Basic cellular activities and coordinated cell actions are governed by intracellular signals, among which the Wnt signaling cascade plays an important role in tissue polarity and cell adhesion or movement through the activation of c-Jun N-terminal kinase (JNK) pathway. As one of the central transcriptional factors, Traf2- and Nck-interacting kinase (TNIK) mediates the transactivation of Wnt target genes and promotes the activity of c-Jun N-terminus kinase (JNK)2 when overexpressed. To further understand the function of TNIK, changes in intracellular signals were detected in colon cancer cell lines using a knockdown strategy. In this study, we found that the short-hairpin RNA-mediated knockdown of TNIK decreased the expressions of CD44, c-MYC and cyclin D1, which was consistent with the results of a TCF-4 reporter assay. Our data showed, for the first time, that the activation of both JNK1 and JNK2 by TNFα could be blocked through TNIK knockdown, which dampened the AP1 luciferase activity accordingly. In addition, adenovirus mediated the downregulation of TNIK-triggered intrinsic apoptosis in SW480 cells by activating caspase-9 and PARP-1. We conclude that TNIK is essential for the activation of both the canonical Wnt pathway and the JNK pathway, and serves as a pro-survival factor.

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BackgroundZimberelimab (GLS-010) is a novel fully human monoclonal immunoglobulin G4 (IgG4) against the programmed cell death-1 (PD-1) receptor.AimTo evaluate the affinity, competitive blocking capability, T cell activation effect, cytotoxic effector functions by Fc, preliminary anti-tumor activity, and pharmacokinetics of GLS-010.MethodsThe affinity of GLS-010 to PD-1 and the ability of GLS-010 to block the PD-L1/2 to PD-1 interaction on the cell surface were measured. An allogeneic mixed lymphocyte reaction was conducted to evaluate the inhibitory effect of GLS-010 on Tregs and stimulatory effect on T cell proliferation and activation. Pharmacodynamics and pharmacokinetics were evaluated in tumor-bearing mice and cynomolgus monkeys, respectively.ResultsThe equilibrium dissociation constant (KD) for the association between GLS-010 and PD-1 was 1.75×10-10 M. GLS-010 could effectively block the binding of PD-L1/2 to PD-1. GLS-010 showed statistically significant anti-tumor effects in the MC38 model in human PD-1 knock-in mice. The RO rate on in the low-, moderate-, and high-dose groups were 64.50%-48.53% in CD3+T, 58.87%-40.12% in CD8+T, and 66.26%-49.07% in CD4+T, respectively. With the increasing dose from 2 mg/kg to 18 mg/kg, the systemic exposure level of GLS-010 (AUC0-last) and C0 increased proportionally, while the proportion of AUC0-last was higher than the proportion of the increase in the dose.ConclusionsAs a fully human anti-PD-1 monoclonal antibody, GLS-010 has a high affinity to PD-1 and shows potent anti-tumor effects in vivo and in vitro. The results support that GLS-010 could be investigated in clinical trials in tumor patients.