Targeting USP2 induces degradation of PML-RARα with or without drug-resistant mutations in acute promyelocytic leukemia

Abstract

Despite the high efficacy of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL), approximately 10%–20% of patients develop drug resistance due to mutations in PML-RARα and other factors. Here, we find that inhibition of USP2 with ML364 or USP2 silencing reduces PML-RARα protein levels in both ATRA-sensitive and ATRA-resistant APL cells, and this effect is reversed by proteasome inhibition. Conversely, USP2 overexpression enhances PML-RARα stability. Mechanistically, USP2 interacts with and deubiquitinates PML-RARα, including its drug-resistant mutants. Consistent with PML-RARα degradation, ML364 treatment significantly induces apoptosis in APL cell lines and primary leukemia cells. In conclusion, this study identifies USP2 as a novel deubiquitinating enzyme for PML-RARα and highlights USP2 inhibition as a potential therapeutic strategy for APL with PML-RARα mutations.

1. Introduction

PML-RARα is generated by the t(15;17)(q24;q21) chromosomal translocation [1]. The resulting oncoprotein blocks cell differentiation and suppresses apoptosis, ultimately leading to the development of acute promyelocytic leukemia (APL) [2,3]. The introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the overall survival of APL patients [1,4,5]. However, approximately 10%–20% of APL patients develop resistance to treatment with ATRA and/or ATO [6]. Among the known resistance mechanisms, the most prevalent one involve mutations in the PML-RARα fusion gene, particularly in the ligand-binding domain (LBD) of RARα and the PML-B2 domain, the primary target site of ATO [7–9]. Mutations in the PML-B2 domain (e.g., A216V and L218P) may impair ATO therapeutic efficacy by disrupting proper ATO binding, interfering with post-translational modifications (such as SUMOylation and multimerization), and causing aberrant subcellular localization [10–12]. These defects collectively lead to insufficient degradation and pathological retention of oncoproteins, ultimately sustaining their stability and driving therapeutic resistance by evading ATO-mediated clearance mechanisms. When mutations occur in the LBD of RARα, conformational changes can weaken its binding affinity to ATRA. Owing to this reduced binding capacity, the LBD may fail to properly dissociate nuclear receptor corepressor complexes and recruit activator complexes, leading to aberrant transcriptional regulation and subsequent ATRA resistance [13–15]. One promising strategy to overcome this mutation-induced drug resistance is to induce protein degradation via alternative pathways. For example, targeting HDAC3 has been shown to induce the degradation of PML-RARα in ATRA- and ATO-resistant APLs [16].

Deubiquitinases (DUBs) regulate protein stability and function by removing ubiquitin chains from substrate proteins, modifying ubiquitin linkages, and processing ubiquitin precursors [17]. The stability of PML-RARα can also be influenced by DUB activity, suggesting that inhibiting specific DUBs may induce PML-RARα degradation and overcome ATRA- and ATO-resistance in patients with PML-RARα mutations. For example, the inhibition of YOD1, a member of the ovarian tumor protease (OTU) family, has been shown to promote PML-RARα degradation and effectively eliminate APL cells, including drug-resistant subtypes [18]. This work suggests that DUB inhibition is a potential therapeutic strategy for degrading PML-RARα.

In the present study, we identified USP2 as a novel DUB for PML-RARα. USP2 knockdown or the use of the USP2 inhibitor ML364 induced the degradation of PML-RARα and the apoptosis of APL cells. Importantly, inhibition of USP2 could also degrade drug-resistant PML-RARα mutants. Our study demonstrated that USP2 is a novel target for the treatment of APL, particularly in patients with drug-resistant APL driven by PML-RARα mutations.