Interleukin-6 Receptor Expression Research Articles

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells

BackgroundDysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC.MethodsThe clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells.ResultsHCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells.ConclusionsIL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

IL-7R Expression Correlates with Prognosis in Breast Cancer.

The tumor microenvironment (TME) exerts a significant influence on the development, invasion, metastasis, and drug resistance of breast cancer. Therefore, this study sought to investigate potential prognostic factors and markers indicative of TME remodeling in breast cancer, utilizing data from the TCGA database. In this study, transcriptome RNA-seq data from 1222 breast cancer samples were processed using CIBERSORT and ESTIMATE algorithms. We conducted a differential gene expression analysis utilizing COX regression analysis and constructed protein-protein interaction (PPI) networks for enhanced visualization. Through univariate COX analysis and cross-analysis within PPI networks, the Interleukin-7 receptor (IL-7R) emerged as a potential predictor. Subsequently, we performed a comprehensive investigation encompassing single-gene survival analysis, clinical correlation assessment, and GSEA enrichment analysis targeting IL-7R as a core gene associated with prognosis. We examined the expression of IL-7R in human breast cancer and normal breast tissue through clinical studies and cytology experiments, followed by an indepth analysis of the relationship between IL-7R and breast cancer. The survival analysis revealed that breast cancer patients with elevated IL-7R expression experienced prolonged survival compared to those with lower IL-7R levels. Results obtained from the Wilcoxon rank-sum test, along with clinical and cellular experiments, indicated higher IL-7R expression in tumor samples compared to normal samples. Correlation tests conducted between IL-7R expression and clinicopathological stage characteristics highlighted statistically significant associations between IL-7R expression and the T and M stages. Additionally, cell classification analysis of tumor-infiltrating immune cells (TIC) proportion showed that activated CD4+ T cells and CD8 T cells of memory B cells were positively correlated with IL-7R expression. These findings further underscored the impact of IL-7R levels on the tumor microenvironment (TME). IL-7R emerges as a potential prognostic indicator for breast cancer patients, particularly in sustaining the immunoactive status of the tumor microenvironment (TME) and contributing to immune reconstitution. These findings offer novel insights into breast cancer treatment strategies.

Identification of the grass carp interleukin-23 receptor and its proinflammatory role in intestinal inflammation

The interleukin 23 receptor (IL-23R) is associated with a variety of inflammatory diseases in humans and other mammals. However, whether IL-23R is involved in inflammatory diseases in teleost fish is less understood. Thus, to investigate the potential involvement of IL-23R in fish inflammatory diseases, the full-length cDNA of IL-23R from grass carp Ctenopharyngodon idella was cloned and used to generate a recombinant protein (rgcIL-23R) containing the extracellular domain of IL-23R, against which a polyclonal antibody (rgcIL-23R pAb) was then developed. qPCR analysis revealed that IL-23R mRNA was significantly upregulated in most grass carp tissues in response to infection with Gram-negative Aeromonas hydrophila. Treatment with rgcIL-23R significantly induced IL-17A/F1 expression in C. idella kidney (CIK) cells. By contrast, knockdown of IL-23R caused significant decreases in IL-23R, STAT3, and IL-17N expression in CIK cells after lipopolysaccharide (LPS) stimulation. Similarly, rgcIL-23R pAb treatment effectively inhibited the LPS-induced increase in the expression of IL-23 subunit genes and those of the IL-23/IL-17 pathway in CIK cells. Furthermore, intestinal symptoms identical to those caused by A. hydrophila were induced by anal intubation with rgcIL-23R, but suppressed by rgcIL-23R pAb. Therefore, these results suggest that IL-23R has a crucial role in the regulation of intestinal inflammation and, thus, is a promising target for controlling inflammatory diseases in farmed fish.

B-Cell Activation Biomarkers in Salivary Glands Are Related to Lymphomagenesis in Primary Sjögren's Disease: A Pilot Monocentric Exploratory Study.

Primary Sjögren's disease is primarily driven by B-cell activation and is associated with a high risk of developing non-Hodgkin's lymphoma (NHL). Over the last few decades, microRNA-155 (miR-155) has arisen as a key regulator of B-cells. Nevertheless, its role in primary Sjögren's disease remains elusive. Thus, the purpose of this study was (i) to explore miR-155, B-cell activating factor (BAFF)-receptor (BAFF-R), and Interleukin 6 receptor (IL-6R) expression in the labial salivary glands (LSG) of patients with primary Sjögren's disease, aiming to identify potential B-cell activation biomarkers related to NHL development. Twenty-four patients with primary Sjögren's disease, and with available tissue blocks from a LSG biopsy performed at diagnosis, were enrolled. Among them, five patients developed B-cell NHL during follow-up (7.3 ± 3.1 years). A comparison group of 20 individuals with sicca disease was included. Clinical and laboratory parameters were recorded and the LSG biopsies were evaluated to assess local inflammation in terms of miR-155/BAFF-R and IL-6R expression. Stratifying the primary Sjögren's disease cohort according to lymphomagenesis, miR-155 was upregulated in primary Sjögren's disease patients who experienced NHL, more so than those who did not experience NHL. Moreover, miR-155 expression correlated with the focus score (FS), as well as BAFF-R and IL-6R expression, which were increased in primary Sjögren's disease patients and in turn related to neoplastic evolution. In conclusion, epigenetic modulation may play a crucial role in the aberrant activation of B-cells in primary Sjögren's disease, profoundly impacting the risk of NHL development.

OP02 IL23R-CAR-Tregs: creating a therapeutic breakthrough for Crohn’s Disease

Abstract Background Regulatory T cells (Tregs) are key immunomodulators to maintain immune homeostasis and self-tolerance, including in the intestine. Disrupted immune homeostasis is associated with Crohn’s Disease (CD) pathogenesis. As a living drug, Treg therapy is a promising treatment for restoring immune balance and achieving durable tissue tolerance in the diseased intestine. By targeting a disease-relevant protein, Interleukin-23 receptor (IL23R), we engineered Tregs expressing IL23R-chimeric antigen receptor (CAR) to create IL23R-CAR-Tregs for treating CD. Methods IL23R protein expression in CD intestine was verified by immunohistochemical analysis. Tonic signaling and CAR-specific activation were quantified during the IL23R-CAR screening. The IL23R-CAR-Treg phenotype was assessed by monitoring the expression of FoxP3 and Helios transcription factors, and the demethylation status of FoxP3 Treg-specific demethylated region (TSDR). The suppressive function of IL23R-CAR-Tregs was evaluated using in vitro assays and a colitis mouse model. The functionality of IL23R-CAR-Tregs were further assessed using intestinal biopsy-derived cells from CD patients in an in vitro activation assay. Transcriptomic and proteomic analyses were performed to associate molecular profiles with IL23R-CAR-Treg activation. Results Our highly potent IL23R-CAR lead candidate displays negligible tonic signalling and a strong signal-to-noise ratio. The resulting IL23R-CAR-Tregs maintain their regulatory phenotype with high FoxP3 and Helios expression and TSDR demethylation during in vitro expansion, CAR-specific stimulation, and in the presence of proinflammatory cytokines. In accordance with their regulatory phenotype, IL23R-CAR-Tregs exhibit CAR-dependent suppressive activity, promote a tolerogenic environment, and protect mice from colitis. The expression of IL23R was also confirmed in the intestinal mucosa from CD patients. Importantly, IL23R-CAR-Tregs elicit activation following exposure to intestinal biopsy-derived cells from CD, demonstrating the engagement and potency of IL23R-CAR towards their target in CD. We also identified molecular profiles that correlated with IL23R-CAR activation and, more broadly, showed their potential to monitor IL23R-CAR-Treg activity in CD patients. Conclusion Our results demonstrate that an IL23R-CAR-Treg product could be a promising therapy for CD patients, with the potential long-term benefits of immune tolerance.

Tocilizumab Exerts Anti-Tumor Effects on Colorectal Carcinoma Cell Xenografts Corresponding to Expression Levels of Interleukin-6 Receptor.

The use of tocilizumab against the interleukin-6 receptor (IL-6R) has been demonstrated as inhibiting the progression of diverse cancers in vitro and in vivo. Nonetheless, evidence regarding the anti-tumor effects of tocilizumab on human colorectal carcinoma (CRC) corresponding to IL-6R expression levels remains scarce. To investigate the influence of IL-6R expression, SW480 and HT-29 cells inoculated subcutaneously into NU/NU mice were used as human CRC xenograft models with anti-IL-6R antibody (tocilizumab) therapy. The IL-6R expression levels, histology of CRC growth/invasiveness, and tumor growth-related signaling pathway were estimated by H&E and immunohistochemical staining. SW480 tumor cells with higher IL-6R expression levels showed better responsiveness in tocilizumab therapy than in the treated HT-29 group. Likewise, therapeutic effects of tocilizumab on the proliferative ability with mitotic index and Ki-67 expressions, invasiveness with MMP-9 proteinase expressions, and ERK 1/2 and STAT3 signaling transduction in the SW480 treatment group were superior to the HT-29 treatment group. In light of our results, IL-6R is the key indicator for the efficacy of tocilizumab treatment in CRC xenografts. From the perspective of precision medicine, tumor response to anti-IL-6R antibody therapy could be predicted on the basis of IL-6R expression levels. In this manner, tocilizumab may serve as a targeted and promising anti-CRC therapy.

Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis.

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

Myricetin ameliorates the IL-21-induced tumorigenic phenotype of adjuvant-induced arthritis FLS by modulating the choline kinase signaling cascade.

The synovial intimal lining is mainly governed by fibroblast-like synoviocytes (FLS), which portray a transformed tumor-like phenotype in rheumatoid arthritis (RA). Among the diverse cytokines that engender FLS, interleukin-21 (IL-21) was reported to stimulate hyperproliferation and perpetuate inflammation. Recently, choline kinase (ChoKα) has been reported to be an essential enzyme aiding RA-FLS hyperproliferation by altering phosphatidylcholine biosynthesis. The current study aimed to elucidate the therapeutic efficacy of myricetin, a flavonoid, in abating the IL-21-induced tumor-like phenotype of adjuvant-induced arthritis (AIA)-FLS via the ChoKα signaling cascade. Our results showed that myricetin suppressed IL-21 receptor expression and activation of the ChoKα signaling cascade (N-Ras, Ral-GDS, and PI3K) in IL-21-induced AIA-FLS. Consequently, myricetin treatment decreased ChoKα and PLD2 enzymatic activity and inhibited the proliferative, migratory, and invasive properties of AIA-FLSs. Our results demonstrated that myricetin could be a promising anti-arthritic compound by abating IL-21-induced hyperproliferation, migration, and invasive behavior of AIA-FLS by downregulating the ChoKα signaling cascade.

EGFR stimulation enables IL-6 trans-signalling via iRhom2-dependent ADAM17 activation in mammary epithelial cells.

The cytokine interleukin-6 (IL-6) has considerable pro-inflammatory properties and is a driver of many physiological and pathophysiological processes. Cellular responses to IL-6 are mediated by membrane-bound or soluble forms of the IL-6 receptor (IL-6R) complexed with the signal-transducing subunit gp130. While expression of the membrane-bound IL-6R is restricted to selected cell types, soluble IL-6R (sIL-6R) enables gp130 engagement on all cells, a process termed IL-6 trans-signalling and considered to be pro-inflammatory. sIL-6R is predominantly generated through proteolytic processing by the metalloproteinase ADAM17. ADAM17 also liberates ligands of the epidermal growth factor receptor (EGFR), which is a prerequisite for EGFR activation and results in stimulation of proliferative signals. Hyperactivation of EGFR mostly due to activating mutations drives cancer development. Here, we reveal an important link between overshooting EGFR signalling and the IL-6 trans-signalling pathway. In epithelial cells, EGFR activity induces not only IL-6 expression but also the proteolytic release of sIL-6R from the cell membrane by increasing ADAM17 surface activity. We find that this derives from the transcriptional upregulation of iRhom2, a crucial regulator of ADAM17 trafficking and activation, upon EGFR engagement, which results in increased surface localization of ADAM17. Also, phosphorylation of the EGFR-downstream mediator ERK mediates ADAM17 activity via interaction with iRhom2. In sum, our study reveals an unforeseen interplay between EGFR activation and IL-6 trans-signalling, which has been shown to be fundamental in inflammation and cancer.

Diminished Interleukin-7 receptor expression on T-cell subsets in tuberculosis patients

Immunopathology in human tuberculosis affects T-cell phenotype and functions. Previous studies identified impaired T-cell sensitivity to Interleukin (IL)-7 accompanied by lower IL-7 receptor α-chain (IL-7Rα) expression in patients with acute tuberculosis. In the present study, we characterized affected T-cell subsets and determined the influence of tuberculosis disease severity and treatment response.Tuberculosis patients (n = 89) as well as age- and gender-matched asymptomatic contacts (controls, n = 47) were recruited in Ghana. Mycobacterium (M.) tuberculosis sputum burden was monitored prior to and during treatment. Blood samples from all patients and controls were analyzed for IL-7Rα expression and T-cell markers by multi-colour flow cytometry.CD4+ and CD8+ T-cells of tuberculosis patients showed generally lower IL-7Rα expression as compared to controls. Concomitantly, tuberculosis patients had higher proportions of naïve and lower proportions of memory CD4+ T-cells. Notably, a subset of CD27 positive central memory T-cells (Tcm), which lacked IL-7Rα expression was enriched in tuberculosis patients as compared to controls. M. tuberculosis sputum burden was not associated with differences in IL-7Rα expression. Treatment duration and response showed no clear effects although IL-7Rα expression patterns were highly variable.These results suggested generally impaired generation of memory CD4+ T-cells and enrichment of a Tcm subset without IL-7Rα expression in patients with tuberculosis.

β2-adrenergic receptor expression in patients receiving bevacizumab therapy for metastatic melanoma.

Vascular endothelial growth factor (VEGF) was initially known as vascular permeability factor and identified as a driver of tumour angiogenesis. Recently, its role in supporting an immunosuppressive tumour microenvironment was demonstrated, and anti-VEGF treatment combined with immune checkpoint blockade is currently investigated. Further, beta-adrenergic signalling as a modifier of cancer hallmarks like immune response, angiogenesis and metastasis gained increased attention during past years. Focusing on the aspect of immunosuppression in upregulated beta-adrenergic signalling, we investigated predictive markers in patients with metastatic melanoma who received bevacizumab monotherapy, a specific VEGF-A binding antibody. We explored the expression of beta-2 adrenergic receptor (β2-AR), interleukin 6-receptor (IL6-R), cyclooxygenase 2 (COX2) and VEGF-A by immunohistochemistry in melanoma to assess the correlation between these proteins in melanoma cells and response to treatment. Strong β2-AR expression in metastases was associated with clinical benefit of bevacizumab. Furthermore, expression of the latter was positively linked to expression of VEGF-A and COX2. β2-AR expression in melanoma metastasis appears to distinguish a subgroup of patients that might benefit from anti-VEGF treatment. Our results strengthen further exploration of anti-VEGF therapy in combination with immune checkpoint blockade in clinical studies and the investigation of β2-AR as predictive marker.

Effects of IL-9 on TRAF3-deficient B cell activation

Abstract The pleiotropic cytokine interleukin-9 (IL-9) signals to target cells by binding to a heterodimeric receptor consisting of the IL-9 receptor (IL-9R) and the common subunit γ chain. In the present study, we found that the expression of IL-9R was strikingly up-regulated in mouse naïve follicular B cells genetically deficient in TRAF3, a critical regulator of B cell survival and function. The highly up-regulated IL-9R on Traf3 −/−follicular B cells conferred responsiveness to IL-9, including IgM production and STAT3 phosphorylation. Interestingly, IL-9 significantly enhanced class switch recombination to IgG1 induced by B cell antigen receptor crosslinking plus IL-4 in Traf3 −/−B cells but not in wild type B cells. Blocking the JAK-STAT3 pathway abrogated the effects of IL-9 on Traf3 −/−B cells. Our study thus revealed a novel pathway that TRAF3 suppresses B cell activation and Ig isotype switching by inhibiting IL-9R-JAK-STAT3 signaling. Our findings provide new insights into the TRAF3-IL-9R axis in B cell function and have significant implications for the understanding and treatment of a variety of human diseases involving aberrant B cell activation. This study was supported by the National Institutes of Health grants R01 CA158402 (P. Xie) and R21 AI128264 (P. Xie), the Department of Defense grant W81XWH-13-1-0242 (P. Xie), and a Busch Biomedical Grant (P. Xie). FACS analyses were supported by the Flow Cytometry Core Facility with funding from NCI-CCSG P30CA072720.

Neuronal growth regulator 1 may modulate interleukin-6 signaling in adipocytes.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays both anti- and pro-inflammatory roles. Due to the restricted expression of membrane IL-6 receptor (IL-6R), most pro-inflammatory functions of IL-6 are attributed to its association with soluble IL-6R (sIL-6R). Neuronal growth regulator 1 (NEGR1) is a brain-enriched membrane protein that has recently been recognized as a risk factor for many human diseases including obesity, depression, and autism. In the present study, we report that the expression levels of IL-6 and IL-6R, as well as the phosphorylation of signal transducer and activator of transcription (STAT) 3, were significantly elevated in white adipose tissues of Negr1 knockout mice. Elevated levels of circulating IL-6 and sIL-6R have also been observed in Negr1 -/- mice. Furthermore, NEGR1 interacted with IL-6R, which was supported by subcellular fractionation and an in situ proximity ligation assay. Importantly, NEGR1 expression attenuated the phosphorylation of STAT3 by sIL-6R, suggesting that NEGR1 negatively regulates IL-6 trans-signaling. Taken together, we propose that NEGR1 may play a regulatory role in IL-6 signaling by interacting with IL-6R, which may contribute to a molecular link underlying obesity, inflammation, and the depression cycle.

IL-6 upregulates the expression of IL-6R through the JAK2/STAT3 signalling pathway to promote progression of hepatocellular carcinoma.

The progression of hepatocellular carcinoma (HCC) involves multifactor, multistep interactions. High expression of interleukin-6 receptor (IL-6R) plays an important role in the occurrence and development of tumours, but the regulatory mechanism of IL-6R expression and its function in HCC have not been fully defined. Western blot was used to evaluate the phosphorylation of key kinases in the JAK2/STAT3 pathway and the protein expression levels of related proliferation molecules, migration molecules and apoptotic molecules. The antiapoptosis, migration and proliferation of cells of each group were analysed with JC-1 to judge the cell apoptosis rate, the EdU method to determine the proliferation vitality of the cells, clone formation experiments and Transwell experiments. High expression of IL-6R in cell lines, lower protein levels of the apoptotic molecules c-Caspase7 and c-Caspase3 and higher protein levels of the proliferative molecules p-P70S6K and migration molecules MMP9 and MMP2 were consistent with stronger antiapoptosis, proliferation and migration. Interestingly, IL-6 upregulated the expression of IL-6R by activating the JAK2/STAT3 signalling pathway. Also, the expression of IL-6R protein was downregulated after lentivirus knockdown of STAT3. In nude mice bearing subcutaneous tumours, upregulation of IL-6R expression after activation of the JAK2/STAT3 signalling pathway by IL-6 significantly increased tumour growth. Moreover, the expression of IL-6R protein was downregulated, and the terminal tumour volume was significantly downregulated in the lentiviral STAT3 knockdown group. IL-6 regulated the transcription of IL-6R through the activation of the JAK2/STAT3 signalling pathway.

IL-6–GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

Abstract 333: Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability

Abstract Classically, the interleukin-23 receptor (IL-23R) is expressed on the surface of T cells where activation by its ligand, interleukin-23 (IL-23) leads to T cell activation through the JAK/STAT pathway. Here, we identify a novel localization and function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation. IL-23R was identified as a potential target through InterLinked Therapeutics’ ITX machine learning platform. IL-23R was identified through this unbiased in silico screen as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed that IL-23R protein was overexpressed &amp;gt;2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression in normal hematopoietic cells. Consistent with prior studies, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were detected on the surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. We confirmed intracellular localization of IL-23R in AML cells using 4 different antibodies targeted against 4 different epitopes of the receptor. We used BioID mass spectrometry to identify novel protein interactors with intracellular IL-23R and identified 61 interacting proteins. 36 of those 61 were nuclear or cytoplasmic in localization and the largest subset was associated with mitotic spindle regulation. By Proximity Ligation Assay, we confirmed the interactions between endogenous IL-23R with known mitotic spindle proteins NUMA, TMEM201, BAG6, and TACC1 in OCI-AML2 cells and primary AML samples. By confocal microscopy, we discovered that IL-23R co-localized with the mitotic spindle and centrosomes in AML cells. Genetic knockout of IL-23R increased spindle defects with multipolarity, lagging chromosomes, and spindle orientation errors. Interestingly, heterodimeric partner of IL-23R, IL12Rβ1 was also detected to be co-localized with mitotic spindles but the cytokine, IL-23, could not be detected. Knockdown of IL-23R also reduced cell growth in OCI-AML2, TEX, K562, NB4, and U937 cells. IL-23R knockdown also reduced the clonogenic growth and engraftment efficiency of TEX leukemia cells into murine bone marrow. In summary, intracellular IL-23R is a critical regulator of mitosis in AML and is necessary for AML viability. This study highlights a novel function and localization of IL-23R in AML and identifies a potential therapeutic target. Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil MacLean, Marcela Gronda, Brandon D. Brown, Brian Raught, Andrea Arruda, Hong Chang, Mark D. Minden, Li Zhang, Steven M. Kornblau, Vito Spadavecchio, Aaron D. Schimmer. Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability [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 333.

Serum albumin is the strongest predictor of anti-tumor necrosis factor nonresponse in inflammatory bowel disease in resource-constrained regions lacking therapeutic drug monitoring.

Evidence on predictors of primary nonresponse (PNR), and secondary loss of response (SLR) to anti-tumor necrosis factor (anti-TNF) agents in inflammatory bowel disease is scarce from Asia. We evaluated clinical/biochemical/molecular markers of PNR/SLR in ulcerative colitis (UC) and Crohn's disease (CD). Inflammatory bowel disease patients treated with anti-TNF agents (January 2005-October 2020) were ambispectively included. Data concerning clinical and biochemical predictors was retrieved from a prospectively maintained database. Immunohistochemistry for expression of oncostatin M (OSM), OSM receptor (OSM-R), and interleukin-7 receptor (IL-7R) were done on pre anti-TNF initiation mucosal biopsies. One-hundred eighty-six patients (118 CD, 68 UC: mean age, 34.1±13.7 years; median disease duration at anti-TNF initiation, 60 months; interquartile range, 28-100.5 months) were included. PNR was seen in 17% and 26.5% and SLR in 47% and 28% CD and UC patients, respectively. In CD, predictors of PNR were low albumin (P<0.001), postoperative recurrence (P=0.001) and high IL-7R expression (P<0.027) on univariate; and low albumin alone (hazard ratio [HR], 0.09; 95% confidence interval [CI], 0.03-0.28; P<0.001) on multivariate analysis respectively. Low albumin (HR, 0.31; 95% CI, 0.15-0.62; P=0.001) also predicted SLR. In UC, predictors of PNR were low albumin (P<0.001), and high C-reactive protein (P<0.001), OSM (P<0.04) and OSM-R (P=0.07) stromal expression on univariate; and low albumin alone (HR, 0.11; 95% CI, 0.03-0.39; P=0.001) on multivariate analysis respectively. Low serum albumin at baseline significantly predicted PNR in UC and PNR/SLR in CD patients. Mucosal markers of PNR were high stromal OSM/OSM-R in UC and high IL-7R in CD patients.

Megakaryocyte-Derived IL-8 Acts as a Paracrine Factor for Prostate Cancer Aggressiveness through CXCR2 Activation and Antagonistic AR Downregulation.

Prostate cancer is the fifth leading cause of cancer-related mortality in men, primarily because of treatment resistance, recurrence, and metastasis. In the present study, we investigated the role of paracrine interleukin-8 (IL-8) in the antagonistic expression of IL-8 and androgen receptor (AR), and the contribution of IL-8 to prostate cancer aggressiveness. In hormone-responsive LNCaP cells that do not express IL-8, recombinant IL-8 treatment significantly increased expressions of IL-8, CXC chemokine receptor 2 (CXCR2), matrix metalloproteinase (MMP)-2/9, Snail, and vimentin. IL-8 treatment significantly decreased AR and E-cadherin expression. IL-8-induced gene expression changes were suppressed by navarixin, a CXCR1/2 inhibitor, and gallein, a Gβγ inhibitor. In PC-3 androgen-refractory prostate cancer cells, IL-8 knockdown reduced expressions of CXCR2, MMP-2/9, Snail, and vimentin, and increased AR and E-cadherin expressions at the mRNA and protein levels. Co-culture with MEG-01 human megakaryocytic cells secreting high levels of IL-8 induced gene expression changes in both LNCaP and PC-3 cells, similar to those induced by IL-8 treatment. The altered gene expressions were accompanied by significant activation of transcription factor Snail in LNCaP and PC-3 cells. Treatment with the CXCR blocker navarixin inhibited the invasion of PC-3 cells but not LNCaP cells. However, invasion induced by MEG-01 was inhibited by navarixin in both LNCaP and PC-3 cells. The collective findings demonstrate that IL-8 enhances CXCR2 expression, which antagonistically regulates AR expression. More importantly, through changes in IL-8/CXCR2-regulated gene expression, IL-8 induces antiandrogen therapy resistance and epithelial-mesenchymal transition in prostate cancer.

Targeting the IL4 receptor with MDNA55 in patients with recurrent glioblastoma: Results of a phase IIb trial.

MDNA55 is an interleukin 4 receptor (IL4R)-targeting toxin in development for recurrent GBM, a universally fatal disease. IL4R is overexpressed in GBM as well as cells of the tumor microenvironment. High expression of IL4R is associated with poor clinical outcomes. MDNA55-05 is an open-label, single-arm phase IIb study of MDNA55 in recurrent GBM (rGBM) patients with an aggressive form of GBM (de novo GBM, IDH wild-type, and nonresectable at recurrence) on their 1st or 2nd recurrence. MDNA55 was administered intratumorally as a single dose treatment (dose range of 18 to 240 ug) using convection-enhanced delivery (CED) with up to 4 stereo-tactically placed catheters. It was co-infused with a contrast agent (Gd-DTPA, Magnevist®) to assess distribution in and around the tumor margins. The flow rate of each catheter did not exceed 10μL/min to ensure that the infusion duration did not exceed 48 h. The primary endpoint was mOS, with secondary endpoints determining the effects of IL4R status on mOS and PFS. MDNA55 showed an acceptable safety profile at doses up to 240 μg. In all evaluable patients (n = 44) mOS was 11.64 months (80% one-sided CI 8.62, 15.02) and OS-12 was 46%. A subgroup (n = 32) consisting of IL4R High and IL4R Low patients treated with high-dose MDNA55 (>180 ug) showed the best benefit with mOS of 15 months, OS-12 of 55%. Based on mRANO criteria, tumor control was observed in 81% (26/32), including those patients who exhibited pseudo-progression (15/26). MDNA55 demonstrated tumor control and promising survival and may benefit rGBM patients when treated at high-dose irrespective of IL4R expression level.Trial Registration: Clinicaltrials.gov NCT02858895.

Roles of IL-7R Induced by Interactions between Cancer Cells and Macrophages in the Progression of Esophageal Squamous Cell Carcinoma.

High infiltration of tumor-associated macrophages (TAMs), which contribute to the progression of several cancer types, is correlated with poor prognosis of esophageal squamous cell carcinoma (ESCC). In addition to the previously reported increase in migration and invasion, ESCC cells co-cultured directly with macrophages exhibited enhanced survival and growth. Furthermore, interleukin-related molecules are associated with ESCC; however, the precise mechanism underlying this association is unclear. Therefore, we explored the role of interleukin-related molecules in ESCC progression. A cDNA microarray analysis of monocultured and co-cultured ESCC cells revealed that the interleukin 7 receptor (IL-7R) was upregulated in ESCC cells co-cultured with macrophages. Overexpression of IL-7R promoted the survival and growth of ESCC cells by activating the Akt and Erk1/2 signaling pathways. The IL-7/IL-7R axis also contributed to the promotion of ESCC cell migration via the Akt and Erk1/2 signaling pathways. Furthermore, immunohistochemistry showed that ESCC patients with high IL-7R expression in cancer nests exhibited a trend toward poor prognosis in terms of disease-free survival, and showed significant correlation with increased numbers of infiltrating macrophages and cancer-associated fibroblasts. Therefore, IL-7R, which is upregulated when directly co-cultured with macrophages, may contribute to ESCC progression by promoting the development of various malignant phenotypes in cancer cells.