The USP22-NAP1L1 deubiquitination axis in the pathogenesis of myelodysplastic syndromes

Abstract Background:Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic system diseases with significant heterogeneity. Its pathogenesis is mostly related to abnormal clonal proliferation of hematopoietic stem/progenitor cells, and abnormal epigenetic regulation is the core mechanism driving the occurrence and development of the disease. As a chromatin remodeling regulator, nucleosome assembly protein 1-like 1 (NAP1L1) plays a key carcinogenic role in the occurrence and development of various malignant tumors, but its role in MDS remains unclear. Aims: To explore the pathogenic mechanism of NAP1L1 in MDS, the molecular pathway through which it is regulated by USP22-mediated deubiquitination, and to validate the therapeutic potential of targeting the USP22-NAP1L1 deubiquitination axis. Methods:This study collected bone marrow samples from myelodysplastic syndrome (MDS) patients and healthy controls. Mononuclear cells were isolated, and CD34⁺ cells were immunopurified using magnetic beads, followed by qPCR quantification of NAP1L1 expression differences. A NAP1L1-knockdown model was then established in MDS cell lines. Cell proliferation, cell cycle distribution, and apoptosis levels were assessed using EdU assays and flow cytometry. To investigate NAP1L1 regulatory mechanisms, interacting proteins were screened by affinity purification-mass spectrometry (AP-MS), with interactions validated through endogenous and exogenous co-immunoprecipitation (Co-IP). The dynamics of NAP1L1 ubiquitination-dependent degradation were further assessed using proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide (CHX). Finally, the impact of targeting the USP22-NAP1L1 axis on MDS cell viability was systematically evaluated by treating cells with a USP22 small-molecule inhibitor and overexpressing NAP1L1 under inhibitor conditions. Results:Single-cell sequencing results revealed that the expression level of NAP1L1 in CD34⁺ cells from MDS patients was significantly elevated compared with that in the normal control group (P<0.001). To further validate this finding, we detected the mRNA level of NAP1L1 in bone marrow CD34⁺ cells from 17 MDS patients and 5 healthy controls. The results showed that the expression level of this gene in MDS patient samples was significantly higher than that in healthy donor samples (P<0.01). Subsequently, after knocking down NAP1L1 in MDS-L and SKM-1 cells, the viability of MDS cells was significantly reduced, which was specifically manifested as inhibited cell proliferation (P<0.01), increased apoptosis level (P<0.01), and cell cycle arrest at the G0/G1 phase (P<0.05). Mechanistic studies confirmed that NAP1L1 interacts with USP22 in cells, and USP22 maintains the protein stability of NAP1L1 through its deubiquitinase activity. After adding a USP22 inhibitor to the MDS cell line, cell viability decreased significantly, accompanied by inhibited proliferation (P<0.05) and increased apoptosis level (P<0.05), and this phenomenon could be reversed by overexpressing NAP1L1. Conclusion:USP22 stabilizes the NAP1L1 protein through deubiquitination modification, thereby promoting the malignant progression of MDS. Targeting the USP22-NAP1L1 regulatory axis can effectively inhibit disease progression, providing a potential novel therapeutic strategy for MDS.