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NEDD8-conjugating enzyme E2s: critical targets for cancer therapy

NEDD8-conjugating enzyme E2s: critical targets for cancer therapy www.nature.com/cddiscovery REVIEW ARTICLE OPEN NEDD8-conjugating enzyme E2s: critical targets for cancer therapy ✉ ✉ ✉ ✉ 1,6 2,6 3,6 1 1 4 5 1 Lisha Zhou , Xiongzhi Lin , Jin Zhu , Luyi Zhang , Siyuan Chen , Hui Yang , Lijun Jia and Baofu Chen © The Author(s) 2023 NEDD8-conjugating enzymes, E2s, include the well-studied ubiquitin-conjugating enzyme E2 M (UBE2M) and the poorly characterized ubiquitin-conjugating enzyme E2 F (UBE2F). UBE2M and UBE2F have distinct and prominent roles in catalyzing the neddylation of Cullin or non-Cullin substrates. These enzymes are overexpressed in various malignancies, conferring a worse overall survival. Targeting UBE2M to influence tumor growth by either modulating several biological responses of tumor cells (such as DNA-damage response, apoptosis, or senescence) or regulating the anti-tumor immunity holds strong therapeutic potential. Multiple inhibitors that target the interaction between UBE2M and defective cullin neddylation protein 1 (DCN1), a co-E3 for neddylation, exhibit promising anti-tumor effects. By contrast, the potential benefits of targeting UBE2F are still to be explored. It is currently reported to inhibit apoptosis and then induce cell growth; hence, targeting UBE2F serves as an effective chemo-/ radiosensitizing strategy by triggering apoptosis. This review highlights the most recent advances in the roles of UBE2M and UBE2F in tumor progression, indicating these E2s as two promising anti-tumor targets. Cell Death Discovery (2023) 9:23 ; https://doi.org/10.1038/s41420-023-01337-w FACTS Whether these UBE2M-DCN1 inhibitors have any biological function? More research is being devoted to finding specific inhibitors Whether to promote clinical trials of these E2s inhibitors alone against neddylation E2s to address the limitations of or in combination? neddylation E1 inhibitor MLN4924. UBE2M and UBE2F are overexpressed in various malignancies, conferring a worse overall survival. Targeting UBE2M to influence tumor growth by either INTRODUCTION modulating several biological responses of tumor cells (such Protein neddylation involves the conjugation of a ubiquitin-like as DNA-damage response, apoptosis, or senescence) or regulat- molecule known as neuronal precursor cell-expressed develop- ing the anti-tumor immunity holds strong therapeutic potential. mentally down-regulated protein 8 (NEDD8) to the lysine residue A few inhibitors targeting UBE2M-DCN1 interaction have been of targeted substrate proteins [1, 2]. Like ubiquitin, NEDD8 binds identified. to substrates by forming an isopeptide chain between its UBE2F appears to be promising as both an anti-tumor target C-terminal glycine residue (Gly76) and a lysine residue on targeted and a chemo-/radiosensitizing target. proteins. In the initial step of the reaction, NEDD8 is produced as a precursor containing five additional residues downstream from Gly76 cleaved by the C-terminal hydrolases [3, 4]. The next step involves activation of the mature NEDD8 in an ATP-dependent OPEN QUESTIONS manner by the NEDD8-activating enzyme (NAE) E1, a heterodimer ● composed of NAE1 and ubiquitin-like modifier activating enzyme The induction of drug-resistant mutations in UBA3 inhibits the 3 (UBA3) [5, 6]. After that, a trans-thiolation process occurs during formation of the MLN4924-NEDD8 adduct, and hence there is which the NEDD8-loaded NAE is transferred to either UBE2M, also a need to explore possible alternative targets against the known as UBC12, or UBE2F (two NEDD8-conjugating enzyme E2s) neddylation pathway. ● [7–9]. Finally, covalent attachment drives a substrate-specificE3 How do the two E2s regulate anti-tumor immunity? In-depth ligase, such as defective in cullin neddylation 1 (DCN1) and RING- elucidation of the underlying mechanisms may provide vital box protein 1/2 (RBX1/2), to transfer NEDD8 from the charged E2 cues for targeting UBE2M and UBE2F. ● to a lysine residue in its target protein (Fig. 1)[10–14]. The best Deciphering the cellular potency of these UBE2M-DCN1 characterized principal substrates of neddylation are the cullin inhibitors compared to MLN4924? 1 2 Taizhou Central Hospital (Taizhou University Hospital), Taizhou University, Taizhou, Zhejiang, China. Graduate School of Medicine, Hebei North University, Zhangjiakou, Hebei, 3 4 China. Department of Surgical Oncology, Jiangxi Cancer Hospital, Nanchang, Jiangxi, China. Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China. 5 6 Cancer Institute of Traditional Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China. These authors contributed equally: Lisha Zhou, Xiongzhi Lin and Jin Zhu. email: lishazhou@tzc.edu.cn; hui_yang@fudan.edu.cn; ljjia@shutcm.edu.cn; chenbf@tzc.edu.cn Received: 8 October 2022 Revised: 11 January 2023 Accepted: 13 January 2023 Official journal of CDDpress 1234567890();,: L. Zhou et al. BIOLOGICAL CHARACTERISTICS AND CORRELATION OF UBE2M AND UBE2F UBE2M and UBE2F bind to the ubiquitin-fold domain and the UBA3 hydrophobic groove of E1 via the core domain and the N-terminal motif, respectively [9]. Acetylation of the N-terminal methionine occurs in both E2s, which facilitates their binding to the PONY domain pocket of neddylation E3 DCN-like (DCNL), thereby increasing the efficiency of the cullin neddylation process [49–51]. In addition to these similarities, protein structure assessment of both UBE2M and UBE2F reveals unique character- istics. UBE2F is very specific to the neddylation of RBX2-related CUL5, while UBE2M can pair with RBX1 to modulate the neddylation of CUL1, 2, 3, 4A, and 4B [50]. It is noteworthy that glycyl-tRNA synthetase, an enzyme necessary for protein produc- tion, binds to the NAE1 subunit of E1 to capture and protect activated UBE2M before it reaches the downstream target [52]. The enzymes UBE2M and UBE2F can activate distinct cullins by enhancing neddylation modification, and interestingly, there is a cross-talk that leads to UBE2M targeting UBE2F for degradation [53]. Specifically, UBE2M serves as a stress-inducible protein and a dual E2 for neddylation and ubiquitylation to degrade UBE2F. Under physiological conditions, UBE2M acts as a neddylation E2 to promote CUL3 neddylation, which triggers polyubiquitylation and Fig. 1 The process of protein neddylation. Neddylation is a degradation of UBE2F through CUL3-KEAP1 E3 ligase. However, process that conjugates NEDD8 to cullins or non-cullin substrates UBE2M is transcriptionally activated under stress by hypoxia- via a three-step reaction, catalyzed by NEDD8-activating enzyme E1 inducible factor 1α (HIF-1α) or transcription factor AP-1 (AP-1). (a heterodimer of NAE1 and UBA3), NEDD8-conjugating enzyme E2 (UBE2M or UBE2F) and substrate-specific NEDD8-E3 ligases (e.g., UBE2M performs ubiquitylation E2 for Parkin-DJ-1 E3, followed by RBX1 and RBX2). MLN4924: an inhibitor of UBA3; N8: neuronal ubiquitylation and degradation of UBE2F. Ultimately, the degrada- precursor cell-expressed developmentally downregulated protein 8. tion of UBE2F is accompanied by the inactivation of CRL5. Collectively, these findings provide evidence that one neddylation family members (CUL1, 2, 3, 4A, 4B, and 5), which function as core E2 (UBE2M) acts as a dual E2 for both neddylation and components of Cullin-RING E3 ubiquitin ligases (CRLs) [4]. The ubiquitylation to decrease the protein levels of the other (UBE2F), activation of CRLs requires NEDD8 to attach to a C-terminal lysine leading to one CRL E3 (CRL3) inactivating the other (CRL5) (Fig. 2). residue of cullins, inducing structural changes in the CRLs complex and leading to open conformation to facilitate substrate ubiquitylation [15–19]. As the most prominent family of E3 UBE2M AND UBE2F AS ATTRACTIVE ANTI-TUMOR TARGETS ubiquitin ligases, CRLs noticeably regulate various essential Most studies reveal that both two neddylation E2s act as biological functions, including tumorigenesis, accomplished by oncogenes, evidenced mainly by their significant upregulation in enhancing ubiquitylation and consequent degradation of a wide various human cancers, including esophageal squamous cell range of critical modulatory proteins [4, 20]. Besides cullins, several carcinoma, osteosarcoma, lung cancer, and hepatocellular carci- non-cullin proteins have been identified as the substrates of noma [54–59]. In addition, the upregulation of the two neddyla- neddylation, such as p53, MDM2, and EGFR [21–24]. However, tion E2s correlates closely with illness progression [55–58]. To regulatory mechanisms and physiological functions of non-cullin attain a greater understanding of the expression profile and substrates still await experimental validation. prognostic significance of UBE2M and UBE2F in human cancers, In multiple cancers, overactivation of the neddylation pathway we examined their expression with the aid of Tumor Immune leads to elevated global neddylation of substrates, such as cullins, Estimation Resource (TIMER) online database, an interactive and consequent accumulation of tumor suppressors, thereby platform that allows users to conduct in-depth studies of TCGA promoting tumorigenesis and development [25–28]. Targeting the gene expression profiles (http://timer.cistrome.org/). Compared to overactivated protein neddylation pathway has been proven to be normal tissues, the mRNA levels of UBE2M are elevated in 17 an effective anti-tumor strategy. MLN4924, commonly referred to different kinds of human cancers, while the mRNA levels of UBE2F as pevonedistat, is an effective and highly selective small- are elevated in 12 different types of human cancers (Fig. 3A, B). molecule inhibitor of NAE adopted to inhibit protein neddylation Kaplan–Meier analysis shows that increased mRNA levels of both via inactivation of the initial stage of the neddylation cascade [29]. enzymes correlated to worse survival for patients with lung When bound to the active site of UBA3, MLN4924 forms a stable adenocarcinoma (LUAD) or with liver hepatocellular carcinoma covalent adduct with NEDD8 to block further enzymatic processes (LIHC) (Fig. 3C, D). They exert oncogenic effects by enhancing the [30]. It achieves potent anti-tumor effects by inducing cell cycle neddylation of certain substrates to mediate a variety of signaling arrest, apoptosis, or senescence of tumor cells, or affecting the pathways and modulate multiple biological activities, such as functions of multiple components of the tumor microenvironment apoptosis or senescence (Fig. 4). These findings suggest that the [28, 29, 31–37]. MLN4924 has been evaluated in Phase I/II clinical overactivation of these two enzymes could be an oncogenic event trials for treating various solid tumors and hematologic cancers throughout the process of tumor occurrence and development. [38–44]. Nevertheless, the induction of drug-resistant mutations in UBA3 inhibits the formation of the MLN4924-NEDD8 adduct, necessitating the need to explore alternative targets against the TARGETING UBE2M FOR ANTI-TUMOR THERAPY neddylation pathway [45, 46]. To address the limitations of In recent decades, increasing studies have shown UBE2M, also MLN4924, specific inhibitors against neddylation E2s are being known as UBC12, to be an attractive anti-tumor target. Compared investigated [47, 48]. This review summarizes the latest progress with E1 subunits (NAE1 and UBA3), UBE2M is significantly more on validating the neddylation of these enzymes as promising anti- consistent with the global protein neddylation levels [56]. tumor targets. Targeting UBE2M suppresses the growth of MLN4924-resistant Cell Death Discovery (2023) 9:23 L. Zhou et al. cells by inhibiting cullins neddylation and inducing the accumula- tion of CRLs substrates [56]. Moreover, UBE2M-mediated protein neddylation is essential for multiple cellular responses, such as DNA-damage response (DDR), apoptosis, senescence, and anti- tumor immunity. These findings validate UBE2M as an attractive alternative anti-tumor target to efficiently inhibit the neddylation pathway. UBE2M PARTICIPATES IN DDR DNA double-strand break (DSB), contributing significantly to genomic stability [60, 61], is sensed and repaired by DDR, warranting the recruitment and post-translational modification of multiple proteins at the damaged DNA sites. This phenomenon induces checkpoint signaling or essential repair steps [62, 63]. Recent studies have shown that UBE2M-mediated neddylation of cullins, or non-cullins, is involved in the DDR pathway (Fig. 4). UBE2M affects DDR and the integrity of the genome by regulating several CRLs substrates, such as CDT1, p21, and claspin, which play complex roles in the increased DNA-damage in UBE2M-silenced cells [64]. Another study supported the accumulation effects of CRLs substrates CDT1 and ORC1 caused by silenced UBE2M. This phenomenon subsequently induces DSBs, evidenced by the upregulated expression of γ-H2AX [65]. Generally, Ku70/Ku80 (Ku) heterodimer is first recruited to the DSB sites, after which, DNA-dependent protein kinase catalytic Fig. 2 UBE2M acts as a dual E2 for neddylation and ubiquitylation subunit (DNA-PKcs) is recruited into the process, facilitating to degrade UBE2F. Under physiological conditions, UBE2M serves nonhomologous end-joining (NHEJ) repair [66, 67]. Upon DNA- as a neddylation E2 to promote CUL3 neddylation, triggering damage, UBE2M-mediated cullins neddylation promotes ubiqui- polyubiquitylation and degradation of UBE2F through CUL3-KEAP1 tylation of Ku, releasing it and its associated proteins from the E3 ligase. Under stressed conditions, UBE2M is transcriptionally activated by HIF-1α or AP-1, which makes it serve as a ubiquitylation damaged sites after repair. After NHEJ, ionizing radiation E2 to complex with DJ-1/Parkin to promote the ubiquitylation and hypersensitivity and decreased cell survival occur in UBE2M- degradation of UBE2F. Ultimately, the degradation of UBE2F is depleted cells [68]. Moreover, UBE2M also regulates the neddyla- accompanied by the inactivation of CRL5. TPA: a typical tumor tion of DNA-PKcs, which promotes DNA-PKc autophosphorylation, promoter and mitogen stimulator, inducing c-JUN. preferentially activating the NHEJ pathway and facilitating its Fig. 3 Expression of UBE2M and UBE2F in cancers and their effects on the prognosis of patients with LUAD and LIHC. A, B The expression of UBE2M and UBE2F in different types of cancer was investigated with the TIMER database. The variance was similar between the groups that were being compared. P < 0.05 was considered as statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 for the indicated comparison. C, D Kaplan-Meier analysis shows that increased levels of both UBE2M and UBE2F are related to worse survival for patients with lung adenocarcinoma (LUAD) or with liver hepatocellular carcinoma (LIHC). Cell Death Discovery (2023) 9:23 L. Zhou et al. Fig. 4 The function of UBE2M and UBE2F in tumor growth. UBE2M influences tumor growth by modulating several biological responses of tumor cells, such as DNA-damage response, cell cycle arrest, apoptosis, or senescence. UBE2F promotes the degradation of NOXA and then inhibits apoptosis and induces tumor growth. release from DNA-damage sites after Ku [69]. Consistently, the UBE2M INHIBITION INDUCES APOPTOSIS OR SENESCENCE knockdown of UBE2M significantly enhances the sensitivity of UBE2M plays an essential role in regulating apoptosis and hormone-resistant prostate cancer cells to radiation-induced DNA senescence (Fig. 4). In intrahepatic cholangiocarcinoma cells, damage [70]. UBE2M knockdown induces apoptosis, demonstrated by shrinkage Furthermore, in response to various stimuli, NEDD8 may of cellular morphology and the upregulation of cleaved PARP and accumulate at DNA-damage sites by relying on UBE2M rather caspase-3/-9 [65]. In hepatocellular cells, UBE2M knockdown than UBE2F. UBE2M, together with an E3 ubiquitin ligase RNF111, promotes apoptosis by inducing the accumulation of cleaved promotes ionizing radiation-induced histone H4 neddylation and PARP and caspase-3 and increasing the mRNA levels of apoptosis- links another E3 ligase RNF168 to DNA-damage sites. This associated proteins, including p53, PUMA, and Bax [76]. Interest- occurrence leads to the recruitment of BRCA1 and other down- ingly, UBE2M knockdown leads to p53-mediated apoptosis by stream DDR factors to repair the damaged DNA [71]. Notably, activating and stabilizing the protein [76]. In esophageal squamous another group has reported that UBE2M/RNF111-mediated cell carcinoma cells, UBE2M knockdown triggers apoptosis or neddylation inhibits BRCA1 and CtIP-regulated DNA end resection, senescence in a cell line-dependent manner. EC1 cells with UBE2M an essential mechanism aiding the selection of an appropriate knockdown exhibit typical senescent morphology, well- repair pathway [72]. characterized by an enlarged and flattened cellular shape and positive staining for senescence-related β-galactosidase [55]. In contrast, KYSE450 cells with UBE2M knockdown show prominent UBE2M INHIBITION INDUCES CELL CYCLE ARREST apoptotic features, shrunk morphology, and a substantial increase The aberrant functioning of cell cycle regulators leads to in cell numbers positive for annexin V [55]. Moreover, UBE2M unregulated cell proliferation, making them promising therapeutic knockdown increases the accumulation of CRL substrate activating targets for cancer treatment [73, 74]. UBE2M has been shown to transcription factor 4 (ATF4), activating death receptor 5 (DR5)- crucially regulate the tumor-suppressive cell cycle inhibitors (Fig. mediated extrinsic apoptosis and proapoptotic protein NOXA- 4). In lung cancer and esophageal squamous cell carcinoma cells, mediated intrinsic apoptosis [55, 77, 78]. Thus, induction of UBE2M knockdown disturbs cell cycle progression by triggering apoptosis or senescence establishes UBE2M as a promising anti- G2 phase cell cycle arrest, specifically by inhibiting cullins tumor target. neddylation and upregulating CRL substrates (p21, p27, and Wee1) [55, 56]. In hepatocellular carcinoma cells, UBE2M-mediated stabilization of β-catenin, leading to the upregulation of its UBE2M IS INVOLVED IN ANTI-TUMOR IMMUNITY downstream effectors, known as cyclin D1, promotes the G1/S Immune evasion is a hallmark of cancer. Anti-tumorigenic immune transition of cells [75]. These data suggest that suppression of cells are often exhausted or repressed by immune suppressive cell UBE2M can potentially trigger cell cycle arrest at distinct stages in populations, such as tumor-associated macrophages (TAMs), a cell type-dependent manner via multiple mechanisms. regulatory T (Treg), and myeloid-derived suppressor cells (MDSCs), Cell Death Discovery (2023) 9:23 L. Zhou et al. Fig. 5 The correlation of UBE2M and immune suppressive cell populations. A Graphs generated from TIMER database show the correlations between UBE2M and the abundance of macrophage, Treg, and MDSCs. B, C The expression of UBE2M was significantly correlated with multiple genes that are related to macrophage M2, MDSCs, or Treg in liver hepatocellular carcinoma. Spearman correlation analysis was used to assess the correlation. P < 0.05 was considered as statistical significance. leading to tumor immune evasion [79–81]. Hence, targeting these SPOP E3 ligase impairs ubiquitination-mediated PD-L1 degrada- immune suppressive cells is a promising anti-tumor immunother- tion, increasing PD-L1 protein levels and reducing the numbers of apy. It is known that UBE2M knockdown inhibits the expression of tumor-infiltrating lymphocytes in mouse tumors and primary proinflammatory cytokines triggered by exposure to lipopolysac- human prostate cancer [87]. A recent study also revealed that charides (LPS) (e.g., IL-6 and TNF-α) in macrophages. UBE2M inhibition of the neddylation pathway by MLN4924 activates ERK knockdown suppresses CUL1 neddylation, inactivates CRL1, and and JNK signals, leading to AP-1 activation. Activated AP-1 induces the accumulation of phosphorylated IκBα and the transactivates PD-L1 expression, inducing tumor immune evasion subsequent suppression of NF-κB nuclear translocation. This to fight the anti-tumor activity of MLN4924 [88]. These findings phenomenon transcriptionally inhibits macrophage-associated offer novel insights for further clinical experiments on tumor cytokines [82]. Recent reports suggest that the Ube2m/Rbx1 axis, patients using a combined method of UBE2M targeting and anti- rather than the Ube2f/Rbx2 one, is crucial to the homeostasis and PD-L1/PD-1 therapy. Altogether, the identification of the role of survival of Treg cells [83]. We then investigated the association UBE2M in modulating anti-tumor immunity deserves further between UBE2M expression and the abundance of immune cell research. populations in tumors using the TIMER database to assess further the effects of UBE2M on these immune suppressive cells (Fig. 5A). This analysis revealed that in hepatocellular carcinoma, the TARGETING UBE2M-DCN1 INTERACTION FOR REGULATION OF expression of UBE2M was significantly positively correlated with NEDDYLATION PATHWAY the abundance of Treg, MDSCs, and macrophage M2 (Fig. 5A). Efforts have been directed to develop more specific small- Moreover, the expression of UBE2M was also significantly molecule inhibitors that preferentially target neddylation E2s to correlated with multiple genes related to macrophage M2, MDSCs, address the shortcomings of MLN4924. The co-crystal structure or Treg (Fig. 5B, C). These results collectively suggest that UBE2M assessment of DCN1, which has no RING finger domain and acts as may drive immunosuppression in hepatocellular carcinoma. a co-NEDD8-E3 with RBX1, and its binding partners UBE2M, However, future investigation of detailed mechanisms is harbors the potential to develop strong small-molecule inhibitors warranted. [47, 51, 89–91]. Over the past few years, several different research Targeting immune checkpoints mediated by programmed cell facilities have developed small-molecule inhibitors for suppressing death 1 (PD-1) and its ligand PD-L1 is a practical approach to interactions between UBE2M and DCN1. Zhou et al. created enhance anti-tumor immunity. This line of therapy has been potent peptidomimetics, such as DI-591 and DI-404, by signifi- approved for treating various human cancers with durable clinical cantly modifying the N-terminal 12-residue peptide of UBE2M to benefits [84, 85]. Notably, inhibition of UBE2M-mediated neddyla- inhibit UBE2M-DCN1 interaction [92, 93]. Subsequently, Guy et al. tion significantly upregulates the expression of PD-L1 by identified non-peptidic and potent small-molecule UBE2M-DCN1 inactivating CUL1 and CUL3 in glioblastoma cancer cells. This inhibitors, such as NAcM-HIT, by high-throughput screening of phenomenon is mainly achieved by the transcriptional activation over 600,000 compounds. The chemical optimization of NAcM-HIT of PD-L1 by dysregulating the CUL1-FBXW7/c-MYC axis and led to designing two more potent inhibitors, NAcM-OPT and stabilizing PD-L1 protein by inhibiting CUL3 E3 ligase activity, NAcM-COV [94–96]. Liu et al. discovered the triazolo[1,5-α] leading to T-cell exhaustion [86]. Moreover, inhibition of CUL3- pyrimidine-based inhibitor WS-383, which targets the UBE2M- Cell Death Discovery (2023) 9:23 L. Zhou et al. DCN1 interaction [97]. In addition, Zhao et al. discovered small- neddylation factors, such as UBE2M, are not affected following molecular DCN1 inhibitors DC-1 and DC-2 based on pyrimidines platinum treatment [100]. [98]. Besides chemosensitization, targeting UBE2F also displays a Such studies have successfully discovered potent small- sensitizing effect on radiotherapy [102]. Elevated UBE2F levels can molecule inhibitors that are either covalent or non-covalent with be attributed to oxidative stress induced by irradiation or other powerful affinities to DCN1. These UBE2M-DCN1 inhibitors have stimuli, causing the degradation of ROS-induced NOXA, conse- been shown to efficiently block the neddylation of CUL1 and/or quently inducing apoptotic resistance to radiotherapy. Moreover, CUL3 while exerting no or minimal impact on the other members silencing UBE2F suppresses NOXA degradation and increases of the cullin family. These UBE2M-DCN1 inhibitors, as predicted, cancer cells’ susceptibility to irradiation-mediated apoptosis [102]. cause an accumulation of the CUL1 or CUL3 substrates, such as Taken together, UBE2F-mediated activation of CRL5 and subse- p21, p27, and NRF2 [92, 95–98]. However, compared to MLN4924, quent ubiquitylation and degradation of NOXA potentially hold the UBE2M-DCN1 inhibitors have moderate cellular potency, great promise as both an anti-tumor target and a chemo-/ suggesting the need for further potency improvement. radiosensitizing target. TARGETING UBE2F FOR ANTI-TUMOR THERAPY CONCLUSIONS In contrast with many studies done on UBE2M, UBE2F receives little Targeting the overactivated neddylation pathway has been attention. Recently, UBE2F has been shown to inhibit apoptosis demonstrated as a promising anti-tumor strategy, supported by and induce cell growth. UBE2F can be efficiently targeted as a the development of MLN4924, a potent inhibitor of the chemo-/radiosensitizing strategy by triggering apoptosis. neddylation E1 subunit UBA3. However, the emergence of drug- resistant mutations in UBA3 warrants the identification of alternative targets against the neddylation pathway. Recent and UBE2F INHIBITION INDUCES APOPTOSIS ongoing research has revealed that UBE2M and UBE2F perform an Zhou et al. reported that by coupling to RBX2, UBE2F neddylates integral function in the biology of tumors. Overexpression of both CUL5, which activates CRL5, eventually leading to the ubiquityla- UBE2M and UBE2F in cancer cells is associated with increased cell tion and degradation of NOXA through the K11-linkage [57]. In proliferation and poor survival. UBE2M regulates tumor growth by lung cancer cells, overexpression of UBE2F activates CRL5 and modulating several cellular responses, such as DDR, senescence, or promotes NOXA degradation, leading to inhibition of apoptosis apoptosis. The functional role of UBE2F, on the other hand, and improvement of cell survival [57]. HA-9104 is recently remains poorly characterized. Recent studies have shown that discovered as a novel small-molecule inhibitor targeting UBE2F- UBE2F inhibits apoptosis, induces cell growth, and serves as an CRL5 axis. HA-9104 interacts with UBE2F to reduce its protein effective chemo-/radiosensitizing target. However, the effects of levels (via a yet-to-defined mechanism), thereby inhibiting CUL5 both E2s on anti-tumor immunity undoubtedly demand more neddylation. Blockage of CUL5 neddylation results in CRL5 experimental investigations. In-depth elucidation of the mechan- inhibition and NOXA accumulation to trigger apoptosis [48]. Since isms of these E2s may provide more in-depth knowledge for UBE2F inhibition promotes apoptosis and suppresses cancer cell targeting UBE2M and UBE2F as attractive anti-tumor therapy. growth (Fig. 4), it is a promising target for anti-tumor therapy. Many inhibitors targeting UBE2M-DCN1 interaction have been Moreover, peroxiredoxin PRDX1 binds to UBE2F and CUL5 to form discovered to overcome the limitations of MLN4924 and tackle the a triple-molecule complex, PRDX1-UBE2F-CUL5, essential for CUL5 crucial role of UBE2M on the neddylation pathway and tumor neddylation. Silencing PRDX1 or blocking PRDX1 oligomerization growth. However, compared to MLN4924, the tumor cell-killing significantly inhibits CUL5 neddylation, suppressing NOXA ubiquiti- potency of these UBE2M-DCN1 inhibitors is moderate, suggesting nation and degradation. Etoposide, an anti-cancer chemotherapeu- the need for further potency improvement. Promoting anti-tumor tic DNA damaging agent, increases NOXA transcription, leading to clinical trials of these E2 inhibitors alone or in combination may apoptosis. Colorectal cancer cells increase CUL5 neddylation to direct further research. Recent studies reported that, in addition to accelerate NOXA degradation, which prevents etoposide-induced effective tumor treatment, MLN4924 also plays a potential role in apoptosis. At the same time, the knockdown of PRDX1 eliminates the treatment of obesity [103], insulin resistance [103, 104], etoposide-induced CUL5 neddylation and increases the sensitivity of nonalcoholic fatty liver [105], and ischemia-reperfusion injury colorectal cancer cells to etoposide therapy [99]. [106–108]. Hence, it would be interesting to identify the function of these UBE2M-DCN1 inhibitors in these non-tumor diseases. In summary, the current research offers profound insights into UBE2F SERVES AS A TARGET FOR CHEMO-/ the role of UBE2M and UBE2F in tumor progression, which is RADIOSENSITIZATION highly conducive to aiding the development of targeted inhibitors UBE2F, an apoptotic regulatory protein, is a probable viable target with higher potency and selectivity. for chemosensitization [100, 101]. In lung cancer cells, depletion of UBE2F renders the cells more sensitive to multiple anti-tumor agents (e.g., an inhibitor of anti-apoptotic protein MCL1) by DATA AVAILABILITY The corresponding author will provide the data and materials upon reasonable accumulating NOXA [101]. UBE2F upregulation allows lung cancer request. cells to evade apoptosis caused by platinum exposure. Mechan- istically, platinum prevents the generation of the complex required for proteasome-mediated UBE2F degradation, which REFERENCES ultimately results in UBE2F accumulation. This phenomenon 1. Kamitani T, Kito K, Nguyen HP, Yeh ET. 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LY21H160008), National Key Research and Reprints and permission information is available at http://www.nature.com/ Development Program of China (2020YFA0804200), Shanghai Municipal Science and reprints Technology Major Project (2018SHZDZX01), the Program for Professors of Special Appointment (Eastern Scholar) at the Shanghai Institutions of Higher Learning Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims (SSF151005), and the Open Fund for Scientific Research of Jiangxi Cancer Hospital in published maps and institutional affiliations. (2021J03). Open Access This article is licensed under a Creative Commons AUTHOR CONTRIBUTIONS Attribution 4.0 International License, which permits use, sharing, LSZ, XZL, and JZ collected the related paper and drafted the manuscript. LYZ and SYC adaptation, distribution and reproduction in any medium or format, as long as you give searched the databases and analyzed the data. 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NEDD8-conjugating enzyme E2s: critical targets for cancer therapy

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www.nature.com/cddiscovery REVIEW ARTICLE OPEN NEDD8-conjugating enzyme E2s: critical targets for cancer therapy ✉ ✉ ✉ ✉ 1,6 2,6 3,6 1 1 4 5 1 Lisha Zhou , Xiongzhi Lin , Jin Zhu , Luyi Zhang , Siyuan Chen , Hui Yang , Lijun Jia and Baofu Chen © The Author(s) 2023 NEDD8-conjugating enzymes, E2s, include the well-studied ubiquitin-conjugating enzyme E2 M (UBE2M) and the poorly characterized ubiquitin-conjugating enzyme E2 F (UBE2F). UBE2M and UBE2F have distinct and prominent roles in catalyzing the neddylation of Cullin or non-Cullin substrates. These enzymes are overexpressed in various malignancies, conferring a worse overall survival. Targeting UBE2M to influence tumor growth by either modulating several biological responses of tumor cells (such as DNA-damage response, apoptosis, or senescence) or regulating the anti-tumor immunity holds strong therapeutic potential. Multiple inhibitors that target the interaction between UBE2M and defective cullin neddylation protein 1 (DCN1), a co-E3 for neddylation, exhibit promising anti-tumor effects. By contrast, the potential benefits of targeting UBE2F are still to be explored. It is currently reported to inhibit apoptosis and then induce cell growth; hence, targeting UBE2F serves as an effective chemo-/ radiosensitizing strategy by triggering apoptosis. This review highlights the most recent advances in the roles of UBE2M and UBE2F in tumor progression, indicating these E2s as two promising anti-tumor targets. Cell Death Discovery (2023) 9:23 ; https://doi.org/10.1038/s41420-023-01337-w FACTS Whether these UBE2M-DCN1 inhibitors have any biological function? More research is being devoted to finding specific inhibitors Whether to promote clinical trials of these E2s inhibitors alone against neddylation E2s to address the limitations of or in combination? neddylation E1 inhibitor MLN4924. UBE2M and UBE2F are overexpressed in various malignancies, conferring a worse overall survival. Targeting UBE2M to influence tumor growth by either INTRODUCTION modulating several biological responses of tumor cells (such Protein neddylation involves the conjugation of a ubiquitin-like as DNA-damage response, apoptosis, or senescence) or regulat- molecule known as neuronal precursor cell-expressed develop- ing the anti-tumor immunity holds strong therapeutic potential. mentally down-regulated protein 8 (NEDD8) to the lysine residue A few inhibitors targeting UBE2M-DCN1 interaction have been of targeted substrate proteins [1, 2]. Like ubiquitin, NEDD8 binds identified. to substrates by forming an isopeptide chain between its UBE2F appears to be promising as both an anti-tumor target C-terminal glycine residue (Gly76) and a lysine residue on targeted and a chemo-/radiosensitizing target. proteins. In the initial step of the reaction, NEDD8 is produced as a precursor containing five additional residues downstream from Gly76 cleaved by the C-terminal hydrolases [3, 4]. The next step involves activation of the mature NEDD8 in an ATP-dependent OPEN QUESTIONS manner by the NEDD8-activating enzyme (NAE) E1, a heterodimer ● composed of NAE1 and ubiquitin-like modifier activating enzyme The induction of drug-resistant mutations in UBA3 inhibits the 3 (UBA3) [5, 6]. After that, a trans-thiolation process occurs during formation of the MLN4924-NEDD8 adduct, and hence there is which the NEDD8-loaded NAE is transferred to either UBE2M, also a need to explore possible alternative targets against the known as UBC12, or UBE2F (two NEDD8-conjugating enzyme E2s) neddylation pathway. ● [7–9]. Finally, covalent attachment drives a substrate-specificE3 How do the two E2s regulate anti-tumor immunity? In-depth ligase, such as defective in cullin neddylation 1 (DCN1) and RING- elucidation of the underlying mechanisms may provide vital box protein 1/2 (RBX1/2), to transfer NEDD8 from the charged E2 cues for targeting UBE2M and UBE2F. ● to a lysine residue in its target protein (Fig. 1)[10–14]. The best Deciphering the cellular potency of these UBE2M-DCN1 characterized principal substrates of neddylation are the cullin inhibitors compared to MLN4924? 1 2 Taizhou Central Hospital (Taizhou University Hospital), Taizhou University, Taizhou, Zhejiang, China. Graduate School of Medicine, Hebei North University, Zhangjiakou, Hebei, 3 4 China. Department of Surgical Oncology, Jiangxi Cancer Hospital, Nanchang, Jiangxi, China. Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China. 5 6 Cancer Institute of Traditional Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China. These authors contributed equally: Lisha Zhou, Xiongzhi Lin and Jin Zhu. email: lishazhou@tzc.edu.cn; hui_yang@fudan.edu.cn; ljjia@shutcm.edu.cn; chenbf@tzc.edu.cn Received: 8 October 2022 Revised: 11 January 2023 Accepted: 13 January 2023 Official journal of CDDpress 1234567890();,: L. Zhou et al. BIOLOGICAL CHARACTERISTICS AND CORRELATION OF UBE2M AND UBE2F UBE2M and UBE2F bind to the ubiquitin-fold domain and the UBA3 hydrophobic groove of E1 via the core domain and the N-terminal motif, respectively [9]. Acetylation of the N-terminal methionine occurs in both E2s, which facilitates their binding to the PONY domain pocket of neddylation E3 DCN-like (DCNL), thereby increasing the efficiency of the cullin neddylation process [49–51]. In addition to these similarities, protein structure assessment of both UBE2M and UBE2F reveals unique character- istics. UBE2F is very specific to the neddylation of RBX2-related CUL5, while UBE2M can pair with RBX1 to modulate the neddylation of CUL1, 2, 3, 4A, and 4B [50]. It is noteworthy that glycyl-tRNA synthetase, an enzyme necessary for protein produc- tion, binds to the NAE1 subunit of E1 to capture and protect activated UBE2M before it reaches the downstream target [52]. The enzymes UBE2M and UBE2F can activate distinct cullins by enhancing neddylation modification, and interestingly, there is a cross-talk that leads to UBE2M targeting UBE2F for degradation [53]. Specifically, UBE2M serves as a stress-inducible protein and a dual E2 for neddylation and ubiquitylation to degrade UBE2F. Under physiological conditions, UBE2M acts as a neddylation E2 to promote CUL3 neddylation, which triggers polyubiquitylation and Fig. 1 The process of protein neddylation. Neddylation is a degradation of UBE2F through CUL3-KEAP1 E3 ligase. However, process that conjugates NEDD8 to cullins or non-cullin substrates UBE2M is transcriptionally activated under stress by hypoxia- via a three-step reaction, catalyzed by NEDD8-activating enzyme E1 inducible factor 1α (HIF-1α) or transcription factor AP-1 (AP-1). (a heterodimer of NAE1 and UBA3), NEDD8-conjugating enzyme E2 (UBE2M or UBE2F) and substrate-specific NEDD8-E3 ligases (e.g., UBE2M performs ubiquitylation E2 for Parkin-DJ-1 E3, followed by RBX1 and RBX2). MLN4924: an inhibitor of UBA3; N8: neuronal ubiquitylation and degradation of UBE2F. Ultimately, the degrada- precursor cell-expressed developmentally downregulated protein 8. tion of UBE2F is accompanied by the inactivation of CRL5. Collectively, these findings provide evidence that one neddylation family members (CUL1, 2, 3, 4A, 4B, and 5), which function as core E2 (UBE2M) acts as a dual E2 for both neddylation and components of Cullin-RING E3 ubiquitin ligases (CRLs) [4]. The ubiquitylation to decrease the protein levels of the other (UBE2F), activation of CRLs requires NEDD8 to attach to a C-terminal lysine leading to one CRL E3 (CRL3) inactivating the other (CRL5) (Fig. 2). residue of cullins, inducing structural changes in the CRLs complex and leading to open conformation to facilitate substrate ubiquitylation [15–19]. As the most prominent family of E3 UBE2M AND UBE2F AS ATTRACTIVE ANTI-TUMOR TARGETS ubiquitin ligases, CRLs noticeably regulate various essential Most studies reveal that both two neddylation E2s act as biological functions, including tumorigenesis, accomplished by oncogenes, evidenced mainly by their significant upregulation in enhancing ubiquitylation and consequent degradation of a wide various human cancers, including esophageal squamous cell range of critical modulatory proteins [4, 20]. Besides cullins, several carcinoma, osteosarcoma, lung cancer, and hepatocellular carci- non-cullin proteins have been identified as the substrates of noma [54–59]. In addition, the upregulation of the two neddyla- neddylation, such as p53, MDM2, and EGFR [21–24]. However, tion E2s correlates closely with illness progression [55–58]. To regulatory mechanisms and physiological functions of non-cullin attain a greater understanding of the expression profile and substrates still await experimental validation. prognostic significance of UBE2M and UBE2F in human cancers, In multiple cancers, overactivation of the neddylation pathway we examined their expression with the aid of Tumor Immune leads to elevated global neddylation of substrates, such as cullins, Estimation Resource (TIMER) online database, an interactive and consequent accumulation of tumor suppressors, thereby platform that allows users to conduct in-depth studies of TCGA promoting tumorigenesis and development [25–28]. Targeting the gene expression profiles (http://timer.cistrome.org/). Compared to overactivated protein neddylation pathway has been proven to be normal tissues, the mRNA levels of UBE2M are elevated in 17 an effective anti-tumor strategy. MLN4924, commonly referred to different kinds of human cancers, while the mRNA levels of UBE2F as pevonedistat, is an effective and highly selective small- are elevated in 12 different types of human cancers (Fig. 3A, B). molecule inhibitor of NAE adopted to inhibit protein neddylation Kaplan–Meier analysis shows that increased mRNA levels of both via inactivation of the initial stage of the neddylation cascade [29]. enzymes correlated to worse survival for patients with lung When bound to the active site of UBA3, MLN4924 forms a stable adenocarcinoma (LUAD) or with liver hepatocellular carcinoma covalent adduct with NEDD8 to block further enzymatic processes (LIHC) (Fig. 3C, D). They exert oncogenic effects by enhancing the [30]. It achieves potent anti-tumor effects by inducing cell cycle neddylation of certain substrates to mediate a variety of signaling arrest, apoptosis, or senescence of tumor cells, or affecting the pathways and modulate multiple biological activities, such as functions of multiple components of the tumor microenvironment apoptosis or senescence (Fig. 4). These findings suggest that the [28, 29, 31–37]. MLN4924 has been evaluated in Phase I/II clinical overactivation of these two enzymes could be an oncogenic event trials for treating various solid tumors and hematologic cancers throughout the process of tumor occurrence and development. [38–44]. Nevertheless, the induction of drug-resistant mutations in UBA3 inhibits the formation of the MLN4924-NEDD8 adduct, necessitating the need to explore alternative targets against the TARGETING UBE2M FOR ANTI-TUMOR THERAPY neddylation pathway [45, 46]. To address the limitations of In recent decades, increasing studies have shown UBE2M, also MLN4924, specific inhibitors against neddylation E2s are being known as UBC12, to be an attractive anti-tumor target. Compared investigated [47, 48]. This review summarizes the latest progress with E1 subunits (NAE1 and UBA3), UBE2M is significantly more on validating the neddylation of these enzymes as promising anti- consistent with the global protein neddylation levels [56]. tumor targets. Targeting UBE2M suppresses the growth of MLN4924-resistant Cell Death Discovery (2023) 9:23 L. Zhou et al. cells by inhibiting cullins neddylation and inducing the accumula- tion of CRLs substrates [56]. Moreover, UBE2M-mediated protein neddylation is essential for multiple cellular responses, such as DNA-damage response (DDR), apoptosis, senescence, and anti- tumor immunity. These findings validate UBE2M as an attractive alternative anti-tumor target to efficiently inhibit the neddylation pathway. UBE2M PARTICIPATES IN DDR DNA double-strand break (DSB), contributing significantly to genomic stability [60, 61], is sensed and repaired by DDR, warranting the recruitment and post-translational modification of multiple proteins at the damaged DNA sites. This phenomenon induces checkpoint signaling or essential repair steps [62, 63]. Recent studies have shown that UBE2M-mediated neddylation of cullins, or non-cullins, is involved in the DDR pathway (Fig. 4). UBE2M affects DDR and the integrity of the genome by regulating several CRLs substrates, such as CDT1, p21, and claspin, which play complex roles in the increased DNA-damage in UBE2M-silenced cells [64]. Another study supported the accumulation effects of CRLs substrates CDT1 and ORC1 caused by silenced UBE2M. This phenomenon subsequently induces DSBs, evidenced by the upregulated expression of γ-H2AX [65]. Generally, Ku70/Ku80 (Ku) heterodimer is first recruited to the DSB sites, after which, DNA-dependent protein kinase catalytic Fig. 2 UBE2M acts as a dual E2 for neddylation and ubiquitylation subunit (DNA-PKcs) is recruited into the process, facilitating to degrade UBE2F. Under physiological conditions, UBE2M serves nonhomologous end-joining (NHEJ) repair [66, 67]. Upon DNA- as a neddylation E2 to promote CUL3 neddylation, triggering damage, UBE2M-mediated cullins neddylation promotes ubiqui- polyubiquitylation and degradation of UBE2F through CUL3-KEAP1 tylation of Ku, releasing it and its associated proteins from the E3 ligase. Under stressed conditions, UBE2M is transcriptionally activated by HIF-1α or AP-1, which makes it serve as a ubiquitylation damaged sites after repair. After NHEJ, ionizing radiation E2 to complex with DJ-1/Parkin to promote the ubiquitylation and hypersensitivity and decreased cell survival occur in UBE2M- degradation of UBE2F. Ultimately, the degradation of UBE2F is depleted cells [68]. Moreover, UBE2M also regulates the neddyla- accompanied by the inactivation of CRL5. TPA: a typical tumor tion of DNA-PKcs, which promotes DNA-PKc autophosphorylation, promoter and mitogen stimulator, inducing c-JUN. preferentially activating the NHEJ pathway and facilitating its Fig. 3 Expression of UBE2M and UBE2F in cancers and their effects on the prognosis of patients with LUAD and LIHC. A, B The expression of UBE2M and UBE2F in different types of cancer was investigated with the TIMER database. The variance was similar between the groups that were being compared. P < 0.05 was considered as statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 for the indicated comparison. C, D Kaplan-Meier analysis shows that increased levels of both UBE2M and UBE2F are related to worse survival for patients with lung adenocarcinoma (LUAD) or with liver hepatocellular carcinoma (LIHC). Cell Death Discovery (2023) 9:23 L. Zhou et al. Fig. 4 The function of UBE2M and UBE2F in tumor growth. UBE2M influences tumor growth by modulating several biological responses of tumor cells, such as DNA-damage response, cell cycle arrest, apoptosis, or senescence. UBE2F promotes the degradation of NOXA and then inhibits apoptosis and induces tumor growth. release from DNA-damage sites after Ku [69]. Consistently, the UBE2M INHIBITION INDUCES APOPTOSIS OR SENESCENCE knockdown of UBE2M significantly enhances the sensitivity of UBE2M plays an essential role in regulating apoptosis and hormone-resistant prostate cancer cells to radiation-induced DNA senescence (Fig. 4). In intrahepatic cholangiocarcinoma cells, damage [70]. UBE2M knockdown induces apoptosis, demonstrated by shrinkage Furthermore, in response to various stimuli, NEDD8 may of cellular morphology and the upregulation of cleaved PARP and accumulate at DNA-damage sites by relying on UBE2M rather caspase-3/-9 [65]. In hepatocellular cells, UBE2M knockdown than UBE2F. UBE2M, together with an E3 ubiquitin ligase RNF111, promotes apoptosis by inducing the accumulation of cleaved promotes ionizing radiation-induced histone H4 neddylation and PARP and caspase-3 and increasing the mRNA levels of apoptosis- links another E3 ligase RNF168 to DNA-damage sites. This associated proteins, including p53, PUMA, and Bax [76]. Interest- occurrence leads to the recruitment of BRCA1 and other down- ingly, UBE2M knockdown leads to p53-mediated apoptosis by stream DDR factors to repair the damaged DNA [71]. Notably, activating and stabilizing the protein [76]. In esophageal squamous another group has reported that UBE2M/RNF111-mediated cell carcinoma cells, UBE2M knockdown triggers apoptosis or neddylation inhibits BRCA1 and CtIP-regulated DNA end resection, senescence in a cell line-dependent manner. EC1 cells with UBE2M an essential mechanism aiding the selection of an appropriate knockdown exhibit typical senescent morphology, well- repair pathway [72]. characterized by an enlarged and flattened cellular shape and positive staining for senescence-related β-galactosidase [55]. In contrast, KYSE450 cells with UBE2M knockdown show prominent UBE2M INHIBITION INDUCES CELL CYCLE ARREST apoptotic features, shrunk morphology, and a substantial increase The aberrant functioning of cell cycle regulators leads to in cell numbers positive for annexin V [55]. Moreover, UBE2M unregulated cell proliferation, making them promising therapeutic knockdown increases the accumulation of CRL substrate activating targets for cancer treatment [73, 74]. UBE2M has been shown to transcription factor 4 (ATF4), activating death receptor 5 (DR5)- crucially regulate the tumor-suppressive cell cycle inhibitors (Fig. mediated extrinsic apoptosis and proapoptotic protein NOXA- 4). In lung cancer and esophageal squamous cell carcinoma cells, mediated intrinsic apoptosis [55, 77, 78]. Thus, induction of UBE2M knockdown disturbs cell cycle progression by triggering apoptosis or senescence establishes UBE2M as a promising anti- G2 phase cell cycle arrest, specifically by inhibiting cullins tumor target. neddylation and upregulating CRL substrates (p21, p27, and Wee1) [55, 56]. In hepatocellular carcinoma cells, UBE2M-mediated stabilization of β-catenin, leading to the upregulation of its UBE2M IS INVOLVED IN ANTI-TUMOR IMMUNITY downstream effectors, known as cyclin D1, promotes the G1/S Immune evasion is a hallmark of cancer. Anti-tumorigenic immune transition of cells [75]. These data suggest that suppression of cells are often exhausted or repressed by immune suppressive cell UBE2M can potentially trigger cell cycle arrest at distinct stages in populations, such as tumor-associated macrophages (TAMs), a cell type-dependent manner via multiple mechanisms. regulatory T (Treg), and myeloid-derived suppressor cells (MDSCs), Cell Death Discovery (2023) 9:23 L. Zhou et al. Fig. 5 The correlation of UBE2M and immune suppressive cell populations. A Graphs generated from TIMER database show the correlations between UBE2M and the abundance of macrophage, Treg, and MDSCs. B, C The expression of UBE2M was significantly correlated with multiple genes that are related to macrophage M2, MDSCs, or Treg in liver hepatocellular carcinoma. Spearman correlation analysis was used to assess the correlation. P < 0.05 was considered as statistical significance. leading to tumor immune evasion [79–81]. Hence, targeting these SPOP E3 ligase impairs ubiquitination-mediated PD-L1 degrada- immune suppressive cells is a promising anti-tumor immunother- tion, increasing PD-L1 protein levels and reducing the numbers of apy. It is known that UBE2M knockdown inhibits the expression of tumor-infiltrating lymphocytes in mouse tumors and primary proinflammatory cytokines triggered by exposure to lipopolysac- human prostate cancer [87]. A recent study also revealed that charides (LPS) (e.g., IL-6 and TNF-α) in macrophages. UBE2M inhibition of the neddylation pathway by MLN4924 activates ERK knockdown suppresses CUL1 neddylation, inactivates CRL1, and and JNK signals, leading to AP-1 activation. Activated AP-1 induces the accumulation of phosphorylated IκBα and the transactivates PD-L1 expression, inducing tumor immune evasion subsequent suppression of NF-κB nuclear translocation. This to fight the anti-tumor activity of MLN4924 [88]. These findings phenomenon transcriptionally inhibits macrophage-associated offer novel insights for further clinical experiments on tumor cytokines [82]. Recent reports suggest that the Ube2m/Rbx1 axis, patients using a combined method of UBE2M targeting and anti- rather than the Ube2f/Rbx2 one, is crucial to the homeostasis and PD-L1/PD-1 therapy. Altogether, the identification of the role of survival of Treg cells [83]. We then investigated the association UBE2M in modulating anti-tumor immunity deserves further between UBE2M expression and the abundance of immune cell research. populations in tumors using the TIMER database to assess further the effects of UBE2M on these immune suppressive cells (Fig. 5A). This analysis revealed that in hepatocellular carcinoma, the TARGETING UBE2M-DCN1 INTERACTION FOR REGULATION OF expression of UBE2M was significantly positively correlated with NEDDYLATION PATHWAY the abundance of Treg, MDSCs, and macrophage M2 (Fig. 5A). Efforts have been directed to develop more specific small- Moreover, the expression of UBE2M was also significantly molecule inhibitors that preferentially target neddylation E2s to correlated with multiple genes related to macrophage M2, MDSCs, address the shortcomings of MLN4924. The co-crystal structure or Treg (Fig. 5B, C). These results collectively suggest that UBE2M assessment of DCN1, which has no RING finger domain and acts as may drive immunosuppression in hepatocellular carcinoma. a co-NEDD8-E3 with RBX1, and its binding partners UBE2M, However, future investigation of detailed mechanisms is harbors the potential to develop strong small-molecule inhibitors warranted. [47, 51, 89–91]. Over the past few years, several different research Targeting immune checkpoints mediated by programmed cell facilities have developed small-molecule inhibitors for suppressing death 1 (PD-1) and its ligand PD-L1 is a practical approach to interactions between UBE2M and DCN1. Zhou et al. created enhance anti-tumor immunity. This line of therapy has been potent peptidomimetics, such as DI-591 and DI-404, by signifi- approved for treating various human cancers with durable clinical cantly modifying the N-terminal 12-residue peptide of UBE2M to benefits [84, 85]. Notably, inhibition of UBE2M-mediated neddyla- inhibit UBE2M-DCN1 interaction [92, 93]. Subsequently, Guy et al. tion significantly upregulates the expression of PD-L1 by identified non-peptidic and potent small-molecule UBE2M-DCN1 inactivating CUL1 and CUL3 in glioblastoma cancer cells. This inhibitors, such as NAcM-HIT, by high-throughput screening of phenomenon is mainly achieved by the transcriptional activation over 600,000 compounds. The chemical optimization of NAcM-HIT of PD-L1 by dysregulating the CUL1-FBXW7/c-MYC axis and led to designing two more potent inhibitors, NAcM-OPT and stabilizing PD-L1 protein by inhibiting CUL3 E3 ligase activity, NAcM-COV [94–96]. Liu et al. discovered the triazolo[1,5-α] leading to T-cell exhaustion [86]. Moreover, inhibition of CUL3- pyrimidine-based inhibitor WS-383, which targets the UBE2M- Cell Death Discovery (2023) 9:23 L. Zhou et al. DCN1 interaction [97]. In addition, Zhao et al. discovered small- neddylation factors, such as UBE2M, are not affected following molecular DCN1 inhibitors DC-1 and DC-2 based on pyrimidines platinum treatment [100]. [98]. Besides chemosensitization, targeting UBE2F also displays a Such studies have successfully discovered potent small- sensitizing effect on radiotherapy [102]. Elevated UBE2F levels can molecule inhibitors that are either covalent or non-covalent with be attributed to oxidative stress induced by irradiation or other powerful affinities to DCN1. These UBE2M-DCN1 inhibitors have stimuli, causing the degradation of ROS-induced NOXA, conse- been shown to efficiently block the neddylation of CUL1 and/or quently inducing apoptotic resistance to radiotherapy. Moreover, CUL3 while exerting no or minimal impact on the other members silencing UBE2F suppresses NOXA degradation and increases of the cullin family. These UBE2M-DCN1 inhibitors, as predicted, cancer cells’ susceptibility to irradiation-mediated apoptosis [102]. cause an accumulation of the CUL1 or CUL3 substrates, such as Taken together, UBE2F-mediated activation of CRL5 and subse- p21, p27, and NRF2 [92, 95–98]. However, compared to MLN4924, quent ubiquitylation and degradation of NOXA potentially hold the UBE2M-DCN1 inhibitors have moderate cellular potency, great promise as both an anti-tumor target and a chemo-/ suggesting the need for further potency improvement. radiosensitizing target. TARGETING UBE2F FOR ANTI-TUMOR THERAPY CONCLUSIONS In contrast with many studies done on UBE2M, UBE2F receives little Targeting the overactivated neddylation pathway has been attention. Recently, UBE2F has been shown to inhibit apoptosis demonstrated as a promising anti-tumor strategy, supported by and induce cell growth. UBE2F can be efficiently targeted as a the development of MLN4924, a potent inhibitor of the chemo-/radiosensitizing strategy by triggering apoptosis. neddylation E1 subunit UBA3. However, the emergence of drug- resistant mutations in UBA3 warrants the identification of alternative targets against the neddylation pathway. Recent and UBE2F INHIBITION INDUCES APOPTOSIS ongoing research has revealed that UBE2M and UBE2F perform an Zhou et al. reported that by coupling to RBX2, UBE2F neddylates integral function in the biology of tumors. Overexpression of both CUL5, which activates CRL5, eventually leading to the ubiquityla- UBE2M and UBE2F in cancer cells is associated with increased cell tion and degradation of NOXA through the K11-linkage [57]. In proliferation and poor survival. UBE2M regulates tumor growth by lung cancer cells, overexpression of UBE2F activates CRL5 and modulating several cellular responses, such as DDR, senescence, or promotes NOXA degradation, leading to inhibition of apoptosis apoptosis. The functional role of UBE2F, on the other hand, and improvement of cell survival [57]. HA-9104 is recently remains poorly characterized. Recent studies have shown that discovered as a novel small-molecule inhibitor targeting UBE2F- UBE2F inhibits apoptosis, induces cell growth, and serves as an CRL5 axis. HA-9104 interacts with UBE2F to reduce its protein effective chemo-/radiosensitizing target. However, the effects of levels (via a yet-to-defined mechanism), thereby inhibiting CUL5 both E2s on anti-tumor immunity undoubtedly demand more neddylation. Blockage of CUL5 neddylation results in CRL5 experimental investigations. In-depth elucidation of the mechan- inhibition and NOXA accumulation to trigger apoptosis [48]. Since isms of these E2s may provide more in-depth knowledge for UBE2F inhibition promotes apoptosis and suppresses cancer cell targeting UBE2M and UBE2F as attractive anti-tumor therapy. growth (Fig. 4), it is a promising target for anti-tumor therapy. Many inhibitors targeting UBE2M-DCN1 interaction have been Moreover, peroxiredoxin PRDX1 binds to UBE2F and CUL5 to form discovered to overcome the limitations of MLN4924 and tackle the a triple-molecule complex, PRDX1-UBE2F-CUL5, essential for CUL5 crucial role of UBE2M on the neddylation pathway and tumor neddylation. Silencing PRDX1 or blocking PRDX1 oligomerization growth. However, compared to MLN4924, the tumor cell-killing significantly inhibits CUL5 neddylation, suppressing NOXA ubiquiti- potency of these UBE2M-DCN1 inhibitors is moderate, suggesting nation and degradation. Etoposide, an anti-cancer chemotherapeu- the need for further potency improvement. Promoting anti-tumor tic DNA damaging agent, increases NOXA transcription, leading to clinical trials of these E2 inhibitors alone or in combination may apoptosis. Colorectal cancer cells increase CUL5 neddylation to direct further research. Recent studies reported that, in addition to accelerate NOXA degradation, which prevents etoposide-induced effective tumor treatment, MLN4924 also plays a potential role in apoptosis. At the same time, the knockdown of PRDX1 eliminates the treatment of obesity [103], insulin resistance [103, 104], etoposide-induced CUL5 neddylation and increases the sensitivity of nonalcoholic fatty liver [105], and ischemia-reperfusion injury colorectal cancer cells to etoposide therapy [99]. [106–108]. Hence, it would be interesting to identify the function of these UBE2M-DCN1 inhibitors in these non-tumor diseases. In summary, the current research offers profound insights into UBE2F SERVES AS A TARGET FOR CHEMO-/ the role of UBE2M and UBE2F in tumor progression, which is RADIOSENSITIZATION highly conducive to aiding the development of targeted inhibitors UBE2F, an apoptotic regulatory protein, is a probable viable target with higher potency and selectivity. for chemosensitization [100, 101]. In lung cancer cells, depletion of UBE2F renders the cells more sensitive to multiple anti-tumor agents (e.g., an inhibitor of anti-apoptotic protein MCL1) by DATA AVAILABILITY The corresponding author will provide the data and materials upon reasonable accumulating NOXA [101]. UBE2F upregulation allows lung cancer request. cells to evade apoptosis caused by platinum exposure. Mechan- istically, platinum prevents the generation of the complex required for proteasome-mediated UBE2F degradation, which REFERENCES ultimately results in UBE2F accumulation. This phenomenon 1. Kamitani T, Kito K, Nguyen HP, Yeh ET. 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Wu D, Li H, Liu M, Qin J, Sun Y. The Ube2m-Rbx1 neddylation-Cullin-RING-Ligase oxidative stress via sirt1 in spinal cord ischemia-reperfusion injury. Oxid Med proteins are essential for the maintenance of Regulatory T cell fitness. Nat Cell Longev. 2019;2019:7283639. Commun. 2022;13:3021. 84. Topalian SL, Taube JM, Pardoll DM. Neoadjuvant checkpoint blockade for cancer immunotherapy. Science. 2020; 367:eaax0182. ACKNOWLEDGEMENTS 85. Yi M, Zheng X, Niu M, Zhu S, Ge H, Wu K. Combination strategies with PD-1/PD- This work was supported by the National Natural Science Foundation of China (nos. L1 blockade: current advances and future directions. Mol Cancer. 2022;21:28. 81871870, 81820108022, 82073069, 82073166, and 82273203), the Zhejiang Province Cell Death Discovery (2023) 9:23 L. Zhou et al. Natural Science Foundation of China (no. LY21H160008), National Key Research and Reprints and permission information is available at http://www.nature.com/ Development Program of China (2020YFA0804200), Shanghai Municipal Science and reprints Technology Major Project (2018SHZDZX01), the Program for Professors of Special Appointment (Eastern Scholar) at the Shanghai Institutions of Higher Learning Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims (SSF151005), and the Open Fund for Scientific Research of Jiangxi Cancer Hospital in published maps and institutional affiliations. (2021J03). Open Access This article is licensed under a Creative Commons AUTHOR CONTRIBUTIONS Attribution 4.0 International License, which permits use, sharing, LSZ, XZL, and JZ collected the related paper and drafted the manuscript. LYZ and SYC adaptation, distribution and reproduction in any medium or format, as long as you give searched the databases and analyzed the data. 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