) target-enriched gene signature in circulating CD4+ T cells of patients with early rheumatoid arthritis (RA) was prominent in autoantibody-negative individuals....

O-032 Increased Pro-Fibrotic miR-21 and Decreased Anti-Fibrotic miR-29b Regulate TGF-β1 Signaling, TGF-β1-Dependent Collagen-I Expression and Fibrosis in Fibrostenotic (B2) Crohn’s Disease

We have shown that in patients with B2 Crohn’s disease constitutive p-STAT3 activation in mesenchymal cells results in increased TGF-β1 expression, TGF-β1-dependent Collagen-I production and fibrosis. In these cells, as expected p-Smad3 activation increases, yet the expected inhibition of increased Smad3-dependent TGF-β1 signaling by upregulation of inhibitory Smad-7 paradoxically does not occur. TargetScan analysis predicts Smad3 and STAT3 to regulate pre-miR-21 and pre-miR29b maturation and STAT3 to regulate pre-miR-21 maturation; Collagen I mRNA is a predicted target of miR-21 and miR-29b and Smad7 is a predicted target of miR-21. To determine the mechanisms regulating pre-miR-21 and pre-miR-29b maturation in mesenchymal cells from patients with fibrostenotic B2 Crohn’s disease and identify the miR-21-dependent and miR-29b-dependent mechanisms that could lead to fibrosis. Mesenchymal cells (smooth muscle cells and subepithelial myofibroblasts) were isolated from the affected ileum and normal resection margin in the same patient with inflammatory (B1), fibrostenotic (B2), or penetrating (B3) Crohn’s disease or non-Crohn’s subjects. Isolated cells were used to prepare miRNA, mRNA, protein lysates, nuclear fractions or initiate primary cell cultures. miR-21, miR-29b, Smad7, Collagen-I expression were measured by qRT-PCR. Factors regulating maturation of pre-miRs were determined using RNA-ChIP. The regulation of miRNA target mRNA was determined using transfection of miR-21, miR-29b, antagomiR-21, antagomiR-29b or scrambled sense. STAT3 and Smad3 knock-down was accomplished using siRNA. The role of miR-21 was investigated using the 6-week chronic TNBS colitis model in C56Bl/6J mice treated with antagomiR-21 oligonucleotide (10 mg/kg/wk i.p weekly). Expression of miR-21 increased and miR-29b decreased in mesenchymal cells of affected ileum compared to normal ileum in the same patient and to non-Crohn’s subjects. The opposite was seen in B3 patients with no change in B1 patients. In cells from B2 patients, p-STAT3 bound to pre-miR21; binding was inhibited by the STAT3 inhibitor, Stattic. p-Smad3 bound to both pre-miR-21 and pre-miR-29b; binding to both was inhibited by the Smad3 inhibitor, SIS3. Knock-down of Smad3 increased miR-29b and decreased miR-21, whereas, knockdown of STAT3 decreased miR-21 but did not affect miR-29b. Transfection of miR-21 decreased Smad7 and increased collagen I expression, whereas transfection of miR-29b decreased collagen-I expression but did not affect Smad7. The opposite pattern was seen following transfection of antagomiR-21 and antagomiR-29b. In mice treated with antagomir-21, i-Smad7 expression was restored, TGF-β1-dependent collagen I production was inhibited along with the development of fibrosis. Expression of pro-fibrotic miR-21 is increased by p-STAT3 and p-Smad3 in affected regions of patients with fibrostenotic Crohn’s disease. Increased miR-21 decreases abundance of Smad7 transcripts accounting for paradoxically decreased inhibitory Smad7. Inhibition of anti-fibrotic miR-29b by p-Smad3 caused loss of negative regulation of Collagen-I. Taken together the observed increase in miR-21 and decrease in miR-29b expression can result in overactive and unopposed TGF-β1 signaling and TGF-β1-dependent collagen-I expression and fibrosis in patients with B2 fibrostenotic Crohn’s disease.

Phospho-flow detection of constitutive and cytokine-induced pSTAT3/5, pAKT and pERK expression highlights novel prognostic biomarkers for patients with multiple myeloma.

Identifying check points in cell signal transduction pathways has led to the development of new cancer therapies; however, relatively few studies have determined the diagnostic and prognostic significance of analysing phosphorylated signaling proteins in patient blood and bone marrow (BM) samples. This is the first comprehensive phospho-flow study of both constitutive and cytokine-induced pSTAT3, pSTAT5, pAKT and phosphorylated extracellular signal-regulated kinase (pERK) expression in malignant plasma cells of patients with monoclonal gammopathies. In diagnostic BM samples from 65 patients with multiple myeloma (MM), interleukin (IL)-6-induced pSTAT3 proved to be a new and independent prognostic biomarker for improved survival. When combined with the International Staging System, 6 subgroups demonstrated stratified median survivals from 9 to 72 months (χ(2)=34.3; P<0.0001). In contrast, constitutive expression of pSTAT3, pSTAT5, pAKT and pERK did not assist the differential diagnosis nor determine prognosis. High pSTAT3 expression was dependent on existing CD45 expression and pSTAT5 appeared to regulate IgG production. Phospho-flow cytometry could be used to screen for personalized therapy, although the lack of clinical significance of constitutive pSTAT3 levels suggests that pSTAT3 blockade may not be clinically relevant in MM. This study has revealed novel prognostic biomarkers and insights into the biology of signaling pathways in patients with MM.

Polyphenol tri-vanillic ester 13c inhibits P-JAK2V617F and Bcr–Abl oncokinase expression in correlation with STAT3/STAT5 inactivation and apoptosis induction in human leukemia cells

Constitutive activity of kinases has been reported in many types of cancers, so that inhibition of “onco-kinases” became a validated anti-cancer strategy. We found that the polyphenol 13c, a tri-vanillate derivative, inhibited kinase phosphorylation in leukemia cells. P-JAK2, P-Src and P-PI3Kp85 inhibition occurred independently of phosphatase involvement in JAK2V617F expressing HEL cells while 13c inhibited Bcr–Abl expression without inhibition of phosphorylation in chronic myelogenous leukemia cell lines (K562, MEG-01). In correlation with kinase inhibition, 13c abolished constitutive P-STAT3/P-STAT5 expression, down-regulated Mcl-1 and c-Myc gene expression and induced apoptosis. Altogether, polyphenol 13c displays potential antitumor activities by affecting onco-kinases and STAT activities.

Autocrine insulin-like growth factor 1 and stem cell factor but not interleukin 6 support self-renewal of human myeloma cells

In this study, we have identified the growth factors supporting myeloma self-renewal in eight myeloma cell lines. All cell lines able to form self-colonies displayed constitutive P-AKT and P-ERK1,2 but not P-STAT3 and did not express CD45, suggesting the presence of an insulin-like growth factor 1 (IGF1) loop. We showed that a blocking anti-insulin-like growth factor 1 receptor (IGF1R) monoclonal antibody (mAb) inhibited colony formation in correlation with IGF1R expression and decreased P-AKT. Imatinib or a blocking anti-stem cell factor (SCF) mAb also inhibited colony formation of two cell lines expressing C-KIT and SCF, and decreased P-AKT. Moreover, the PI3K/AKT pathway inhibitor wortmannin inhibited colony formation. Blocking interleukin (IL)6R did not inhibit colony formation in good agreement with a lack of constitutive P-STAT3. We showed that primary cells frequently co-expressed IGF1R/IGF1 but not C-KIT/SCF or IL6R/IL6, suggesting that in vivo autonomous growth could be possible via IGF1R. Despite their similar role in clonogenic growth and shared signaling pathway, IGF1R and C-KIT had opposite prognostic values, suggesting that they were surrogate markers. Indeed, we showed that both C-KIT and IGF1R prognostic values were not independent of MMSET expression. This study highlights the autocrine role of IGF1 in myeloma cells and reinforces the interest in targeting IGF1R in IGFR1+ CD45+/− patients, such as MMSET+ patients.

Abstract 1791: Stat3 signaling in acute myeloid leukemia: Ligand-dependent and -independent activation, impact on prognosis, and induction of apoptosis by a novel Stat3 inhibitor

Abstract Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite very toxic chemotherapy. Newly developed therapies for AML are targeted at signal transduction pathways known to support blast survival. One such potential target is Stat3, a signaling intermediate that is activated by tyrosine phosphorylation in response to a number of cytokines active in myeloid cells, such as G-CSF. Aberrant activation of Stat3 has been demonstrated in many malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were to 1) characterize Stat3 signaling patterns in AML cells lines and primary pediatric AML samples, and 2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We measured tyrosine-phosphorylated Stat3 (pStat3) in 7 AML cell lines by FACS and Western blot. We found that in 6 of the 7 cell lines, at least 20% of cells had pStat3 (by FACS) in the absence of stimulation, and thus were considered constitutively activated. Likewise, 6 of 7 cell lines demonstrated at least a 2-fold increase in the mean fluorescence intensity (MFI) of pStat3 after G-CSF stimulation, and thus were considered responsive to G-CSF. We next analyzed primary pediatric AML samples (from the Children's Oncology Group AML Reference Lab) and normal bone marrow mononuclear cells (BMMCs) for constitutive and G-CSF-induced pStat3. We found 6 of 18 AML samples and 1of 5 BMMCs showed constitutive pStat3 (pStat3+), and 16/18 AML and 3/5 BMMC samples were responsive to G-CSF. Event-free survival analysis revealed that constitutive pStat3 was significantly associated with inferior outcome. Specifically, 4/6 pStat3+ patients died of relapsed or refractory disease, while 2/12 pStat3- patients died (p = 0.019). These results support the hypothesis that aberrant Stat3 activity promotes an aggressive phenotype in pediatric AML. In this regard, we evaluated the effect of a novel small molecule Stat3 inhibitor that has been developed by us, C188-9, to block G-CSF-induced Stat3 phosphorylation and to induce apoptosis in AML cells. The IC50s of C188-9 to inhibit Stat3 activation in AML cell lines were in the range of 4-7 μM, and in primary AML samples the IC50s were in the range of 8-18 μM. For apoptosis studies, AML cell lines and primary samples were treated for 24 hours with the compound, then apoptotic cells were quantified by FACS analysis for annexin V-labeled cells. The EC50s for apoptosis induction were quite variable, ranging from 6 μM to over 50 μM. These results indicate that there is a subset of AML samples that are dependent on Stat3 signaling for survival and are killed by inhibition of Stat3. Our data suggest that aberrant Stat3 activity may be both a valuable new prognostic indicator and an important target for novel therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1791.

Abstract 2432: Fully human anti-interleukin-6 (IL-6) monoclonal antibody suppresses the growth of human tumor xenograft in vivo

Abstract Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a major role in the response to injury or infection and is involved in the immune response, inflammation, and hematopoiesis. Aberrant expression of IL-6 has been implicated in many human diseases. In cancer, high levels of circulating IL-6 are observed in many tumor types and associated with poor outcome. We hypothesized that a monoclonal antibody that neutralizes IL-6 activity may be effective in inhibiting the progression of cancer. A fully humanized monoclonal antibody against human IL-6 (CAT-6001) was isolated by screening phage-display libraries. CAT-6001 completely neutralizes IL-6 activities via binding to the IL-6 receptor and signal transducer gp130 binding sites of human IL-6. We demonstrate that several cancer cell lines secrete high levels of IL-6 and soluble IL-6 receptor, and express gp130. High constitutive pStat3 activation is seen in several of these cell lines suggesting an IL-6 autocrine loop. Treatment of these cell lines with CAT-6001 effectively blocked phosphorylation of Stat3 and in some cell lines, functionally inhibited IL-6 induced cell proliferation. In vivo, CAT-6001 inhibited the growth of DU145, a human prostate cancer xenograft model, and significantly blocked the release of human IL-6 and inhibited Stat3 phosphorylation. Taken together, our data suggest that IL-6 signaling contributes to tumor growth, and provide support for the further development of CAT-6001 for the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2432.