Dasatinib

Manufactured by LC Laboratories

Sourced in United States, Macao, United Kingdom

Dasatinib is a tyrosine kinase inhibitor. It is a small-molecule drug designed to inhibit the activity of several protein kinases, including BCR-ABL, SRC family kinases, c-KIT, PDGFR, and EPHA2.

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115 protocols using dasatinib

Collagen-Based 3D Cell Culture Assay

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Cells were brought to suspension, counted, and centrifuged (4 min, 200 rcf, 25 °C). The cell pellets were resuspended, mixed with collagen solution on ice, and plated in wells. After 15 min incubation at 37 °C, the gelled samples were overlaid with cultivation medium containing 1% FBS. The resulting composition of the collagen matrix was 1 mg/mL rat-tail collagen, 1×RPMI medium, 15 mM HEPES, 1% fetal bovine serum, and 50 μg/mL gentamicin. For details about the number of cells and the amount of collagen matrix for respective approaches, see the respective part of the Methods. For induction of inducible constructs (icaRhoA), 250 nM doxycycline (Sigma, Piscataway, NJ, USA) was used. For induction of MAT by dasatinib treatment, 1 μM dasatinib (LC Laboratories, Woburn, MA, USA) was used. The concentration of ROCK inhibitor Y-27632 (Sigma, Piscataway, NJ, USA) was 10 μM.

Merta L., Gandalovičová A., Čermák V., Dibus M., Gutschner T., Diederichs S., Rösel D, & Brábek J. (2020). Increased Level of Long Non-Coding RNA MALAT1 Is a Common Feature of Amoeboid Invasion. Cancers, 12(5), 1136.

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Dasatinib Inhibits C4-2B Cell Invasion

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For cells to be treated with Dasatinib (Catalog # D3307, LC Laboratories, Woburn, MA), C4-2B cells were plated on the upper chamber of Matrigel-coated transwell filters (2 × 10⁵ cells/filter) in growth media, supplemented with 1 % FBS, containing 0.5 μM Dasatinib or vehicle control. For Pertussis toxin (PTX) (Catalog # 516561, Calbiochem, La Jolla, CA) studies, cells were pretreated with 200 ng/ml PTX for 3 h prior to cell invasion studies. For cells to be treated with Src siRNA, C4-2B cells were transfected with scrambled or Src siRNA using Lipofectamine 2000 (Invitrogen) 24 h prior to seeding 2 × 10⁵ cells in serum free medium on Matrigel coated inserts. For both Dasatinib and siRNA conditions, cells were allowed to invade for 24 h in the presence or absence of 200 ng/mL CXCL12 added to the bottom chamber. Cotton swabs were used to remove non-migrated cells from the upper chamber, and inserts were stained with 0.9 % crystal violet. Total number of migrated cells was counted under 10X magnification in five fields. Assays were performed in triplicate. *: p < 0.05; **: p ≤ 0.005. For protein analysis, cells were treated with Dasatinib or Src siRNA as performed for the invasion assays, in the presence or absence of CXCL12. Cell lysate was collected after 24 h and analyzed by Western blot.

Conley-LaComb M.K., Semaan L., Singareddy R., Li Y., Heath E.I., Kim S., Cher M.L, & Chinni S.R. (2016). Pharmacological targeting of CXCL12/CXCR4 signaling in prostate cancer bone metastasis. Molecular Cancer, 15, 68.

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Inhibitor Dissolution and Storage

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AZD2014, MLN0128 and dasatinib were purchased from LC laboratories. Each inhibitor was dissolved in DMSO to the final concentration of 1 mM and stored at –80°C.

Gotesman M., Vo T.T., Herzog L.O., Tea T., Mallya S., Tasian S.K., Konopleva M, & Fruman D.A. (2018). mTOR inhibition enhances efficacy of dasatinib in ABL-rearranged Ph-like B-ALL. Oncotarget, 9(5), 6562-6571.

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Dasatinib Treatment for Leukemic Engraftment

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After substantial engraftment of leukemic cells was confirmed by BM aspiration, mice (n = 2 per group) were treated with dasatinib (LC laboratories, Woburn, MA) or vehicle. dasatinib was formulated in 0.5% (w/v) methylcellulose solution (Wako, Japan) and delivered orally at 35 mg/kg for 5 days (Monday to Friday) on/2 days off. Leukemic cell chimerism was monitored by flow cytometry analysis of BM aspirates, as described above. Once leukemic cell chimerism in BM reached <5%, the dasatinib dose was reduced to 20 mg/kg. After chimerism had recovered to >90%, mice were sacrificed by cervical dislocation, the femur, tibia, ilium, and brain were removed and mechanically dispersed, and leukemic cells were then isolated by Ficoll–Hypaque density gradient centrifugation and used for FISH analysis.

Tanaka K., Kato I., Dobashi Y., Imai J., Mikami T., Kubota H., Ueno H., Ito M., Ogawa S., Nakahata T., Takita J., Toyoda H., Ogawa C., Adachi S., Watanabe S, & Goto H. (2022). The first Japanese biobank of patient‐derived pediatric acute lymphoblastic leukemia xenograft models. Cancer Science, 113(11), 3814-3825.

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Establishing BTZ-resistant MCL cell lines

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Human MCL cell lines Jeko1 and Mino were purchased from the American Type Culture Collection (Manassas, VA, USA). We established BTZ-resistant Jeko1 and Mino cell lines by continuous exposure to increasing concentrations of BTZ over 6 months. The resulting stable BTZ-resistant cell lines were designated Jeko1/BTZ and Mino/BTZ. All cells were cultured in RPMI-1640 medium with 10% fetal bovine serum. BTZ and dasatinib were purchased from LC Laboratories (Boston, MA, USA) and stored as 10 mM stock solutions at −70°C. The Src kinase inhibitor PP2 was purchased from Calbiochem (San Diego, CA, USA).

Kim A., Seong K.M., Kang H.J., Park S, & Lee S.S. (2015). Inhibition of Lyn is a promising treatment for mantle cell lymphoma with bortezomib resistance. Oncotarget, 6(35), 38225-38238.

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In vivo Evaluation of SJ11646 in T-ALL

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For in vivo efficacy evaluation of SJ11646, primary human T-ALL cells from each case were injected into female NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice between 8–12 weeks of age through tail vein (2 million cells/mouse, resuspended in 200μl sterile PBS). Health status of all injected mice were monitored every day. SJ11646 or Dasatinib was given to T-ALL bearing NSG mice starting at three days post injection. SJ11646 was dissolved in 5% NMP + 40% PEG400 + 5% Solutol HS-15 + 0.5% polyvinyl alcohol (50%) + 49.5% saline; Dasatinib (LC Laboratories, #D-3307) was dissolved in 4% DMSO + 30% PEG300 + 5% Tween 80 + 61%. SJ11646 and Dasatinib were administrated once daily through intraperitoneal injection at 15 mg/kg and 10 mg/kg, respectively. Starting from two weeks after injection, peripheral blood was obtained by retro-orbital bleeding and subjected to flow cytometry to determine the level of human leukemia (humanCD45 and humanCD7 double positive) weekly. Mice with human blast percentage over 75% in blood or sign of moribund were considered lethal in survival analysis and euthanized.

Hu J., Jarusiewicz J., Du G., Nishiguchi G., Yoshimura S., Panetta J.C., Li Z., Min J., Yang L., Chepyala D., Actis M., Reyes N., Smart B., Pui C.H., Teachey D.T., Rankovic Z, & Yang J.J. (2022). Preclinical Evaluation of Proteolytic Targeting of LCK as a Therapeutic Approach in T-cell Acute Lymphoblastic Leukemia. Science translational medicine, 14(659), eabo5228.

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FRA-1 Modulation in Cancer Cells

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Dasatinib (LC Laboratories, Woburn, MA, USA), trametinib/dabrafenib/selumetinib/sorafenib (Selleckchem, Houston, TX, USA) and PP2 (Calbiochem, Gibbstown, NJ, USA) were dissolved in dimethylsulfoxide (DMSO) (Bioshop Canada, Burlington, ON, Canada) and added to fresh medium at the indicated concentrations.
Ten nanomoles of siRNA duplex (fosl1 Smart pool: L-004341-00 (GE Healthcare Dharmacon Inc, Lafayette, CO, USA), plaur [29 (link)] or Scrambled (sequences listed in Additional file 1: Table S1) was transfected into cells using RNAiMax according to the manufacturers protocol (Life Technologies Inc., Burlington, ON, Canada). For the rescue of FRA-1 expression, two fosl1 small interfering RNAs (siRNAs) that target the 3’ UTR were used (Additional file 1: Table S1).
The cDNA for fosl1 was purchased from GE Healthcare Bio-Sciences Company (Lafayette, CO, USA) and cloned into an expression vector to add an HA-tag to the N-terminus. Phospho-deficient and phospho-mimetic versions were created using Quick-change mutagenesis (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s directions. Sequences for the oligonucleotides used to make these mutants are listed in Additional file 1: Table S1.

Annis M.G., Ouellet V., Rennhack J.P., L’Esperance S., Rancourt C., Mes-Masson A.M., Andrechek E.R, & Siegel P.M. (2018). Integrin-uPAR signaling leads to FRA-1 phosphorylation and enhanced breast cancer invasion. Breast Cancer Research : BCR, 20, 9.

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Melanoma Cell Lines and Drug Responses

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Cells were obtained as authenticated stocks from the ATCC (VA, USA) (WM115, SK-MEL-1, SK-MEL-5, Malme-3 M, MeWo, SK-MEL-2), the Wistar Institute (PA, USA) (WM35, WM3211, WM1366, WM1791C, WM3060) and the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) (Mel Juso). Five cell lines carried activating BRAF mutations (WM115, WM35, SK-MEL-5, SK-MEL-1, Malme-3 M), four cell lines NRAS mutations (Mel Juso, WM3060, SK-MEL-2, WM1366), one cell line a c-KIT mutation (WM3211), and two cell lines were BRAF/NRAS/c-KIT wildtype (WM1791C, MeWo). Cells were cultured in DMEM (Lonza, Slough, UK) supplemented with 4 mM L-glutamine, 4.5 g/l glucose, 10% (w/v) heat inactivated fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich). Cells were grown at 5% CO₂ and 37 °C. To study treatment responses, cells were seeded into flat bottom 96 well plates and treated with dasatinib (LC Laboratories, MA, USA), dacarbazine (Medac GmbH, Germany) or combinations of both drugs. Drugs were added simultaneously.

Skoko J., Rožanc J., Charles E.M., Alexopoulos L.G, & Rehm M. (2018). Post-treatment de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma. BMC Cell Biology, 19, 28.

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Establishing CML Cell Line Models

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CML cell lines KCL-22, K562 and KU812 were purchased from German Collection of Cell Cultures, Braunschweig, Germany, and grown in RPMI 1640 medium with 10% fetal bovine serum (Hyclone, SH30071.03). KCL-22M cells harboring T315I BCR-ABL mutation were derived from KCL-22 cells [11] (link). Imatinib, nilotinib and dasatinib were purchased from LC Laboratories (Woburn, MA). ATRA was purchased from Sigma.

Wang Z., Liu Z., Wu X., Chu S., Wang J., Yuan H., Roth M., Yuan Y.C., Bhatia R, & Chen W. (2014). ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance. PLoS Genetics, 10(6), e1004414.

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Inhibitor Compound Preparation Protocol

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ARQ 531 was synthesized and provided by ArQule, Inc., (Burlington, MA). Quizartinib, midostaurin, and dasatinib were purchased from LC Laboratories (Woburn, MA). Gilteritinib and venetoclax were purchased from Chemietek (Indianapolis, IN). Entospletinib (GS-9973) was purchased from Selleckchem (Houston, TX). All chemical compounds were dissolved in Dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution.

Elgamal O.A., Mehmood A., Jeon J.Y., Carmichael B., Lehman A., Orwick S.J., Truxall J., Goettl V.M., Wasmuth R., Tran M., Mitchell S., Lapalombella R., Eathiraj S., Schwartz B., Stegmaier K., Baker S.D., Hertlein E, & Byrd J.C. (2020). Preclinical efficacy for a novel tyrosine kinase inhibitor, ArQule 531 against acute myeloid leukemia. Journal of Hematology & Oncology, 13, 8.

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