Wnt/β-Catenin Inhibitor Combined with Venetoclax Synergistically Induce Apoptosis in Acute Myeloid Leukemia By Increasing the Degradation of MCL-1 Protein
Authors: Pan Mengya , Sun Menghua , Zhou Junjie , Jiao Changqing , Chen Enbo , Wu Zhonghui , Ma Xiaoyu , Liu Xiaoying , Ge Jian 
Published: December 2024

Acute myeloid leukemia (AML) is a highly heterogeneous malignant proliferative disease. Venetoclax is a highly selective inhibitor of BCL-2, and it has been proved that it can effectively induce the elderly patients who can not tolerate standard intensive chemotherapy to alleviate the disease when combined with low-dose cytarabine or hypomethylated medicines. However, many patients have acquired venetoclax resistance during the treatment.

The mechanism of drug resistance in venetoclax is complex. Therefore, finding a new adjuvant drug combined with venetoclax will improve the therapeutic effect of AML patients more effectively. Wnt signaling pathway is highly conserved in evolution and can regulate the proliferation and differentiation of cells and the occurrence and development of cancer. The dysregulation of the Wnt/β-catenin signaling pathway is closely related to the development of leukemia and the occurrence of chemotherapy resistance. Yet, it is not clear whether targeting Wnt/β-catenin pathway can enhance the anti-leukemia activity of venetoclax.

In this study, we found significant differences in β-catenin expression between healthy people and AML patients by analyzing GSE30029 and BeatAML datasets. In addition, we collected bone marrow blood samples from healthy people and peripheral/bone marrow blood from AML patients, which showed the mRNA and protein expression levels of β-catenin is abnormally increased. We selected MOLM13 parental cell line which is sensitive to venetoclax to construct MOLM13 resistant cell line. Western blotting showed that compared with MOLM13-P cells, the protein expression of MCL-1 and BCL-xL is increased, and the protein expression level of β-catenin is also significantly increased. Notably, knockdown the expression of β-catenin using siRNA in MOLM13-R cells increased cellular sensitivity to venetoclax and the levels of cell apoptosis.

Based on the above results, we treated the AML cell lines and primary cells with C-82 and VEN alone or in combination. The data suggested that C-82 combined with VEN have strong synergistic pro-apoptotic effects on AML cell lines and primary cells. In further, we detected mRNA and protein expression levels of anti-apoptotic protein molecules. We observed that C-82/VEN group is more able to reduce the protein expression of MCL-1. Interestingly, we found that the mRNA expression is inconsistent with the trend of protein expression in MCL-1. Next, we found that the protein expression of MCL-1 decreased gradually with the prolongation of C-82 in different time, while the mRNA expression of MCL-1 remained unchanged or increased at the same time. Therefore, we hypothesized that C-82 does not affect mRNA transcription of MCL-1, but rather post-transcriptional translational modification of MCL-1.

Next, CHX and MG-132 experiments showed that the action of C-82 on AML cells affects the stability of MCL-1 protein and reduces the half-life of MCL-1 protein. What's more, MCL-1 protein was degraded by ubiquitin-proteasome pathway. Besides, many studies have shown that phosphorylation of different sites of MCL-1 protein can regulate protein stability. Our data showed that with the increasing concentration of C-82, the protein expression of MCL-1 gradually decreased, the phosphorylation of MCL-1 Thr163 also gradually decreased, but the phosphorylation of MCL-1 Ser159 remained unchanged.

A lot of studies have shown that cell apoptosis mediated by Caspase-3 induced by chemotherapy drugs can be transformed into cell pyroptosis, which enhances the effect of anti-tumor treatment. Interestingly, under the light microscope, we observed pyroptosis-like cell morphology in the combined group. The result of levels of LDH release showed that the release level of LDH in the C-82/VEN group is significantly increased when it compared with single agent group and the control group. In addition, western blotting also showed that the more Cleaved-Caspase3 increased in the combined group, the more protein expression of the GSDME-N increased.

In Conclusion. The expression of β-catenin was up-regulated in AML patients and venetoclax resistance cell line. Wnt/β-catenin inhibitor C-82 combined with venetoclax has synergistic anti-leukemic activity. Therefore, the research results indicated that targeting Wnt/β-catenin signaling pathway combination with venetoclax may be a new strategy for the treatment of AML patients in the future.