Traf2- And Nck-interacting Kinase Inhibitor Research Articles

Discovery of Bis-imidazolecarboxamide Derivatives as Novel, Potent, and Selective TNIK Inhibitors for the Treatment of Idiopathic Pulmonary Fibrosis.

Traf2- and Nck-interacting kinase (TNIK) has been identified as a promising therapeutic target for the treatment of fibrosis-driven diseases. Utilizing a structure-based drug design workflow, we developed a series of potent TNIK inhibitors that modulate the conformation of the gatekeeper Met105 side chain and access the TNIK back pocket. The lead optimization efforts culminated in the discovery of the recently reported compound 4 (INS018_055), a novel TNIK inhibitor. This molecule demonstrated excellent activity in both enzymatic and cell-based assays, along with high selectivity in a kinome panel. Further, in vitro and in vivo preclinical studies revealed favorable in vitro and in vivo DMPK properties. Results from multiple cell-based and animal models proved that compound 4 exhibits considerable antifibrotic and anti-inflammatory efficacy. Currently, phase II clinical trials of compound 4 are underway for the treatment of idiopathic pulmonary fibrosis (IPF).

Inhibiting renal Traf2 and NCK interacting kinase (TNIK) decreases NKKC2 phosphorylation and blunts salt-sensitive hypertension

The Na/K/2Cl cotransporter NKCC2 mediates the NaCl reabsorption in the thick ascending limb (TAL). Phosphorylation at Thr-96,101 and traffcking to the surface are increased in the Dahl salt-sensitive (DSS) rat together with SPAK phosphorylation, an upstream kinase for Thr-96,101 in NKCC2. We previously found that deletion of SPAK in the DSS, lowers NKCC2 phosphorylation and blunts salt-sensitive hypertension, but doesn’t completely abolish NKCC2 phosphorylation. Therefore, other kinases may be involved in NKCC2 phosphorylation. We recently found the Traf2 and NCK interacting kinase (TNIK) phosphorylates NKCC2 and is overexpressed in the TALs of DSS rats. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 hyperphosphorylation in DSS rats. We approached TNIK inhibition in two ways, using a pharmacological inhibitor and its genomic deletion with CRISPR/Cas9. First, we studied the small molecule TNIK inhibitor NCB-0846, and whether it could decrease baseline NKCC2 phosphorylation in TALs from DSS rats. Treating TALs from DSS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr-96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.001, n=3). To study in vivo effcacy of NCB-0846, we directly infused NCB (or vehicle) into the left kidney via implanted renal medullary catheters connected to minipumps in DSS rats fed 4% NaCl diet. We found that NCB-0846 decreased SBP by 30 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs control). To decrease TNIK gene expression we used Cas9 mediated gene editing. For this, we designed gRNA sequences targeting TNIK and tested their effcacy in C6 cells. One of the gRNAs designed decreased TNIK expression by 48 ± 22% compared to control (p=0.101, n=3). To decrease TNIK in TALs in vivo, we injected the renal medulla of the left kidney with AAV-gRNA-TNIK together with AAV-NKCC2 promoter-Cas9. After 2 weeks, we isolated medullary TALs and measured TNIK expression and NKCC2 phosphorylation. TNIK expression was decreased by 50 ± 5.5% (n=4, p<0.0001), and pThr-96,101-NKCC2 was decreased by 38.1 ± 10.6% in transduced TALs compared to kidneys transduced with AV-control (n=4, p<0.05). The expression of related kinases SPAK and OSR1 was not modified (SPAK 79.2 ± 18.3%, OSR1 81.3 ± 13.3%, n=4, ns). We conclude that NKCC2 phosphorylation in the DSS rat is in part caused by TNIK and that this new kinase could be a target for salt sensitive hypertension. Future experiments will test the effect of gene editing TNIK in salt sensitive hypertension. 23POST1019882 AHA15GRNT25710369. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A small-molecule TNIK inhibitor targets fibrosis in preclinical and clinical models.

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease with a high mortality rate. Putative drug targets in IPF have failed to translate into effective therapies at the clinical level. We identify TRAF2- and NCK-interacting kinase (TNIK) as an anti-fibrotic target using a predictive artificial intelligence (AI) approach. Using AI-driven methodology, we generated INS018_055, a small-molecule TNIK inhibitor, which exhibits desirable drug-like properties and anti-fibrotic activity across different organs in vivo through oral, inhaled or topical administration. INS018_055 possesses anti-inflammatory effects in addition to its anti-fibrotic profile, validated in multiple in vivo studies. Its safety and tolerability as well as pharmacokinetics were validated in a randomized, double-blinded, placebo-controlled phase I clinical trial (NCT05154240) involving 78 healthy participants. A separate phase I trial in China, CTR20221542, also demonstrated comparable safety and pharmacokinetic profiles. This work was completed in roughly 18 months from target discovery to preclinical candidate nomination and demonstrates the capabilities of our generative AI-driven drug-discovery pipeline.

The Traf2 and NcK interacting kinase inhibitor NCB-0846 suppresses seizure activity involving the decrease of GRIA1

Epilepsy, one of the most common neurological disorders, is characterized by spontaneous recurrent seizures. Temporal lobe epilepsy (TLE) is one of the most common medically intractable seizure disorders. Traf2-and NcK-interacting kinase (TNIK) has recently attracted attention as a critical modulation target of many neurological and psychiatric disorders, but its role in epilepsy remains unclear. In this study, we hypothesized the involvement of TNIK in epilepsy and investigated TNIK expression in patients with intractable TLE and in a pilocarpine-induced rat model of epilepsy by western blotting, immunofluorescence, and immunohistochemistry. A pentylenetetrazole (PTZ)-induced epilepsy rat model was used to determine the effect of the TNIK inhibitor NCB-0846 on behavioral manifestations of epilepsy. Coimmunoprecipitation (Co-IP)/mass spectrometry (MS) was used to identify the potential mechanism. Through Co-IP, we detected and confirmed the main potential TNIK interactors. Subcellular fractionation was used to establish the effect of NCB-0846 on the expression of the main interactors in postsynaptic density (PSD) fractions. We found that TNIK was primarily located in neurons and decreased significantly in epilepsy model rats and TLE patients compared with controls. NCB-0846 delayed kindling progression and decreased seizure severity. Co-IP/MS identified 63 candidate TNIK interactors in rat hippocampi, notably CaMKII. Co-IP showed that TNIK might correlate with endogenous GRIA1, SYN2, PSD-95, CaMKIV, GABRG1, and GABRG2. In addition, the significant decrease in GRIA1 in hippocampal total lysate and PSDs after NCB-0846 treatment might help modify the progression of PTZ kindling. Our results suggest that TNIK contributes to epileptic pathology and is a potential antiepileptic drug target.

CAMP stimulated NKCC2 phosphorylation at Thr-96,101 is in part independent from SPAK: a Role for TNIK

Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitivity. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at Thr 96,101 by upstream kinases SPAK or OSR1. We found that Thr 96,101 phosphorylation is enhanced in TALs of Dahl Salt Sensitive rats (Dahl SS) on normal or high salt diet. Genetic deletion of SPAK in Dahl SS lowers NKCC2 phosphorylation by 40% and blunts salt-sensitive hypertension but does not completely restore these to baseline. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thr 96,101 in NKCC2 in rats and mice (manuscript under revision). In addition, we and others found that cAMP is a potent stimulus for NKCC2 Thr-96,101 phosphorylation. We hypothesize that TNIK is in part responsible for cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. First, we tested the ability of a novel TNIK inhibitor (NCB-0846) to decrease baseline NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr 96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.0001, n=3). Next, we measured NKCC2 phosphorylation in rats with genetic deletion of SPAK in a Dahl SS background. In SPAK KO rats, the cAMP analogue db-cAMP (500 μM) increased NKCC2 phosphorylation at Thr 96,101 by 626 ± 46% (p<0.0001, n=3) and the TNIK inhibitor NCB-0846, blunted cAMP-stimulated NKCC2 phosphorylation by 56 ± 4.3% (p=0.0013, n=3). There was no significant decrease in phosphorylation at phospho-Ser 126 NKCC2 (cAMP=46 ± 7-fold vs cAMP+NCB= 31±5-fold, n.s). These data indicate that in the absence of SPAK, TNIK mediates cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. To further support this, we studied TALs from whole animal TNIK knock-out mice (TNIK KO). db-cAMP (500 μM) increased NKCC2 phosphorylation by 1330 ± 268% in wild-type mice (p<0.01) whereas cAMP-stimulated NKCC2 phosphorylation was blunted by 49 ± 15% in TNIK KO TALs (p<0.05, n=3). No significant difference was found in cAMP-stimulated NKCC2-Ser 126 phosphorylation in TNIK KO (WT=23 ± 12-fold vs TNIK KO=15 ± 4-fold, n=3). We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK and that cAMP-stimulated NKCC2 Thr-96,101 phosphorylation in TALs is in part independent from SPAK and involves TNIK. Our data point to potential role for TNIK in renal ion transport, blood pressure regulation and urine concentration. AHA15GRNT25710369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of Wnt Signaling in Colon Cancer Cells via an Oral Drug that Facilitates TNIK Degradation.

We have synthesized an oxetane derivative of the benzimidazole compound mebendazole (OBD9) with enhanced solubility and strong anticancer activity in multiple types of cancer cells, especially colorectal cancer. In this report, we provide evidence that OBD9 suppresses colorectal cancer growth by interfering with the Wnt signaling pathway, a main driver of cell growth in colorectal cancer. Specifically, we find that OBD9 induces autophagic degradation of TNIK (traf2 and Nck-interacting kinase), which promotes T-cell factor-4 (TCF4)/beta-catenin-mediated gene expression. Thus, OBD9 as a TNIK inhibitor blocks Wnt/beta-catenin signaling at the final step of transcriptional activation. We suggest that OBD9 provides a potential novel autophagy-mediated, Wnt-damping therapeutic strategy for the treatment of colorectal cancer.

SPAK‐Independent NKCC2 Phosphorylation in Dahl SS Rats: A Role for TNIK in NKCC2 Hyperphosphorylation

Increased NaCl reabsorption in the Thick Ascending Limb (TAL) is implicated in salt sensitive hypertension. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. We reported that NKCC2 phosphorylation at Tre 96,101 and surface expression are enhanced in Dahl Salt sensitive rats (Dahl SS) on normal or high salt diets. The phosphorylation of SPS1‐related proline/alanine‐rich kinase (SPAK), an upstream kinase for Thre 96,101 in NKCC2, was found to be higher in Dahl SS TALs. Deletion of SPAK in Dahl SS, lowers NKCC2 phosphorylation and expression and blunts salt‐sensitive hypertension, but does not completely prevent this. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thre 96,101 in NKCC2. We hypothesize that TNIK is in part responsible for enhanced NKCC2 phosphorylation in Dahl SS rats. We first measured total expression of SPAK, OSR1 and TNIK by Western blot in TALs isolated form Dahl SS and control rats. SPAK and OSR1 expression (normalized to GAPDH) was similar between strains whereas TNIK expression was 3‐fold higher in Dahl SS TALs (control: 100, Dahl SS: 396±80%, n =5, p<.025). We then tested whether a novel TNIK inhibitor (KY‐05009) decreased NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with KY‐05009 (1 µM) for 25 min decreased NKCC2 Thre 96,101 phosphorylation by 40±4% (p<0.05, n=5). To study whether NKCC2 phosphorylation can be stimulated in the absence of SPAK, we used SPAK knockout Dahl SS rats. Baseline NKCC2 expression and phosphorylation in TALs was 50% lower in Dahl SS‐SPAK KO TALs, however, stimulation of Beta‐adrenergic receptors with Isoproterenol increased NKCC2 phosphorylation by 192±49% (p<0.01, n =4) in SPAK‐KO. Similarly, the cAMP analogue db‐cAMP (500 µM) increased NKCC2 phosphorylation by 939±183% in SPAK KO (p<0.01 n=4) and by 497±105% (p<0.01 n=4) in control rat TALs, indicating that a kinase other than SPAK is essential for baseline and cAMP‐stimulated NKCC2 phosphorylation in Dahl SS rats. We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK, which show 3‐fold higher expression in this strain. SPAK also contributes to higher baseline NKCC2 phosphorylation, whereas a kinase other than SPAK (either TNIK or OSR1) is involved in cAMP‐stimulated NKCC2 Thre 96,101 phosphorylation in Dahl SS TALs.

Feasibility of Targeting Traf2-and-Nck-Interacting Kinase in Synovial Sarcoma.

Background: The treatment of patients with metastatic synovial sarcoma is still challenging, and the development of new molecular therapeutics is desirable. Dysregulation of Wnt signaling has been implicated in synovial sarcoma. Traf2-and-Nck-interacting kinase (TNIK) is an essential transcriptional co-regulator of Wnt target genes. We examined the efficacy of a small interfering RNA (siRNA) to TNIK and a small-molecule TNIK inhibitor, NCB-0846, for synovial sarcoma. Methods: The expression of TNIK was determined in 20 clinical samples of synovial sarcoma. The efficacy of NCB-0846 was evaluated in four synovial sarcoma cell lines and a mouse xenograft model. Results: We found that synovial sarcoma cell lines with Wnt activation were highly dependent upon the expression of TNIK for proliferation and survival. NCB-0846 induced apoptotic cell death in synovial sarcoma cells through blocking of Wnt target genes including MYC, and oral administration of NCB-846 induced regression of xenografts established by inoculation of synovial sarcoma cells. Discussion: It has become evident that activation of Wnt signaling is causatively involved in the pathogenesis of synovial sarcoma, but no molecular therapeutics targeting the pathway have been approved. This study revealed for the first time the therapeutic potential of TNIK inhibition in synovial sarcoma.

Tool inhibitors and assays to interrogate the biology of the TRAF2 and NCK interacting kinase.

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.

Characterization of the ERG-regulated Kinome in Prostate Cancer Identifies TNIK as a Potential Therapeutic Target

Approximately 50% of prostate cancers harbor the TMPRSS2:ERG fusion, resulting in elevated expression of the ERG transcription factor. Despite the identification of this subclass of prostate cancers, no personalized therapeutic strategies have achieved clinical implementation. Kinases are attractive therapeutic targets as signaling networks are commonly perturbed in cancers. The impact of elevated ERG expression on kinase signaling networks in prostate cancer has not been investigated. Resolution of this issue may identify novel therapeutic approaches for ERG-positive prostate cancers. In this study, we used quantitative mass spectrometry-based kinomic profiling to identify ERG-mediated changes to cellular signaling networks. We identified 76 kinases that were differentially expressed and/or phosphorylated in DU145 cells engineered to express ERG. In particular, the Traf2 and Nck-interacting kinase (TNIK) was markedly upregulated and phosphorylated on multiple sites upon ERG overexpression. Importantly, TNIK has not previously been implicated in prostate cancer. To validate the clinical relevance of these findings, we characterized expression of TNIK and TNIK phosphorylated at serine 764 (pS764) in a localized prostate cancer patient cohort and showed that nuclear enrichment of TNIK (pS764) was significantly positively correlated with ERG expression. Moreover, TNIK protein levels were dependent upon ERG expression in VCaP cells and primary cells established from a prostate cancer patient-derived xenograft. Furthermore, reduction of TNIK expression and activity by silencing TNIK expression or using the TNIK inhibitor NCB-0846 reduced cell viability, colony formation and anchorage independent growth. Therefore, TNIK represents a novel and actionable therapeutic target for ERG-positive prostate cancers that could be exploited to develop new treatments for these patients.

Abstract B146: A novel TNIK inhibitor potently downregulates cancer stem cell population through attenuation of Wnt signaling

Abstract Background: TNIK (Traf2- and Nck-interacting kinase) has been identified as a component of the β-catenin and T-cell factor 4 (TCF4) complex that regulates the downstream of Wnt signaling. Recent studies indicated that the Wnt pathway is highly activated in cancer stem cells (CSCs), which have been recognized as the driving force of tumorigenesis and mediating tumor recurrence. Thus, eradicating CSCs by inhibiting Wnt signaling may become a new therapeutic option for a complete cure of cancers. Recently we have demonstrated that TNIK is required for the tumor-initiating function of colorectal CSCs, and therefore TNIK is considered to be attractive targets for drug discovery. We have discovered a thiazole-based TNIK inhibitor having potent inhibitory activity for the Wnt signaling. Further optimization of the thiazole-based TNIK inhibitor resulted in an orally available novel TNIK inhibitor, NC-1. In this study, we evaluated in vivo efficacy of the TNIK inhibitor in mouse models as well as its effects on CSCs in vitro. Materials and Methods: To evaluate the effects of NC-1 on the CSCs in SW620, human colon cancer cells, the enzymatic activity of aldehyde dehydrogenase (ALDH) was analyzed by flow cytometry using an Aldefluor assay. The expression of CSCs surface marker protein, CD133, was analyzed using flow cytometry. Antitumor efficacy of NC-1 was evaluated in a human colorectal tumor xenograft mouse model. Results: We demonstrated that treatment of cancer cells with NC-1 significantly downregulated ALDH positive cell population in a dose-dependent manner. In addition, NC-1 suppressed cell surface expression of CD133 in a dose-dependent manner, suggesting that NC-1 preferentially killed CSCs population in vitro. Furthermore, NC-1 inhibited tumor growth in the HCT116 xenograft mouse model. Citation Format: Yuko Uno, Hideki Moriyama, Shigeki Kashimoto, Mari Masuda, Masaaki Sawa, Tesshi Yamada. A novel TNIK inhibitor potently downregulates cancer stem cell population through attenuation of Wnt signaling [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B146.

Synergistic inhibition effect of TNIK inhibitor KY-05009 and receptor tyrosine kinase inhibitor dovitinib on IL-6-induced proliferation and Wnt signaling pathway in human multiple myeloma cells.

Multiple myeloma is a fetal form of plasma cell malignancy characterized by abnormal clonal proliferation of plasma cells. Especially, the canonical Wnt signaling pathway mediated by β-catenin is activated in multiple myeloma cells, stimulating their proliferation. Here, we investigated the relationship between interleukin-6-induced proliferation of multiple myeloma cells and Traf2- and Nck-interacting kinase (TNIK) expression in Wnt signaling. Interleukin-6 increased the proliferation of multiple myeloma cells and TNIK mRNA and protein expression. In addition, we examined the effect on TNIK of TNIK inhibitor KY-05009 and receptor tyrosine kinase inhibitor dovitinib and whether inhibition of TNIK suppresses the interleukin-6-induced proliferation of multiple myeloma cells. KY-05009 and dovitinib synergistically inhibited interleukin-6-stimulated proliferation and induced apoptosis through the inhibition of Wnt signaling in MM cells. Our results provide crucial information that TNIK is involved in the interleukin-6-dependent proliferation of multiple myeloma cells and inhibition of Wnt signaling involving TNIK could be a therapeutic strategy for the treatment of interleukin-6-dependent multiple myeloma.

TNIK inhibition abrogates colorectal cancer stemness.

Canonical Wnt/β-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and β-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik−/−/Apcmin/+ mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apcmin/+ mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach.

Abstract B17: Traf2- and NCK-interacting kinase (TNIK) inhibitor down-regulates Wnt signaling pathway in cancer cells

Abstract Introduction: Aberrant activation of the Wnt signaling pathway has been implicated as the key driver of carcinogenesis, particularly in colorectal cancers. Because nearly 90% of colorectal cancers carry mutations either APC or β-catenin, downstream signaling molecules of the β-catenin destructive complex are considered as more attractive therapeutic targets in drug discovery research for the treatment of colorectal cancers. Recently, Traf2- and NCK-interacting kinase (TNIK) has been identified as one of components of the T-cell factor-4 (TCF4) transcriptional complex and essential for the expression of Wnt target genes. Knockdown of TNIK gene expression suppressed the transcriptional activity of the Wnt signaling pathway, and decreased in proliferation of colorectal cancer cells. Therefore inhibitors of TNIK may represent a promising therapeutic approach for treating Wnt-signal activated cancers. Previously we reported a thiazol-based TNIK inhibitor, N5355 have single digit nM IC50 value and the compound significantly down-regulated the Wnt target genes such as AXIN2 and cMYC in colorectal cancer cell line, HCT116. Further evaluations of N5355 have been implemented to characterize the effects of TNIK inhibition on cancer cell lines. Methods: To study the effects of N5355 on the Wnt signaling pathway, the expression levels of Wnt target proteins were analyzed by Western blotting using Wnt-signal activated cancer cell lines. The ability of N5355 for blocking of nuclear translocation of phosphorylated TNIK was also analyzed by Western blotting using HCT116 cells. Anti-proliferative activity profile of N5355 was studied using a panel of 44 cancer cell lines. The pharmacokinetic profile of TNIK inhibitor was also analyzed. Results: The Wnt signal inhibition was validated by significant suppression of Wnt target protein expressions by treating with N5355 in several cancer cells. In addition, Wnt co-receptor proteins LRP6 and LRP5 were found to be down-regulated by N5355 treatment. N5355 exhibited strong anti-proliferation activities against Wnt-driven cancer cell lines. Interestingly in DLD1 colorectal cancer cell lines, N5355 showed no anti-proliferative effect in conventional 2D cell culture assay, but displayed strong inhibitory activity in 3D cell culture assay using soft agar, which is biologically relevant assay system. The fact that N5355 inhibited the autophosphorylation of TNIK in the cytosol and subsequent nuclear translocation of TNIK in HCT116 cells suggested the depletion of TNIK proteins in nucleus resulted in the observed Wnt signal inhibition effect. Detailed results will be presented in the conference. Citation Format: Yuko Uno, Hideki Moriyama, Shigeki Kashimoto, Mari Masuda, Masaaki Sawa, Tesshi Yamada. Traf2- and NCK-interacting kinase (TNIK) inhibitor down-regulates Wnt signaling pathway in cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B17.

Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders.

The essential role of TNIK gene amplification in gastric cancer growth

Traf2- and Nck-interacting kinase (TNIK) is one of the germinal center kinase family members involved in cytoskeleton organization and neuronal dendrite extension. Emerging evidence supports that TNIK is essential for activation of WNT signaling pathway in colon cancer growth. To search for novel genetic aberrations that drive carcinogenesis, we performed microarray-based comparative hybridization assay for gene copy number variations in primary tumor samples. Our data showed that TNIK gene was amplified in 7% (8/106) of Chinese gastric cancer patients. Theses amplifications were confirmed by fluorescence in situ hybridization analysis. PAMC82 human gastric cancer and T47D human breast cancer cell lines with TNIK amplification were identified to further understand the function of TNIK gene amplification. RNA-interference-mediated silencing of TNIK resulted in significant inhibition of cell growth and induction of cell death in TNIK-amplified, but not in TNIK-non-amplified, cell lines tested. This selective sensitivity to the TNIK inhibition was also observed under the effect of a small-molecule TNIK inhibitor. Furthermore, our data indicated that TNIK's role in gastric cancer growth was not dependent on Wnt signaling but rather was involved in AKT activation and cell autophagy. Together, our results suggest that TNIK is a novel therapeutic target in gastric cancer and TNIK amplification can be potentially used for patient selection.