Delineation of the distinct inflammatory signaling roles of TAK1 and JAK1/3 in the CIA model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complex autoimmune disease characterized by hyperactive immune cells within the joints, which leads to inflammation, bone degeneration, and chronic pain. For several decades, frontline immunomodulators such as the anti-tumor necrosis factor (TNF) biologics adalimumab (Humira), etanercept (Enbrel), and infliximab (Remicade) have successfully managed disease progression for many patients. However, over time, patients become refractory to these treatments requiring chronic disease to be managed with conventional and more problematic disease modifying antirheumatic drugs such as methotrexate and hydroxychloroquine, and corticosteroids. Due to the large proportion of patients who continue to fail on frontline biologic therapies, there remains an unmet need to derive novel alternative targets with improved efficacy and safety profiles to treat RA. Recent advances in the field have defined novel targets that play important roles in RA pathology, including the Janus activated kinase (JAK) and transforming growth factor beta activated kinase-1 (TAK1). Although three inhibitors of the JAK signaling pathway have been approved for the treatment of moderately to severely active RA in patients who failed on one or more anti-TNFs, at present, no FDA approved TAK1 treatments exist. Our recent discovery of a highly potent and selective, orally bioavailable TAK1 inhibitor has provided insight into the therapeutic potential of this protein kinase as a novel target for RA. Here, we show the distinct cytokine signaling of tofacitnib (Xeljanz; JAK1/3 inhibitor) compared to HS-276 (TAK1 inhibitor) in lipopolysaccharide (LPS) challenged THP-1 cells. Furthermore, in the collagen induced arthritis pre-clinical mouse model of RA, both tofacintib and HS-276 attenuated disease activity score and inflammatory cytokines in the serum. Overall, our results delineate the distinct cytokine signaling of JAK1/3 and TAK1 targeted therapies in vitro and in vivo and suggest that selective TAK1 inhibitors may provide superior therapeutic relief in RA with fewer adverse events.

Expression of membrane Hsp90 is a molecular signature of T cell activation.

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells. In cell-based assays, stimulation of human T cells induced a 20-fold increase in mHsp90 expression at the plasma membrane, suggesting trafficking of mHsp90 is regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in reducing the mean clinical score in the CIA arthritis model. Our results suggest mHsp90 expression on T cells is a molecular marker of T cell activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis.

Expression of ectopic heat shock protein 90 in male and female primary afferent nociceptors regulates inflammatory pain.

ABSTRACT: Heat shock protein 90 (Hsp90) is a ubiquitously expressed integral cellular protein essential for regulating proteomic stress. Previous research has shown that Hsp90 regulates critical signaling pathways underlying chronic pain and inflammation. Recent discovery of membrane bound ectopic Hsp90 (eHsp90) on tumor cells has shown that Hsp90 induction to the plasma membrane can stabilize disease-relevant proteins. Here, we characterize eHsp90 expression in a mouse model of inflammation and demonstrate its role in nociception and pain. We found that intraplantar complete Freund adjuvant (CFA) induced robust expression of eHsp90 on the cell membranes of primary afferent nociceptors located in the L3-L5 dorsal root ganglia (DRG), bilaterally, with minimal to no expression in other tissues. Complete Freund adjuvant-induced increases in eHsp90 expression on lumbar DRG were significantly greater in females compared with males. Furthermore, exogenous Hsp90 applied to primary Pirt-GCaMP3 nociceptors induced increases in calcium responses. Responses were estrogen-dependent such that greater activity was observed in female or estrogen-primed male nociceptors compared with unprimed male nociceptors. Treatment of mice with the selective eHsp90 inhibitor HS-131 (10 nmol) significantly reversed CFA-induced mechanical pain, thermal heat pain, and hind paw edema. Notably, a higher dose (20 nmol) of HS-131 was required to achieve analgesic and anti-inflammatory effects in females. Here, we provide the first demonstration that inflammation leads to an upregulation of eHsp90 on DRG nociceptors in a sex-dependent manner and that inhibition of eHsp90 reduces nociceptor activity, pain, and inflammation. Thus, eHsp90 represents a novel therapeutic axis for the development of gender-tailored treatments for inflammatory pain.

TAK1: a potent tumour necrosis factor inhibitor for the treatment of inflammatory diseases.

Aberrant tumour necrosis factor (TNF) signalling is a hallmark of many inflammatory diseases including rheumatoid arthritis (RA), irritable bowel disease and lupus. Maladaptive TNF signalling can lead to hyper active downstream nuclear factor (NF)-κß signalling in turn amplifying a cell's inflammatory response and exacerbating disease. Within the TNF intracellular inflammatory signalling cascade, transforming growth factor-ß-activated kinase 1 (TAK1) has been shown to play a critical role in mediating signal transduction and downstream NF-κß activation. Owing to its role in TNF inflammatory signalling, TAK1 has become a potential therapeutic target for the treatment of inflammatory diseases such as RA. This review highlights the current development of targeting the TNF-TAK1 signalling axis as a novel therapeutic strategy for the treatment of inflammatory diseases.

TAK1 regulates the tumor microenvironment through inflammatory, angiogenetic and apoptotic signaling cascades.

Transforming growth factor beta-activated kinase 1 (TAK1) has been implicated for its role in inflammatory signaling and as an important mediator of cellular apoptosis and necroptosis in various cell types. Our recent discovery of a first-in-class, potent and selective TAK1 inhibitor, takinib, represents a novel pharmacological tool to evaluate TAK1's role in cancer. In this study we evaluated the potential therapeutic capacity of TAK1 inhibition on tumor growth and on tumor microenvironment remodeling. In a screen of 16 cancer cell lines, takinib in combination with tumor necrosis factor (TNF) was found to induce cell death (>20%) in 6 out of 16 cell lines. Furthermore, knocking out of TAK1 in MDA-MB-231 cells dramatically increased their sensitization to TNF-mediated apoptosis. In vivo xenographs of MDA-MB-231 TAK1KO tumors displayed delayed tumor growth and increased overall survival compared to TAK1WT controls. Histological and proteomic analysis of TAK1KO tumors showed altered angiogenic signaling and inflammatory signaling via immune cells. Overall, these findings suggest that the targeting of TAK1 in immune mediated cancers may be a novel therapeutic axis.

A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase.

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

Pharmacological inhibition of TAK1, with the selective inhibitor takinib, alleviates clinical manifestation of arthritis in CIA mice.

OBJECTIVES: To examine the ability of takinib, a selective transforming growth factor beta-activated kinase 1 (TAK1) inhibitor, to reduce the severity of murine type II collagen-induced arthritis (CIA), and to affect function of synovial cells. METHODS: Following the induction of CIA, mice were treated daily with takinib (50 mg/kg) and clinical scores assessed. Thirty-six days post-CIA induction, histology was performed on various joints of treated and vehicle-treated animals. Inflammation, pannus, cartilage damage, bone resorption, and periosteal bone formation were quantified. Furthermore, pharmacokinetics of takinib were evaluated by LC-MS in various tissues. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) cells were cultured with 10 µM takinib and cytokine secretion analyzed by cytokine/chemokine proteome array. Cytotoxicity of takinib for RA-FLS was measured with 24 to 48 h cultures in the presence or absence of tumor necrosis factor (TNF). RESULTS: Here, we show takinib's ability to reduce the clinical score in the CIA mouse model of rheumatoid arthritis (RA) (p < 0.001). TAK1 inhibition reduced inflammation (p < 0.01), cartilage damage (p < 0.01), pannus, bone resorption, and periosteal bone formation and periosteal bone width in all joints of treated mice compared to vehicle treated. Significant reduction of inflammation (p < 0.004) and cartilage damage (p < 0.004) were observed in the knees of diseased treated animals, with moderate reduction seen in the forepaws and hind paws. Furthermore, the pharmacokinetics of takinib show rapid plasma clearance (t½ = 21 min). In stimulated RA-FLS cells, takinib reduced GROα, G-CSF, and ICAM-1 pro-inflammatory cytokine signaling. CONCLUSION: Our findings support the hypothesis that TAK1 targeted therapy represents a novel therapeutic axis to treat RA and other inflammatory diseases.