Entrectinib

Manufactured by Selleck Chemicals

Sourced in United States

Entrectinib is a small-molecule tyrosine kinase inhibitor. It targets the NTRK, ROS1, and ALK genes.

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Entrectinib (RXDX-101, (2 citations)

14 protocols using entrectinib

Establishment and Characterization of Entrectinib-Resistant Cell Line

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HCC78 cell was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere containing 5% CO₂. Cell line identity was authenticated by short-tandem-repeat analysis. Entrectinib-resistant HCC78 (HCC78ER) cells were newly established in our laboratory through the exposure of HCC78 cells to gradually increasing concentrations of Entrectinib (starting at 100 nM and ending with 5 μM) over 6 months. The established cells maintained resistance to Entrectinib even after the withdrawal of Entrectinib from the culture medium.
Entrectinib was provided by Ignyta, Inc./F.Hoffmann-La Roche Ltd. Crizotinib, ceritinib, lorlatinib and selumetinib were purchased from Selleckchem. All drugs were dissolved at a 10 mM concentration in dimethyl sulfoxide (DMSO) and stored in small aliquots at −20 °C until further use. Antibodies specific for p-ROS1 (Tyr1068), ROS1, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-STAT3 (Tyr705), STAT3, p-p53 (Ser15), p53, p-H2AX (Ser139), H2AX and PARP were obtained from Cell Signaling Technologies. Anti-FGF3 and β-actin antibodies were obtained from Santa Cruz Biotechnology.

Ku B.M., Bae Y.H., Lee K.Y., Sun J.M., Lee S.H., Ahn J.S., Park K, & Ahn M.J. (2019). Entrectinib resistance mechanisms in ROS1-rearranged non-small cell lung cancer. Investigational New Drugs, 38(2), 360-368.

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Functional Regulation of miRNA Targets

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Hsa-miR-483-5p mimic, YM00473215-ADA, mimic control, YM00479902-ADA, inhibitors hsa-miR-675-5p, 427419-00, hsa-miR-675-3p, 427420-00, hsa-miR-483-5p, 427155-00, hsa-miR-483-3p, 427154-00, hsa-miR-204-5p, 426934-00, hsa-miR-135-5p, 426790-00, hsa-miR-10a-5p, 426651-00, hsa-miR-9-5p, 427460-00, hsa-miR-9-3p, 427461-00, Control B, 199021-00 are from Qiagen, Germantown, MD, USA. Ceritinib, S7083, linsitinib, S1091, entrectinib, S7998, GSK1904529A, S1093, picropodophyllin, S7668 were obtained from Selleck Chemicals, Houston, TX, USA; GSK1838705A, SML0995, was purchased from Millipore-Sigma, Burlington, MA, USA. The primers used for miRNA and mRNA quantification were miR-99b-5p, 4,427,975, miR-483-5p, 4,427,975, miR-675-5p, 4,427,975, IGF-2, 4,331,182, GAPDH, 4,331,182, ThermoFisher Scientific, Waltham, MA, USA.

Uhlmann E.J., Mackel C.E., Deforzh E., Rabinovsky R., Brastianos P.K., Varma H., Vega R.A, & Krichevsky A.M. (2023). Inhibition of the epigenetically activated miR-483-5p/IGF-2 pathway results in rapid loss of meningioma tumor cell viability. Journal of Neuro-Oncology, 162(1), 109-118.

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Comprehensive Drug Screening in Organoids

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All compounds were dissolved in DMSO at 10 mM (except for cisplatin and carboplatin) and aliquots were stored at −20 °C, 4 °C or room temperature according to the manufacturer’s recommendations. Sorafenib tosylate, lenvatinib mesylate, cabozantinib mesylate, regorafenib, octreotide acetate, lanreotide acetate, etoposide, sunitinib malate, everolimus, entrectinib, larotrectinib: all from Selleckchem; pasireotide ditrifuloroacetate (MedChem Express); cisplatin (Sandoz); carboplatin (Labatec). For drug screenings, organoids were dissociated with 0.25% Trypsin-EDTA (Gibco) and seeded at 1000 cells/well in 384-well plates in organoid expansion medium supplemented with 10% BME2. Two days later, compounds were added in a 2-fold dilution series ranging from 0.02 nM to 10 μM. After 6 days of treatment, cell viability was measured using CellTiter-Glo 3D (Promega). Luminescence was measured on a Synergy H1 Multi-Mode Reader (BioTek Instruments). Results were normalized to vehicle control (100% DMSO or 100% water). All experiments were performed twice. Dose-response curves were calculated using Prism 9.3.1 (GraphPad), nonlinear regression algorithm was used with a constrain of 0 for the bottom and 100 for the top.

Meier M.A., Nuciforo S., Coto-Llerena M., Gallon J., Matter M.S., Ercan C., Vosbeck J., Terracciano L.M., Soysal S.D., Boll D., Kollmar O., Delaloye R., Piscuoglio S, & Heim M.H. (2022). Patient-derived tumor organoids for personalized medicine in a patient with rare hepatocellular carcinoma with neuroendocrine differentiation: a case report. Communications Medicine, 2, 80.

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Characterization of NTRK Fusion Cell Lines

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The NTRK fusion human colorectal cancer cell line KM12SM was kindly gifted by Dr. I. J. Fidler (MD Anderson Cancer Center) to our laboratory in 1999. KM12SM cells, a highly metastatic variant of KM12C cells, have the TPM3‐NTRK1 gene rearrangement. HCC78 cells were obtained from American Type Culture Collection. The PC‐9 cell line with an EGFR exon 19 deletion was obtained from the RIKEN Cell Bank. A925L cells with EML4‐ALK variant 5a (E2:A20) were kindly provided by Dr. H. Uramoto (Kanazawa Medical University). Human lung fibroblasts MRC‐5 and IMR‐90 were obtained from RIKEN Cell Bank. The KM12SM and HCC78 cell lines were cultured in RPMI 1640 with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (10 mg/ml) in a humidified, 5% CO2 incubator at 37°C. The IMR‐90 and MRC‐5 cell lines were maintained in DMEM containing 10% FBS, penicillin (100 U/ml), and streptomycin (10 mg/ml). Entrectinib and capmatinib were obtained from Selleck Chemicals (Houston, TX, USA). Recombinant NRG, HGF, IGF‐1, FGF2, and amphiregulin were purchased from R&D Systems (Minneapolis, MN, USA). Goat anti‐human HGF neutralizing antibody and control goat IgG were obtained from R&D Systems as well.

Takumi Y., Arai S., Suzuki C., Fukuda K., Nishiyama A., Takeuchi S., Sato H., Matsumoto K., Sugio K, & Yano S. (2022). MET kinase inhibitor reverses resistance to entrectinib induced by hepatocyte growth factor in tumors with NTRK1 or ROS1 rearrangements. Cancer Medicine, 12(5), 5809-5820.

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Anticancer Potential of Kinase Inhibitors

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ALK inhibitors (crizotinib, ceritinib, entrectinib, alectinib, and ASP3026) and MEK inhibitor (trametinib) were purchased from Selleck Chemicals. Cells were treated with inhibitor concentrations ranging from 0.3 nmol/L to 10,000 nmol/L for either 72 hours (viability assays) or 2 hours (immunoblots). Controls were treated with DMSO alone.

Couts K.L., Bemis J., Turner J.A., Bagby S.M., Murphy D., Christiansen J., Hintzsche J.D., Le A., Pitts T.M., Wells K., Applegate A., Amato C., Multani P., Chow-Maneval E., Tentler J.J., Shellman Y.G., Rioth M.J., Tan A.C., Gonzalez R., Medina T., Doebele R.C, & Robinson W.A. (2017). ALK inhibitor response in melanomas expressing EML4-ALK fusions and alternate ALK isoforms. Molecular cancer therapeutics, 17(1), 222-231.

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Cell Line Maintenance and Compound Screening

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NIH3T3 cells were purchased from American Type Culture Collection (ATCC). Ba/F3, WEHI, and BOSC23 cells were kindly provided by Dr. Daniel G. Tenen (Harvard Medical School). Crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, entrectinib, gilteritinib, and repotrectinib were purchased from Selleck. NIH3T3 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin (P/S). Parental Ba/F3 cells were maintained in RPMI1640 supplemented with 5% WEHI (as a source of IL-3), 10% FBS, and P/S. Ba/F3 cells expressing CLIP1-LTK mutants were maintained in RPMI1640 supplemented with 10% FBS and P/S. All cell lines were routinely tested for mycoplasma infection and negative for mycoplasma infection.

Mori S., Izumi H., Araki M., Liu J., Tanaka Y., Kagawa Y., Sagae Y., Ma B., Isaka Y., Sasakura Y., Kumagai S., Sakae Y., Tanaka K., Shibata Y., Udagawa H., Matsumoto S., Yoh K., Okuno Y., Goto K, & Kobayashi S.S. (2024). LTK mutations responsible for resistance to lorlatinib in non-small cell lung cancer harboring CLIP1-LTK fusion. Communications Biology, 7, 412.

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Colon Cancer Cell Line Apoptosis Assay

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Human colon cancer cell lines KM12SM, HT29, and HCT116 were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum. The cells were cultured at 100% humidity and 5% CO₂ at 37 °C. The NTRK inhibitors LOXO-101, entrectinib, dovitinib, dovitinib lactate, dovitinib dilactic acid, regorafenib, and crizotinib were purchased from Selleck Chemicals (Houston, TX, USA). We used the annexin V-APC/propidium iodide (PI) apoptosis detection kit (Thermo Fisher Scientific, Rockford, IL, USA).

Sohn S.H., Sul H.J., Kim B., Kim B.J., Kim H.S, & Zang D.Y. (2021). TRK inhibitors block NFKB and induce NRF2 in TRK fusion-positive colon cancer. Journal of Cancer, 12(21), 6356-6362.

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GC Cell Line Maintenance and Compound Testing

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The human GC cell lines NCI-N87, SNU16, MKN7, MKN28, and AGS were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and maintained in RPMI-1640 supplemented with 10% fetal bovine serum. The cells were cultured at 37 °C with 100% humidity and 5% CO₂. LOXO-101, entrectinib, dovitinib, dovitinib lactate, dovitinib dilactic acid, regorafenib, cabozantinib, and crizotinib were purchased from Selleck Chemicals (Houston, TX, USA).

Sohn S.H., Sul H.J., Kim B.J., Kim H.S, & Zang D.Y. (2021). Entrectinib Induces Apoptosis and Inhibits the Epithelial–Mesenchymal Transition in Gastric Cancer with NTRK Overexpression. International Journal of Molecular Sciences, 23(1), 395.

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Clonal Growth Analysis of Mast Cell Lines

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To analyze clonal growth, HMC-1 cells and HMC-1.2 (purchased from Millipore) were plated in H4230 media (StemCell Technologies, Vancouver, Canada) in the presence of kinase inhibitors. The cells were plated as duplicates or quadruplicates. HMC-1 cells with ectopic expression of TRKB were generated by retroviral transduction using a vector expressing human TRKB [12 (link)]. We performed PCR for detection of mycoplasma in cell cultures [57 (link)] and did not detect contamination of mycoplasma in tested cell lines. Primary mast cells were cultured in the presence of inhibitors for 48 hours before apoptosis analysis. Cell viability was analyzed using the Annexin-V assay (BD Pharmingen, Heidelberg, Germany). Inhibitors dasatinib and entrectinib were purchased from Selleckchem (Houston, TX). NGF and BDNF were purchased from PeproTech. We chose dasatinib for KIT inhibition in this study, since dasatinib treatment showed benefit in some patients with SM [5 (link), 58 (link)], and dasatinib is currently being tested in our Phase III clinical trial for CBF AML including KIT D816V mutated patients (ClinicalTrials.gov number, NCT02013648).

Yang M., Pan Z., Huang K., Büsche G., Feuerhake F., Chaturvedi A., Nie D., Heuser M., Thol F., von Neuhoff N., Ganser A, & Li Z. (2017). Activation of TRKA receptor elicits mastocytosis in mice and is involved in the development of resistance to KIT-targeted therapy. Oncotarget, 8(43), 73871-73883.

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Molecular Profiling of Receptor Tyrosine Kinases

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Entrectinib, crizotinib, capmatinib, and afatinib were purchased from Selleck Chemicals. Antibodies to total AKT, AKT pS473, total ERK, ERK pT202/Y204, total ROS1, ROS1 pY2274, total MET, MET pY1234/1235, total AXL, total EGFR, EGFR pY1068, EGFR pY1086, EGFR pY1173, total HER2, HER2 pY1196, HER2 pY1221/1222, HER3 pY1289 were purchased from Cell Signaling Technologies. Antibody to GAPDH was purchased from Santa Cruz Biotechnologies Inc. MET and CEP7 interphase FISH probes were purchased from Abbott Molecular Inc. MET, CEP7, MYC, and CEP8 metaphase FISH probes were purchased from Empire Genomics.

Tyler L.C., Le A.T., Chen N., Nijmeh H., Bao L., Wilson T.R., Chen D., Simmons B., Turner K.M., Perusse D., Kasibhatla S., Christiansen J., Dudek A.Z, & Doebele R.C. (2022). MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC. Thoracic Cancer, 13(21), 3032-3041.

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