Recruitment Of STAT3 Research Articles

USP7 inhibits the progression of nasopharyngeal carcinoma via promoting SPLUNC1-mediated M1 macrophage polarization through TRIM24

Reprogramming of macrophages toward an M1 phenotype is a novel strategy to induce anticancer immunity. However, the regulatory mechanisms of M1 macrophage polarization and its functional roles in nasopharyngeal carcinoma (NPC) progression need to be further explored. Here we found that SPLUNC1 was highly expressed and responsible for M1 macrophage polarization. JAK/STATs pathway activation was involved in SPLUNC1-mediated M1 macrophage polarization. Importantly, regulation of SPLUNC1 in macrophages affected CM-mediated influence on NPC cell proliferation and migration. Mechanistically, USP7 deubiquitinated and stabilized TRIM24, which promoted SPLUNC1 expression via recruitment of STAT3 in M1 macrophages. Depletion of TRIM24 inhibited M1 macrophage polarization, which facilitated NPC cell growth and migration. However, over-expression of USP7 exhibited the opposite results and counteracted the tumorigenic effect of TRIM24 silencing. Finally, the growth and metastasis of NPC cells in vivo were repressed by USP7-induced M1 macrophage polarization via modulating TRIM24/SPLUNC1 axis. USP7 delayed NPC progression via promoting macrophage polarization toward M1 through regulating TRIM24/SPLUNC1 pathway, providing evidence for the development of effective antitumor immunotherapies for NPC.

Abstract 1461: C-terminally phosphorylated p27 modulates chromatin remodeling in breast cancer

Abstract Breast Cancer (BC) is the leading cause of cancer-related death in women. Transcriptional deregulation is a frequent characteristic of cancers. Chromatin remodeling determines the accessibility of the chromatin through dynamic modification of chromatin architecture, and thereby regulates gene expression. Alteration in chromatin remodeling is associated with cancer. Therefore, a greater understanding of chromatin remodeling and its role in tumorigenesis could provide a significant cornerstone to develop more effective therapeutic strategies. p27 is an integral regulator of the cell cycle. It plays a dual role in tumorigenesis. p27 acts as a tumor suppressor to restrain the cell cycle, but upon C-terminal phosphorylation at T157 and T198 by PI3K activated kinases, it acts as an oncogene to promote cancer metastasis. We previously showed that p27pT157pT198 (p27pTpT) acts as a transcriptional co-regulator of cJun. Our new data now indicate that p27pTpT also binds to and transcriptionally co-regulates STAT3 to drive cancer stem cell gene programs. We also found that p27 is widely recruited to consensus motifs shared by many other transcription factors, suggesting a broader role in transcriptional regulation. Here we investigated if p27pTpT regulates transcriptional programs in BC through modulating chromatin accessibility. ATACseq was carried out in 231 cells, 231 with overexpression of a cell cycle-defective phosphomimetic p27 (231p27CK-DD), and in 1883 cells with high phospho-activated p27 levels, and 1833 with p27 knockdown and analyzed together with our prior p27 ChIPseq. p27pTpT increased ATAC-seq signals at the promoter of a sub-set of STAT3 target genes, where p27 ChIPseq and ATACseq signals overlapped. These genes are upregulated in 231p27CK-DD and in 1833 compared to 231. Thus, p27 appears to potentiate a more open chromatin state at these binding sites to modulate gene expression. p27 and STAT3 co-occupy at the promoters of the STAT3 target genes including MYC, IL6, and VEGFA, in which p27 knockdown decreased STAT3 recruitment to these sites. Using ENCODE p300 and CBP ChIP-seq data, we found potential co-localization of histone acetyltransferase (HAT), CBP and p300, with p27 and STAT3 on MYC, IL6, and VEGFA promoters. Thus, p27 might recruit HATs to the chromatin to increase chromatin accessibility through H3K27 acetylation (H3K27ac). ChIP-qPCR assays validated that p27, STAT3 and CBP recruitment to the MYC promoter was greater in p27-activated 231p27CK-DD and 1833 lines than in 231, and decreased upon p27 depletion in 1833. Notably, ChIP assays also showed CBP recruitment increased H3K27ac at this site. These data support a model in which p27 enrichment to the promoter of a subset of STAT3 target genes increases both CBP and STAT3 recruitment, to increase chromatin accessibility through H3K27ac and upregulate their expression. This work revealed a novel genome-wide action of p27 in chromatin remodeling. Citation Format: Seyedeh Fatemeh Razavipour, Hyunho Yoon, Miyoung Shin, Alec McIntosh, Dekuang Zhao, Amir Bagheri, Derek Van Booven, Chunling Yi, Joyce Slingerland. C-terminally phosphorylated p27 modulates chromatin remodeling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1461.

Src-FAK Signaling Mediates Interleukin 6-Induced HCT116 Colorectal Cancer Epithelial–Mesenchymal Transition

Colorectal cancer is one of the most prevalent and lethal malignancies, affecting approximately 900,000 individuals each year worldwide. Patients with colorectal cancer are found with elevated serum interleukin-6 (IL-6), which is associated with advanced tumor grades and is related to their poor survival outcomes. Although IL-6 is recognized as a potent inducer of colorectal cancer progression, the detail mechanisms underlying IL-6-induced colorectal cancer epithelial-mesenchymal transition (EMT), one of the major process of tumor metastasis, remain unclear. In the present study, we investigated the regulatory role of IL-6 signaling in colorectal cancer EMT using HCT116 human colorectal cancer cells. We noted that the expression of epithelial marker E-cadherin was reduced in HCT116 cells exposed to IL-6, along with the increase in a set of mesenchymal cell markers including vimentin and α-smooth muscle actin (α-SMA), as well as EMT transcription regulators-twist, snail and slug. The changes of EMT phenotype were related to the activation of Src, FAK, ERK1/2, p38 mitogen-activated protein kinase (p38MAPK), as well as transcription factors STAT3, κB and C/EBPβ. IL-6 treatment has promoted the recruitment of STAT3, κB and C/EBPβ toward the Twist promoter region. Furthermore, the Src-FAK signaling blockade resulted in the decline of IL-6 induced activation of ERK1/2, p38MAPK, κB, C/EBPβ and STAT3, as well as the decreasing mesenchymal state of HCT116 cells. These results suggested that IL-6 activates the Src-FAK-ERK/p38MAPK signaling cascade to cause the EMT of colorectal cancer cells. Pharmacological approaches targeting Src-FAK signaling may provide potential therapeutic strategies for rescuing colorectal cancer progression.

Batf stabilizes Th17 cell development via impaired Stat5 recruitment of Ets1‐Runx1 complexes

Although the activator protein‐1 (AP‐1) factor Batf is required for Th17 cell development, its mechanisms of action to underpin the Th17 program are incompletely understood. Here, we find that Batf ensures Th17 cell identity in part by restricting alternative gene programs through its actions to restrain IL‐2 expression and IL‐2‐induced Stat5 activation. This, in turn, limits Stat5‐dependent recruitment of Ets1‐Runx1 factors to Th1‐ and Treg‐cell‐specific gene loci. Thus, in addition to pioneering regulatory elements in Th17‐specific loci, Batf acts indirectly to inhibit the assembly of a Stat5‐Ets1‐Runx1 complex that enhances the transcription of Th1‐ and Treg‐cell‐specific genes. These findings unveil an important role for Stat5‐Ets1‐Runx1 interactions in transcriptional networks that define alternate T cell fates and indicate that Batf plays an indispensable role in both inducing and maintaining the Th17 program through its actions to regulate the competing actions of Stat5‐assembled enhanceosomes that promote Th1‐ and Treg‐cell developmental programs.

Maternal High Fat Diet Anticipates the AD-like Phenotype in 3xTg-AD Mice by Epigenetic Dysregulation of Aβ Metabolism.

Maternal overnutrition has been reported to affect brain plasticity of the offspring by altering gene expression, regulating both synaptic plasticity and adult neurogenesis. However, whether perinatal metabolic stress may influence the accumulation of misfolded proteins and the development of neurodegeneration remains to be clarified. We investigated the impact of maternal high fat diet (HFD) in an experimental model of Alzheimer's disease (AD). The 3xTg-AD mice born to overfed mothers showed an impairment of synaptic plasticity and cognitive deficits earlier than controls. Maternal HFD also altered the expression of genes regulating amyloid-β-protein (Aβ) metabolism (i.e., Bace1, Ern1, Ide and Nicastrin) and enhanced Aβ deposition in the hippocampus. Finally, we found an epigenetic derangement and an aberrant recruitment of transcription factors NF-kB and STAT3 and chromatin remodeler HDAC2 on the regulatory sequences of the same genes. Collectively, our data indicate that early life metabolic stress worsens the AD phenotype via epigenetic alteration of genes regulating Aβ synthesis and clearance.

Interleukin-7 receptor signaling is crucial for enhancer-dependent TCRδ germline transcription mediated through STAT5 recruitment.

γδ T cells play important roles in immune responses by rapidly producing large quantities of cytokines. Recently, γδ T cells have been found to be involved in tissue homeostatic regulation, playing roles in thermogenesis, bone regeneration and synaptic plasticity. Nonetheless, the mechanisms involved in γδ T-cell development, especially the regulation of TCRδ gene transcription, have not yet been clarified. Previous studies have established that NOTCH1 signaling plays an important role in the Tcrg and Tcrd germline transcriptional regulation induced by enhancer activation, which is mediated through the recruitment of RUNX1 and MYB. In addition, interleukin-7 signaling has been shown to be required for Tcrg germline transcription, VγJγ rearrangement and γδ T-lymphocyte generation as well as for promoting T-cell survival. In this study, we discovered that interleukin-7 is required for the activation of enhancer-dependent Tcrd germline transcription during thymocyte development. These results indicate that the activation of both Tcrg and Tcrd enhancers during γδ T-cell development in the thymus depends on the same NOTCH1- and interleukin-7-mediated signaling pathways. Understanding the regulation of the Tcrd enhancer during thymocyte development might lead to a better understanding of the enhancer-dependent mechanisms involved in the genomic instability and chromosomal translocations that cause leukemia.

IFNγ-induced PD-L1 expression in ovarian cancer cells is regulated by JAK1, STAT1 and IRF1 signaling

Expression of the immune checkpoint programmed death ligand-1 (PD-L1) is increased in ovarian cancer (OC) and correlates with poor prognosis. Interferon-γ (IFNγ) induces PD-L1 expression in OC cells, resulting in their increased proliferation and tumor growth, but the mechanisms that regulate the PD-L1 expression in OC remain unclear. Here, we show that the IFNγ-induced PD-L1 expression in OC cells is associated with increased levels of STAT1, Tyr-701 pSTAT1 and Ser-727 pSTAT1. Suppression of JAK1 and STAT1 significantly decreases the IFNγ-induced PD-L1 expression in OC cells, and STAT1 overexpression increases the IFNγ-induced PD-L1 expression. In addition, IFNγ induces expression of the transcription factor interferon regulatory factor 1 (IRF1) and IRF1 suppression attenuates the IFNγ-induced gene and protein levels of PD-L1. Chromatin immunoprecipitation results show that IFNγ induces PD-L1 promoter acetylation and recruitment of STAT1, Ser-727 pSTAT1 and IRF1 in OC cells. Together, these findings demonstrate that the IFNγ-induced PD-L1 expression in OC cells is regulated by JAK1, STAT1, and IRF1 signaling, and suggest that targeting the JAK1/ STAT1/IRF1 pathway may provide a leverage to regulate the PD-L1 levels in ovarian cancer.

Histone deacetylase 2 knockout suppresses immune escape of triple-negative breast cancer cells via downregulating PD-L1 expression

The PD-L1 overexpression is an important event of immune escape and metastasis in triple-negative breast cancer (TNBC), but the molecular mechanism remains to be determined. Interferon gamma (IFNγ) represents a major driving force behind PD-L1 expression in tumor microenvironment, and histone deacetylase 2 (HDAC2) is required for IFN signaling. Here, we investigated the regulation of HDAC2 on the IFNγ-induced PD-L1 expression in TNBC cells. We found the HDAC2 and PD-L1 expression in TNBC was significantly higher than that in non-TNBC, and HDAC2 was positively correlated with PD-L1 expression. HDAC2 promoted PD-L1 induction by upregulating the phosphorylation of JAK1, JAK2, and STAT1, as well as the translocation of STAT1 to the nucleus and the recruitment of STAT1 to the PD-L1 promoter. Meanwhile, HDAC2 was recruited to the PD-L1 promoter by STAT1, and HDAC2 knockout compromised IFNγ-induced upregulation of H3K27, H3K9 acetylation, and the BRD4 recruitment in PD-L1 promoter. In addition, significant inhibition of proliferation, colony formation, migration, and cell cycle of TNBC cells were observed following knockout of HDAC2 in vitro. Furthermore, HDAC2 knockout reduced IFNγ-induced PD-L1 expression, lymphocyte infiltration, and retarded tumor growth and metastasis in the breast cancer mouse models. This study may provide evidence that HDAC2 promotes IFNγ-induced PD-L1 expression, suggesting a way for enhanced antitumor immunity when targeting the HDAC2 in TNBC.

IFN-α Upregulates the Expression of C/EBPβ and Induces Myeloid Differentiation and Exhaustion of CD34+ CML Stem Cells

It is widely accepted that chronic myeloid leukemia (CML) stem cells survive independently of BCR-ABL signaling, and maintain their immature and quiescent status in the BM niche. As this population could give rise to relapse after long-term remission with tyrosine kinase inhibitor (TKI) therapy, eradication of CML stem cells is still necessary for the complete cure of the disease. The efficacy of interferon-α (IFN-α), which has been used for the treatment for CML-chronic phase (CP), has recently been re-evaluated. Several clinical trials have shown that combination treatment with IFN-α significantly improves the therapeutic effects of TKI. In some ‘Stop TKI’ trials, a history of IFN-α therapy was associated with a higher incidence of treatment-free remission. However, the molecular mechanisms underlying the action of IFN-α on CML stem cells have not been fully elucidated yet.At the annual meeting of ASH last year, we have shown that IFN-α induced differentiation and exhaustion of CML stem cells in a CML mouse model, and that IFN-α significantly upregulated CCAAT/Enhancer Binding Protein β (C/EBPβ), a transcription factor required for stress-induced “on-demand” granulocyte production under hematopoietic stresses, such as infection and cytokine stimulation (Hirai H et al, Nat Immunol 2006; Satake S et al, J Immunol 2012). C/EBPβ was responsible for the IFN-α-mediated differentiation and exhaustion of CML stem cells both in vitro and in vivo . We have also identified the Cebpb 3' distal enhancer region, which was required for efficient upregulation of C/EBPβ in response to IFN-α, and that IFN-α upregulated C/EBPβ at least in part through recruiting STAT5 to this enhancer. Here we investigated the involvement of a major downstream signaling molecule of IFN-α, STAT1, in the effect of IFN-α on CML stem cells, and then verified the relevance of our hypothesis that IFN-α induces exhaustion of CML stem cells through upregulation of C/EBPβ, by using CD34+ cells derived from CML patients.First, we examined whether STAT1 is recruited to the 3' distal Cebpb enhancer region in response to IFN-α. When a mouse hematopoietic stem/progenitor cell line, EML was treated with IFN-α, recruitment of STAT1 to this enhancer was observed by ChIP PCR. IFN-α treatment of BCR-ABL-transduced EML cells also enriched STAT1 strongly in this region, indicating that the STAT1-mediated pathway downstream of IFNα remains intact in cells expressing BCR-ABL. Furthermore, whereas BCR-ABL signaling induced the recruitment of STAT5 to this enhancer region, IFN-α treatment further increased the binding of STAT5. These data suggest that when BCR-ABL expressing CML cells were stimulated with IFN-α, recruitment of STAT1 to the enhancer is responsible for the upregulation of C/EBPβ in addition to the recruitment of STAT5.Next, we explored the effect of IFN-α on CD34+ CML stem cells obtained from CML patients (4 CP and 1 accelerated phase). This protocol was approved by the IRB at Kyoto University, and the written informed consent was obtained from patients prior to this study. Patient-derived lineage- CD34+ cells were treated with 100 or 500 U/mL recombinant human IFN-α in vitro for either 0.5 or 3 hours. IFN-α significantly upregulated CEBPB mRNAexpression in a dose- and time-dependent manner, while the transcript for CEBPA, the transcription factor required for steady-state granulopoiesis, was slightly downregulated. This result suggests that IFN-α directly acts on patient-derived CML stem cells and induces upregulation of C/EBPβ. Then, we performed the colony-forming assay to evaluate the impact of IFN-α on the function of patient-derived CML stem cells. Purified lineage- CD34+ cells were subjected to this assay, and colonies were counted and classified on day 14. The cells harvested from the primary colonies were subjected to another round of colony-forming assay or analyzed by flow cytometry. Notably, IFN-α significantly decreased the total colony number in both first- and second-round of plating. Furthermore, IFN-α strongly promoted differentiation of CML stem cells toward CD66-expressing myeloid cells, and induced their exhaustion in a dose-dependent manner (Figure).Collectively, these data suggest that STAT1/STAT5-C/EBPβ axis plays an important role in the IFN-α-mediated exhaustion of CML stem cells not only in mouse CML model, but also in patient-derived CML stem cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vitamin C Potentiates Plasma Cell Differentiation via TET-mediated DNA Modifications at a Novel Prdm1 Distal Element

Abstract Ascorbate (vitamin C) is an essential micronutrient in humans. Chronic severe ascorbate deficiency results in a condition termed Scurvy with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. In this study, we showed that ascorbate is pivotal for the IL-21-dependent differentiation of BLIMP1+ plasma cells. Ascorbate significantly increases the proportion of B cells expressing BLIMP1 upon IL-21 stimulation without affecting the IL-21-STAT3 signaling. As a co-factor for TET DNA 5-methylcytosine oxidases, ascorbate boosters the enzymatic activity of TET, promoting the oxidation of 5-methylcytosines into 5-hydroxymethylcytosine (5hmC), an intermediate for DNA demethylation. Correspondingly, TET2/3 are required for the enhancement of plasma cell differentiation by ascorbate. Using 5hmC enrichment analysis, we identified ascorbate promotes plasma cell lineage via a novel distal element at the BLIMP1/Prdm1 locus. This ascorbate-responsive element (EAR) is normally methylated and is hydroxymethylated in the presence of ascorbate. Notably, the 5hmC-modified CpG exists within the STAT3 binding motif and the pattern is evolutionarily conserved, suggesting the ascorbate-assisted DNA modification is required for STAT3 recruitment to EAR and the subsequent Prdm1 activation. This study provides an example of how micronutrients may influence immune response via epigenetic regulation. Our finding also implies that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell-fate decisions.

Inhibition of relaxin autocrine signaling confers therapeutic vulnerability in ovarian cancer.

Ovarian cancer (OC) is the most deadly gynecological malignancy, with unmet clinical need for new therapeutic approaches. The relaxin peptide is a pleiotropic hormone with reproductive functions in the ovary. Relaxin induces cell growth in several types of cancer, but the role of relaxin in OC is poorly understood. Here, using cell lines and xenograft models, we demonstrate that relaxin and its associated GPCR RXFP1 form an autocrine signaling loop essential for OC in vivo tumorigenesis, cell proliferation, and viability. We determined that relaxin signaling activates expression of prooncogenic pathways, including RHO, MAPK, Wnt, and Notch. We found that relaxin is detectable in patient-derived OC tumors, ascites, and serum. Further, inflammatory cytokines IL-6 and TNF-α activated transcription of relaxin via recruitment of STAT3 and NF-κB to the proximal promoter, initiating an autocrine feedback loop that potentiated expression. Inhibition of RXFP1 or relaxin increased cisplatin sensitivity of OC cell lines and abrogated in vivo tumor formation. Finally, we demonstrate that a relaxin-neutralizing antibody reduced OC cell viability and sensitized cells to cisplatin. Collectively, these data identify the relaxin/RXFP1 autocrine loop as a therapeutic vulnerability in OC.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

SummaryProlactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

LncRNA PVT1 induces aggressive vasculogenic mimicry formation through activating the STAT3/Slug axis and epithelial-to-mesenchymal transition in gastric cancer.

Vasculogenic mimicry (VM), a vessel-like network formed by highly aggressive tumor cells, plays an important role in accelerating cancer progression. This special vascularization pattern is closely associated with a poor prognosis in various cancers. As yet, however, the regulatory mechanism of VM formation is largely unknown. In this study, we assess whether the long noncoding RNA PVT1 is involved in VM generation in gastric cancer. VM formation was determined by immunohistochemistry using PAS/CD31 double staining in gastric cancers and Matrigel tube formation in vitro. qRT-PCR and Western blotting were used to assess mRNA and protein expression. Interaction between PVT1 and STAT3 was determined using a RNA pull-down assay. Luciferase reporter and chromatin immunoprecipitation assays were performed to evaluate transcriptional activity of STAT3 on the Slug gene promoter. We found that PVT1 can induce VM generation both in vitro and in vivo. Mechanistically, we found that PVT1 interacted with and activated STAT3 through a 850-1770nt fragment. PVT1 facilitated STAT3 recruitment to the Slug promoter and transcriptionally enhanced Slug expression, thereby triggering epithelial-to-mesenchymal transition (EMT) and VM capillary formation. STAT3 inhibition effectively blocked PVT1-mediated VM. In primary gastric cancer samples, a positive correlation was found between PVT1 and Slug upregulation, and patients with a high PVT1 and Slug expression exhibited markedly shorter survival times. Our results shed light on the role of PVT1 in gastric cancer cell-dependent VM formation. Our findings provide valuable clues for the design of new anti-angiogenic therapeutic strategies. The PVT1/STAT3 axis may serve as a potential target in gastric cancer treatment.

Identification of goldfish (Carassius auratus L.) leukocyte immune-type receptors shows alternative splicing as a potential mechanism for receptor diversification

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.

Association of the Polymorphism rs13259960 in SLEAR With Predisposition to Systemic Lupus Erythematosus.

Genome-wide association studies have identified many susceptibility loci for systemic lupus erythematosus (SLE). However, most of these loci are located in noncoding regions of the genome. Long noncoding RNAs (lncRNAs) are pervasively expressed and have been reported to be involved in various diseases. This study aimed to explore the genetic significance of lncRNAs in SLE. A genome-wide survey of SLE risk variants in lncRNA gene loci was performed in Han Chinese subjects (4,556 with SLE and 9,451 healthy controls). The functional relevance of an SLE risk variant in one of the lncRNA genes was explored using biochemical and molecular cell biology analyses. In vitro loss-of-function and gain-of-function strategies were used to clarify the functional and phenotypic relevance of this SLE susceptibility lncRNA. Moreover, correlation of this lncRNA with the degree of apoptosis in the peripheral blood of SLE patients was evaluated. A novel SLE susceptibility locus in a lncRNA gene, designated SLEAR (for SLE-associated RNA), was identified at the single-nucleotide polymorphism rs13259960 (odds ratio 1.35, Pcombined = 1.03 × 10-11 ). The A>G variation at rs13259960, located in an intronic enhancer, was found to impair STAT1 recruitment to the enhancer that loops to the SLEAR promoter, resulting in decreased SLEAR production in peripheral blood mononuclear cells from patients with SLE (3 with the G/G genotype, 22 with A/G, and 103 with A/A at rs13259960; P = 0.0241). Moreover, SLEAR interacted with the RNA binding proteins interleukin enhancer binding factor 2, heterogeneous nuclear RNP F, and TATA-binding protein-associated factor 15, to form a complex for transcriptional activation of the downstream antiapoptotic genes. In addition, SLEAR regulated apoptosis of Jurkat cells in vitro, and its expression level was correlated with the degree of cell death in the peripheral blood of patients with SLE (r = 0.824, P = 2.15 × 10-8 ; n = 30). These findings suggest a mechanism by which the risk variant at rs13259960 modulates SLEAR expression and confers a predisposition to SLE. Taken together, these results may give insights into the etiology of SLE.

Rotenone-induced reactive oxygen species signal the recruitment of STAT3 to mitochondria.

STAT3, a transcription factor involved in various physiological and pathological processes, is also present in mitochondria. Mitochondrial STAT3 regulates complex I activity and reactive oxygen species (ROS) production, yet the mechanisms governing its translocation to mitochondria remain poorly understood. In this study, we show that rotenone-induced ROS triggers the Ser727 phosphorylation of STAT3 and its increased mitochondrial localisation. Furthermore, we show that STAT3-depleted cells display increased ROS levels during rotenone treatment. Targeted expression in mitochondria of wild-type STAT3 - but not S727A mutant - lowers ROS levels, indicating the importance of Ser727 phosphorylation, both in rotenone-induced mitochondrial targeting and quenching of ROS levels. Together, our results demonstrate a novel STAT3-mediated feedback mechanism to maintain redox homeostasis during stress.

Benzodiazepines Drive Alteration of Chromatin at the Integrated HIV-1 LTR.

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.

Correction: PRMT1 Plays a Critical Role in Th17 Differentiation by Regulating Reciprocal Recruitment of STAT3 and STAT5.

Th17 cells are a class of Th cells that secrete IL-17 and mediate pathogenic immunity responsible for autoimmunity including experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. Retinoic acid-related orphan receptor γ t (RORγt) is the critical transcription factor that controls the differentiation of Th17 cells. However, little is known about the transcriptional cofactors for RORγt in the regulation of Th17 differentiation. In this study, we demonstrate that protein arginine N-methyltransferase 1 (PRMT1) associates with RORγt and regulates mouse Th17 differentiation. Overexpression of PRMT1 promoted Th17 differentiation, whereas inactivation or knockdown of PRMT1 decreased Th17 differentiation while expanding Foxp3+ regulatory T cells. Consistently, pharmacological inhibition of PRMT1 impaired the generation of Th17 cells and prevented induction of experimental autoimmune encephalomyelitis in mice. Mechanistically, PRMT1-dependent modification of asymmetric histone 4 arginine 3 dimethylation is required to stabilize the stimulatory STAT3 to displace the inhibitory STAT5 at IL-17 locus, resulting in the activation of IL-17 gene. Furthermore, PRMT1-facilitated recruitment of STAT3 overcame the inhibition of Th17 differentiation exerted by IL-2-induced STAT5 activation. PRMT1 thus regulates Th17 differentiation by controlling the reciprocal recruitment of STAT3 and STAT5. Our study thus reveals PRMT1 as a novel target for alleviating Th17-mediated autoimmunity by decreasing RORγt-dependent generation of pathogenic Th17 cells.

IFN-\u03b3 selectively suppresses a subset of TLR4-activated genes and enhancers to potentiate macrophage activation

Activation of macrophage proinflammatory and antimicrobial phenotypes is regulated by IFN-γ and LPS via synergistic induction of canonical, inflammatory NF-κB target genes. However, whether IFN-γ negatively regulates components of the LPS response, and how this may affect macrophage activation, is still unclear. Here we use combined transcriptomic and epigenomic approaches to find that IFN-γ selectively abrogates LPS-induced feedback and alters macrophage metabolic pathways by suppressing TLR4-mediated gene activation. In contrast to superinduction of inflammatory genes via enhancers that bind IRF1 and STAT1, IFN-γ represses target enhancers that bind STAT3. TLR4-activated but IFN-γ-suppressed enhancers comprise two subsets discernable by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin. Our findings thus show that IFN-γ suppresses feedback inhibitory and metabolic components of TLR responses to enhance macrophage activation; they also provide insights for IFN-γ-mediated selective inhibition of TLR4-induced transcription. Such inhibition can contribute to severe and sustained inflammatory responses.

Abstract 4508: Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis

Abstract Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2). Jak2 associates with PRLr, phosphorylates c-terminus of the PRLr, which leads to Stat5 recruitment and activation. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase (PPI), which is constitutively bound to the PRLr that catalyze the cis-trans interconversion of proline imide bonds. Treatment with Cyclosporine (CsA) inhibited CypA binding to the PRLr and blocked PRLr-driven activation of Jak2/Stat5. Recently, Waters et al., utilizing Fluorescence Resonance Energy Transfer (FRET) with transfectants expressing CFP and YFP-tagged forms of the growth hormone receptor (GHR) showed that GHR activation induced a rotational movement in C-terminus of the GHR, resulting in a loss of baseline FRET signal. Like the GHR, PRL stimulation of transfectants expressing CFP/YFP-tagged PRLr constructs resulted in a loss of FRET efficiency. In contrast, treatment with NIM811 (a non-immunosuppressive form of CsA) or siRNA knockdown of CypA resulted in a return of FRET signal in the presence of PRL. These studies reveal that ligand stimulation of the PRLr results in a conformational change as measured by FRET signal of the receptor that is reversed by CypA inhibition or knockdown, implicating CypA as the mediator of this conformational change and ligand-induced signaling. To further assess the consequences of CypA inhibition or knockdown on the PRLr/Jak2 mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated in breast cancer cells. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates in ER+/PR+ T47D cells in a time dependent manner. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL induced genes. NIM811 inhibited ER-/+, and HER2+ breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent pre-clinical testing of NIM811 in relevant mouse mammary cancer models has found that NIM811 treatment of a TNBC xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA (by constitutive genetic deletion) in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates conformational change in the C-terminus of the PRLr through its PPI activity, and alters PRLr/Jak2 complex signaling/functions in breast cancer and mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention as a chemo-preventive and as an inhibitor of metastasis. Citation Format: Shawn Hakim, Shannon E. Hedrick, Justin M. Craig, Charles V. Clevenger. Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis [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 4508.