Novel human-derived EML4-ALK fusion cell lines identify ribonucleotide reductase RRM2 as a target of activated ALK in NSCLC

Lung Cancer. 2022 Sep;171:103-114. doi: 10.1016/j.lungcan.2022.07.010. Epub 2022 Jul 25.

Abstract

Introduction: Echinoderm microtubule-associated protein-like 4 (EML4)-Anaplastic Lymphoma Kinase (ALK) rearrangements occur in 3% to 7% of lung adenocarcinomas and are targets for treatment with tyrosine kinase inhibitors (TKIs). Here we have developed three novel EML4-ALK-positive patient-derived Non-Small-Cell-Lung-Cancer (NSCLC) cancer cell lines, CUTO8 (variant 1), CUTO9 (variant 1) and CUTO29 (variant 3) and included a fourth ALK-positive cell line YU1077 (variant 3) to study ALK-positive signaling and responses. Variants 1 and 3 are the most common EML4-ALK variants expressed in ALK-positive NSCLC, and currently cell lines representing these EML4-ALK variants are limited.

Materials and methods: Resazurin assay was performed to evaluate cell viability. Protein levels were determined using western blotting. RNA sequencing was performed in all four cell lines to identify differentially expressed genes. Whole-genome sequencing was performed to determine the presence of EML4-ALK fusion and ALK tyrosine kinase inhibitor resistance mutations.

Results: In this study, we have confirmed expression of the corresponding ALK fusion protein and assessed their sensitivity to a range of ALK tyrosine kinase inhibitors. These patient derived cell lines exhibit differential sensitivity to lorlatinib, brigatinib and alectinib, with EML4-ALK variant 3 containing cell lines exhibiting increased sensitivity to lorlatinib and brigatinib as compared to alectinib. These cell lines were further characterized by whole genome sequencing and RNA-seq analysis that identified the ribonucleotide reductase regulatory subunit 2 (RRM2) as a downstream and potential therapeutic target in ALK-positive NSCLC.

Conclusion: We provide a characterization of four novel EML4-ALK-positive NSCLC cell lines, highlighting genomic heterogeneity and differential responses to ALK TKI treatment. The RNA-Seq characterization of ALK-positive NSCLC CUTO8, CUTO9, CUTO29 and YU1077 cell lines reported here, has been compiled in an interactive ShinyApp resource for public data exploration (https://ccgg.ugent.be/shiny/nsclc_rrm2_2022/).

Keywords: Alectinib; Anaplastic lymphoma kinase; Brigatinib; EML4-ALK; Echinoderm microtubule associated protein like 4; Lorlatinib; Lung adenocarcinoma; NSCLC; RRM2; Tyrosine kinase inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaplastic Lymphoma Kinase / genetics
  • Carcinoma, Non-Small-Cell Lung* / drug therapy
  • Carcinoma, Non-Small-Cell Lung* / genetics
  • Carcinoma, Non-Small-Cell Lung* / pathology
  • Cell Line
  • Humans
  • Lung Neoplasms* / drug therapy
  • Lung Neoplasms* / genetics
  • Lung Neoplasms* / pathology
  • Oncogene Proteins, Fusion / genetics
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinase Inhibitors / therapeutic use
  • Ribonucleoside Diphosphate Reductase* / metabolism

Substances

  • EML4-ALK fusion protein, human
  • Oncogene Proteins, Fusion
  • Protein Kinase Inhibitors
  • ribonucleotide reductase M2
  • Ribonucleoside Diphosphate Reductase
  • Anaplastic Lymphoma Kinase