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

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/).

Dopamine transporter SPECT using fast kinetic ligands: 123I-FP-beta-CIT versus 99mTc-TRODAT-1.

A comparative study was carried out on two promising presynaptic dopamine transporter single-photon emission tomography (SPECT) radioligands with a fast pharmacokinetic profile, 123I-FP-beta-CIT (FP) and 99mTc-TRODAT-1 (TR), in order to assess their differential diagnostic power in early parkinsonism and their sensitivity for detection of disease progression. This cross-sectional study was conducted on 96 patients with early-stage parkinsonism referred in a tertiary clinical setting. Mean disease duration was 2.0+/-1.3 years, and patients had a modified Hoehn and Yahr (H&Y) stage of 1-2 (average 1.2). Forty-seven patients received TR, and 49 received FP. In both groups, ten patients with normal presynaptic function were included as a control population; all other patients were clinically diagnosed as having idiopathic Parkinson's disease. Groups were matched for gender, age, disease duration and modified H&Y stage. Triple-head gamma camera SPECT was analysed using a semiquantitative index of transporter binding (BI). Discriminant analysis with cross-validation resulted in a maximal classification accuracy for FP of 93% (sensitivity 95% and specificity 86%) for the contralateral putamen BI. For TR, the corresponding values were 87% accuracy, 92% sensitivity and 70% specificity. For FP, disease duration was correlated with both the putamen BI (-8.8%/year, rho=-0.41, P=0.025) and the putamen/caudate ratio (-7.4%/year, rho=-0.51, P=0.004), but for TR no significant correlation was found (all P values >0.5). In conclusion, both FP and TR show high sensitivity in a clinically relevant setting, but FP has superior accuracy for early differential diagnosis of idiopathic parkinsonism and non-degenerative extrapyramidal disorders, as well as better sensitivity for disease follow-up.