Targeting Cullin-RING E3 Ligases for Radiosensitization: From NEDDylation Inhibition to PROTACs

Abstract

As a dynamic regulator for short-lived protein degradation and turnover, the ubiquitin-proteasome system (UPS) plays important roles in various biological processes, including response to cellular stress, regulation of cell cycle progression, and carcinogenesis. Over the past decade, research on targeting the cullin-RING (really interesting new gene) E3 ligases (CRLs) in the UPS has gained great momentum with the entry of late-phase clinical trials of its novel inhibitors MLN4924 (pevonedistat) and TAS4464. Several preclinical studies have demonstrated the efficacy of MLN4924 as a radiosensitizer, mainly due to its unique cytotoxic properties, including induction of DNA damage response, cell cycle checkpoints dysregulation, and inhibition of NF-κB and mTOR pathways. Recently, the PROteolysis TArgeting Chimeras (PROTACs) technology was developed to recruit the target proteins for CRL-mediated polyubiquitination, overcoming the resistance that develops inevitably with traditional targeted therapies. First-in-class cell-permeable PROTACs against critical radioresistance conferring proteins, including the epidermal growth factor receptor (EGFR), androgen receptor (AR) and estrogen receptor (ER), cyclin-dependent kinases (CDKs), MAP kinase kinase 1 (MEK1), and MEK2, have emerged in the past 5 years. In this review article, we will summarize the most important research findings of targeting CRLs for radiosensitization.

Keywords: EGFR; MLN4924; NEDDylation; PROTAC; cullin-RING E3 ligase.

Figure 1

A schematic overview of cullin-RING E3 ligase (CRL) and NEDD8 conjugation. Conjugation of NEDD8 to the scaffold cullin protein in the CRL is carried out in three enzymatic steps involving NEDD8-activating enzyme (NAE, E1^N), UBC12, and UBE2F (E2^N). The substrate receptor protein, docked in the CRL complex by binding to the adaptor protein, recruits substrates for ubiquitin conjugation. MLN4924 and TAS4464 are specific NAE inhibitors, prohibiting NEDD8 conjugation and thus inhibit CRL activity. The 2-D structure of MLN4924 and TAS4464 was derived from Yu et al. (34). N, NEDD8; U, ubiquitin; Rbx, RING box protein.

Figure 2

Overview of the major cytotoxic mechanism of NEDDylation inhibition. All the characters in “Red” color are CRL substrates. Accumulation of Cdt1 can trigger DNA rereplication, resulting in DNA damages. Stabilization of CDC25A can potentially cause dysregulation of both early (G1 phase to S phase) and late (G2 phase to M phase) cell cycle checkpoints (dash arrow). Meanwhile, the accumulation of Wee1, p21, and p27 will trigger cell cycle arrest at the G1 or G2 phase. Stabilization of IκBα will lead to sequestration NF-κB p65 and p50 heterodimer in the cytosol, leading to inhibition of its transcriptional activities. Stabilization of DEPTOR can directly inhibit mTOR. Ionizing radiation also triggers cell death via induction of DNA damages. p, phosphor group.

Figure 3

An overview of the PROTAC targeting EGFR for CRL2^VHL-mediated polyubiquitination. (A) 2D structure of the PROTAC MS39 that targets EGFR. The “warhead” potion of the PROTAC is based on EGFR inhibitor (EGFRi) gefitinib. (B) MS39 can recruit EGFR for polyubiquitin conjugation by the CRL2^VHL. MS39 mediated EGFR degradation is a catalytic process, as evidenced with dissociation of the PROTAC from the CRL2^VHL complex after EGFR polyubiquitination.

Figure 4

A non-exhaustive list of PROTACs targeting proteins related with the development of radioresistance. Corresponding CRLs that mediate polyubiquitination of these proteins were also identified. EGFR, epidermal growth factor receptor; ER, estrogen receptor; AR, androgen receptor; CDK, cyclin-dependent kinase; ALK, anaplastic lymphoma kinase; MEK, mitogen-activated protein kinase kinase; BTK, Bruton tyrosine kinase; BET, bromodomain and Extra-Terminal motif; BRD, bromodomain.