pSTAT3 Signaling Research Articles

Abstract 4263: Autophagic ovarian cancer cells exhibit substantially enhanced sensitivity to ALK inhibition

Abstract One of the major factors contributing to poor outcomes for patients with ovarian cancer is the persistence of dormant, drug resistant cancer cells after primary surgery and chemotherapy. The persistent cancer found in positive second look operations is poorly vascularized and autophagy is widespread in more than 80% of cases. Consequently, drugs that regulate survival in autophagic cancer cells may be much more active when administered as maintenance therapy than when used to treat gross recurrent disease. Using unbiased siRNA screens, we have identified target genes that regulate the survival of ovarian cancer cells that are undergoing autophagy-induced by the re-expression of DIRAS3 (ARHI) or by serum starvation. Knockdown of the anaplastic lymphoma kinase (ALK) significantly reduced survival of ovarian cancer cells that were undergoing autophagy. Importantly, FDA-approved ALK inhibitors, including crizotinib, exhibited significantly greater toxicity after induction of autophagy by upregulation of DIRAS3 or serum starvation in 5 ovarian cancer cell lines. Induction of autophagy by upregulation of DIRAS3 or serum starvation in ovarian cancer cells reduced the IC50 of crizotinib and other ALK inhibitors ranging from 1.5 to 20-fold (all p-values <0.05). Crizotinib treatment in autophagic cancer cells enhanced autophagy and apoptosis via a decrease in p-STAT3 and BCL-2 signaling. Selective targeting of p-STAT3 or BCL-2 also resulted in significantly greater toxicity after induction of autophagy by upregulation of DIRAS3 or serum starvation in multiple ovarian cancer cells. Inducible upregulation of DIRAS3 by doxycycline in the first 6 weeks prompted autophagy and dormancy in two separate xenograft models (SKOV3 and OVCAR8). Crizotinib treatment of dormant autophagic SKOV3 xenografts prolonged median survival by 9.7 weeks (p<0.0032) and cured a subset of mice. Thirteen of fifteen mice (87%) bearing SKOV3 xenografts and nine of eleven mice (82%) bearing OVCAR8 xenografts, were tumor free at 200 days compared to the other three control arms (overall survival, p<0.0001 and p<0.05, respectively). Our studies suggest that ALK inhibitors might provide an effective agent to eliminate autophagic, dormant, drug resistant ovarian cancer cells that remain after conventional cytoreductive surgery and combination chemotherapy. Citation Format: Alicia M. Blessing, Weiqun Mao, Lan Pang, Philip Rask, Zhen Lu, Robert C. Bast. Autophagic ovarian cancer cells exhibit substantially enhanced sensitivity to ALK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4263.

IFN-I and IL-22 mediate protective effects of intestinal viral infection.

Products derived from bacterial members of the gut microbiota evoke immune signaling pathways from the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. Recently, we demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities upon depletion of bacteria, indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here, we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and IL-22 producing innate lymphoid cells (ILCs), which in turn promote pSTAT3 signaling in intestinal epithelial cells and protection from intestinal injury. Additionally, we demonstrate that MNV provides a striking IL-22 dependent protection against early life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges.

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.

Euphorbia factor L2 inhibits TGF-β-induced cell growth and migration of hepatocellular carcinoma through AKT/STAT3

BackgroundEuphorbia factor L2 has potent effects on ascites, hydropsy and cancers. PurposeWe investigated the pharmacological effects of Euphorbia factor L2 (EFL2) on hepatocellular carcinoma (HCC). MethodsMTT assay was conducted to determine the proliferative activity of EFL2 on Hep G2 and SMMC-7721 cells. Wound-healing assay, colony formation assay, western blotting and quantitative PCR were carried out to examine the cell migration, p-AKT and p-STAT3 signaling. Moreover, we used human tumor xenograft BALB/c nude mice to detect the effect of EFL2 on HCC in vivo. ResultsEFL2 inhibited the proliferation of SMMC-7721 and Hep G2 cells in concentration- and time-dependent manners. EFL2 also suppressed the cell migration and colony formation of hepatocellular carcinoma cells. Using a transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) model, we provided evidences that EFL2 could also inhibit TGF-β induced cell growth, vimentin, N-cadherin expressions, activation of p-AKT and p-STAT3, whereas up-regulate E-cadherin expression. Furthermore, EFL2 inhibited tumor growth and STAT3 phosphorylation in vivo. ConclusionIn conclusion, EFL2 has the potential to be explored as a candidate treatment agent for HCC by inhibiting cell growth and migration both in vitro and in vivo.

Molecular mechanisms of interleukin-38 inhibiting inflammatory bowel disease in children by regulating nuclear factor-κB and signal transduction and activator of transcription 3 pathway

Objective To explore the role of interleukin (IL)-38 in inhibiting inflammatory bowel disease (IBD) in children and to investigate the potential molecular mechanisms. Methods From January 2014 to October 2017, 67 patients with ulcerative colitis (UC) and 115 patients with Crohn′s disease (CD) admitted to Wuhan Children′s Hospital were recruited, and 40 individuals with normal endoscopic findings were selected as control. Serum levels of IL-38 of IBD patients and healthy control were determined by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining (IHC) was used to detect the expression level of IL-38, nuclear factor κB (NF-κB), phosphorylated signal transduction and activator of transcription 3 (p-STAT3), C-reaction protein (CRP) and erythrocyte sedimentation rate (ESR) in the intestinal mucosa of IBD patients and healthy controls. The extent of disease, therapeutic agents and disease activity scores (Mayo score system for UC patients, Crohn′s disease activity index (CDAI) for CD patients) were evaluated. IL-38-C57BL/6 transgenic mice model was established, and dextran sulfate sodium was used to induce IBD mice model. The intestinal inflammation levels were compared between the wild type IBD mice and IL-38 transgenic IBD mice. The levels of IL-38, NF-κB and p-STAT3 in intestinal mucosa of mice of different groups were determined by IHC. The ratio of CD4+ IL-17+ T helper (Th) 17 cells in peripheral blood of mice of different groups was detected by flow cytometry. Independent sample t test, chi square test and Pearson correlation were performed for statistical analysis. Results The results of ELISA showed that the serum levels of IL-38 of UC and CD patients were (6.1±1.9) ng/L and (9.8±2.1) ng/L, respectively, which both were lower than that of healthy controls ((16.4±2.7) ng/L), and the differences were statistically significant (t=23.107 and 15.853, both P<0.05). The results of IHC indicated that the levels of IL-38 in the intestinal mucosal tissues of UC and CD patients were 0.04±0.01 and 0.03±0.01, respectively, which were both lower than that of healthy controls (0.18±0.02), and the differences were statistically significant (t=48.186 and 69.443, both P<0.05). The levels of NF-κB and p-STAT3 of UC and CD patients were 0.150±0.030, 0.160±0.040 and 0.130±0.030, 0.110±0.010, which were all higher than those of healthy controls (0.020±0.003 and 0.010±0.002), and the differences were statistically significant (tUC=27.273 and 23.078, tCD=23.657 and 62.684; all P<0.05). The number of patients with disease at active phase, CRP level, ESR and disease activity scores of UC and CD patients with low IL-38 expression were all significantly higher than those of patients with high IL-38 expression (χUC2=11.552, tUC=7.118, 8.991 and 7.086; χCD2=5.675, tCD=9.559, 9.358 and 11.268; all P<0.05). The results of Pearson correlation analysis demonstrated that the level of IL-38 in the intestinal mucosal tissue of UC patients was negatively correlated with CRP, ESR and Mayo scores (r=-0.291, -0.672 and -0.639; all P<0.05). And the level of IL-38 in the intestinal mucosal tissue of CD patients was negatively correlated with CRP, ESR and CDAI (r=-0.559, -0.471 and -0.353; all P<0.05). The IHC results showed that the levels of NF-κB and p-STAT3 of IL-38 transgenic IBD mice were lower than those of wild type IBD mice (0.14±0.02 vs. 0.32±0.06, 0.12±0.02 vs. 0.44±0.07), and the differences were statistically significant (t=6.971 and 10.767, both P<0.05). The results of flow cytometry showed that the ratio of CD4+ IL-17+ Th17 cells in the peripheral blood of IL-38 transgenic IBD mice was lower than that of wild type IBD mice (0.030±0.006 vs. 0.280±0.050), and the difference was statistically significant (t=12.160, P<0.05). Conclusions The expression level of IL-38 significantly decreases in the intestinal mucosal tissues of IBD patients, while the level of NF-κB and p-STAT3 significantly increases. IL-38 may inhibit IBD by regulating NF-κB and p-STAT3 signaling pathway to alleviate intestinal immune reaction. Key words: Inflammatory bowel diseases; NF-kappa B; interleukin-38; STAT3

Transforming growth factor-β dampens interleukin-6 signaling in chondrocytes by decreasing the interleukin-6 receptor

Purpose: Interleukin-6 (IL-6) is a pro-inflammatory cytokine present in increased levels in serum and synovial fluid from patients with osteoarthritis (OA) and is even considered to be predictive for disease progression. Previous studies implicate IL-6 in articular cartilage destruction, which is the main hallmark of OA. In contrast, transforming growth factor-β (TGF-β) is an important homeostatic regulator of cartilage that can decrease the catabolic effects of pro-inflammatory cytokines in chondrocytes. Multiple mechanisms of cross-talk between TGF-β and IL-6 are reported in other tissues. In this study, we investigated whether TGF-β is able to control IL-6 signaling in chondrocytes. Methods: Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with rhTGF-β1 (1.0 ng/ml). We determined mRNA expression levels of IL-6 and IL-6 receptor (IL-6R) using qPCR, measured IL-6 protein release using Luminex multianalyte technology and IL-6R protein levels by Western Blot. rhIL-1β was used as a positive control for IL-6 induction. Induction of signal transducer and activator of transcription (STAT)3 phosphorylation by TGF-β was determined using Western Blot, in presence of the IL-6R inhibitor Tocilizumab to study IL-6R dependency. Induction of suppressor of cytokine signaling (SOCS)3 gene expression was used as a read-out for IL-6/STAT3 mediated transcriptional activity. Results: TGF-β increased both IL-6 mRNA expression and protein release in G6 and primary chondrocytes. Moreover, we observed activation of the main IL-6R downstream signaling protein STAT3 after TGF-β exposure, which could be completely prevented by blockade of the IL-6R, suggesting IL-6 dependency. We further explored whether TGF-β mediated IL-6/p-STAT3 signaling resulted in activation of target genes, and measured expression of SOCS3, which is a well-known IL-6 target gene as well as an important regulator of IL-6 signaling. Surprisingly however, TGF-β did not increase SOCS3 expression, despite the increased IL-6 and p-STAT3 levels we observed in these cells. This unexpected finding suggests regulation of STAT3 mediated transcriptional activity by TGF-β. The effect of exogenously added rhIL-6 was also inhibited by TGF-β, as 6h pre-treatment with TGF-β prevented rhIL-6-induced SOCS3 expression. At the level of STAT3, TGF-β decreased the response to rhIL-6 as well, although p-STAT3 levels were not reduced to baseline level. This points towards a crucial role for TGF-β in the regulation of IL-6 effects upstream of STAT3. Interestingly, we observed decreased expression of the IL-6R in both primary and G6 chondrocytes after exposure to TGF-β, which might explain the decreased intracellular IL-6 signaling. This effect was not dependent on IL-6 itself, as IL-1β did not affect IL-6R expression, despite being a strong inducer of IL-6. Finally, we showed that rescuing IL-6R levels using soluble IL-6R dose-dependently restored induction of SOCS3 and p-STAT3 by rhIL-6 in the presence of TGF-β. This confirms that TGF-β inhibition of IL-6 effects is indeed dependent on reduced IL-6R levels. Conclusions: Our study reveals a novel, protective effect of TGF-β that could help preserve cartilage homeostasis under inflammatory conditions by dampening IL-6 signaling via IL-6R down regulation.

Antiproliferative and Anti-invasion Effects of Carvacrol on PC3 Human Prostate Cancer Cells through Reducing pSTAT3, pAKT, and pERK1/2 Signaling Proteins.

Background:One of the most effective parameters in the progression of the prostate cancer is interleukin (IL)-6 through affecting pSTAT3, pERK1/2, and pAKT cell signaling proteins. Carvacrol is an herbal antioxidant with antitumor effects. The purpose of this study was to investigate the effects of carvacrol on IL-6 gene expression, pSTAT3, pAKT, pERK1/2 cellular signaling proteins, and invasion in human prostate cancer PC3 cells.Methods:PC3 cell viability was evaluated by MTT assay with different concentrations of carvacrol (0–800 μM). IL-6 gene expression and cellular concentration of pSTAT3, pERK1/2, and pAKT were investigated using the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting technic, respectively. PC3 cell invasion was determined by invasion assay test.Results:Carvacrol IC50 for PC3 prostate cancer cells was 360 μM. Carvacrol led to a significant reduction (P < 0.05) for IL-6 gene expression in a dose-dependent manner compared to control. IL-6 protein reduced 41.5% and 52.7% when compared with control cells at 360 and 420 μM of carvacrol, respectively. Carvacrol led to a decline in pSTAT3, pAKT, and pERK1/2 above 360 μM compared to control. PC3 potential invasion was significantly reduced after treatment with carvacrol in a dose-dependent manner.Conclusions:Decreased IL-6 protein level by carvacrol resulted in diminishing of pSTAT3, pERK1/2, and pAKT signaling proteins, which leads to the reduction of the cell survival, proliferation, and invasion in PC3 cells.

The miR-561-5p/CX3CL1 Signaling Axis Regulates Pulmonary Metastasis in Hepatocellular Carcinoma Involving CX3CR1+ Natural Killer Cells Infiltration

Natural killer (NK) cell can inhibit tumor initiation and regulates metastatic dissemination, acting as key mediators of the innate immune response. Intrinsic factors modulating NK cells infiltration and its anticancer activity remain poorly characterized. We investigated the roles of dysregulation of micro(mi)RNAs and NK cells in progression of hepatocellular carcinoma (HCC).Methods: Small RNA sequencing were used to detect the miRNA profiles of tumor tissues from HCC patients with (n=14) or without (n=13) pulmonary metastasis and HCC cell lines with different pulmonary metastatic potentials. Chemokine expression profiling and bioinformatics were used to detect the downstream target of candidate target. In gain- and loss-of-function assays were used to investigate the role of miRNA in HCC progression. Different subsets of NK cells were isolated and used for chemotaxis and functional assays in vivo and in vitro. In situ hybridization and immunohistochemical analyses were performed to detect the expression of miRNA in tumor tissues from 242 HCC patients undergoing curative resection from 2010.Results: Three miRNAs (miR-137, miR-149-5p, and miR-561-5p) were identified to be associated with pulmonary metastasis in patients with HCC. miR-561-5p was most highly overexpressed in metastatic HCC tissues and high-metastatic-potential HCC cell lines. In gain- and loss-of-function assays in a murine model, miR-561-5p promoted tumor growth and spread to the lungs. Yet, miR-561-5p did not appear to affect cellular proliferation and migration in vitro. Bioinformatics and chemokine expression profiling identified chemokine (C-X3-C motif) ligand 1 (CX3CL1) as a potential target of miR-561-5p. Furthermore, miR-561-5p promoted tumorigenesis and metastasis via CX3CL1-dependent regulation of CX3CR1+ NK cell infiltration and function. CX3CR1+ NK cells demonstrated stronger in vivo anti-metastatic activity relative to CX3CR1- NK cells. CX3CL1 stimulated chemotactic migration and cytotoxicity in CX3CR1+ NK cells via STAT3 signaling. Blockade of CX3CL1, CX3CR1, or of pSTAT3 signaling pathways attenuated the antitumor responses. Clinical samples exhibited a negative correlation between miR-561-5p expression and levels of CX3CL1 and CX3CR1+ NK cells. High miR-561-5p abundance, low CX3CL1 levels, and low numbers of CX3CR1+ NK cells were associated with adverse prognosis.Conclusion: We delineated a miR-561-5p/CX3CL1/NK cell axis that drives HCC metastasis and demonstrated that CX3CR1+ NK cells serve as potent antitumor therapeutic effectors.

MYD88 Triggered SYK Activation Promotes BCR Cross-Talk, and Identifies SYK As a Novel Therapeutic Target of Mutated MYD88 Signaling

Activating mutations in MYD88, a component of the Toll-receptor (TLR) pathway, trigger Myddosome self-assembly and multiple downstream pro-survival signaling through BTK and IRAK4/IRAK1 (Yang et al, Blood 2013). Activation of B-cell receptor (BCR) signaling has also been observed in WM, though the mechanism(s) remain to be clarified (Argyropoulos et al, Leukemia 2016). While activating mutations in the BCR components CD79A/B are common in MYD88 mutated ABC DLBCL, they are uncommon in WM (Ngo et al, Nature 2011; Hunter et al, Blood 2014). We therefore sought to clarify if TLR/MYD88 crosstalk could explain aberrant BCR signaling in WM. We performed PhosFlow analysis of MYD88 and BCR signaling components of MYD88 mutated and wild-type WM and ABC DLBCL cell lines. These studies showed high levels for expression of the BCR component p-SYK (Y525-526) in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cell lines versus MYD88 wild-type cell lines. High levels of p-SYK were also observed in primary MYD88 mutated WM cells compared to healthy donor peripheral blood B-cells. Following treatment with a MYD88 blocking peptide, p-SYK was robustly reduced in WM cell lines, while only modest reduction was observed in ABC DLBCL cell lines which also carry activating CD79B mutations. Use of MYD88 inhibitor also blocked the p-SYK in primary MYD88 mutated WM cells. Lentiviral over-expression of MYD88 L265P but not wild-type MYD88 or vector control augmented p-SYK expression in MYD88 mutated BCWM.1 WM cells, as well as MYD88 wild-type Ramos and OCI-Ly7 cells. Conversely, knockdown of MYD88 in BCWM.1 cells confirmed the dependence of p-SYK on mutated MYD88. p-SYK was also confirmed to be in complex with the Myddosome in BCWM.1 cells by co-immunoprecipitation (co-IP) using anti-MYD88 and anti-SYK/anti-p-SYK specific antibodies. Over-expression and knockdown studies of mutated MYD88 also showed an association of downstream p-STAT3 (Y705) signaling on MYD88 triggered p-SYK, a finding confirmed by use of SYK inhibitors which blocked p-STAT3 in a dose-dependent manner. CellTiter-Glo® viability assays showed the SYK inhibitors, R406 and Entospletinib (GS-9973), produced higher cytotoxicity in MYD88 mutated versus wild-type B-cell lines. (Figure 1A) Since BCR/SYK kinase triggers divergent signaling from TLR/BTK/IRAK pathways, we examined the combined effect of BTK and SYK inhibition. Combination index and normalized isobologram analysis demonstrated synergistic effects with the combination of ibrutinib with either R406 or Entospletinib in MYD88 mutated WM and ABC DLBCL cell lines (Figure 1B, 1C). The combination of ibrutinib with either R406 or Entospletinib also produced more robust tumor cell apoptosis in primary MYD88 mutated WM cells. Our findings show that MYD88 can cross-talk with BCR pathway through SYK tyrosine kinase, and trigger aberrant p-STAT3 signaling. The inhibition of both TLR/BTK and BCR/SYK activation by use of ibrutinib and SYK inhibitors produced synergistic tumor cell killing of MYD88 mutated WM and ABC DLBCL lymphomas, and provides a framework for clinical studies aimed at extinguishing aberrant MYD88 signaling. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Hunter:Pharmacyclics: Consultancy. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

IDH2 Mutations Induce Altered STAT Signaling and Cytokine Responses Which Are Restored By Enasidenib

Acute myeloid leukemia (AML) is a heterogeneous myeloid malignancy characterized by mutational and clonal heterogeneity. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are common, occurring in approximately 15-20% of patients, and actionable, with recently approved inhibitors for both mutations. These inhibitors lead to leukemia cell differentiation in vitro, in vivo and in patients. Healthy myeloid differentiation is governed by precise regulation of intracellular signaling, but this regulation is disrupted in AML. Given that signal transducer and activator of transcription (STAT) proteins are involved in both leukemogenesis and myeloid differentiation, we sought to determine the role of phosphorylated STATs and other signaling proteins in mIDH AML and inhibitor-induced differentiation of mIDH2 AML.To simultaneously dissect single cell signaling, differentiation and epigenetic changes, we employed fluorescence flow cytometry and mass cytometry to study an in vitro model of mIDH2R140Q leukemia, consisting of a parental TF-1 leukemia cell line and a CRISPR/Cas9 gene-edited mIDH2R140Q-mutated cell line. In order to generate provoked signaling profiles, we stimulated both the wild-type IDH2 and mutant IDH2 cells with nine cytokines. Additionally, we treated both cell lines and primary mIDH2 AML samples ex vivo with enasidenib and measured changes in high-dimensional single cell phenotype and phospho-protein expression via mass cytometry.mIDH2 leukemia cells displayed elevated basal pSTAT1, pSTAT3 and pNFkB-s529 expression, and concomitant low basal IkBa levels when compared to parental cells. Further, mIDH2 cells had increased PMA induced pS6 and IL1β induced pNFkB-s529 than the parental cell line, while wtIDH2 cells had higher levels of cytokine-induced pSTAT1, pSTAT3 and pSTAT5.After prolonged enasidenib treatment (28 days), model mIDH2 cells expressed lower basal pSTAT1, pSTAT3 and pNFkB-s529 than vehicle-treated mIDH2 cells. Further, when stimulated with GM-CSF, enasidenib-treated mIDH2 leukemia cells showed increased response at pSTAT3 and pSTAT5 as compared to vehicle-treated leukemia cells. We dissected enasidenib-induced differentiation using 18 cell surface markers and visualized results using t-distributed stochastic neighbor embedding (tSNE). Presence of mIDH2 leads to baseline expression differences including higher CD90 and CD71. Following treatment with enasidenib, mIDH2 leukemia cells had increased CD45 and CD11b expression as compared to vehicle-treated controls. Additionally, prolonged treatment with enasidenib increased proliferation as shown by increased Ki67 and decreased histone hypermethylation at suppressive histone marks, H3K27 and H3K9, while 7 days of enasidenib did not result in changes to these marks.We further explored the impact of mIDH2 in primary leukemia samples. First, we developed an ex vivo stromal co-culture system that allowed for treatment and expansion of four primary mIDH2 AML samples for 16 days. While the basal levels of signaling markers were sample dependent, consistently across all samples enasidenib-treated primary AML was more sensitive to IL-6 and GM-CSF-induced pSTAT1, pSTAT3 and pSTAT5 signaling. The enasidenib-treated AML also showed increased expression of mature myeloid markers, including CD33 and CD11c.Here we have shown the presence of mIDH2 mutations leads to decreased STAT signaling in response to cytokine stimulation as compared to wtIDH2 AML cells. We have also demonstrated increased response of pNFkB-s529 and pS6 to cytokine stimulation in mIDH2 AML cells as compared to wtIDH2 AML cells. Moreover, in both mIDH2 AML cell line and primary samples, IDH2 inhibitor-induced differentiation restored sensitivity to cytokine responses and reduced histone hypermethylation. Future work exploring these aberrant signaling events may reveal precise connections between mutated IDH, associated epigenetic changes, and intracellular signaling, potentially uncovering synergistic therapeutic strategies for mIDH AML. DisclosuresFerrell:Incyte: Research Funding.

The protective microRNA-199a-5p-mediated unfolded protein response in hypoxic cardiomyocytes is regulated by STAT3 pathway.

The protective effects of downregulated miR-199a-5p on ischemic and hypoxic cardiomyocytes were well recognized, but the underlying mechanism of inhibited miR-199a-5p is not yet clear. The present study explored the relationship between enhanced signal transducer and activator of transcription 3 (STAT3) signaling and lowered production of miR-199a-5p in hypoxic cardiomyocytes. This study firstly found the correlation between elevated interleukin (IL)-6 and IL-11, as well as subsequent STAT3 signaling activation and the downregulation of miR-199a-5p in hypoxic myocardial samples from children with congenital heart disease. Then, using model of hypoxic mice and the intervention of phosphorylated STAT3 (pSTAT3), it was observed that pSTAT3 affected the expression of miR-199a-5p and modulated the expression of its target genes, including endoplasmic reticulum stress (ERS)-related activating transcription factor 6 (ATF6) and 78kDa glucose-regulated protein (GRP78). Further observation revealed that the pSTAT3 signal in cardiac tissue could affect the expression of pri-miR-199a-2, a precursor of miR-199a-5p. And the chromatin immunoprecipitation (ChIP) assay also confirmed that pSTAT3 could bind to the promoter region of miR-199a-2 gene, which is more significant under hypoxic conditions. In conclusion, the activation of STAT3 signaling in cardiomyocytes during chronic hypoxia leads to downregulation of miR-199a-5p, which promotes the expression of many downstream target genes. This is an important pathway in the adaptive protection mechanism of myocardium during hypoxia.

Preventive effect of bergenin against the development of TNBS-induced acute colitis in rats is associated with inflammatory mediators inhibition and NLRP3/ASC inflammasome signaling pathways

Ulcerative colitis is an idiopathic inflammatory bowel disease characterized by intestinal inflammation; blocking this inflammatory process may be the key to the development of new naturally occurring anti-inflammatory drugs, with greater efficiency and lower side effects. The objective of this study is to explore the effects of bergenin (BG) in TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced acute colitis model in rats in order to assist in the studies for the development of novel natural product therapies for inflammatory bowel disease. 48 Wistar rats were randomized into six groups: (i) Control and (ii) TNBS control; (iii) 5-ASA 100 mg/kg/day (iv) BG 12 mg/kg/day (v) BG 25 mg/kg/day and (vi) BG 50 mg/kg/day. Colitis was induced by instillation of TNBS. Colitis was evaluated by an independent observer who was blinded to the treatment. Our results revealed that bergenin decreased the macroscopic and microscopic damage signs of colitis, and reduced the degree of neutrophilic infiltration in the colon tissue; also, it was capable to down-regulate COX-2, iNOS, IkB-α, and pSTAT3 protein expression. Similarly, using a protocol for indirect ELISA quantification of cytokines, bergenin treatment reduced IL-1β, IFN-γ and IL-10 levels, and inhibited both canonical (IL-1) and non-canonical (IL-11) NLRP3/ASC inflammasome signaling pathways in TNBS-induced acute colitis. Conclusion: Our study has provided evidence that administration of bergenin reduced the damage caused by TNBS in an experimental model of acute colitis in rats, reduced levels of pro-inflammatory proteins and cytokines probably by modulation of pSTAT3 and NF-κB signaling and blocking canonical and non-canonical NLRP3/ASC inflammasome pathways.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Enhanced IL-6/phosphorylated STAT3 signaling is related to the imbalance of circulating T follicular helper/T follicular regulatory cells in patients with rheumatoid arthritis

BackgroundFollicular helper T (Tfh) cells are specialized in helping B lymphocytes, which play a central role in autoimmune diseases that have a major B cell component, such as in rheumatoid arthritis (RA). Follicular regulatory T (Tfr) cells control the over-activation of Tfh and B cells in germinal centers. Dysregulation of Tfh cells and Tfr cells has been reported to be involved in the pathogenesis of some autoimmune diseases. However, the balance of Tfh and Tfr cells, and their roles in the development and progression of RA are still not clear.MethodsIn this study, we enrolled 44 patients with RA (20 patients with active RA and 24 patients with inactive RA) and 20 healthy controls, and analyzed the frequencies of circulating Tfh and Tfr cells, expression of programmed death-1 (PD-1), inducible co-stimulator (ICOS), intracellular IL-21, and pSTAT3 in Tfh cells, and serum levels of IL-6. The correlation among these parameters and that of Tfh or Tfr cells with disease activity were also analyzed.ResultsPatients with RA (especially active RA) had higher frequencies of Tfh cells, but lower percentages of Tfr cells, thereby resulting in elevated ratios of Tfh/Tfr. Expression levels of PD-1 and IL-21 in Tfh cells were higher in patients with RA than in healthy subjects, while no difference in ICOS expression was observed between patients and controls. Both pSTAT3 expression and serum IL-6 levels increased in patients with RA, and positive correlation between them was observed. Additionally, pSTAT3 expression was positively correlated with Tfh cell frequency. The Disease Activity Score in 28 joints based on C-reactive protein (DAS28-CRP) was negatively correlated with Tfr cell frequency, but was positively correlated with both Tfh/Tfr ratio and PD-1 expression.ConclusionsResults demonstrated that enhanced IL-6/pSTAT3 signaling may contribute to promotion of Tfh cells, consequently skewing the ratio of Tfh to Tfr cells, which may be crucial for disease progression in RA.

Desmoglein 2, but not desmocollin 2, protects intestinal epithelia from injury.

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.

Abstract B023: Identification and biochemical validation of piperlongumine as a potent therapeutic against neuroendocrine prostate cancer

Abstract Introduction and Objectives: Recent studies have highlighted the existence of a highly lethal and drug-resistant variant of prostate cancer (PCa) termed neuroendocrine prostate cancer (NEPC). We have used a genomics-based drug-repositioning approach to identify piperlongumine, a natural product constituent of the fruit of the long pepper (Piper longum), as a potential therapeutic against NEPC. The efficacy of this drug was tested in the NEPC cell line model NCI-H660 and compared to several other PCa cell lines in a modified WST-1 assay. Preclinical testing in mouse xenograft models of NEPC was also undertaken. Finally, the ability of piperlongumine to inhibit p-STAT3 signaling and promote apoptosis in cell lines and extracted tumors were measured by Western blot analyses. Methods: PCa cell lines (LNCaP, 22Rv1, Du145, PC3, H660, and RWPE) were grown in complete media (RPM1 10% FBS, Advanced DMEM 5% FBS, or Keratinocyte-SFM) and treated with piperlongumine for 3 or 7 days. IC50 values were generated from WST assay data using Prism6. Nude mice (n=5) were injected with 1.5x106 H660 cells on each flank, and intraperitoneally administered with either 2.5mg/kg piperlongumine (n=3) or DMSO (n=2) daily for 3 weeks after tumors formed 200mm3 in volume. Tumor volume was measured daily with calipers. Western blot analyses were performed on protein lysates extracted from tumors and PCa cells treated with 0-10 μM of drug. Results: Piperlongumine was highly effective in inhibiting the growth of H660 cells (IC50 = 0.4 μM) compared to LNCaP, 22Rv1, Du145, PC3, and RWPE cells. On average, the growth rate of untreated tumors was 2.7 times greater than those treated was piperlongumine. Treated H660 cells exhibited significant inhibition of p-STAT3 and increases in cPARP1 levels while other cell lines displayed little to no fluctuation. Conclusions: Using drug-repositioning approaches we have identified a lead compound that inhibits the growth of the highly drug-resistant NEPC cell line NCI-H660. Remarkably, piperlongumine happens to be a water-soluble plant product with no known side effects. Citation Format: Kamlesh K. Yadav, Khader Shameer, Jennifer A. Stockert, Cordelia Elaiho, Ben Readhead, Shalini S. Yadav, Joel Dudley, Ashutosh K. Tewari. Identification and biochemical validation of piperlongumine as a potent therapeutic against neuroendocrine prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B023.

Augmented Insulin and Leptin Resistance of High Fat Diet-Fed APPswe/PS1dE9 Transgenic Mice Exacerbate Obesity and Glycemic Dysregulation.

Alzheimer’s disease (AD), a progressive neurodegenerative disease is highly associated with metabolic syndromes. We previously demonstrated that glycemic dysregulation and obesity are augmented in high fat diet (HFD)-treated APPswe/PS1dE9 (APP/PS1) transgenic mice. In the current study, the underlying mechanism mediating exacerbated metabolic stresses in HFD APP/PS1 transgenic mice was further examined. APP/PS1 mice developed insulin resistance and, consequently, impaired glucose homeostasis after 10 weeks on HFD. [18F]-2-fluoro-2-deoxy-d-glucose ([18F]-FDG) positron emission tomography showed that interscapular brown adipose tissue is vulnerable to HFD and AD-related pathology. Chronic HFD induced hyperphagia, with limited effects on basal metabolic rates in APP/PS1 transgenic mice. Excessive food intake may be caused by impairment of leptin signaling in the hypothalamus because leptin failed to suppress the food intake of HFD APP/PS1 transgenic mice. Leptin-induced pSTAT3 signaling in the arcuate nucleus was attenuated. Dysregulated energy homeostasis including hyperphagia and exacerbated obesity was elicited prior to the presence of the amyloid pathology in the hypothalamus of HFD APP/PS1 transgenic mice; nevertheless, cortical neuroinflammation and the level of serum Aβ and IL-6 were significantly elevated. Our study demonstrates the pivotal role of AD-related pathology in augmenting HFD-induced insulin and leptin resistance and impairing hypothalamic regulation of energy homeostasis.

Abstract 2514: Most clinically approved anti-EGFR antibodies fail to neutralize EGFRvIII, explaining their lack of efficacy in high-grade glioma

Abstract The epidermal growth factor receptor (EGFR), a cell-surface receptor tyrosine kinase, is amplified or overexpressed in 60% of gliomas. Furthermore, in 30% of gliomas, an autoactive, disulphide-bonded truncation mutant, EGFRvIII, is co-expressed. Both the wild-type (wt) and EGFRvIII are central to driving gliomagenesis. Multiple anti-EGFR antibodies have been developed to neutralise these receptors; however, despite promising preclinical results, their efficacy against glioma in the clinic has been disappointing for reasons that are not understood. We have tested the efficacy and mechanism of action of the majority of known FDA-approved or preclinical anti-EGFR antibodies [panitumumab, cetuximab, necitumumab, nimotuzumab, matuzumab and chimeric 806 (ch806)] against five primary EGFRvIII-bearing glioma lines. We find that cetuximab, necitumumab, nimotuzumab and matuzumab all fail to fully neutralize EGFRvIII activity, leading to sustained downstream signalling and modest antiproliferative effects. ch806 neutralized EGFRvIII activity but did not engage and neutralize wtEGFR. Only panitumumab was able to completely neutralize both wtEGFR and EGFRvIII activation, resulting in a superior antiproliferative response. Not all EGFRvIII-bearing cell lines were responsive to panitumumab (3 out of 5), suggesting that the expression of EGFRvIII is not indicative of sensitivity to anti-EGFR therapy and that additional mechanisms of resistance are present when EGFRvIII is expressed. Only when cetuximab or necitumumab was combined with ch806, to neutralize both wtEGFR and EGFRvIII, was an antiproliferative response similar to panitumumab achieved. Mechanistically, panitumumab neutralized p-ERK but not p-AKT or p-STAT3 signaling. Panitumumab, cetuximab and ch806 induced EGFRvIII internalization and recycling by Rab11-positive endosomes back to the cell surface, while wtEGFR was degraded. Subsequent wtEGFR translation replaced the degraded wtEGFR, ensuring receptor pool replenishment. Finally, panitumumab does not exert its effects by breaking the active EGFRvIII disulphide-bonded dimer; rather, it may work by trapping EGFRvIII into an inactive high molecular weight receptor cluster, an effect that was not observed for cetuximab. Animal trials to compare the efficacy of these antibodies against one another in intracranial orthografts of patient-derived cell lines are ongoing. Collectively, our results challenge the existing premise that EGFRvIII is neutralized and degraded by EGFR antibodies and demonstrate that both wtEGFR and EGFRvIII must be neutralized for maximum antitumor effect. These results may help explain why existing antibodies such as cetuximab and nimotuzumab have failed in clinical trials in glioma and provide rationale that panitumumab may offer a superior and effective candidate for a further clinical trial in glioma. Citation Format: Sameer A. Greenall, Timothy E. Adams, Terrance G. Johns. Most clinically approved anti-EGFR antibodies fail to neutralize EGFRvIII, explaining their lack of efficacy in high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2514.

Abstract 261: Chemopreventive effects of flavonoids on pancreatic carcinogenesis

Abstract Although clinical therapies are being developed for combating pancreatic cancer, its prognosis remains dismal; consequently, chemopreventive strategies may be needed to manage this disease. Luteolin (Lut), a flavone contained in perilla, is one of the most effective antioxidant flavonoids and has demonstrated potential for the treatment of inflammatory and neoplastic disease. Hesperetin (Hes) is a flavone contained in citrus, and the Ohsaki cohort study showed that citrus consumption was correlated with reduced pancreatic cancer (RR=0.62, 95%CI=0.38-1.00) (Int.J.Cancer, 2010: Li.W). However, there are no studies dealing with their antineoplastic effects against pancreatic cancer. In the present study, to assess the chemopreventive effects of Lut and Hes for pancreatic carcinogenesis, 6 week-old syrian golden hamsters were subcutaneously injected with N-nitrsobis(2-oxopropyl)amine four times and then fed Quick Fat Diet (control, n=14) or Quick Fat Diets with Lut 100 ppm (n=13) or Hes 100 ppm, 1000 ppm (n=13,13) for 13 weeks. There were no differences in body weights or liver or kidney weights among the groups. Serum cholesterol was significantly decreased in the Lut group. The incidence and multiplicity of pancreatic cancer was significantly suppressed in the Lut group (Incidence/ Multiplicity: 23%/ 0.23±0.44; P&amp;lt;0.05), but not the Hes groups (100ppm: 54%/ 0.69±0.75, 1000ppm: 62%/ 0.77±0.73), compared to the controls (71%/ 0.93±0.73). The Ki67 labeling index in pancreastic intraepithelial neoplasias (PanINs) was significantly decreased by Lut, but not Hes. Therefore, we focused further investigation on Lut. In vitro, cell proliferation of both hamster (HPD1NR) and human (MiaPaCa, PANC1) pancreatic cancer cell lines was inhibited by Lut in a dose-dependent manner. Multi-blot analysis for phosphorylated proteins related to cell signaling revealed that phosphorylated forms of Stat3 (tyr705) were down-regulated by Lut in MiaPaCa and PANC1 cells. We confirmed them in a dose-dependent manner in the three pancreatic cell lines and also found that Lut down-regulated cyclin D1. In vivo, the nuclear positive proportion of pStat3 (tyr705) in both PanINs and adenocarcinomas was significantly decreased by Lut. Because pStat3 (tyr705) is regulated by cytokines such as Il6, we examined mRNA levels of several cytokines including Il6 in hamster pancreatic tissues. Lut decreased the mRNA expression of Tgfb, Tnfa, Il10, and Il6. Immunohistochemistry of alpha smooth muscle actin revealed that the positive area of activated pancreatic stellate cells, which can produce these cytokines in cancerous tissue, tended to be decreased by Lut. In conclusion, these results indicate that Lut, but not Hes, has potential as a chemopreventive agent for pancreatic cancer and inhibition of pStat3 signaling may be involved in its mechanism of action. Citation Format: Hiroyuki Kato, Aya Naiki, Shugo Suzuki, Yoriko Yamashita, Satoru Takahashi. Chemopreventive effects of flavonoids on pancreatic carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 261.

Abstract P3-05-19: Regorafenib induces immunogenic cell death via p-stat3 inhibition in triple negative breast cancer cells

Abstract Background Metastatic triple-negative breast cancer (TNBC), despite being chemo-sensitive, remains poor prognostic. Rising data have suggested some anti-cancer agents exert immunostimulatory activities through inducing immunogenic cell death (ICD). ICD can be detected by presence of damage-associated molecular patterns (DAMPs) such as cell surface exposure of calreticulin (CRT), secretion of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells. Signal transducer and activator of transcription 3 (STAT3) activity is activated in TNBC and regorafenib has been shown to suppress p-STAT3 signal. We tested whether regorafenib induces ICD in TNBC. Methods Mice TNBC cell line 4T1 cells were treated with regorafenib and cell survival was examined by MTT assay and flow cytometric analysis. DAMPs were examined by western blot analysis, immunofluorescence microscopy and luminescent assay. The correlation between regorafenib-mediated ICD and STAT3 inhibition were validated in ectopic STAT3 transfected 4T1 cells. Moreover, we investigated the combination strategies of regorafenib with immune checkpoint blockade in syngeneic 4T1 tumor bearing mice (TNBC animal model) with treatment of regorafenib or mPD1 treatment alone/ combination of mPD1 and regorafenib. Results The results demonstrated that regorafenib reduced cell survival and induced cell apoptosis in a dose-dependent pattern in 4T1 cell line. Regorafenib induced ICD, as evidenced by it triggered the release of HMGB1 and ATP, as well as the exposure of CRT on the cell surface. Moreover, regorafenib-induced ICD was attenuated by ectopic expression of STAT3 (thus increased phosphorylation of STAT3) in STAT3-overexpressed 4T1 cells. Last but not the least, we observed 4T1 tumor bearing mice with treatment of regorafenib alone, or anti-PD1 monoclonal antibody (mAb) alone, or in combination of regorafenib and anti–PD1 mAb resulted in reduced size of primary tumors, increased survival and fewer lung metastases. Flow cytometric analysis revealed regorafenib treatment increased activated CD8+ T cells, increased antigen-presenting dendritic cells, and suppressed regulatory T cells (Tregs) in mice spleen. Conclusions Regorafenib induced ICD in 4T1 cells via the inhibition of p-STAT3. Regorafenib also promoted CD8+ T cells activation and antigen presenting ability of dendritic cells, and suppressed Tregs. Our study demonstrated regorafenib as an ICD-inducer and immunomodulator and its potential combination with immune checkpoint blockade in TNBC. Citation Format: Liu C-Y, Lau K-Y, Huang T-T, Chen J-L, Chu P-Y, Huang C-T, Wang W-L, Tseng L-M. Regorafenib induces immunogenic cell death via p-stat3 inhibition in triple negative breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-19.