1. Department of Histology and Embryology, Xiang Ya School of Medicine, Central South University, Changsha, Hunan, China
2. The affiliated Hospital of Guilin Medical University, Guangxi Key Laboratory of Brain and Cognitive Neuroscience, Guangxi Neurological Diseases Clinical Research Center, Guilin, Guangxi, China
3. Department of Cardiology, Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, Shanghai, China
4. National Center for International Research of Biological Targeting Diagnosis and Therapy (Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy Research), Guangxi Medical University, Nanning, China
5. Department of Surgery, Robert-Wood-Johnson Medical School University Hospital, The State University of New Jersey, New Brunswick, New Jersey, USA
6. Pharmacy Department, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
7. Department of Gastrointestinal Surgery, Zhongshan Hospital of Xiamen University, Xiamen, Fujian, China
Resistance to the chemotherapeutic drug 5'-azacytidine (5'-AZA) is a major obstacle in the treatment of patients with acute myeloid leukemia (AML). The uridine-cytidine kinase 1 (UCK1) has an established role in activating 5'-AZA and its protein level is significantly downregulated in patients resistant to the drug. However, the underlying molecular mechanism for the reduced UCK1 expression remains to be elucidated.
Methods: Using mass spectrometry and molecular biochemistry analyses, we identified specific enzymes mediating UCK1 degradation. Human AML cell lines and murine AML model were used to characterize the effects of these enzymes on 5'-AZA resistance.
Results: We demonstrated that the ubiquitin E3 ligase KLHL2 interacted with UCK1 and mediated its polyubiquitination at the K81 residue and degradation. We showed that deubiquitinase USP28 antagonized KLHL2-mediated polyubiquitylation of UCK1. We also provided evidence that ATM-mediated phosphorylation of USP28 resulted in its disassociation from KLHL2 and UCK1 destabilization. Conversely, UCK1 phosphorylation by 5'-AZA-activated ATM enhanced the KLHL2-UCK1 complex formation. Importantly, silencing KLHL2 or USP28 overexpression not only inhibited AML cell proliferation but also sensitized AML cells to 5'-AZA-induced apoptosis in vitro and in vivo. These results were no longer observed in USP28-deficient cells.
Conclusions: Our study revealed a novel mechanism by which the KLHL2/USP28/ATM axis mediates resistance of AML cells to 5'-AZA by regulating UCK1 ubiquitination and phosphorylation. These results have direct clinical implications and provide a rationale for the combination drug treatment of AML patients.
Keywords: 5'-AZA, UCK1, USP28, KLHL2, ubiquitination