Phase separation of EML4\u2013ALK in firing downstream signaling and promoting lung tumorigenesis

Zhen Qin,Honghua Sun,Meiting Yue,Xueliang Zhu,Xinwen Pan,Xiumin Yan,Xinhua Feng,Hongbin Ji,Liang Chen

doi:10.1038/s41421-021-00270-5

Abstract

EML4–ALK fusion, observed in about 3%–7% of human lung adenocarcinoma, is one of the most important oncogenic drivers in initiating lung tumorigenesis. However, it still remains largely unknown about how EML4–ALK fusion exactly fires downstream signaling and drives lung cancer formation. We here find that EML4–ALK variant 1 (exon 1–13 of EML4 fused to exon 20–29 of ALK) forms condensates via phase separation in the cytoplasm of various human cancer cell lines. Using two genetically engineered mouse models (GEMMs), we find that EML4–ALK variant 1 can drive lung tumorigenesis and these murine tumors, as well as primary tumor-derived organoids, clearly show the condensates of EML4–ALK protein, further supporting the findings from in vitro study. Mutation of multiple aromatic residues in EML4 region significantly impairs the phase separation of EML4–ALK and dampens the activation of the downstream signaling pathways, especially the STAT3 phosphorylation. Importantly, it also significantly decreases cancer malignant transformation and tumor formation. These data together highlight an important role of phase separation in orchestrating EML4–ALK signaling and promoting tumorigenesis, which might provide new clues for the development of clinical therapeutic strategies in treating lung cancer patients with the EML4–ALK fusion.

Highlights

Non-small cell lung cancer (NSCLC) is one of the most common cancers worldwide with high incidence and mortality[1]
Through living cell imaging analyses, we found that the GFP–echinoderm microtubuleassociated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK) condensates were able to undergo fusion, indicative of their liquid properties (Fig. 1b and Supplementary Movie S1)
As EML4–ALK is known as an important oncogenic driver in lung tumorigenesis, we overexpressed GFP–EML4–ALK variant 1 in BEAS-2B cells, a non-transformed human bronchial epithelial cell line and observed similar condensate formation (Fig. 1e)

Summary

Introduction

Non-small cell lung cancer (NSCLC) is one of the most common cancers worldwide with high incidence and mortality[1]. NSCLC is frequently associated with oncogenic driver mutations which significantly contribute to tumorigenesis and cancer progression. Oncogenic mutations of epidermal growth factor receptor (EGFR), v-Ki-ras[2] Kirsten. The EML4–ALK fusion was initially discovered in Japanese NSCLC patients by Soda and colleagues in 20074. Later study showed that about 3%–7% of NSCLC patients harbor EML4–ALK fusion[5]. ALK belongs to the receptor tyrosine kinase (RTK) family and the full length of ALK protein contains 1620 amino acids[6]. The ALK protein is comprised of three domains, including an extracellular domain (ECD), a transmembrane domain, and an intracellular domain (ICD)[7]. As a fusion partner of ALK, echinoderm microtubuleassociated protein-like 4 (EML4) belongs to the echinoderm microtubule-associated protein-like family.

Methods

Results

Conclusion