Nck-interacting Kinase Research Articles

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.

Identification of TNIK as a novel potential drug target in thyroid cancer based on protein druggability prediction.

Thyroid cancer is a common endocrine malignancy; however, surgery remains its primary treatment option. A novel targeted drug for the development and application of targeted therapy in thyroid cancer treatment remain underexplored.We obtained RNA sequence data of thyroid cancer from The Cancer Genome Atlas database and identified differentially expressed genes (DEGs). Then, we constructed co-expression network with DEGs and combined it with differentially methylation analysis to screen the key genes in thyroid cancer. PockDrug-Server, an online tool, was applied to predict the druggability of the key genes. Finally, we constructed protein-protein interaction (PPI) network to observe potential targeted drugs for thyroid cancer.We identified 3 genes correlated with altered DNA methylation level and oncogenesis of thyroid cancer. According to the druggable analysis and PPI network, we predicted TRAF2 and NCK-interacting protein kinase (TNIK) sever as the drug targeted for thyroid cancer. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that genes in protein-protein interaction network of TNIK enriched in mitogen-activated protein kinase signaling pathway. For drug repositioning, we identified a targeted drug of genes in PPI network.Our study provides a bioinformatics method for screening drug targets and provides a theoretical basis for thyroid cancer targeted therapy.

Direct conversion of osteosarcoma to adipocytes by targeting TNIK.

Osteosarcoma (OS) is an aggressive mesenchymal tumor for which no molecularly targeted therapies are available. We have previously identified TRAF2- and NCK-interacting protein kinase (TNIK) as an essential factor for the transactivation of Wnt signal target genes and shown that its inhibition leads to eradication of colorectal cancer stem cells. The involvement of Wnt signaling in the pathogenesis of OS has been implicated. The aim of the present study was to examine the potential of TNIK as a therapeutic target in OS. RNA interference or pharmacological inhibition of TNIK suppressed the proliferation of OS cells. Transcriptome analysis suggested that a small-molecule inhibitor of TNIK upregulated the expression of genes involved in OS cell metabolism and downregulated transcription factors essential for maintaining the stem cell phenotype. Metabolome analysis revealed that this TNIK inhibitor redirected the metabolic network from carbon flux toward lipid accumulation in OS cells. Using in vitro and in vivo OS models, we confirmed that TNIK inhibition abrogated the OS stem cell phenotype, simultaneously driving conversion of OS cells to adipocyte-like cells through induction of PPARγ. In relation to potential therapeutic targeting in clinical practice, TNIK was confirmed to be in an active state in OS cell lines and clinical specimens. From these findings, we conclude that TNIK is applicable as a potential target for treatment of OS, affecting cell fate determination.

Pharmacological blockage of transforming growth factor-\u03b2 signalling by a Traf2- and Nck-interacting kinase inhibitor, NCB-0846

BackgroundMetastasis is the primary cause of death in cancer patients, and its management is still a major challenge. Epithelial to mesenchymal transition (EMT) has been implicated in the process of cancer metastasis, and its pharmacological interference holds therapeutic promise.MethodsTraf2- and Nck-interacting kinase (TNIK) functions as a transcriptional coregulator of Wnt target genes. Given the convergence of Wnt and transforming growth factor-β (TGFβ) signalling, we examined the effects of a small-molecule TNIK inhibitor (named NCB-0846) on the TGFβ1-induced EMT of lung cancer cells.ResultsNCB-0846 inhibited the TGFβ1-induced EMT of A549 cells. This inhibition was associated with inhibition of Sma- and Mad-Related Protein-2/3 (SMAD2/3) phosphorylation and nuclear translocation. NCB-0846 abolished the lung metastasis of TGFβ1-treated A549 cells injected into the tail veins of immunodeficient mice. The inhibition of EMT was mediated by suppression of the TGFβ receptor type-I (TGFBR1) gene, at least partly through the induction of microRNAs targeting the TGFBR1 transcript [miR-320 (a, b and d) and miR-186].ConclusionsNCB-0846 pharmacologically blocks the TGFβ/SMAD signalling and EMT induction of lung cancer cells by transcriptionally downregulating TGFBRI expression, representing a potentially promising approach for prevention of metastasis in lung cancer patients.

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.

Effect of TNIK upregulation on JQ1-resistant human colorectal cancer HCT116 cells

JQ1 disrupts the binding of bromodomain and extra-terminal (BET) family of proteins to acetylated histones, modulates the expression of various genes, and inhibits the proliferation of cancer cells. We established two JQ1-resistant sublines from human colorectal cancer HCT116 cells. These resistant cells showed an 8- to 9-fold higher resistance to JQ1, and a 2- to 4-fold higher resistance to various anti-cancer agents, such as doxorubicin, etoposide, mitoxantrone, SN-38, cisplatin, and methotrexate than the parental HCT116 cells. The JQ1-resistant cells expressed higher levels of TRAF2 and NCK-interacting protein kinase (TNIK), cyclin D1 (CCND1), cyclin E1 (CCNE1), and their corresponding mRNAs than the parental cells. TNIK is a regulator of Wnt/β-catenin signaling and is known to transactivate CCND1. Transient transfection of HCT116 cells with a TNIK expression plasmid resulted in the upregulation of cyclin D1, cyclin E1, and their corresponding mRNAs, as well as an increase in CCNE1 promoter activity. Furthermore, luciferase assay revealed that the JQ1-resistant cells showed high CCNE1 promoter activity. These results suggest that TNIK also transactivates CCNE1. Three stable TNIK transfectant clones of HEK293 cells expressed 1.5- to 2-fold higher levels of TNIK, cyclin D1, and cyclin E1 than the parental cells. The 293/TNIK-6 cells, which expressed the highest level of TNIK among the transfectants, showed a 2.3-fold higher resistance to JQ1 than the parental cells. These results suggest the possible involvement of TNIK in cellular resistance to JQ1.

TNIK (TRAF2 and NCK interacting kinase)

TNIK (TRAF2 and NCK interacting kinase)

TNIK signaling imprints CD8+ T cell memory formation early after priming

Co-stimulatory signals, cytokines and transcription factors regulate the balance between effector and memory cell differentiation during T cell activation. Here, we analyse the role of the TRAF2-/NCK-interacting kinase (TNIK), a signaling molecule downstream of the tumor necrosis factor superfamily receptors such as CD27, in the regulation of CD8+ T cell fate during acute infection with lymphocytic choriomeningitis virus. Priming of CD8+ T cells induces a TNIK-dependent nuclear translocation of β-catenin with consecutive Wnt pathway activation. TNIK-deficiency during T cell activation results in enhanced differentiation towards effector cells, glycolysis and apoptosis. TNIK signaling enriches for memory precursors by favouring symmetric over asymmetric cell division. This enlarges the pool of memory CD8+ T cells and increases their capacity to expand after re-infection in serial re-transplantation experiments. These findings reveal that TNIK is an important regulator of effector and memory T cell differentiation and induces a population of stem cell-like memory T cells.

Disease-associated synaptic scaffold protein CNK2 modulates PSD size and influences localisation of the regulatory kinase TNIK

Scaffold proteins are responsible for structural organisation within cells; they form complexes with other proteins to facilitate signalling pathways and catalytic reactions. The scaffold protein connector enhancer of kinase suppressor of Ras 2 (CNK2) is predominantly expressed in neural tissues and was recently implicated in X-linked intellectual disability (ID). We have investigated the role of CNK2 in neurons in order to contribute to our understanding of how CNK2 alterations might cause developmental defects, and we have elucidated a functional role for CNK2 in the molecular processes that govern morphology of the postsynaptic density (PSD). We have also identified novel CNK2 interaction partners and explored their functional interdependency with CNK2. We focussed on the novel interaction partner TRAF2- and NCK-interacting kinase TNIK, which is also associated with ID. Both CNK2 and TNIK are expressed in neuronal dendrites and concentrated in dendritic spines, and staining with synaptic markers indicates a clear postsynaptic localisation. Importantly, our data highlight that CNK2 plays a role in directing TNIK subcellular localisation, and in neurons, CNK2 participates in ensuring that this multifunctional kinase is present in the correct place at desirable levels. In summary, our data indicate that CNK2 expression is critical for modulating PSD morphology; moreover, our study highlights that CNK2 functions as a scaffold with the potential to direct the localisation of regulatory proteins within the cell. Importantly, we describe a novel link between CNK2 and the regulatory kinase TNIK, and provide evidence supporting the idea that alterations in CNK2 localisation and expression have the potential to influence the behaviour of TNIK and other important regulatory molecules in neurons.

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 1123: TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells

Abstract [Introduction] Osteosarcoma (OS) is a rare malignant bone tumor and affects predominantly adolescents and young adults. There is no molecular targeted therapy for OS and breakthrough intervention has long been awaited for decades. Disturbances in differentiation towards mature osteoblast from mesenchymal stem cell are related to OS pathogenesis. Wnt signaling is a key component in managing this differentiation process. We previously reported that TNIK was a positive regulator of Wnt signaling. To develop the first molecular targeted therapy for OS patients, we focused on Wnt pathway-targeted therapy in OS and initiated this study to demonstrate TNIK as a novel therapeutic target for OS treatment. [Methods] Eleven different osteosarcoma cell lines were used in the present study. Twenty clinical samples were obtained at biopsy and surgical resection from ten OS patients. TNIK expression was assessed by western blotting, immunofluorescence and immunohistochemical staining. The generation of the novel TNIK inhibitor was previously described (Masuda et al., 2016). Cells were cultured with TNIK inhibitor or siTNIK. Cell viability was evaluated by real-time cell analysis system or ATP quantitation assay. The effect of TNIK inhibitor in vivo was also assessed by a mouse xenograft model. RNA-seq was performed using the RNA extracted from vehicle or TNIK inhibitor-treated OS cells. Cancer stem cell (CSC)-like properties were assessed by soft-agar colony-formation assay, limiting dilution assay, ALDH activities and the expression of CSC markers. Adipogenic phenotypes were qualitatively and quantitatively analyzed by Oil red O staining and the expression of adipogenesis markers. [Results] TNIK was identified in all cell lines and most of cases. Immunohistochemistry of OS tissue microarray also revealed that 52 out of 55 OS samples were positive for TNIK protein. TNIK inhibitor suppressed OS cell proliferation both in vitro and in vivo. SiTNIK also inhibited OS cell growth. Global gene expression analysis revealed a significant decrease in the expression of the genes relating to Wnt signaling pathway and maintenance of stem cell pluripotency. Soft-agar colony-formation assay and limiting dilution assay revealed that TNIK inhibitor decreased sphere formation activities in OS cells. Accordingly, TNIK inhibitor significantly suppressed ALDH activities and the expression of the proteins that are involved in the maintenance of CSCs, including NANOG, SOX2, OCT4A and MYC. Concomitantly, TNIK inhibitor drove adipogenic transdifferentiation of OS cells with PPARG activation. TNIK knock-down U2OS cells created by shRNAs showed nearly identical results. [Conclusion] We present that TNIK inhibition promotes adipogenic transdifferentiation of OS cells and eliminates CSCs. Our study thus suggests the feasibility of TNIK as a target for OS treatment. Citation Format: Toru Hirozane, Mari Masuda, Naoko Goto, Teppei Sugano, Naofumi Asano, Eisuke Kobayashi, Keisuke Horiuchi, Hideo Morioka, Akira Kawai, Masaaki Sawa, Morio Matsumoto, Masaya Nakamura, Tesshi Yamada. TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells [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 1123.

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.