Wnt; Traf2- and Nck-interacting kinase (TNIK) (IC50 = 21 nM)

NCB-0846 exhibits anti-Wnt properties. TNIK is bound by NCB-0846 in an inactive conformation; this binding mode seems to be essential for Wnt suppression. With an IC50 of 21 nM, NCB-0846 has inhibitory action against TNIK. Moreover, FLT3, JAK3, PDGFRα, TRKA, CDK2/CycA2, and HGK are inhibited by NCB-0846. At concentrations between 0.1 and 0.3 μM, NCB-0846 accelerates the migration of TNIK-phosphorylated TCF4, and at 3 μM, it totally prevents TCF4 phosphorylation. In addition to inhibiting HCT116 cell growth, NCB-0846 has a greater (-20-fold) inhibitory action when it comes to the same cells' ability to form colonies in soft agar [1].

---

NCB-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. 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]. Conclusions: NCB-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 [2].

The formation of tumors created by grafting HCT116 cells into immunocompromised mice is inhibited by NCB-0846. Wnt target gene expression (AXIN2, MYC, and CCND1) was downregulated in xenografts following NCB-0846 treatment. There is an increase in sub-G1 cell populations caused by NCB-0846. Apoptosis is induced when poly(ADP-ribose) polymerase 1 cleaves [1].

---

NCB-0846 was administrable orally and suppressed the growth of tumours established by inoculating HCT116 cells into immunodeficient mice (Fig. 3c, left). The body weight of mice fell at the beginning of NCB-0846 administration, but gradually recovered (Fig. 3c, right). The expression of Wnt-target genes (AXIN2, MYC and CCND1) in xenografts was reduced following the administration of NCB-0846 (Fig. 3d). The effect of NCB-0846 on Wnt-driven tumorigenesis was then investigated in Apcmin/+ mice. The hydrochloride salt of NCB-0846 was water-soluble and used for oral administration subsequently. Administration of NCB-0846 was started 10 weeks after birth, and the mice were scarified at the age of 15 weeks. NCB-0846 dose dependently reduced the multiplicity and dimensions of tumours that developed in the small intestine (Fig. 3g). Although the effect of NCB-0846 on colon tumorigenesis was not statistically significant, this finding may not have been conclusive in view of the small number of animals examined and the general paucity of colon tumour development in the Apcmin/+ mice. Active Wnt signalling transforms a single intestinal epithelial cell into adenoma, and NCB-0846 is thought to suppress this tumour initiation process. The number of tumours that developed in the colon was also reduced, but the difference failed to reach statistical significance due to the paucity of colon tumours and the number of animals examined.[1]

---

Potential of NCB-0846 to inhibit metastasis [2]
Finally, we explored whether the EMT inhibitory activity of NCB-0846 affects metastasis. A549 cells were treated with TGFβ1 in the absence or presence of either NCB-0846 or NCB-0970 for 48 h in vitro, and then injected into immunodeficient mice (eight per group) via the tail vein. Seven weeks after injection, the mice were sacrificed, and their lung metastases were digitally quantified in tissue sections (Fig. 5a). This animal experiment is used mainly to evaluate the trans-endothelial migration/extravasation capability of cancer cells embolised in peripheral lung vessels immediately after systemic injection.
Metastatic lesions occupied 38% of the lung area in mice injected with TGFβ1-treated cells but were significantly (P < 0.001) reduced in the lungs of mice injected with NCB-0846-treated cells (Fig. 5b). We confirmed the complete absence of metastasis by microscopic inspection of tissue sections. In parallel, the average lung weights of mice injected with NCB-0846-treated cells were significantly decreased due to lack of the pulmonary metastatic burden, compared with those of mice injected with cells that had been treated with DMSO (control) or NCB-0970 (Fig. 5c). These results support the notion that inhibition of EMT by NCB-0846 compromises the TGFβ1-induced metastatic potential of lung cancer cells.

Mobility shift assay [1]
Enzymatic activity of TNIK was measured by mobility shift assay52 using a QuickScout Screening Assist Kit. The reaction product was quantified using a LabChip EZ Reader II. The IC50 values were calculated from the dose–response curves using nonlinear regression analysis (Fig. 2b).

---

Kinase selectivity profiling [1]
The selectivity of compounds against a panel of 50 human protein kinases was assessed using a non-radiometric assay. Percentage inhibition was determined at an inhibitor concentration of 0.1 M with ATP at the Km concentration (Fig. 2c and Supplementary Table 1).

Immunofluorescence microscopy [2]
Cells were seeded at 2.5 or 5.0 × 104 per well onto collagen-coated 8-well culture slides (BioCoat). Following 24-h serum starvation, the cells were cultured with dimethyl sulfoxide (DMSO) alone (control), TGFβ1 (5 ng/ml) and DMSO, TGFβ1 and NCB-0846 (1 µM) or TGFβ1 and NCB-970 (1 µM) for 48 h and fixed in 4% paraformaldehyde. The fixed cells were immunostained with primary antibodies (listed in Supplementary Table S1) as described previously.18 Following overnight incubation at 4 °C, the cells were incubated with relevant secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 568 and co-stained with TOTO3 or phalloidin for visualisation of nuclei or filamentous actin, respectively. Cells were examined with the LSM 5 PASCAL laser confocal microscopy system.

---

Wound-closure assay [2]
A549 cells were seeded at 5.0 × 105 per well in a 6-well plate and serum-starved for 24 h prior to creation of a scratch on the confluent cell monolayer. The wounded monolayers were then treated with DMSO alone (control), TGFβ1 (5 ng/ml) and DMSO, TGFβ1 and NCB-0846 (3 µM), or TGFβ1 and NCB-970 (3 µM) for 48 h. The degree of migration was determined by measuring the width of cell-free areas in triplicate.

---

Automated real-time cell migration assay [2]
Cells were plated (1.0 × 104 cells/well) in triplicate onto a microelectrodes-embedded xCelligence E-culture plate. When cells reached confluence (indicated by steady-state impedance), scratches were created with sterile P200 micropipette tips and the cells were treated with DMSO alone (control), TGFβ1 (5 ng/ml) and DMSO, TGFβ1 and NCB-0846 (3 µM), or TGFβ1 and NCB-970 (3 µM) for 40 h. The impedance of cell monolayers before and after scratching as well as the recovery of full impedance were monitored in real time with the xCelligence instrument.

---

Cell invasion assay [2]
Cell invasion was assessed using 16-well transwell plates with 8-µm pores (CIM plate 16) precoated with 20 µl of Matrigel diluted 1:40 in MEM medium. Microelectrodes were located on the underside of membranes in the upper chambers. Medium containing 10% FCS was added to the lower chamber, and cells were seeded into the upper chamber at 4 × 104 per well in serum-free medium. A549 cells were treated with DMSO alone (control), TGFβ1 (5 ng/ml) and DMSO, TGFβ1 and NCB-0846 (3 µM), or TGFβ1 and NCB-970 (3 µM). Impedance of migrated cells was monitored in real time with the xCelligence instrument. Data analysis was carried out using RTCA software 2.0 supplied with the instrument.

---

Quantitative PCR [2]
Total RNA was isolated with a RNeasy Mini Kit or miRNeasy Mini Kit in accordance with the manufacturer’s instructions. cDNA was prepared using a High-Capacity cDNA reverse transcription kit or MicroRNA Reverse Transcription Kit. The relative expression of mRNA and miRNA was measured using the TaqMan Gene Expression Assay or TaqMan MicroRNA Assay, respectively. Amplification was quantified using the comparative threshold cycle (CT) method.19 β-Actin (ACTB) or U6 was used as an internal control. Predesigned primer and probe sets are listed in Supplementary Table Supplementary Table S2.

---

miRNA microarray analysis [2]
Total RNA was prepared from the A549 cells treated with DMSO alone (control), TGFβ1 (5 ng/ml) and DMSO, TGFβ1 and NCB-0846 (3 µM) or TGFβ1 and NCB-970 (3 µM) using a miRNeasy Mini Kit and subjected to miRNA expression profiling with the use of an Agilent Human miRNA Microarray kit 8 × 60 K rel.21.0 in accordance with the manufacturer’s protocol. The scanned images were analysed with Feature Extraction Software 11.5.1.1 using default parameters to obtain the background subtracted value. The GeneView files were generated using Agilent’s Feature Extraction software version 11.5.1.1. The microarray data were deposited in the NCBIs Gene Expression Omnibus database under the accession number GSE95766 (released on March 08, 2017).

---

Oligonucleotide transfection [2]
siTβRI or siNC was transfected into cells at a final concentration of 5 nM with Lipofectamine 2000, and the transfection medium was replaced after 12-h serum starvation. The cells were then incubated with or without TGFβ1 (5 ng/ml) for 48 h. All mimics and inhibitors were transfected with Lipofectamine 2000 at a final concentration of 50 nM and incubated at 37 °C for 48 h.

---

Luciferase reporter gene assay [2]
A549 cells transfected with a luciferase reporter plasmid carrying three copies of a SMAD-binding element (SBE), pGL4.48-luc2P/SBE/Hygro, were treated with DMSO (control) or TGF-β1 (2 ng/ml) for 60 min and with NCB-0846 or NCB-970 at various concentrations for 5 h, and their luciferase activities were measured.

Five million HCT116 cells suspended in medium containing 25% Matrigel (BD Biosciences) were inoculated into the subcutaneous tissues of 9-week-old female BALB/c nude mice. When the tumour volume reached ∼80 mm3, the mice were randomized according to tumour volume (9 mice per group). NCB-0846 suspended in DMSO/polyethylene glycol#400/30% 2-hydroxypropyl-β-cyclodextrin solution (10:45:45v/v) was administered daily by oral gavage at 0 (vehicle alone), 40 or 80 mg kg–1 (body weight) BID (bis in die) for 14 days (Fig. 3c).

Patient-derived colorectal cancer spheroids were dissociated, resuspended in medium containing 50% Matrigel, and inoculated subcutaneously (#6; 1 × 104 cells per injection, #19; 1 × 105 cells per injection) into the flank of female NOD/ShiJic-scid mice (4-week-old females). When the tumour volume reached ∼200 mm3, the mice were randomized according tumour volume (10 mice per group) and treatment was started. NCB-1026 (strong>hydrochloride salt of ) was dissolved in sterile saline solution and administered BID by oral gavage at 0 (vehicle alone), 50 or 100 mg kg−1 (body weight) on a 6-days-on, 1-day-off schedule (Fig. 6f,g).

Patient-derived colorectal cancer xenografts COX021 and COX021 were established by Shanghai ChemPartner. Female Nu/Nu nude mice aged 5–6 weeks were implanted subcutaneously with the xenografts and randomized into three treatment groups (10 mice per group) based on the developed tumour volume (∼200 mm3). NCB-1026 (hydrochloride salt of NCB-0846) dissolved in sterile water was administered BID by oral gavage at 0 (vehicle alone), 45, or 90 mg kg−1 (body weight) on a 5-days-on, 2-day-off schedule. [1]

---

Evaluation of metastatic potential [2]
Eight-week-old male severe combined immunodeficiency (SCID) mice (C.B-17/Icr-scid) were housed in open-top cages under a 12-h light-dark cycle with free access to standard chow and water ad libitum and maintained in a specific pathogen-free environment. A549 cells were serum-starved for 24 h and stimulated with TGF-β (5 ng/ml) and DMSO (control), NCB-0846 (3 µM) or NCB-970 (3 µM) for 48 h, and 5.0 × 105 cells in 100 µL of PBS were injected into the anesthetised mice through the tail vein. Seven weeks after the injection of cells treated as indicated, the mice were euthanatised by cervical dislocation, and the digital images of entire lung tissue sections (HE stained) were captured with a BZ-X700 auto-microscope. The areas occupied by metastases were quantified using the Hybrid Cell Count software.

---

Suspended in DMSO/polyethylene glycol#400/30% 2-hydroxypropyl-β-cyclodextrin solution (10:45:45v/v); oral gavage at 0 (vehicle alone), 40 or 80 mg kg–1 (body weight) BID (bis in die) for 14 days.

9-week-old female BALB/c nude mice

[1]. TNIK inhibition abrogates colorectal cancer stemness. Nat Commun. 2016 Aug 26;7:12586.

[2]. Pharmacological blockage of transforming growth factor-β signalling by a Traf2- and Nck-interacting kinase inhibitor, NCB-0846. Br J Cancer. 2021 Jan;124(1):228-236.

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(-/-)/Apc(min/+) 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 Apc(min/+) 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. [1]

---

Background: Metastasis 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. Methods: Traf2- 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. Results: NCB-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].[2]

C21H21N5O2

375.42

375.17

C, 67.18; H, 5.64; N, 18.65; O, 8.52

1792999-26-8

2749881-54-5; 1792999-26-8

91801204

Light yellow to yellow solid powder

3.6

3

6

4

28

515

0

C1CC(CCC1O)OC2=CC=CC3=CN=C(N=C32)NC4=CC5=C(C=C4)N=CN5

FYWRWBSYRGSWIQ-UHFFFAOYSA-N

InChI=1S/C21H21N5O2/c27-15-5-7-16(8-6-15)28-19-3-1-2-13-11-22-21(26-20(13)19)25-14-4-9-17-18(10-14)24-12-23-17/h1-4,9-12,15-16,27H,5-8H2,(H,23,24)(H,22,25,26)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.25 mg/mL (5.99 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.25 mg/mL (5.99 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)