Neuroblastoma (NB) is an embryonal deadly cancer in childhood driven by MYC or MYCN-driven oncogenic signaling. The amplification of MYCN leads to malignant progression of NB and poor prognosis. Traditional chemotherapy is still a standard treatment for NB. Most of the cytostatic drugs take function in anti-neuroblastoma. However, children with NB exhibit different clinical outcome variability and biological characteristics compared to adult patients. Given this, it is an urgent need to explore novel, safer and more efficacious treatments.

MYCN is one of the MYC family members (MYC, NMYC and LMYC), taking an essential part in cellular growth control, cell transformation, and tumorigenesis. It is associated with 70% of human cancers, and the amplification of the MYCN always indicates a poor prognosis. MYC proteins are composed of basic helix-loop-helix leucine zipper DNA binding proteins, which can bind with MYC-associated protein X (MAX). The MYC-MAX complex further interacts with E-box regulatory DNA elements to involve in the transcription of genes.¹ However, the MYCN protein is intrinsically disordered in nature, with unclear protein interactions, which made the direct inhibition of MYCN-amplificated expression difficult.² Nevertheless, the inhibition and activation of transcription factors have a significant impact on the overexpression of MYCN-amplificated protein. Hence, in the past several years, there were mainly three methods for inhibiting MYCN, such as impairing MYC transcription, interfering with MYC mRNA, and reducing MYC stability and function.

Ubiquitination has been valued for its extensive and complex roles during many cellular functions, such as protein degradation, cell survival and differentiation, innate and adaptive immunity, and signal transduction.² These pathways were considered anti-cancer targets for many years. The ubiquitin-proteasome system can degrade protein rapidly, which is very important to control the normal physiological function of MYC.³

Depending on this mechanism, there were five types of indirect inhibition therapy for MYCN-amplified cancer. (1) Targeting regulators of MYC protein stability: Aurora-A inhibitors and Aurora-B inhibitors,⁴ the inhibitors of HUWE1 (HECT domain-containing ubiquitin E3 ligase),² the inhibitors of PLK1 (Polo-like kinase 1),^{5, 6} and silencing the protein arginine methyltransferase 5 (PRMT5)^{7, 8}; (2) Targeting for the transcription of MYC: the Bromodomain and Extra-Terminal (BET) bromodomain and extra-terminal domain inhibitors,^9-11 the inhibitor of CDK9^12-14; (3) Targeting for MYCN cofactors/coregulators: the inhibitors of LSD1 (Lysine-specific histone demethylase 1)¹⁵; (4) Targeting the MYCN downstream targets: the inhibitor of ornithine decarboxylase 1 (ODC1)¹⁶; (5) Targeting MYCN Synthetic lethal approach: the antiapoptotic protein BCL2 and cyclin-dependent kinase 2 (CDK2).¹⁷ Moreover, using inhibitors in combination, targeting transcription and translation of MYCN, can improve the therapeutic effects.

It is regrettable that, although the BET protein inhibitors can function in early adult trials, their effect was short-term and unsustainable, revealing that tumor cells can tolerate the BET inhibition. Moreover, the Aurora Kinase A inhibitors also showed substantial toxicity and disappointing responses in subsequent clinical testing.⁴

Once considered undruggable and its potential side effects with inhibitory MYCN. Directly targeting the MYCN was considered difficult before. Nevertheless, the paradigm shift in our understanding came with the successful trial of the MYC dominant negative mutant Omomyc, which indicated that transient inhibition of MYC is worth exploring. As the direct inhibitor of protein/protein interaction or binding to DNA, the small molecule inhibitor of Myc/Max dimerization can prevent the binding of Myc and DNA, disrupting the interaction of Myc/Max.¹ Moreover, Omomyc has been used in clinical trials and has shown a favorable safety profile. Further treatment about dose expansion cohorts (Phase IIa) will be conducted in the future.¹⁸ With the discovery of the structural and the basic understanding of the MYC protein's biochemical and biological properties, it is worth waiting to demystify the MYC's druggability.

In addition to negative mutant Omomyc, the direct targeting of the c-Myc promoter was also shown to be a promising therapy by preventing the interaction between transcription and the G-quadruplexes, which are located in the nuclease hypersensitive element taking part in the expression of MYC. Moreover, G-quadruplex has been explored in clinical trials, and the curative effect of downregulating the transcription of the MYCN oncogene was verified.¹⁹

Another appealing approach may be to identify small molecules that can combine with MYCN in complex, and then guide the design and/or optimization of the drug by profoundly understanding the structure. Linking this small molecule with an E3 ligase binder promotes the degradation of the MYCN. For example, they bind E3 ligase with proteolysis-targeting chimeras (PROTACs) and the protein of interest to become a stable ternary complex, which is then recognized/degraded by the proteasome.²⁰ The first oral PROTACs: ARV-110 (NCT03888612) and ARV-471 (NCT04072952) have confirmed encouraging curative effects in clinical trials among breast and prostate cancer patients, which arouse tremendous enthusiasm for PROTAC research. However, there are hundreds of MYCN interacting protein complexes, and we should maintain the physiologic functions while inhibiting the oncogenic function. In the future, advanced techniques like X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM) will be needed to discover the structure of MYCN protein and aid drug development. Here we do not go into details about the presence of some small molecules inhibiting MYCN, acting on other pathways like promoting the degradation of MYC mRNA: Antisense oligonucleotides, lentiviral delivery of N-myc-siRNA.

In conclusion, the inhibition of MYCN protein includes indirect inhibition of MYC expression, direct inhibition of MYC, knockdown, protein interaction inhibitors, inhibitors preventing protein and DNA interaction, and the regulation of translation/expression. The inhibitors also have developed rapidly, encompassing a broad spectrum ranging from traditional small molecules to organic and inorganic nanoparticles, from RNA and antisense agents to peptides and small protein inhibitors. However, there are few treatments for NB patients targeting MYCN-amplified. Most of the trials targeting MYCN-amplified treatments have not been conducted among NB cell lines/patients in Table 1 and Table 2. Besides discovering new target candidates, it is also worth using advanced techniques to identify MYCN protein's structure and provide more effective and safe treatments for NB patients.

AUTHOR CONTRIBUTIONS

Drs. Jiang, Zhang, Chang, Zhu, Yang, and He contributed to the preparation and collection of original literatures and figures and the writing and editing of manuscript. Dr. Yang and He responsible for the structural designs, scientific quality and writing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

FUNDING

This work was supported by the National Natural Science Foundation of China (No: 82173593).

ETHICS APPROVAL

Not applicable.