Authors: Xinzhao Wei, Ken Uchibori, Makoto Nishio, Ryohei Katayama
Published: 2025-04-21
DOI: 10.1158/1538-7445.am2025-5531
Source: Full article
RET-fusion positive non-small cell lung cancer (NSCLC) accounts for 1-2% of total population of NSCLC. RET selective tyrosine kinase inhibitors (TKIs) have demonstrated remarkable efficacy, and 2 RET-TKIs have been approved for the treatment of RET-rearranged NSCLC. However, the emergence of acquired resistance which remains unclear largely. To better simulate in vivo tumor conditions, we employed a 3D culture system using LC2/ad (CCDC6-RET) and LCC190 (CCDC6-RET, originally established from RET-TKI naïve patient’s pleural effusion) cell lines. RET-TKIs showed significant increased efficacy in this 3D system compared to standard 2D cultures with increased apoptosis pathway and reduced ERBB-mediated MAPK and PI3K/Akt/mTOR signaling pathways.Further, we conducted a genome-wide CRISPR/Cas9 screening in the 3D condition in LCC190, which identified multiple candidate genes including ERRFI1 (MIG6) contributor to drug-tolerant persister (DTP) cell survival during RET-TKI treatment. Knockout of MIG6, a negative regulator of EGFR signaling, induced significant EGFR activation in the presence of extreme low ligand concentrations (1 ng/mL), leading to resistance through the activation of MAPK and PI3K/Akt/mTOR pathways. These findings highlight the critical role of regulation of MIG6 on EGFR in bypassing RET inhibition. Notably, combining RET-TKIs with EGFR inhibitors (afatinib or cetuximab) markedly reduced the survival of DTP cells in vivo and in vitro suggesting a potential combination strategy to overcome such resistance. Conversely, overexpression of MIG6 restored sensitivity to RET-TKIs under conditions of low EGFR ligands with reduced level of phosphorylation of EGFR. In conclusion, this study identifies MIG6 as a critical mediator of RET-TKI resistance in NSCLC and highlights the utility of 3D culture systems in uncovering key resistance mechanisms in RET-fusion positive NSCLC. Targeting EGFR in combination with RET-TKIs presents a promising therapeutic strategy to overcome drug-tolerant persistence and improve treatment outcomes in RET-rearranged NSCLC. Besides MIG6, we have also found several candidate genes related to DTP cell survival or RET-TKI resistance from the genome-wide Crispr screening. Further studies are needed to clarify the mechanisms of resistance induced by loss of these candidate genes to comprehensively understand the mechanisms how DTP cells survive against TKI treatment and how such resistance could be overcome by novel treatment methods.