Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Anticancer Drugs
2022 Feb 01;332:124-131. doi: 10.1097/CAD.0000000000001249.
Show Gene links
Show Anatomy links
Sensitivity of eight types of ALK fusion variant to alectinib in ALK-transformed cells.
Furugaki K
,
Harada N
,
Yoshimura Y
.
???displayArticle.abstract???
Tyrosine kinase inhibitors of anaplastic lymphoma kinase (ALK-TKIs) including alectinib have been the standard therapy against ALK fusion gene-positive non-small cell lung cancers (NSCLCs). Many ALK fusion variants have been identified in NSCLCs, and the predominant variants are echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) variant 1 (V1), V2 and V3a/b. However, there have been conflicting reports on the clinical responses of these variants to ALK-TKIs, and there are few reports on other less common ALK variants. To examine the influence of ALK variants on the efficacy of ALK-TKIs, we analyzed the sensitivity to alectinib of eight types of ALK variant: three major variants (V1, V2 and V3a) and five less common variants (V4; kinesin family member 5-ALK; kinesin light chain 1-ALK; striatin, calmodulin-binding protein-ALK; and tropomyosin-receptor kinase fused gene-ALK). Analysis was done by cell-free kinase assays using the recombinant proteins and by cell, growth assays using murine Ba/F3 cells expressing ALK variants. The kinase activity of each recombinant protein was significantly inhibited by alectinib. Intracellular ALK phosphorylation levels and its downstream signaling mediators, STAT3 and ERK, were suppressed by alectinib in each ALK variant-expressing Ba/F3 cell. Each cellular proliferation was markedly inhibited by alectinib treatment. There was no significant difference in the IC50 values between cells, with a <3.6-fold difference in responsiveness. In conclusion, these eight ALK variants had similar sensitivity to alectinib in vitro, indicating that it may not be possible to predict the response to alectinib just by determination of the ALK variant type in ALK fusion-positive NSCLCs.
Fig. 1. Effect of alectinib on recombinant ALK fusion variants in vitro. (a) The kinase activities of eight recombinant ALK variants were measured and their activities relative to that of V1 were calculated. NS means no significant difference in any pairwise comparison between each variant by Tukey–Kramer’s HSD test. (b) The kinase activity was measured with alectinib (+) or without alectinib (−), and the relative activity (percentage) in the presence of alectinib with respect to activities in the presence of vehicle alone was calculated. Asterisks indicate a significant difference between them at P<0.05 by Student’s t test. Each point represents the mean + SD of triplicates.
Fig. 2. Effect of alectinib on intracellular ALK fusion variants. (a) RNA was extracted from stable Ba/F3 cells, and mRNA expression level of the neomycin resistance gene relative to that of GAPDH was measured by RT-PCR analysis. NS means no significant difference in any pairwise comparison between each ALK variant cell by Tukey–Kramer’s HSD test. Each point represents the mean + SD of quadruplicates. (b) Immunoblots of cell lysates from the ALK variant cells treated with (+) or without (−) alectinib.
Fig. 3. Effect of alectinib on various signaling molecules of ALK variant cells. Immunoblots of cell lysates from the ALK variant cells treated with (+) or without (−) 100 nM alectinib for 3 h (a) or 24 h (b). Antibodies against phospho-JNK, phospho-P38, phospho-STAT5, phospho-eIF2A, phospho-4E-BP1, phospho-AKT (473), phospho-ULK1 (555 or 757), MCL-1, BCL-XL, BAK, Beclin-1, BIM (Cell Signaling Technology), BAX (SIGMA) and phospho-AKT (308) (Thermo Fisher Scientific) were used.
Fig. 4. Effect of alectinib on cell proliferation of ALK variant cells. (a) ALK-variant-expressing Ba/F3 cells or control Ba/F3 cells were cultured with or without alectinib, and their viability with alectinib relative to their viability with vehicle alone was measured. Each point represents the mean + SD of triplicates. (b) IC50 values of alectinib were calculated as described previously [20]. NS means no significant difference in any pairwise comparison between each ALK variant cell by Tukey–Kramer’s HSD test. Horizontal bars indicate the mean ± SD of triplicates.
Fig. 5. Effect of alectinib on cell death of ALK variant cells. ALK-variant-expressing Ba/F3 cells or control Ba/F3 cells were cultured with or without 10 nM alectinib for 4 days. Then, the cell numbers of viable and nonviable cells were counted with double-fluorescent dye staining acridine orange and DAPI of cell count & viability assay kit using Image Cytometer NucleoCounter NC-3000. The cell death ratio was calculated as follows: Cell death ratio % = 100−100× (viable cell numbers with alectinib treatment/viable cell numbers without treatment). NS means no significant difference in any pairwise comparison between each ALK variant cell by Tukey–Kramer’s HSD test. Horizontal bars indicate the mean ± SD of quadruplicates.
Fig. 6. Effect of crizotinib on cell proliferation of ALK variant cells. (a) ALK-variant-expressing Ba/F3 cells or control Ba/F3 cells were cultured with or without crizotinib, and their viability with crizotinib relative to their viability with vehicle alone was measured. Each point represents the mean + SD of triplicates. (b) IC50 values of crizotinib were calculated as described previously [20]. NS means no significant difference in any pairwise comparison between each ALK variant cell by Tukey–Kramer’s HSD test. Horizontal bars indicate the mean ± SD of triplicates.
Fig. 7. Effect of transient alectinib treatment on colony formation of ALK variant cells. ALK-variant-expressing Ba/F3 cells or control Ba/F3 cells were cultured with or without 100 nM alectinib for 24 h. After washing the cells, they were cultured in semisolid agar media for 6 days using CytoSelect 96-well in vitro tumor sensitivity assay kit (Cell Biolabs). Each point represents the mean + SD of triplicates.
Camidge,
Updated Efficacy and Safety Data and Impact of the EML4-ALK Fusion Variant on the Efficacy of Alectinib in Untreated ALK-Positive Advanced Non-Small Cell Lung Cancer in the Global Phase III ALEX Study.
2019, Pubmed,
Echinobase
Camidge,
Updated Efficacy and Safety Data and Impact of the EML4-ALK Fusion Variant on the Efficacy of Alectinib in Untreated ALK-Positive Advanced Non-Small Cell Lung Cancer in the Global Phase III ALEX Study.
2019,
Pubmed
,
Echinobase
Courtin,
Emergence of resistance to tyrosine kinase inhibitors in non-small-cell lung cancer can be delayed by an upfront combination with the HSP90 inhibitor onalespib.
2016,
Pubmed
Furugaki,
Impact of bevacizumab in combination with erlotinib on EGFR-mutated non-small cell lung cancer xenograft models with T790M mutation or MET amplification.
2016,
Pubmed
Heuckmann,
Differential protein stability and ALK inhibitor sensitivity of EML4-ALK fusion variants.
2012,
Pubmed
Katayama,
Cabozantinib overcomes crizotinib resistance in ROS1 fusion-positive cancer.
2015,
Pubmed
Kim,
AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
2011,
Pubmed
Kodama,
Alectinib shows potent antitumor activity against RET-rearranged non-small cell lung cancer.
2014,
Pubmed
Kris,
Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs.
2014,
Pubmed
Lei,
Anaplastic Lymphoma Kinase Variants and the Percentage of ALK-Positive Tumor Cells and the Efficacy of Crizotinib in Advanced NSCLC.
2016,
Pubmed
,
Echinobase
Lin,
Impact of EML4-ALK Variant on Resistance Mechanisms and Clinical Outcomes in ALK-Positive Lung Cancer.
2018,
Pubmed
Mitiushkina,
Variability in lung cancer response to ALK inhibitors cannot be explained by the diversity of ALK fusion variants.
2018,
Pubmed
Mizushima,
The role of Atg proteins in autophagosome formation.
2011,
Pubmed
Ou,
Catalog of 5' Fusion Partners in ALK-positive NSCLC Circa 2020.
2020,
Pubmed
Sabir,
EML4-ALK Variants: Biological and Molecular Properties, and the Implications for Patients.
2017,
Pubmed
,
Echinobase
Sasaki,
The biology and treatment of EML4-ALK non-small cell lung cancer.
2010,
Pubmed
,
Echinobase
Shaw,
Tyrosine kinase gene rearrangements in epithelial malignancies.
2013,
Pubmed
Woo,
Differential protein stability and clinical responses of EML4-ALK fusion variants to various ALK inhibitors in advanced ALK-rearranged non-small cell lung cancer.
2017,
Pubmed
,
Echinobase
Xia,
How to select the best upfront therapy for metastatic disease? Focus on ALK-rearranged non-small cell lung cancer (NSCLC).
2020,
Pubmed
,
Echinobase
Yoshida,
Differential Crizotinib Response Duration Among ALK Fusion Variants in ALK-Positive Non-Small-Cell Lung Cancer.
2016,
Pubmed
Yoshimura,
Antitumor activity of alectinib, a selective ALK inhibitor, in an ALK-positive NSCLC cell line harboring G1269A mutation: Efficacy of alectinib against ALK G1269A mutated cells.
2016,
Pubmed
