AXIN1, has been initially identified as a prominent antagonist within the WNT/β-catenin signaling pathway, and subsequently unveiled its integral involvement across a diverse spectrum of signaling cascades. These encompass the WNT/β-catenin, Hippo, TGFβ, AMPK, mTOR, MAPK, and antioxidant signaling pathways. The versatile engagement of AXIN1 underscores its pivotal role in the modulation of developmental biological signaling, maintenance of metabolic homeostasis, and coordination of cellular stress responses. The multifaceted functionalities of AXIN1 render it as a compelling candidate for targeted intervention in the realms of degenerative pathologies, systemic metabolic disorders, cancer therapeutics, and anti-aging strategies. This review provides an intricate exploration of the mechanisms governing mammalian AXIN1 gene expression and protein turnover since its initial discovery, while also elucidating its significance in the regulation of signaling pathways, tissue development, and carcinogenesis. Furthermore, we have introduced the innovative concept of the AXIN1-Associated Phosphokinase Complex (AAPC), where the scaffold protein AXIN1 assumes a pivotal role in orchestrating site-specific phosphorylation modifications through interactions with various phosphokinases and their respective substrates.

Background: Inhibitors targeting immune checkpoint were proved effective in cancer immunotherapy, such as PD-1/PD-L1 blockade. The novel immune checkpoint TIGIT/PVR plays critical roles in suppressing the anti-tumor effects of CD8⁺ T and NK cells, and dual blockade of TIGIT/PVR and PD-1/PD-L1 by antibody can elicit synergistic effects in tumor models and clinical trials. However, small molecules for TIGIT/PVR blockade have not been investigated. Methods: The expression of PVR in tumors were analyzed by using TCGA, Oncomine and GEO database, and in cancer cell lines examined by flow cytometry. Natural product compounds were docked to PVR for virtual screening by using the software Molecular Operating Environment (MOE). Candidate compounds were further tested by biolayer interferometry-based binding assay, microscale thermophoresis assay and cell based blocking assay. The in vitro activity of the candidate compound was determined by MTT, peripheral blood mononuclear cells (PBMCs) activation assay and coculture assay. The anti-tumor effects and mechanism were also investigated by using MC38 tumor-bearing mice model and immune cell depletion tumor model. Results: PVR was over-expressed in many tumor tissues and cancer cell lines, making it a promising therapeutic target. Through virtual screening, binding, and blocking assay, liothyronine was discovered to bind PVR and block the interaction of TIGIT/PVR. Liothyronine could enhance the function of CD4⁺ and CD8⁺ T cells in PBMCs. Besides, in the Jurkat-hTIGIT and CHOK1-hPVR coculture assay, liothyronine could reverse the IL-2 secretion inhibition resulted by TIGIT/PVR ligation. Although had no influence on the proliferation of tumor cells in vitro, liothyronine could significantly inhibit tumor growth when administrated in vivo, by enhancing CD8⁺ T cell infiltration and immune responses in the tumor bearing mice. The immune cell depletion model showed that the anti-tumor effects of liothyronine depends on CD4⁺ T cells, CD8⁺ T cells and NK cells. Conclusions: A small molecule liothyronine was discovered to serve as a potential candidate for cancer immunotherapy by blocking the immune checkpoint TIGIT/PVR. Graphical

Programmed cell death protein 1 (PD-1), a negative co-stimulatory molecule, plays crucial roles in immune escape. Blockade of the interaction between PD-1 and PD-L1 shows exciting clinical responses in a fraction of cancer patients and the success makes PD-1 as a valuable target in immune checkpoint therapy. For the rational design of PD-1 targeting modulators, the ligand binding mechanism of PD-1 should be well understood in prior. In this study, we applied 50 ns molecular dynamics simulations to observe the structural properties of PD-1 molecule in both apo and ligand bound states, and we studied the structural features of PD-1 in human and mouse respectively. The results showed that the apo hPD-1 was more flexible than that in PD-L1 bound state. We unexpectedly found that K135 was important for binding energy although it was not at the binding interface. Moreover, the residues which stabilized the interactions with PD-L1 were distinguished. Taking the dynamic features of these residues into account, we identified several residual sites where mutations may gain the function of ligand binding. The in vitro binding experiments revealed the mutants M70I, S87 W, A129L, A132L, and K135 M were better in ligand binding than the wild type PD-1. The structural information from MD simulation combined with in silico mutagenesis provides guidance to design engineered PD-1 mutants to modulate the PD-1/PD-L1 pathway.

Strategies boosting both innate and adaptive immunity have great application prospects in cancer immunotherapy. Antibodies dual blocking the innate checkpoint CD47 and adaptive checkpoint PD-L1 or TIGIT could achieve durable anti-tumor effects. However, a small molecule dual blockade of CD47/SIRPα and TIGIT/PVR pathways has not been investigated. Here, an elevated expression of CD47 and PVR was observed in tumor tissues and cell lines analyzed with the GEO datasets and by flow cytometry, respectively. Compounds approved by the FDA were screened with the software MOE by docking to the potential binding pockets of SIRPα and PVR identified with the corresponding structural analysis. The candidate compounds were screened by blocking and MST binding assays. Azelnidipine was found to dual block CD47/SIRPα and TIGIT/PVR pathways by co-targeting SIRPα and PVR. In vitro, azelnidipine could enhance the macrophage phagocytosis when co-cultured with tumor cells. In vivo, azelnidipine alone or combined with irradiation could significantly inhibit the growth of MC38 tumors. Azelnidipine also significantly inhibits the growth of CT26 tumors, by enhancing the infiltration and function of CD8⁺ T cell in tumor and systematic immune response in the tumor-draining lymph node and spleen in a CD8⁺ T cell dependent manner. Our research suggests that the anti-hypertensive drug azelnidipine could be repositioned for cancer immunotherapy.

Background: Targeting the tumor microenvironment (TME) has emerged as a promising strategy in cancer treatment, particularly through the utilization of immune checkpoint blockade (ICB) agents such as PD-1/PD-L1 inhibitors. Despite partial success, the presence of tumor-associated macrophages (TAMs) contributes to an immunosuppressive TME that fosters tumor progression, and diminishes the therapeutic efficacy of ICB. Blockade of the CD47/SIRPα pathway has proven to be an effective intervention, that restores macrophage phagocytosis and yields substantial antitumor effects, especially when combined with PD-1/PD-L1 blockade. Therefore, the identification of small molecules capable of simultaneously blocking CD47/SIRPα and PD-1/PD-L1 interactions has remained imperative. Methods: SMC18, a small molecule with the capacity of targeting both SIRPα and PD-L1 was obtained using MST. The efficiency of SMC18 in interrupting CD47/SIRPα and PD-1/PD-L1 interactions was tested by the blocking assay. The function of SMC18 in enhancing the activity of macrophages and T cells was tested using phagocytosis assay and co-culture assay. The antitumor effects and mechanisms of SMC18 were investigated in the MC38-bearing mouse model. Results: SMC18, a small molecule that dual-targets both SIRPα and PD-L1 protein, was identified. SMC18 effectively blocked CD47/SIRPα interaction, thereby restoring macrophage phagocytosis, and disrupted PD-1/PD-L1 interactions, thus activating Jurkat cells, as evidenced by increased secretion of IL-2. SMC18 demonstrated substantial inhibition of MC38 tumor growths through promoting the infiltration of CD8⁺ T and M1-type macrophages into tumor sites, while also priming the function of CD8⁺ T cells and macrophages. Moreover, SMC18 in combination with radiotherapy (RT) further improved the therapeutic efficacy. Conclusion: Our findings suggested that the small molecule compound SMC18, which dual-targets the CD47/SIRPα and PD-1/PD-L1 pathways, could be a candidate for promoting macrophage- and T-cell-mediated phagocytosis and immune responses in cancer immunotherapy.