Tumor Progression Locus Research Articles

Luteolin and its analog luteolin-7-methylether from Leonurus japonicus Houtt suppress aromatase-mediated estrogen biosynthesis to alleviate polycystic ovary syndrome by the inhibition of tumor progression locus 2

Ethnopharmacological relevanceLeonurus japonicus Houtt (L. japonicus, Chinese motherwort), known as Yi Mu Cao which means “good for women”, has long been widely used in China and other Asian countries to alleviate gynecological disorders, often characterized by estrogen dysregulation. It has been used for the treatment of polycystic ovary syndrome (PCOS), a common endocrine disorder in women but the underlying mechanism remains unknown. Aim of the studyThe present study was designed to investigate the effect and mechanism of flavonoid luteolin and its analog luteolin-7-methylether contained in L. japonicus on aromatase, a rate-limiting enzyme that catalyzes the conversion of androgens to estrogens and a drug target to induce ovulation in PCOS patients. Materials and methodsEstrogen biosynthesis in human ovarian granulosa cells was examined using ELISA. Western blots were used to explore the signaling pathways in the regulation of aromatase expression. Transcriptomic analysis was conducted to elucidate the potential mechanisms of action of compounds. Finally, animal models were used to assess the therapeutic potential of these compounds in PCOS. ResultsLuteolin potently inhibited estrogen biosynthesis in human ovarian granulosa cells stimulated by follicle-stimulating hormone. This effect was achieved by decreasing cAMP response element-binding protein (CREB)-mediated expression of aromatase. Mechanistically, luteolin and luteolin-7-methylether targeted tumor progression locus 2 (TPL2) to suppress mitogen-activated protein kinase 3/6 (MKK3/6)-p38 MAPK-CREB pathway signaling. Transcriptional analysis showed that these compounds regulated the expression of different genes, with the MAPK signaling pathway being the most significantly affected. Furthermore, luteolin and luteolin-7-methylether effectively alleviated the symptoms of PCOS in mice. ConclusionsThis study demonstrates a previously unrecognized role of TPL2 in estrogen biosynthesis and suggests that luteolin and luteolin-7-methylether have potential as novel therapeutic agents for the treatment of PCOS. The results provide a foundation for further development of these compounds as effective and safe therapies for women with PCOS.

Tpl2-/- macrophages exhibit impaired mitochondrial respiration due to type I interferon signaling in response to LPS stimulation.

Abstract Macrophages are essential to the innate immune system and their dysfunction has been implicated in many diseases, including autoimmunity, obesity, and cancer. Activated macrophages can alter their oxidative phosphorylation, metabolite production, and reactive oxygen species generation to elicit their diverse functions. Tumor progression locus 2 (Tpl2, MAP3K8, or COT) is a serine-threonine kinase involved in macrophage activation and function. In TLR-stimulated macrophages, TPL2 ablation induces high type I interferon (IFN) production. Previous research implicates type I IFNs in metabolic regulation, but their exact mechanism remains unclear. We hypothesize that TPL2 promotes oxidative phosphorylation in LPS-stimulated macrophages by suppressing type I IFN signaling. LPS-stimulated Tpl2-/- bone marrow-derived macrophages had decreased oxygen consumption, ATP production, and maximal respiration compared to LPS-stimulated wild type and Tpl2-/-IFNAR-/- macrophages. LPS-stimulated Tpl2-/- macrophages had reduced antioxidant gene expression compared to wild type macrophages, including SOD2, HMOX-1, and PRDX5. Disrupted IFN signaling in IFNAR-/- and Tpl2-/-IFNAR-/- macrophages significantly increased antioxidant gene expression, suggesting the absence of IFN signaling mitigates macrophage oxidative stress. These data indicate that Tpl2-/- macrophages are metabolically defective and type I IFNs contribute to the underlying dysfunction.

Overexpression of TPL2 may be a predictor of good prognosis in patients with breast invasive ductal carcinoma

The objective of this study was to investigate the clinical significance and roles of tumor progression locus 2 (TPL2) and peptidyl-prolyl cis–trans isomerase 1 (Pin1) in the occurrence and development of breast invasive ductal carcinoma (IDC). Immunohistochemistry was used to detect the expression of TPL2 and Pin1 in human breast tissues, which included normal breast tissues (Normal), tissues with fibrocystic changes (FCC), ductal carcinoma in situ (DCIS), and IDC. The roles of TPL2 and Pin1 in the occurrence and development of IDC, as well as the correlation between their expression levels and clinicopathological parameters, were analyzed. Compared with Normal and FCC groups, the overexpression of TPL2 and Pin1 was significantly increased in DCIS and IDC groups (DCIS vs Normal: P = 0.002/P < 0.001; IDC vs Normal: P = 0.007/P = 0.003; DCIS vs. FCC: P = 0.008/P = 0.004; IDC vs. FCC: P = 0.04/P = 0.043). The expression levels of TPL2 and Pin1 were positively correlated in DCIS and IDC groups (P = 0.001, P = 0.011). In the IDC group, the Ki67 level in the TPL2 overexpression group was significantly lower than that in the TPL2 low expression group (P = 0.02). The TPL2 overexpression rate was significantly higher in IDC with histological grades 1–2 than that in IDC with histological grade 3 (P = 0.029). The TPL2 overexpression rate in IDC with tumor-node-metastasis (TNM) stage I was significantly higher than that in IDC with TNM stages II–III (P = 0.035). We conclude that TPL2 and Pin1 may synergistically promote the occurrence and development of IDC, but TPL2 overexpression may be an early molecular event in IDC development. TPL2 overexpression is significantly related with IDC with lower malignancy or earlier TNM stage, suggesting that the prognosis of IDC patients with TPL2 overexpression may be better and TPL2 overexpression may be a predictor of good prognosis in IDC.

TPL2 kinase activity regulates microglial inflammatory responses and promotes neurodegeneration in tauopathy mice.

Tumor progression locus 2 (TPL2) (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates microglia activation states and brain cytokine levels. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNA sequencing analysis indicates that protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity can promote multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS (inducible nitric oxide synthase) induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions.

Tumor Progression Locus 2 Promotes Mouse Plasmacytoid Dendritic Cell Cytokine Secretion and Signaling

Abstract The autoimmune disorder systemic lupus erythematosus is thought to be caused by the inefficient clearance of dying cells. This releases nucleic acid debris that stimulates type I interferon (T1 IFN) secretion. Plasmacytoid dendritic cells (pDCs) are the major source of T1 IFNs, which are causally linked to lupus pathogenesis. Both T1 IFNs and pDCs are thus the focus of intense studies for immunotherapies. The serine-threonine kinase, tumor progression locus 2 (TPL2), negatively regulates T1 IFN secretion by conventional dendritic cells (cDCs) and macrophages in response to Toll-like receptor 9 (TLR9) stimulation in vitro. In contrast, we and others have shown that TPL2 is required for T1 IFN production in TLR9-stimulated pDCs. The goal of this study is to validate TPL2-dependent regulation of pDC function in vivoand to delineate the mechanisms responsible. Both cDCs and pDCs develop normally in Tpl2−/−mice, but Tpl2−/−mice show defective secretion of T1 IFNs and proinflammatory cytokines in response to administration of CpG (TLR9) or R848 (TLR7) in vivo. The same defect in T1 IFN and proinflammatory cytokine production was observed in bone marrow-derived pDCs and cDCs in vitro. A modest defect in TLR-induced expression of maturation markers CD80, CD86 and MHC class II was seen in Tpl2−/−pDCs, suggesting impaired NFkB activation. Flow cytometric measurement of phosphoproteins demonstrated that TPL2 is also required for TLR-induced activation of MAPK and ribosomal S6 kinase pathways in pDCs. Ongoing studies are examining the role of TPL2 in TLR trafficking and lupus pathogenesis. These data establish TPL2-dependent regulation of pDC functions, which may be exploited for therapeutic intervention in lupus or other IFN-related diseases. Supported by grants from NIH/NIAMS (R21 AR075317) titled "Tpl2 regulation of pDC function and SLE pathogenesis"

Tpl2−/− macrophages exhibit impaired mitochondrial respiration and function in response to LPS stimulation.

Abstract Macrophages are instrumental in maintaining immune homeostasis, and their dysfunction or depletion is tied to many human diseases, including autoimmunity, obesity, and cancer. Activated macrophages modify their cellular metabolic pathways, such as glycolysis and oxidative phosphorylation, to drive their diverse functions. Many metabolic processes occur in the mitochondria, including oxidative phosphorylation, metabolite production, and reactive oxygen species (ROS) generation. Previous research demonstrated that tumor progression locus 2 (Tpl2, MAP3K8, or COT) is required for the induction of oxidative phosphorylation and the alternatively activated phenotype in macrophages during Schistosome infection; however, Tpl2 regulation of cellular metabolism in classically activated macrophages remains unexplored. We hypothesize that Tpl2 normally promotes oxidative phosphorylation, and its absence causes mitochondrial dysfunction. LPS-stimulated Tpl2−/−bone marrow-derived macrophages (BMDMs) had decreased oxygen consumption, basal respiration, ATP production, and maximal respiration compared to LPS-stimulated wild type BMDMs. Additionally, Tpl2−/−BMDMs exhibited higher basal and LPS-stimulated ROS production than wild type BMDMs. These data suggest that Tpl2−/−mitochondria are functionally defective, and the absence of TPL2 increases mitochondrial oxidative stress. Together, this may reveal the underlying mechanism for defective Tpl2−/−macrophage behavior. Understanding the role of Tpl2 in classically activated macrophages will provide a foundation for developing targeted therapies to treat cancer and autoimmune diseases. Supported by grant from NIH (R21 AI147003).

Abstract 2620: TRIM4 is the E3 ubiquitin-ligase for TPL2/MAP3K8 and is modulated by oncogenic KRAS

Abstract TPL2 (tumor progression locus 2, a. k. a. Cot/MAP3K8) kinase becomes oncogenic via disruption of the C-terminus, which abrogates its proteasomal degradation. However, the mechanism underlying degradation of TPL2 remains unclear. Using proximity-dependent biotin identification (BioID), for the first time we have discovered that Tripartite Motif Containing 4 (TRIM4) is the E3-ligase that binds and degrades TPL2 via polyubiquitination of lysine 415 and 439. Notably, naturally-occurring oncogenic R442H and E188K mutations in TPL2 abrogated the binding and polyubiquitination by TRIM4. Furthermore, KRAS oncoprotein stabilizes TPL2 by promoting TRIM4 degradation. Using immunoprecipitation-mass spectrometry, we identified TRIM21 as a ligase that binds and stabilizes TRIM4, in part by reducing TRIM4 polyubiquitination. Therefore, oncogenic KRAS binds to and promotes TRIM21 degradation, destabilizing TRIM4 and prolonging the half-life of TPL2. Overall, we discovered novel mechanisms of TPL2 regulation which are influenced by oncogenic KRAS, providing opportunities for the development of novel therapies. Citation Format: Sapana Prakashrao Bansod. TRIM4 is the E3 ubiquitin-ligase for TPL2/MAP3K8 and is modulated by oncogenic KRAS [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 2620.

TPL2, a New Prognostic Factor and a Potential Therapeutic Target in ABC-DLBCL

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype of DLBCL, characterized by the constitutive activation of NF-κB and JAK/STAT3 pathways on the background of the chronic active BCR signaling. Being featured by mutations in MYD88 and/or CD79B, it potentially comprises the MCD/C5 subtype in the recent genetics-based categorization. In light of the inferior prognosis of this subtype to germinal center B-cell (GCB) like DLBCL, identification of subtype-specific strategies has been sought. Tumor progression locus 2 (TPL2) is an intracellular serine/threonine kinase that is reported to be activated downstream of TLRs/MYD88 signaling and to play roles in oncogenesis. However, the involvement of TPL2 in B-cell malignancies is less defined despite relatively higher transcript expression than in solid tumors. Results: To address the impact of TPL2 protein levels on DLBCL phenotypes, we performed an IHC analysis on 79 FFPE samples of DLBCL diagnosed and treated at our institution (48 non-GCB and 31 GCB according to Hans algorithm). When divided into two groups, one with strong TPL2 expression (TPL-high) and the other with no/weak expression (TPL2-low), the ratio of TPL2-high did not differ between non-GCB and GCB (67% vs 64%). However, TPL2-high had a significantly higher percentage of positive B symptom (42% vs 11%, p=0.005) and extra-nodal involvement (75% vs 52%, p=0.047) compared to TPL2-low, and these trends were observed more specifically in non-GCB. Moreover, it is noteworthy that TPL2-high showed a significantly lower overall survival in non-GCB (p=0.037), but did not in GCB (p=0.999) (Figure 1). Intriguingly, the analysis combined with pSTAT3 (Y705) staining revealed that TPL2-high accompanied by positive pSTAT3 tended to display particularly poor prognosis in non-GCB. Then, we analyzed the mechanism by which TPL2 is involved in the tumorigenesis of ABC-DLBCL using TMD8, a well-described ABC-DLBCL cell line with MYD88L265P and CD79BY196 mutations. A specific TPL2 inhibitor significantly affected the cell proliferation of TMD8 cells, whereas did not on the cells with GCB phenotype. Of note, TPL2 inhibitor induced G1 cell-cycle arrest via downregulation of CyclinE and CDK2 and upregulation of p27 in western blot analysis, which was confirmed by quantitative PCR. Next, we investigated the downstream signaling of TPL2 in ABC-DLBCL. Western blot analysis revealed that TPL2 inhibitor as well as genetic TPL2 knock down suppressed the activation of NF-κB components and STAT3, and the expression of c-MYC. These results implied the involvement of TPL2 in IL-6/IL-10 autocrine loop and resultant JAK/STAT3 activation. As expected, treatment with TPL2 inhibitor downregulated IL-6/IL-10 mRNA by quantitative PCR and accordingly decreased the levels of IL-6/IL-10 in the cell culture supernatant of treated TMD8 cells. Therefore, we speculated that TPL2 promoted the oncogenic activation of NF-κB and STAT3 pathways via IL-6/IL-10 upregulation in ABC-subtype. Lastly, we evaluated the physiological function of TPL2 by western blot analysis using 293T. Although an overexpression TPL2 alone did not elicit NF-κB and STAT3 activation, when co-expressed with MYD88L265P TPL2 obviously activated these pathways, whereas dominant negative TPL2 failed to demonstrate those effects even with MYD88L265P co-expression. These findings indicated that TPL2 could possibly exert its tumorigenicity in MYD88L265P-dependent manner in ABC-DLBCL. Conclusion: In association with the importance as an unfavorable prognostic factor, our results suggested that TPL2 might mediate the crosstalk between the oncogenic MYD88L265P and the activation of NF-κB and STAT3 pathways, coordinately contributing to the tumorigenesis of ABC-DLBCL. From this standpoint, TPL2 could emerge as a new therapeutic target in ABC-DLBCL. Figure legend Overall survival of non-GCB (A) and GCB (B) DLBCL patients, with regard to the TPL2 expression: TPL2-low (red line) or TPL2-high (black line). Overall survival was determined by the Kaplan-Meier method and compared by using the log-rank test. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Influenza-Induced Pulmonary Inflammatory Exudate in Susceptible Tpl2-Deficient Mice Is Dictated by Type I IFN Signaling.

The most prominent host response to viral infection is the production of type 1 interferons (T1 IFNs). One host regulator of the T1 IFNs is the serine-threonine kinase, tumor progression locus 2 (TPL2). We have previously demonstrated that Tpl2−/− mice succumb to infection with a low-pathogenicity influenza A strain (x31), in association with with increased pulmonary levels of interferon-β (IFN-β), chemokine CCL2, and excessive monocyte and neutrophil pulmonary infiltration. TPL2-dependent overexpression of IFN-β has been implicated in enhanced susceptibility to Mycobacterium tuberculosis; therefore, we examined the role of T1 IFNs in susceptibility of Tpl2−/− mice to influenza. CCL2 overexpression and monocyte recruitment were normalized in Ifnar1−/−Tpl2−/− mice, confirming that TPL2 constrains inflammatory monocyte recruitment via inhibition of the T1 IFN/CCL2 axis. Unexpectedly, excessive neutrophil recruitment in Ifnar1−/− strains was further exacerbated by simultaneous TPL2 genetic ablation in Ifnar1−/−Tpl2−/− by 7 dpi, accompanied by overexpression of neutrophil-regulating cytokines, CXCL1 and IFN-λ. Collectively, our data suggest that TPL2 and T1 IFNs synergize to inhibit neutrophil recruitment. However, treatment with the neutrophil-depleting anti-Ly6G antibody showed only a modest improvement in disease. Analysis of sorted innate immune populations revealed redundant expression of inflammatory mediators among neutrophils, inflammatory monocytes and alveolar macrophages. These findings suggest that targeting a single cell type or mediator may be inadequate to control severe disease characterized by a mixed inflammatory exudate. Future studies will consider TPL2-regulated pathways as potential predictors of severe influenza progression as well as investigate novel methods to modulate TPL2 function during viral infection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10753-022-01736-8.

Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

Establishment and characterization of a new activated B-cell-like DLBCL cell line, TMD12

We report the establishment of a novel activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, from a patient with highly refractory DLBCL. ABC-DLBCL is a subtype with a relatively unfavorable prognosis that was originally categorized using gene expression profiling according to its cell of origin. TMD12 cells were isolated from the pleural effusion of the patient at relapse and passaged continuously in vitro for >4 years. The cells displayed cluster of differentiation (CD)19, CD20, CD22, CD38, human leukocyte antigen-DR isotype, and κ positivity and CD5, CD10, CD23, and λ negativity, as detected using flow cytometric analysis. The chromosomal karyotypic analysis, including the spectral karyotyping method, confirmed t(1;19)(q21:q13.1), del(6q23), gain of chromosome 18, and other abnormalities. Mutation analyses, including whole-exome sequencing, revealed that TMD12 cells harbored mutations in MYD88 and CD79B, indicating an ABC subtype. TMD12 cells exhibited chronic active B-cell receptor signaling and constitutive activation of the nuclear factor κB pathway, which is typically associated with sensitivity to a specific Bruton tyrosine kinase inhibitor, ibrutinib. Intriguingly, TMD12 cells displayed moderate resistance to ibrutinib and lacked activation of Janus kinase/signal transducers and activators of transcription 3 signaling, another hallmark of this DLBCL subtype. Treatment with an inhibitor against tumor progression locus 2 (TPL2), a multifunctional intracellular kinase that is activated particularly downstream of Toll-like receptors or MYD88 and IκB kinase α/β (IKKα/β), suppressed the proliferation of TMD12 cells, implying the possible involvement of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. Because only a limited number of ABC-DLBCL cell lines are currently available, TMD12 cells might provide a useful tool in the search for novel druggable targets for this intractable lymphoma.

TPL2 kinase expression is regulated by the p38γ/p38δ-dependent association of aconitase-1 with TPL2 mRNA

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.

Tumor Progression Locus 2 Protects against Acute Respiratory Distress Syndrome in Influenza A Virus-Infected Mice.

ABSTRACTExcessive inflammation in patients with severe influenza disease may lead to acute lung injury that results in acute respiratory distress syndrome (ARDS). ARDS is associated with alveolar damage and pulmonary edema that severely impair gas exchange, leading to hypoxia. With no existing FDA-approved treatment for ARDS, it is important to understand the factors that lead to virus-induced ARDS development to improve prevention, diagnosis, and treatment. We have previously shown that mice deficient in the serine-threonine mitogen-activated protein kinase, Tpl2 (MAP3K8 or COT), succumb to infection with a typically low-pathogenicity strain of influenza A virus (IAV; HKX31, H3N2 [x31]). The goal of the current study was to evaluate influenza A virus-infected Tpl2−/− mice clinically and histopathologically to gain insight into the disease mechanism. We hypothesized that Tpl2−/− mice succumb to IAV infection due to development of ARDS-like disease and pulmonary dysfunction. We observed prominent signs of alveolar septal necrosis, hyaline membranes, pleuritis, edema, and higher lactate dehydrogenase (LDH) levels in the lungs of IAV-infected Tpl2−/− mice compared to wild-type (WT) mice from 7 to 9 days postinfection (dpi). Notably, WT mice showed signs of regenerating epithelium, indicative of repair and recovery, that were reduced in Tpl2−/− mice. Furthermore, biomarkers associated with human ARDS cases were upregulated in Tpl2−/− mice at 7 dpi, demonstrating an ARDS-like phenotype in Tpl2−/− mice in response to IAV infection.IMPORTANCE This study demonstrates the protective role of the serine-threonine mitogen-activated protein kinase, Tpl2, in influenza virus pathogenesis and reveals that host Tpl2 deficiency is sufficient to convert a low-pathogenicity influenza A virus infection into severe influenza disease that resembles ARDS, both histopathologically and transcriptionally. The IAV-infected Tpl2−/− mouse thereby represents a novel murine model for studying ARDS-like disease that could improve our understanding of this aggressive disease and assist in the design of better diagnostics and treatments.

FRI187 - Inhibition of tumour progression locus 2 (TPL2) halts the progression of liver fibrosis in a stringent long term choline deficient high-fat diet (CdHFD) rat model

FRI187 - Inhibition of tumour progression locus 2 (TPL2) halts the progression of liver fibrosis in a stringent long term choline deficient high-fat diet (CdHFD) rat model

Cellular mechanism underlying the facilitation of contractile response induced by IL-25 in mouse tracheal smooth muscle.

Asthma is a common heterogeneous respiratory disease characterized by airway inflammation and airway hyperresponsiveness (AHR) which is associated with abnormality in smooth muscle contractility. The epithelial cell-derived cytokine IL-25 is implicated in type 2 immune pathology including asthma, whereas the underlying mechanisms have not been fully elucidated. This study aims to investigate the effects of IL-25 on mouse tracheal smooth muscle contractility and elucidate the cellular mechanisms. Incubation with IL-25 augmented the contraction of mouse tracheal smooth muscles, which could be suppressed by the L-type voltage-dependent Ca2+ channel (L-VDCC) blocker nifedipine. Furthermore, IL-25 enhanced the cytosolic Ca2+ signals and triggered the upregulation of α1C L-VDCC (CaV1.2) in primary cultured mouse tracheal smooth muscle cells. Knocking down IL-17RA/IL-17RB receptors or inhibiting the transforming growth factor-β-activated kinase 1 (TAK1)-tumor progression locus 2 (TPL2)-MAPK kinase 1/2 (MEK1/2)-ERK1/2-activating protein-1 (AP-1) signaling pathways suppressed the IL-25-elicited upregulation of CaV1.2 and hyperreactivity in tracheal smooth muscles. Moreover, inhibition of TPL2, ERK1/2 or L-VDCC alleviated the AHR symptom induced by IL-25 in a murine model. This study revealed that IL-25 potentiated the contraction of tracheal smooth muscle and evoked AHR via activation of TPL2-ERK1/2-CaV1.2 signaling, providing novel targets for the treatment of asthma with a high-IL-25 phenotype.

Influenza-induced Tpl2 expression within alveolar epithelial cells is dispensable for host viral control and anti-viral immunity.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that regulates the expression of inflammatory mediators in response to Toll-like receptors (TLR) and cytokine receptors. Global ablation of Tpl2 leads to severe disease in response to influenza A virus (IAV) infection, characterized by respiratory distress, and studies in bone marrow chimeric mice implicated Tpl2 in non-hematopoietic cells. Lung epithelial cells are primary targets and replicative niches of influenza viruses; however, the specific regulation of antiviral responses by Tpl2 within lung epithelial cells has not been investigated. Herein, we show that Tpl2 is basally expressed in primary airway epithelial cells and that its expression increases in both type I and type II airway epithelial cells (AECI and AECII) in response to influenza infection. We used Nkx2.1-cre to drive Tpl2 deletion within pulmonary epithelial cells to delineate epithelial cell-specific functions of Tpl2 during influenza infection in mice. Although modest increases in morbidity and mortality were attributed to cre-dependent deletion in lung epithelial cells, no alterations in host cytokine production or lung pathology were observed. In vitro, Tpl2 inhibition within the type I airway epithelial cell line, LET1, as well as genetic ablation in primary airway epithelial cells did not alter cytokine production. Overall, these findings establish that Tpl2-dependent defects in cells other than AECs are primarily responsible for the morbidity and mortality seen in influenza-infected mice with global Tpl2 ablation.

Unravelling the Impact of the Genetic Variant rs1042058 within the TPL2 Risk Gene Locus on Molecular and Clinical Disease Course Patients with Inflammatory Bowel Disease.

Background: The single nucleotide polymorphism (SNP) rs1042058 within the gene locus encoding tumor progression locus 2 (TPL2) has been recently identified as a risk gene for inflammatory bowel disease (IBD). TPL2 has been shown to regulate pro-inflammatory signaling and cytokine secretion, while inhibition of TPL2 decreases intestinal inflammation in vivo. However, the clinical and molecular implications of this disease-associated TPL2 variation in IBD patients have not yet been studied. Methods: We analyzed the impact of the IBD-associated TPL2 variation using clinical data of 2145 genotyped patients from the Swiss IBD Cohort Study (SIBDCS). Furthermore, we assessed the molecular consequences of the TPL2 variation in ulcerative colitis (UC) and Crohn’s disease (CD) patients by real-time PCR and multiplex ELISA of colon biopsies or serum, respectively. Results: We found that presence of the SNP rs1042058 within the TPL2 gene locus results in significantly higher numbers of CD patients suffering from peripheral arthritis. In contrast, UC patients carrying this variant feature a lower risk for intestinal surgery. On a molecular level, the presence of the rs1042058 (GG) IBD-risk polymorphism in TPL2 was associated with decreased mRNA levels of IL-10 in CD patients and decreased levels of IL-18 in the intestine of UC patients. Conclusions: Our data suggest that the presence of the IBD-associated TPL2 variation might indicate a more severe disease course in CD patients. These results reveal a potential therapeutic target and demonstrate the relevance of the IBD-associated TPL2 SNP as a predictive biomarker in IBD.

Tpl2 Ablation Leads to Hypercytokinemia and Excessive Cellular Infiltration to the Lungs During Late Stages of Influenza Infection.

Tumor progression locus 2 (Tpl2) is a serine-threonine kinase known to promote inflammation in response to various pathogen-associated molecular patterns (PAMPs), inflammatory cytokines and G-protein-coupled receptors and consequently aids in host resistance to pathogens. We have recently shown that Tpl2-/- mice succumb to infection with a low-pathogenicity strain of influenza (x31, H3N2) by an unknown mechanism. In this study, we sought to characterize the cytokine and immune cell profile of influenza-infected Tpl2-/- mice to gain insight into its host protective effects. Although Tpl2-/- mice display modestly impaired viral control, no virus was observed in the lungs of Tpl2-/- mice on the day of peak morbidity and mortality suggesting that morbidity is not due to virus cytopathic effects but rather to an overactive antiviral immune response. Indeed, increased levels of interferon-β (IFN-β), the IFN-inducible monocyte chemoattractant protein-1 (MCP-1, CCL2), Macrophage inflammatory protein 1 alpha (MIP-1α; CCL3), MIP-1β (CCL4), RANTES (CCL5), IP-10 (CXCL10) and Interferon-γ (IFN-γ) was observed in the lungs of influenza-infected Tpl2-/- mice at 7 days post infection (dpi). Elevated cytokine and chemokines were accompanied by increased infiltration of the lungs with inflammatory monocytes and neutrophils. Additionally, we noted that increased IFN-β correlated with increased CCL2, CXCL1 and nitric oxide synthase (NOS2) expression in the lungs, which has been associated with severe influenza infections. Bone marrow chimeras with Tpl2 ablation localized to radioresistant cells confirmed that Tpl2 functions, at least in part, within radioresistant cells to limit pro-inflammatory response to viral infection. Collectively, this study suggests that Tpl2 tempers inflammation during influenza infection by constraining the production of interferons and chemokines which are known to promote the recruitment of detrimental inflammatory monocytes and neutrophils.

Map3k8/COT1/Tpl2 Drives Resistance to Bcr-Abl1 Inhibition through Activation of MEK-ERK and NF-Kb Pathways

The introduction of tyrosine kinase inhibitors (TKI) has transformed chronic myeloid leukemia (CML) into a manageable, chronic disease with long-term survival exceeding 85%, but discontinuation of TKI in patients who achieve deep molecular remissions results in a 50% relapse rate. The relapses are believed to be caused by Bcr-Abl1-expressing hematopoietic stem cells (CML stem cells) which are independent of the Bcr-Abl1 kinase activity for their survival. Signaling pathways, including PI3K/AKT, Wnt/β-catenin, JAK/STAT, and Hedgehog/SMO, are crucial for CML stem cell self-renewal and survival in the presence of TKI. Eradication of these cells poses the greatest current challenge.We previously described a Bcr-Abl1-independent model of resistance to imatinib mesylate (IM), in which K562 cells cultured in high concentration of IM displayed reduced Bcr-Abl1 protein and activity levels (Chorzalska et al., Leukemia 2014). Quantitative phosphoproteomic analysis of these IM-resistant cells (IMRC) identified 2,593 phosphopeptides showing significant differences in abundancies in comparison to IM-sensitive cells. Functional analysis of identified phosphopeptides pointed to MEK-ERK pathway as a highly dysregulated cascade in IMRC (Chorzalska et al., Stem Cell Dev, 2017). Within the list of identified phosphopeptides, we assessed the abundancy of 222 MEK-ERK pathway-specific core interactor peptides (as listed in MEK-ERK superpathway in Pathcards), and identified the Map3k8-derived phosphopeptide as the most abundant one in IMRC (Fig. 1A). Map3k8, also known as Tumor Progression Locus (Tpl2) or Cancer Osaka Thyroid (COT1), links the MEK-ERK and NF-κB signaling and drives resistance to Raf inhibition in melanoma, but its role in chemoresistance of CML stem cells has not been explored.Using RT-PCR assays in CML CD34+ cells cultured with IM for 7 days, we found a 2-fold increase in MAP3K8 transcriptsand a 1.7-fold increase in transcripts of its binding partner NFKB1 (NF-κB1; Fig. 1B). IMRC also showed increased expression of Map3k8 and NF-κB1 protein levels, and increased activity of the NF-κB signaling components: NF-κB inhibitor - IκB and IκB kinase - IKKα/β (Fig. 1C). Immunoblotting evaluation of the Map3k8-dependent effect on MEK-ERK signaling indicated no significant difference between IMRC and control cells in the activity of Raf module, with exception of C-Raf Ser289/296/301 inhibitory sites associated with a MEK-ERK negative feedback loop (Fig. 1D). Evaluation of the downstream effectors of Raf showed no difference in MEK1 and decrease in MEK2 abundance, and enhanced phosphorylation of MEK1/2. IMRC also showed higher ERK1 and lower ERK2 abundance, and no significant change in the stoichiometry of ERK1/ERK2 phosphorylation at Thr202/Tyr204 (Fig. 1D). To identify other upstream mediators of MEK phosphorylation, we evaluated the activity of Src Family Kinases (SFKs) and found a marked increase in phospho-SFKs in IMRC (Fig. 1E).Considering the observed upregulation of SFKs, MEK and NF-κB signaling in Map3k8-overexpressing IMRC, we investigated apoptotic effects of the SFKs inhibitor dasatinib, the MEK inhibitor U0126, and the IKK inhibitor PS-1145, individually and in combination. The SFKs and IKK inhibitors, as single agents or combined, induced only a modest (35%) increase in apoptosis. Conversely, the combination of SFKs+MEK inhibitors decreased survival of IMRC by nearly 50%, whereas the triple SFKs+MEK+IKK inhibition killed 65% of IMRC within 48h of incubation (Fig. 1F). In addition, the combination of three inhibitors SFKs+MEK+IKK significantly decreased the phosphorylation of MEK, ERK, SFKs and NF-κB, surpassing the effect of a dual SFKs+MEK or SFKs+IKK inhibition (Fig. 1G). The effect of SFKs, MEK and IKK inhibitors on the capacity of CML CD34+ cells to form colonies in methylcellulose assays was also evaluated. The dual SFKs+MEK inhibition reduced the colony number by 70%, while the triple SFKs+MEK+IKK inhibition decreased it by about 80% (Fig. 1H). Finally, CML CD34+ cells cultured for 7 days in the presence of SFKs+MEK+IKK inhibitors showed significant reduction of MAP3K8 transcript levels (Fig. 1I).In conclusion, combined inhibition of SFKs, MEK and NF-κB attenuates the survival of IM- resistant CML cell line and CML stem/progenitor cells, and a combination of relevant inhibitors may offer a new therapeutic option for overcoming the TKIs resistance in CML patients. [Display omitted] DisclosuresReagan:Teva: Membership on an entity's Board of Directors or advisory committees.

Tpl2 Signaling Regulates Dendritic Cell Activation and Responding T Cell Differentiation Following Mycobacterium tuberculosis Infection

Abstract Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persist inside of host antigen-presenting cells. Dendritic cells (DCs) are important in restricting Mtb growth by migrating to draining lymph nodes and activating antigen-specific T cell responses, but the roles of DCs in Mtb infection require further study. This study investigated DC activation and functional outcomes following Mtb H37Ra-driven tumor progression locus 2 (Tpl2) signaling. The role of Tpl2 was interrogated genetically, utilizing bone marrow-derived DCs from Tpl2−/− mice. We assessed cytokine production via ELISA, mRNA levels via qRT-PCR, and expression of cell surface molecules via flow cytometry. In Mtb-treated DCs, genetic depletion of Tpl2 increased production of pro-inflammatory cytokines such as IL-12p40 and IL-6. Loss of Tpl2 in Mtb-treated DCs also led to decreased E-cadherin expression, and increased expression of Icam-1 (Cd54) and Mmp2, which are molecules involved in DC transmigration. Expression of Ccr7 and Ccr4 was also enhanced in Mtb-treated Tpl2−/− DCs, which correlated with improved migration of Tpl2−/− DCs towards CCL19 and CCL21 in vitro (assessed using a trans-well assay). When antigen-specific CD4+ T cells were co-cultured with Mtb-infected DCs, deletion of Tpl2−/− in the DCs resulted in increased T cell production of IFNγ and IL-2, as well as increased Tbet expression, indicative of enhanced Th1 polarization. Together, these data indicate that Mtb-induced Tpl2 signaling suppresses certain aspects of DC activation, leading to blunted Th1 responses against the pathogen.