Loss-of-function mutations in the C terminus of TPL2 kinase promote oncogenesis by impeding its proteasomal degradation, leading to sustained protein expression. However, the degradation mechanism for TPL2 has remained elusive. Through proximity-dependent biotin identification (BioID), we uncovered tripartite motif-containing 4 (TRIM4) as the E3 ligase that binds and degrades TPL2 by polyubiquitination of lysines 415 and 439. The naturally occurring TPL2 mutants R442H and E188K exhibit impaired TRIM4 binding, enhancing their stability. We further discovered that TRIM4 itself is stabilized by another E3 ligase, TRIM21, which in turn is regulated by KRAS. Mutant KRAS recruits RNF185 to degrade TRIM21 and subsequently TRIM4, thereby stabilizing TPL2. In the presence of mutant KRAS, TPL2 phosphorylates and degrades GSK3β, resulting in β-catenin stabilization and activation of the Wnt pathway. These findings elucidate the physiological mechanisms regulating TPL2 and its exploitation by mutant KRAS, underscoring the need to develop TPL2 inhibitors for KRAS-mutant cancers.

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.

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.

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.

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.

Excessive 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.

Signaling through adhesion-related molecules is important for cancer growth and metastasis and cancer cells are resistant to anoikis, a form of cell death ensued by cell detachment from the extracellular matrix. Herein, we report that detached carcinoma cells and immortalized fibroblasts display defects in TNF and CD40 ligand (CD40L)-induced MEK-ERK signaling. Cell detachment results in reduced basal levels of the MEK kinase TPL2, compromises TPL2 activation and sensitizes carcinoma cells to death-inducing receptor ligands, mimicking the synthetic lethal interactions between TPL2 inactivation and TNF or CD40L stimulation. Focal Adhesion Kinase (FAK), which is activated in focal adhesions and mediates anchorage-dependent survival signaling, was found to sustain steady state TPL2 protein levels and to be required for TNF-induced TPL2 signal transduction. We show that when FAK levels are reduced, as seen in certain types of malignancy or malignant cell populations, the formation of cIAP2:RIPK1 complexes increases, leading to reduced TPL2 expression levels by a dual mechanism: first, by the reduction in the levels of NF-κΒ1 which is required for TPL2 stability; second, by the engagement of an RelA NF-κΒ pathway that elevates interleukin-6 production, leading to activation of STAT3 and its transcriptional target SKP2 which functions as a TPL2 E3 ubiquitin ligase. These data underscore a new mode of regulation of TNF family signal transduction on the TPL2-MEK-ERK branch by adhesion-related molecules that may have important ramifications for cancer therapy.

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.

Pancreatic ductal adenocarcinoma (PDAC) remains highly refractory to treatment. While the KRAS oncogene is present in almost all PDAC cases and accounts for many of the malignant feats of PDAC, targeting KRAS or its canonical, direct effector cascades remains unsuccessful in patients. The recalcitrant nature of PDAC is also heavily influenced by its highly fibro-inflammatory tumor microenvironment (TME), which comprises an acellular extracellular matrix and various types of non-neoplastic cells including fibroblasts, immune cells, and adipocytes, underscoring the critical need to delineate the bidirectional signaling interplay between PDAC cells and the TME in order to develop novel therapeutic strategies. The impact of tumor-cell KRAS signaling on various cell types in the TME has been well covered by several reviews. In this article, we critically reviewed evidence, including work from our group, on how the feedback inflammatory signals from the TME impact and synergize with oncogenic KRAS signaling in PDAC cells, ultimately augmenting their malignant behavior. We discussed past and ongoing clinical trials that target key inflammatory pathways in PDAC and highlight lessons to be learned from outcomes. Lastly, we provided our perspective on the future of developing therapeutic strategies for PDAC through understanding the breadth and complexity of KRAS and the inflammatory signaling network.

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.