Neddylation Regulates Macrophages and Implications for Cancer Therapy

Tumor-associated macrophages (TAMs) promote cancer progression via stimulating angiogenesis, invasion/metastasis, and suppressing anti-cancer immunity. Targeting TAMs is a potential promising cancer therapeutic strategy. Neddylation adds the ubiquitin-like protein NEDD8 to substrates, and thereby regulates diverse biological processes in multiple cell types, including macrophages. By controlling cellular responses, the neddylation pathway regulates the function, migration, survival, and polarization of macrophages. In the present review we summarized how the neddylation pathway modulates Macrophages and its implications for cancer therapy.

In 2009, a specific small molecular inhibitor of NAE, called MLN4924 (also known as pevonedistat), was identified via high throughput screening (Soucy et al., 2009). MLN4924 forms a covalent NEDD8-MLN4924 adduct at the active site of NAE to inhibit the first step of the neddylation enzymatic process (Brownell et al., 2010;Enchev et al., 2015). By doing so, MLN4924 inhibits the entire neddylation pathway and blocks the activation of CRLs, thus inducing the accumulation of various tumorsuppressive CRL substrates which trigger cell-cycle arrest, DNA damage, apoptosis or senescence Liang et al., 2020;Zhou and Jia, 2020). Phase II/III clinical trials of MLN4924 have been conducted for the treatment of several solid tumors and hematologic malignancies (Swords et al., 2015(Swords et al., , 2018Bhatia et al., 2016;Sarantopoulos et al., 2016;Shah et al., 2016).
The neddylation pathway also modulates macrophages and their response to different stimulation Zhou et al., 2019a,b), thus highlighting the connection between neddylation, macrophages, and cancer.

NEDDYLATION REGULATES INFLAMMATION-RELATED SIGNAL PATHWAYS IN MACROPHAGES
Transcription factors are intracellular molecules that modulate the activity of specific genes. When macrophages are stimulated, transcription factors activate related genes to eliminate pathogens or other dangerous elements. Nuclear factor kappa-B (NF-κB), one of the basic inflammatory-related factors, functions as a precursor to increase the concentration of proinflammatory factors and thus coordinates the inflammatory response (DiDonato et al., 2012). In normal conditions, NF-κB is sequestered in the cytoplasm by interacting with its inhibitory protein IκBα (Bhoj and Chen, 2009). When stimulated by various signals, neddylation modification to the C-terminal lysine residue of cullin changes the conformation of CRLs and activates CRLs enzymatic function for IκBα ubiquitination and degradation (Bhoj and Chen, 2009;Chang et al., 2012;Jin et al., 2018). The degradation of IκBα by the ubiquitin proteasome system allows NF-κB entering into nucleus where it binds to DNA promoter regions, thus turning on transcription of a wide spectrum of genes and the release of inflammatory factors (Bhoj and Chen, 2009). This process is triggered by IκBα kinases (IKKα or β), which phosphorylate IκBα at S32 and S36 Tan et al., 1999), thus, highlighting the underlying cooperative relationship between phosphorylation and neddylation whereas. Inactivation of neddylation inhibits the activity of CRLs and induces the accumulation of its substrate IκBα, which sequesters NF-κB in the cytoplasm to block NF-κB transcriptional activity (Chang et al., 2012;Jin et al., 2018; Figure 3A). Moreover, Cullin 5 neddylation following LPS stimulation triggers the interaction with tumor necrosis factor receptor-associated factor 6 (TRAF6), an essential adaptor to promote the activation of NF-κB, thus inducing K63-linked TFAR6 polyubiquitination and leading to NF-κB activation, and eventually facilitating the generation of proinflammatory cytokines (Zhu et al., 2016(Zhu et al., , 2017. Apart from the modulation of transcription factors, the neddylation pathway regulates the maturation and secretion processes of inflammatory factors in macrophages. For example, the association of pro-caspase-1 with NLR family pyrin domain containing 3 (NLRP3)/apoptosis-associated speck-like (ASC) protein via caspase recruitment domain (CARD) promotes the autocatalytic activity of pro-caspase-1 to self-cleavage into caspase-1, and thus leads to the maturation of 31 KD prointerleukin-1β into 17KD IL-1β (Bryant and Fitzgerald, 2009;Dowling and O'Neill, 2012). In this process, neddylation modification to the CARD domain is required for the selfcleavage of pro-caspase-1 to generate its catalytically active subunits (Segovia et al., 2015). NEDD8 silencing or MLN4924 inhibition of neddylation modification of the caspase-1 CARD domain diminishes caspase-1 maturation and inhibits IL-1β maturation and secretion (Segovia et al., 2015; Figure 3B). These  findings demonstrate how neddylation pathway modulates the macrophage inflammation response, which provides a molecular basis for targeting neddylation pathway in macrophages to ameliorate the inflammation microenvironment in tumors.
A recent study from our group showed that the elevated neddylation pathway in cancer cells led to the accumulation of NF-κB-regulated activation of chemokines CCL2 with promotion of macrophage infiltration (Zhou et al., 2019a). Inactivation neddylation in cancer cells, either pharmacologically (MLN4924) or genetically (NEDD8 knock out via Crisp Cas9), inhibited CCL2 expression and macrophage tumor infiltration, thus inhibiting lung metastasis (Zhou et al., 2019a; Figure 3C). MLN4924 also suppressed cancer-associated fibroblasts (CAF)derived and macrophage-derived CCL2 (Zhou et al., 2019b; Figure 3C). Therefore, neddylation activation promotes the migration of macrophages via regulating tumor/CAF-derived CCL2, indicating synergistic inhibition of neddylation in CCL2producing cells to target the CCL2-macrophage axis. MLN4924 can thus reduce macrophage accumulation in tumors, which could be an effective cancer therapy.
Functionally, tumor infiltrated macrophages induce an immunosuppressive and tumorigenic phenotype by neutralizing the function of cytotoxic CD8 + T cells (Cassetta and Pollard, 2018). Neddylation inactivation decreases macrophage tumor infiltration and promotes CD8 + T cell tumor infiltration (Zhou et al., 2019a). Based on these findings, we postulate that targeting the neddylation pathway to inhibit macrophage recruitment in tumors would be tested in clinical trials.

NEDDYLATION REGULATES THE PROLIFERATION AND SURVIVAL OF MACROPHAGES
Similar to cancer cells, the neddylation pathway is required for the proliferation and survival of macrophages. Neddylation inactivation inhibits macrophage viability with the following mechanisms, including: (1) Neddylation inactivation by MLN4924 blocks cullin neddylation and suppresses CRL activity, thus leading to the accumulation of cell-cycle inhibitors (e.g., p21, p27, and Wee1) and inducing G 2 -M-phase cell-cycle arrest in macrophages. (2) MLN4924 activates DNA re-replication stress and DNA damage by inducing the accumulation DNA replication licensing protein of CDT1 and ORC1 in macrophages.
How dose neddylation modification influence survival and the inflammatory response of macrophages? Firstly, partial inhibition of neddylation by MLN4924 inhibits inflammatory response of macrophages at an early stage when cell viability is not significantly blocked. However, continuous inactivation of neddylation by MLN4924 impairs macrophage viability, indicating that the balance of macrophage survival or death depends on the treatment degree of neddylation inactivation by MLN4924 treatment  Figure 4B). Secondly, RBX2overexpressing macrophages maintain viability via degradation of the pro-apoptotic proteins (BAX and SARM), which facilitate the pathogen-associated molecular patterns (PAMPs)stimulated inflammatory response. RBX2 knockdown induces the accumulation of BAX and SARM to trigger intrinsic apoptosis (Chang and Ding, 2014; Figure 4B), suggesting the RBX2dependent ubiquitin-proteasome system serves as a checkpoint between the survival and death of macrophages. These results suggest strategies for targeting neddylation to inhibit tumor infiltration macrophages as potential cancer therapy.
In summary, these results imply that neddylation regulates the polarization of macrophages in a cell-type and microenvironment-type dependent manner. Additional investigation is needed to further decipher the detailed mechanisms. Nevertheless, in either case, a high number of macrophages in tumor is associated with poor overall survival. Therefore, limiting the numbers of macrophages via inactivating neddylation in tumor is a promising therapeutic strategy.

CONCLUSION AND REMARKS
Macrophages are a major component of TME. Neddylation inactivation to suppress the accumulation of macrophages in tumor is a novel and promising cancer therapeutic strategy (Zhou et al., 2019a,b). However, some questions still await further investigation.
Firstly, further studies are needed to fully identify macrophage phenotypes and define the determining factors for macrophage's polarization upon MLN4924 treatment in various tumor models. Secondly, the TME comprises different types of infiltrated immune cells, fibroblasts, endothelial cells as well as cancer cells. The role of the neddylation genes (such as NEDD8, UBA3, NAE1, UBE2M, UBE2F, RBX1, and RBX2) in specific cell subsets of the TME needs to be further clarified. Thirdly, we need to learn how neddylation modulates the proliferation and differentiation of hematopoietic stem cells and myeloid progenitor cells, and how neddylation modulates the production of monocytes in multiple tumor models. Fourthly, the efficacy of MLN4924 in combination with cancer immunotherapy (such as nivolumab, avelumab, ipilimumab) needed to be tested. Finally, identification of the biomarkers indicating the viability of macrophages upon neddylation inhibition could maximize the therapeutic efficacy of MLN4924, and optimize the dose, routine, and schedule.
Once we assure these questions, the regulatory mechanisms of macrophages will be clearly clarified, which would extend our understanding of how neddylation pathway modulates macrophages in fundamental cancer biology, and provide a sound rationale and molecular basis for neddylation-based targeting macrophages therapies for clinical cancer treatment.

AUTHOR CONTRIBUTIONS
YJ, LL, and YL collected the related manuscript and drafted the manuscript. GL and RMH provided the technical or material support. LJ, YJ, and RMH revised and finalized the manuscript. All authors read and approved the final manuscript.