Development of 6-morpholino-1,3,5-triazine Derivatives as Novel Anti-metastatic and Anti-cancer Immune Evasion Drugs Based on Endosomal Trafficking

開發6-嗎啉代-1,3,5-三嗪衍生物作為靶向於內體運輸的新型抗腫瘤轉移和免疫逃避藥物

Student thesis: Doctoral Thesis

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Award date2 Feb 2021

Abstract

Cancer is the second leading cause of death worldwide and it has a major impact on individuals and societies around the globe. Metastasis is the hallmark of cancer, and it accounts for more than 90% of the cancer-related deaths. Although recent therapeutic advances in cancer treatment have contributed to a decline in the overall cancer death rate and a concomitant increase in the survival rate, cancer metastasis remains poorly understood and largely incurable. In addition, despite the vast amount of manpower and material resources invested over the years to fund research on the development of anti-metastatic drugs, there are still no specific anti-metastatic therapeutics available to date. Endocytosis is an evolutionarily conserved cellular process, which regulates the trafficking and sorting of extracellular molecules, plasma membrane proteins, and lipids. It maintains the composition of the cell surface, and thus plays an important role in a wide variety of physiological functions. As efficient cell migration and invasion are key parameters of metastasis, this process requires constant endocytosis and recycling of migration-related proteins, such as integrin.

Here, by combining high-content fluorescence image-based drug screening, virtual drug screening, and chemical synthesis, I have identified a series of efficient and specific endosomal trafficking modulators, including several 6-morpholino-1,3,5-triazine derivatives, e.g., vacuolin-1 (V1) and 6J1. I assessed the anti-cancer metastasis effect of V1 both in vitro and in vivo, and I further studied how V1 is involved in endosomal trafficking to inhibit cell migration and invasion (Chapter3). In addition, I investigate the endosomal trafficking process of the membrane protein PD-L1, and then I applied 6J1 to manipulate the distribution of PD-L1 and promote the anti-cancer immune evasion effect (Chapter4).

In chapter 3, I demonstrated that V1 significantly inhibits colony formation, migration, and the invasion of various cancer cells as well as exhibiting little cell toxicity in vitro. It also compromises the assembly-disassembly dynamics of focal adhesions (FAs) by inhibiting the recycling and degradation of integrins. I showed that V1 potently suppresses breast cancer or melanoma metastasis in various experimental or transgenic mouse models. I further identified the capping protein Zβ (CapZβ) as a V1 binding protein and showed that V1 targets CapZβ to inhibit endosomal trafficking. I finally demonstrated that CapZβ is required for the V1-mediated inhibition of migration and metastasis of cancer cells.

Cancer immunotherapy utilizes the host immune system to fight against cancer. Programmed death ligand-1 (PD-L1), one of the critical immune checkpoints, has been found to be overexpressed in various solid malignancies. The success of anti-CTLA4, anti-PD-L1, and anti-PD1 antibodies against a variety of cancers has revolutionized cancer immunotherapy. However, only a small portion of the patients respond to these antibody therapies, and some patients develop primary or secondary resistance to antibody therapy. PD-L1 undergoes metabolism as well as continuous internalization and dynamic recycling to the cell surface to maintain the amounts required. In Chapter 4, I found that cell surface PD-L1 is internalized via clathrin-dependent endocytosis, after which it is directed to lysosomes (for degradation), recycling endosomes, or extracellular vesicles. I demonstrated that 6J1, a 6-morpholino-1,3,5-triazine derivative, significantly blocked the trafficking of PD-L1 via trapping PD-L1 into the endosomal compartment and inhibiting PD-L1 degradation and recycling, leading to a reduction in the level of PD-L1 at the cell surface. I also showed that 6J1 dramatically induced the discharge of PD-L1 out of the cell by exosome secretion. Moreover, blocking PD-L1 secretion could significantly enhance 6J1 anti-cancer efficiency in the orthotopic mouse model of 4T1 mouse mammary carcinoma by promoting anti-cancer immunity.

In summary, in this thesis, I demonstrated that targeting the endocytosis pathway by two 6-morpholino-1,3,5-triazine derivatives, V1 and 6J1, provides a novel strategy to fight against metastasis and promote anti-cancer immunity.