RLF‐MYCL fusion: A new tool for treatment of small cell lung cancer

Lung cancer is the second most common cancer worldwide, accounting for 2.207 million new cases and 1.796 million deaths in 2020. ¹ The burden of lung cancer is rapidly growing in China. ² , ³ Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. It accounts for about 13% of all lung cancer cases and is characterized by a 5‐year overall survival rate of less than 7%. ⁴ , ⁵ Although SCLC is initially sensitive to standard chemotherapy, almost all SCLC patients develop chemoresistance and recurrent disease within 1 year. ⁴ , ⁶ Even today, whilst immunotherapy can greatly improve the overall survival of patients with a variety of solid tumors, the combination of immunotherapy and chemotherapy has been reported to only prolong the median survival time of SCLC patients by two months. ⁷ In order to develop safer and more effective treatment strategies, improving our understanding of SCLC is necessary.

According to the differential expression of critical transcriptional factors, SCLC can be divided into different subtypes, including SCLC‐A (the SCLC subtype preferentially expressed in achaete‐scute homologue 1), SCLC‐N (the SCLC subtype preferentially expressed in neurogenic differentiation factor 1), SCLC‐P (the SCLC subtype preferentially expressed in POU class 2 homeobox 3), and SCLC‐Y (the SCLC subtype preferentially expressed in yes‐associated protein 1). ⁸ Moreover, MYC family gene amplification, TP53 and RB1 biallelic loss, and NOTCH mutations are common genetic variants in SCLC. ⁹ Some recent research progress on SCLC molecular subtypes has promoted our understanding of SCLC genotype‐specific therapy. ⁵ , ¹⁰ These studies suggest that the genetically engineered murine SCLC model can recapitulate many of the key biomarkers, mutations, and metastasis patterns observed in human SCLC. ¹⁰ , ¹¹ Indeed, the intranasal delivery of Cre recombinase‐expressing adenoviruses can trigger the lung tumor growth in genetically engineered mouse models with Tp53 and Rb1 alleles flanked by loxP sites. ¹¹ In addition, simultaneous amplification of Nfib and Mycl can accelerate the tumor initiation and metastasis in SCLC genetically engineered mouse models. ¹²

Gene fusions caused by insertions, deletions, and chromosomal translocations are common genomic structural aberrancies in SCLC, ¹³ but their pathological significance in the initiation and development of SCLC is not yet clear. Among the reported SCLC intrachromosomal rearrangements, the most notable is the recurrent in‐frame gene fusion between MYCL and RLF. Although previous studies have found that MYCL expression is closely related to SCLC‐A, as well as MYCL has been shown to promote SCLC initiation and tumor cell proliferation, ¹⁴ the role of RLF‐MYCL fusion in SCLC is still unknown. Due to the lack of feasible somatic engineering strategies before, it is very difficult to explore the role of fusion genes in driving tumorigenesis and development. However, the development of genome editing technology, such as CRISPR/Cas9, has made it possible to engineer and construct complex chromosomal rearrangements in vivo. In a study recently published in Cancer Discovery, Ciampricotti et al. ¹⁵ developed an Rlf‐Mycl1 gene fusion‐driven genetically engineered mouse model to investigate the functional role of the in frame RLF‐MYCL1 gene fusion in SCLC.

In this study, the authors induced Rlf‐Mycl fusion in murine cells by using the single guide RNAs promoting Cas9‐mediated double‐strand DNA breaks in the first introns of Rlf and Mycl gene. After that, they tried to use derivatives from Rb1^fl/fl/Rbl2^fl/fl/Trp53^fl/fl SCLC transgenic models to access the role of RLF‐MYCL fusion in the early stages of tumorigenesis. The results showed that the expression of RLF‐MYCL fusion accelerated tumor growth and malignant transformation in preneoplastic neuroendocrine cells. By using an autochthonous SCLC model, the authors further investigated that RLF‐MYCL endogenous induction can both promote tumor formation and accelerate early metastatic dissemination in vivo. With reference to human SCLC with RLF‐MYCL fusion and sgNeosgNeo tumors, the authors determined the transcriptional programs driving Rlf‐Mycl fusion‐mediated tumorigenesis. They found that the Rlf‐Mycl1 gene fusion‐driven genetically engineered mouse model shared most of the hallmarks pointed by both individual gene sequencing data and KEGG pathway analyses with human RLF‐MYCL SCLC.

This study is the first attempt to use CRISPR/Cas9‐mediated in vivo genome editing technology to explore the role of the most common gene fusion in SCLC. Mouse Rlf and Mycl genes and human RLF and MYCL genes have similar local chromosomal environments in the genomes of their respective species, making it possible to construct models effectively. Since the Rlf and Mycl genes on mouse chromosome 4 and the RLF and MYCL genes on human chromosome 1 are in opposite orientations, both of the fusion in these species required an inversion event to generate the target fusion genes. The Rlf‐Mycl fusion was successfully generated in a precursor model and genetically engineered mouse models of SCLC using CRISPR/Cas9‐based genome editing. Based on these models, this study demonstrated that RLF‐MYCL fusion gene accelerated the transformation and proliferation of SCLC cells and increased the spread of metastasis and the diversity of metastasis sites. Additionally, the gene expression profiling of the tumors harvested from the Rlf‐Mycl genetically engineered mouse models was similar to that of human SCLC with RLF‐MYCL fusion.

Chromosomal translocations in tumor cells resulting in gene fusion have been identified as drivers for many solid tumors including SCLC. ¹³ Identification and characterization of oncogenic fusions is extremely important for the development of tumor specific therapeutic targets. For example, the discovery of activating ALK fusion proteins in non‐small cell lung cancer has promoted the development of highly selective targeted therapy drugs. MYC family members have been identified as oncogenic drivers before ALK, but the development of targeted inhibitors for MYC family members has remained stalled. Although inhibiting MYCL activity is still a challenge at present, this study provided a theoretical basis for exploring MYCL fusions as therapeutic targets.

Human SCLC is known for its early and widespread metastases. However, most of the SCLC animal models that have been reported only develop primary tumors, and rarely have distant multiple organ metastases. The Rlf‐Mycl fusion genetically engineered mouse models successfully constructed in this study is more similar to human SCLC due to its broader metastatic tropism. For this reason, it can become a powerful tool in future preclinical study.

Overall, this study successfully constructed a mouse model which harbors recurrent gene fusion of SCLC for the first time and demonstrated that RLF‐MYCL fusion can promote tumorigenesis and multiorgan metastasis in SCLC. Further analysis of this mouse model will help develop safer and more effective therapeutic strategies that can be used for the benefit of SCLC patients.

CONFLICT OF INTEREST

The authors declare no competing interests.