Traf2- And Nck-interacting Kinase Research Articles

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.

Discovery of benzo[d]oxazol-2(3H)-one derivatives as a new class of TNIK inhibitors for the treatment of colorectal cancer

Colorectal cancer (CRC) is one of the most commonly diagnosed cancer types and Traf2- and Nck-interacting kinase (TNIK) has been thought as a potential target for CRC treatment. Herein we report the discovery and structure–activity relationship (SAR) of benzo[d]oxazol-2(3H)-one derivatives as a new class of TNIK inhibitors. The most potent compound 8g showed an IC50 value of 0.050 μM against TNIK. It effectively suppressed proliferation and migration of colorectal cancer cells. Western blot analysis indicated that 8g could inhibit aberrant transcription activation of Wnt signaling. Collectively, this study provides a potential lead compound for subsequent drug discovery targeting TNIK.

The Traf2 and Nck-Interacting Kinase Is a Conserved Regulator of Self-Renewal in Hematopoietic and Leukemia Stem Cells

The Traf2 and Nck-interacting kinase (TNIK) is a member of the germinal center kinase (GCK) family. We have recently found that signaling through the tumor necrosis factor superfamily receptor CD27 activates Wnt target genes through Traf2-TNIK-β-catenin signaling, promoting leukemia development and progression in acute and chronic myeloid leukemia (AML and CML).To explore how TNIK signaling regulates hematopoietic and leukemia stem cells (HSCs and LSCs), we generated a constitutive TNIK-/- knockout mouse.Under steady-state conditions, total bone marrow (BM) cellularity of naïve TNIK-/- mice was slightly reduced (mean BM count (x108): TNIK-/- = 1.42; TNIKwt/wt = 2.15, p < 0.0132). In contrast, no differences in absolute number of lineage negative (Lin-) cells, as well as the more primitive subset Lin-; Sca1+; c-kit+ (LSK) cells could be observed. Similarly, proportions of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs) and multipotent progenitors (MPPs) did not differ between TNIK-/- and TNIKwt/wt mice. Functionally, naïve TNIK-/- and TNIKwt/wt HSCs displayed in vitro a comparable serial colony formation capacity in methylcellulose. Altogether these data suggest that TNIK does not affect the maintenance and self-renewal of HSCs under steady-state conditions.Next the impact of TNIK on hematopoietic recovery after genotoxic stress was analyzed in vivo. TNIK-/- and TNIKwt/wt mice were injected i.p. with 5-Fluorouracil (5-FU) (150mg/g bodyweight) and BM cellularity as well as HSC phenotype and function were assessed 9 days post injection. The total BM cellularity was significantly higher in TNIK-/- mice compared to controls (mean BM count (x106/ml): TNIK-/- =10.25; TNIKwt/wt =4.55, p< 0.0382). Furthermore, LSKs were significantly reduced in frequency in TNIK-/- mice (% LSK of whole BM: TNIK-/- = 2.93; TNIKwt/wt = 7.073, p < 0.0009) but not in absolute numbers. Interestingly, however, the total number LT-HSCs was significantly reduced in the BM of TNIK-/- mice (mean LT-HSC count (x103): TNIK-/- = 5.628; TNIKwt/wt = 16.725, p < 0.0101). These results were functionally confirmed in vivo by competitive BM transplantation (BMTx) of whole BM from 5-FU treated mice into lethally irradiated secondary recipients. Mice receiving BM from 5-FU-treated TNIK-/- mice showed a significantly reduced peripheral engraftment capacity 6 weeks post BMTx compared to controls (mean peripheral blood chimerism: TNIK-/- = 64.6%; TNIKwt/wt = 35.4%, p < 0.0001), suggesting that TNIK signaling mediates HSC maintenance during genotoxic stress conditions in vivo.In parallel the role of TNIK signaling was analyzed in myeloid LSCs. CML-like disease was induced in mice by transplanting pMSCV-BCR-ABL1-IRES-GFP transduced TNIK-/- or TNIKwt/wt LSKs into BL/6 mice. Even though CML developed similarly as indicated by measuring BCR-ABL1-GFP+ granulocytes in the peripheral blood, primary TNIK-/- CML mice survived significantly longer compared to TNIKwt/wt CML mice (Median survival (days): TNIK-/- = 33, TNIKwt/wt = 23.5, p < 0.0216) with 2 out of 7 mice surviving long-term. Next, we determined the effect of TNIK signaling on the functionality of FACS sorted Lin-; GFP+; Sca1+; c-kit+ LSC in vitro in serial colony forming assays and in vivo in secondary transplantations. Serial colony formation capacity of TNIK-/- CML LSCs was significantly reduced after re-plating in vitro (CFUs fold change (TNIK-/- vs TNIKwt/wt): 1st Plating = 0.89, P<0.5149, 2nd Plating = 0.42, 9 < 0.0002). In line with these findings, only 1 out of 9 mice secondarily transplanted with LSCs from primary TNIK-/- CML mice developed CML within 90 days, while all 9 recipients receiving TNIKwt/wt LSCs succumbed to the disease within 23 days after transplantation (p < 0.0001). These results suggest that TNIK signaling also promotes self-renewal of CML LSCs.To summarize, we have described for the first time TNIK signaling as a commonly conserved regulator in stress-induced hematopoiesis and CML, which promotes stem cell self-renewal and maintenance. As TNIK inhibitors are currently developed for the treatment of different kinds of solid tumors, TNIK may be an attractive therapeutic target for LSCs in myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

TRAF2 and NCK-Interacting Kinase Inhibitors for Colorectal Cancer: In Vitro and Theoretical Validations.

TRAF2 and NCK-interacting kinase (TNIK) is a critical factor in colorectal cancer (CRC) proliferation mediated by Wnt signaling. We attempted to identify efficient TNIK inhibitors using computational high-throughput virtual screening (HTVS) from various drug banks and databases. By performing/on performing e-pharmacophore screening and molecular docking methods, from ∼700 000 molecules, compounds LC_222150, LC_112060, and LC_64796 were identified as potential leads, through molecular dynamics (MD) simulations and density functional theory (DFT). These top 3 structures were commercially procured, and their inhibitory activity was assessed in vitro. Significant TNIK inhibition was observed, with an average IC50 of 18.33 ± 0.75 nM. In terms of anticancer activity, the observed average relative % activity (RPA) of 90.28 ± 1.04 for these compounds compared well with doxorubicin (86.75 ± 1.45) as a standard. Compounds LC_222150, LC_112060, and LC_64796, therefore, warrant further evaluation in vivo to assess their CRC therapeutic effects.

Methylome-wide association study of first-episode schizophrenia reveals a hypermethylated CpG site in the promoter region of the TNIK susceptibility gene

Accumulating evidence suggests that epigenetics plays an important role in the etiology of schizophrenia. Here, we performed a methylome-wide association study (MWAS) of first-onset schizophrenia patients and controls from the Han Chinese population using microarray technology. The DNA methylation profiles revealed 4494 differentially methylated CpG sites. Gene ontology (GO) analysis showed that the functions of differentially methylated genes were primarily involved in enzymatic activity, cytoskeleton organization and cell adhesion, and the TNIK (encoding TRAF2- and NCK-interacting kinase) gene was enriched in most of these terms. By combining the MWAS results with those of previous genome-wide association studies (GWASs), we identified 72 candidate genes located in 49 human genome loci. Among the overlapping genes, the most significantly methylated CpG sites were in the transcriptional start site (TSS) 200 region (cg21413905, Punadjusted = 3.20 × 10−5) of TNIK. TNIK was listed in the top 50 differentially methylated loci. The results of pyrosequencing and TNIK mRNA expression were consistent with those of the microarray study. Bioinformatics analyses, dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) studies showed that TNIK interacted with genes associated with schizophrenia and NRF1 was identified as a novel transcription factor (TF) that binds to TNIK in its TSS200 region. Thus, the regulatory function of NRF1 may be influenced by the status of the methylated CpG site in this region. In summary, our study provides new insights into the epigenetic mechanisms that regulate schizophrenia. Studies of the functions of TNIK methylation should be performed in vitro and in vivo to provide a better understanding of the pathophysiology of schizophrenia.

Cyclic pentapeptide cRGDfK enhances the inhibitory effect of sunitinib on TGF-β1-induced epithelial-to-mesenchymal transition in human non-small cell lung cancer cells.

In human lung cancer progression, the EMT process is characterized by the transformation of cancer cells into invasive forms that migrate to other organs. Targeting to EMT-related molecules is emerging as a novel therapeutic approach for the prevention of lung cancer cell migration and invasion. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as an anti-proliferative target molecule to regulate the Wnt signaling pathway in several types of cancer cells. In the present study, we evaluated the inhibitory effect of a tyrosine kinase inhibitor sunitinib and the integrin-αⅤβ3 targeted cyclic peptide (cRGDfK) on EMT in human lung cancer cells. Sunitinib strongly inhibited the TGF-β1-activated EMT through suppression of Wnt signaling, Smad and non-Smad signaling pathways. In addition, the cRGDfK also inhibited the expression of TGFβ1-induced mesenchymal marker genes and proteins. The anti-EMT effect of sunitinib was enhanced when cRGDfK was treated together. When sunitinib was treated with cRGDfK, the mRNA and protein expression levels of mesenchymal markers were decreased compared to the treatment with sunitinib alone. Co-treatment of cRGDfK has shown the potential to improve the efficacy of anticancer agents in combination with therapeutic agents that may be toxic at high concentrations. These results provide new and improved therapies for treating and preventing EMT-related disorders, such as lung fibrosis and cancer metastasis, and relapse.

Abstract 508: A novel orally available Wnt-pathway inhibitor for the treatment of metastatic cancers

Abstract We have developed a novel drug compound (OBD9) which blocks growth of metastatic cancer cells both in vitro and in vivo. We now identify OBD9 as an effective Wnt-signaling inhibitor targeting TNIK (TRAF2 And NCK Interacting Kinase) and we hypothesize that OBD9 represent a novel therapeutic option for patients with tumors that have an activated Wnt signaling pathway. To find novel drug candidates that selectively inhibit metastatic tumor cell viability, a drug screen was firstly performed and results of the screen identified members of the FDA-approved benzimidazole methylcarbamate family (e.g. mebendazole (MBZ) and albendazole (ALB)) as potential therapies for metastatic cancer. Earlier work also supports a role for this chemical family in the potential treatment of multiple cancers, but progress has been stalled by their poor water solubility and poor bioavailability for systemic delivery to disseminated tumors. We therefore synthesized a novel compound (OBD9) containing the scaffold of MBZ coupled to an oxetane group to enhance aqueous solubility to 361μM. OBD9 demonstrates significant cytotoxicity toward a variety of cancer cell types including colon, lung, and prostate cancers (IC50: 0.9-2μM). In a mouse xenograft model using highly aggressive PCMLN3 prostate cancer cells, OBD9 at 30 mg/kg significantly repressed growth of established tumors with no visible toxicity. In a mouse xenograft model of human A549 lung cancer cell line, orally delivered OBD9 also dramatically inhibited the growth of established tumors at 30 or 90μM without noticeable toxicity. Mechanistically, we find that OBD9 treatment significantly reduces TNIK levels as early as 4 hours at 1uM via an autophagy-dependent protein degradation pathway. TNIK functions as an activator of Wnt signaling pathway via phosphorylation of the beta-catenin/TCF4 complex that regulates Wnt downstream targets. We show that OBD9 treatment inhibits colon cancer cell growth and both qPCR and Western blot data suggest that Wnt signaling downstream targets, such as TCF4, AXIN2 and cMyc, are all significantly suppressed by OBD9 via the inhibition of TNIK. Overall, our in vitro and in vivo data suggest that OBD9 potentially represents a novel therapeutic option for multiple cancers including but not limit to colon, lung and prostate cancer. Citation Format: Kun Zhou, Jae Eun Cheong, Catherine Alix-Panabières, Lijun Sun, Bruce R. Zetter. A novel orally available Wnt-pathway inhibitor for the treatment of metastatic cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 508.

TNIK (TRAF2 and NCK interacting kinase)

TNIK (TRAF2 and NCK interacting kinase)

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.

A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b.

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.

The Misshapen subfamily of Ste20 kinases regulate proliferation in the aging mammalian intestinal epithelium.

The intestinal epithelium has a high rate of cell turn over and is an excellent system to study stem cell-mediated tissue homeostasis. The Misshapen subfamily of the Ste20 kinases in mammals consists of misshapen like kinase 1 (MINK1), mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), and TRAF2 and NCK interacting kinase (TNIK). Recent reports suggest that this subfamily has a novel function equal to the Hippo/MST subfamily as upstream kinases for Warts/Large tumor suppressor kinase(LATS) to suppress tissue growth. To study the in vivo functions of Mink1, Map4k4, and Tnik, we generated a compound knockout of these three genes in the mouse intestinal epithelium. The intestinal epithelia of the mutant animals were phenotypically normal up to approximately 12 months. The older animals then exhibited mildly increased proliferation throughout the lower GI tract. We also observed that the normally spatially organized Paneth cells in the crypt base became dispersed. The expression of one of the YAP pathway target genes Sox9 was increased while other target genes including CTGF did not show a significant change. Therefore, the Misshapen and Hippo subfamilies may have highly redundant functions to regulate growth in the intestinal epithelium, as illustrated in recent tissue culture models.

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.

The transcriptional coactivator WBP2 primes triple-negative breast cancer cells for responses to Wnt signaling via the JNK/Jun kinase pathway

The transcriptional coactivator WW domain-binding protein 2 (WBP2) is an emerging oncogene and serves as a node between the signaling protein Wnt and other signaling molecules and pathways, including epidermal growth factor receptor, estrogen receptor/progesterone receptor, and the Hippo pathway. The upstream regulation of WBP2 is well-studied, but its downstream activity remains unclear. Here, we elucidated WBP2's role in triple-negative breast cancer (TNBC), in which Wnt signaling is predominantly activated. Using RNAi coupled with RNA-Seq and MS analyses to identify Wnt/WBP2- and WBP2-dependent targets in MDA-MB-231 TNBC cells, we found that WBP2 is required for the expression of a core set of genes in Wnt signaling. These included AXIN2, which was essential for Wnt/WBP2-mediated breast cancer growth and migration. WBP2 also regulated a much larger set of genes and proteins independently of Wnt, revealing that WBP2 primes cells to Wnt activity by up-regulating G protein pathway suppressor 1 (GPS1) and TRAF2- and NCK-interacting kinase (TNIK). GPS1 activated the c-Jun N-terminal kinase (JNK)/Jun pathway, resulting in a positive feedback loop with TNIK that mediated Wnt-induced AXIN2 expression. WBP2 promoted TNBC growth by integrating JNK with Wnt signaling, and its expression profoundly influenced the sensitivity of TNBC to JNK/TNIK inhibitors. In conclusion, WBP2 links JNK to Wnt signaling in TNBC. GPS1 and TNIK are constituents of a WBP2-initiated cascade that primes responses to Wnt ligands and are also important for TNBC biology. We propose that WBP2 is a potential drug target for JNK/TNIK-based precision medicine for managing TNBC.

Abstract 2034: Traf2-and Nck-interacting kinase (TNIK) inhibitor, NCB-0846, suppresses TGF-β1-induced epithelial-mesenchymal transition in lung cancer

Abstract Epithelial mesenchymal transition (EMT) has been implicated in cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) signal was known to be an essential EMT promotor. Development of therapeutics for targeting EMT through the TGF-β signal may be beneficial for the management of cancer metastasis. Recently, we found that a Traf2- and Nck-interacting kinase (TNIK) inhibitor, named NCB-0846, was capable of attenuating tumor-initiating cells among human colorectal cancer (1, 2). The cross link between EMT and cancer stemness has been revealed in several studies. Therefore we evaluated whether this small-molecule compound could have efficacy to inhibit TGF-β-induced EMT. NCB-0846 reduced the expression of mesenchymal markers (Vimentin and N-cadherin) and upregulated the expression of epithelial marker E-cadherin in A549 and H2228 cells. NCB-0846 suppressed the phosphorylation and nuclear translocation of Smad proteins and also inhibited migration, invasion, and metastasis. NCB-0846 inhibited TGF-β1-induced EMT through the down-regulation of TGFBR1 in mRNA levels. MiR-186-5p and miR-320 family were identified as candidate miRNAs that could target TGFBR1 and inhibited TGFBR1 protein expression.NCB-0846 might be a novel therapeutics drugs that targets the invasion and metastasis through inhibiting TGF-β-induced EMT in lung cancer. 1. Masuda M, Sawa M, Yamada T. Therapeutic targets in the Wnt signaling pathway: Feasibility of targeting TNIK in colorectal cancer. Pharmacology &amp; therapeutics. 2015;156:1-9. Epub 2015/11/07.2. Masuda M, Uno Y, Ohbayashi N, Ohata H, Mimata A, Kukimoto-Niino M, et al. TNIK inhibition abrogates colorectal cancer stemness. Nature communications. 2016;7:12586. Epub 2016/08/27. Citation Format: Teppei Sugano, Mari Masuda, Yuko Uno, Naoko Goto, Masahiro Seike, Masaaki Sawa, Akihiko Gemma, Tesshi Yamada. Traf2-and Nck-interacting kinase (TNIK) inhibitor, NCB-0846, suppresses TGF-β1-induced epithelial-mesenchymal transition in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2034.

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.

Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells.

Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13nM) and the activation of Wnt signaling effectors such as β-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway.