Dasatinib

MMAE (ChemScene, Deerpark, NJ; CS-0837), LY294002 (Abcam; ab120243), dasatinib (Abcam; ab142050), MLR-1023 (Sigma-Aldrich, St. Louis, MO; SML0361), and cabozantinib (Selleck Biotech, Tokyo, Japan; BMS-907351) were dissolved in DMSO (Sigma-Aldrich; 07-4860-5), which was also used as the vehicle control (final concentration 0.1% DMSO).

NSML (Ptpn11^Y279C/+) mice were obtained from The Jackson Laboratory (Stock number: 026759). NSML male mice were crossed with C57BL/6J female, and offspring were genotyped by PCR for the Ptpn11 Y279C allele. Dasatinib (Biovision) was suspended in vehicle (1% DMSO in citrate buffer). Dasatinib was injected daily (i.p.) into mice at the age of 8 weeks for 4 weeks for the PK study and echocardiography, and at 14 weeks of age for 4 weeks for RNA-seq analysis. Animals were housed and cared for in facilities run by the Division of Animal Care and were routinely monitored by the veterinary staffs. Animal handling was approved by Yale University Institutional Animal Care and Use Committee.

Erlotinib was kindly provided by F. Hoffman-La Roche Ltd; TPX-0005 was from TP Therapeutics, Inc.; dasatinib was purchased from Bio Vision; sorafenib was from Toronto Research Chemicals Inc; the other inhibitors were from Selleck Chemicals. Anti-HER2 and anti-pHER2 antibodies were purchased from Upstate Biotechnology; anti-EGFR, anti-pEGFR, anti-pHER3, anti-MET anti-pMET, anti-ERK1/2, anti-pERK1/2, anti-AKT, anti-pAKT (T308 or S473), anti-STAT3, anti-pSTAT3, anti-PTEN, anti-AXL, anti-CDCP1, anti-pCDCP1, anti-SRC, anti-FYN, anti-LYN, anti-YES, anti-LCK, anti-pSFK (Y416), anti-β-catenin, anti-S6K, and anti-pS6K antibodies were from Cell Signaling Technology; anti-E-cadherin antibody was from BD biosciences; anti-HER3 antibody was from Santa Cruz Biotechnology; anti-β-actin antibody was from Abcam, Inc.; Anti-GAPDH antibody was from PROMEGA; α-tubulin antibody was sourced from Sigma-Aldrich.

Peptide–MHC tetramer binding to cognate T-hybridoma was assessed by flow cytometry. Tetramers were prepared via five equal additions of streptavidin-phycoerythrin (Prozyme, Hayward, CA USA) totaling an equimolar amount to biotinylated pMHCs. For tetramer binding assays, 150,000 cells were plated per 96-well plate and pre-treated with 50 nM dasatinib (Bio Vision Inc., Milpitas, CA, USA) for 30 min at 37 °C in complete media. Media formulation for HA5D3.9 and Col_II-26.B6.18 is IMDM supplemented with 20% FBS and for DR4.CII.23.5 is DMEM supplemented with 10% FBS, Glutamax, pen-strep, and 50 mM beta-mercaptoethanol. Tetramers were then added at indicated concentrations and incubated for 1 h at room temperature. Anti-CD3-APC (Biolegend, San Diego, CA USA) was then added and incubated for 30 min at 4 °C. Cells were washed 1× in PBS/BSA before fixing for 10 min at room temperature with Cytofix (BD). Data were acquired on the CytoFLEX LX (Beckman-Coulter, Brea, CA USA) and analyzed with Cytobank software.

HL-60, a human non-APL AML cell line, was obtained from the Health Science Research Resources Bank (Tokyo, Japan). ATRA and VD3 were purchased from Sigma (St. Louis, MO, USA) and dissolved in ethanol to obtain a final concentration of 1 mM and 100 μM, respectively, and stored at −20 °C in the dark. Dasatinib was purchased from BioVision (Mountain View, CA, USA) and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mM. Phycoerythrin (PE)-conjugated mouse anti-human CD11b IgG, fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD11c IgG, and the PE and FITC-conjugated isotype control IgG were obtained from Becton–Dickinson (San Jose, CA, USA). FITC-labeled polyclonal antibodies for total and phosphorylated Lyn (Y396) were purchased from Bioss (Woburn, MA, USA). Rabbit anti-human c-Myc polyclonal antibody and its negative control (non-binding rabbit IgG) were purchased from GeneTex (Irvine, CA, USA). FITC-labeled goat anti-rabbit polyclonal IgG antibody (secondary antibody) was purchased from Jackson ImmunoResearch (West Grove, PA, USA).

Erlotinib was kindly provided by F. Hoffman-La Roche Ltd, gefitinib was provided by AstraZeneca Inc; VS-4718 was provided by Verastem Inc; afatinib, osimertinib, lapatinib, AZD4547, and BIBF1120 were purchased from Selleck Chemicals; SU11274 and picropodophyllin were from Carbiochem; dasatinib was from Bio Vision; SB203580 was from Cayman Chemical; sorafenib was acquired from Toronto Research Chemicals Inc, cisplatin was from Bristol-Myers Squibb Company; and PD173074 was from Sigma-Aldrich.
Anti-HER2 and anti-pHER2 antibodies were purchased from Merck Millipore Corporation, anti-EGFR, anti-pEGFR, anti-pHER3, anti-HER4, anti-pHER4, anti-pc-Met, anti-IGF1Rβ, anti-pIGF1Rβ, anti-PDGFRβ, anti-pPDGFRβ, anti-FGFR1, anti-pFGFR, anti-ERK1/2, anti-pERK1/2, anti-AKT, anti-pAKT, anti-STAT3, anti-pSTAT3, anti-PTEN, anti-SRC, anti-FYN, anti-LYN, anti-YES, anti-LCK, anti-pSRC family (Y416), anti-pSRC (Y527), anti-FAK, anti-pFAK (Y397), anti-pFAK (Y576/577), anti-pFAK (Y925) and anti- EGFR (del E746-A750) antibody were from Cell Signaling Technology, anti-HER3 and anti-c-Met were from Santa Cruz Biotechnology Inc, anti-β-actin was from Abcam, Inc., and anti-α-tubulin was from Sigma-Aldrich.

Dasatinib was purchased from BioVision (Mountain View, CA) and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 300 μmol/L. Imatinib and nilotinib were purchased from Tokyo Chemical Industry (Tokyo, Japan) and ChemScene (Monmouth Junction, NJ) and dissolved in DMSO at a concentration of 2 mmol/L, respectively.
Antibodies for immunophenotyping, including phycoerythrin (PE)‐conjugated anti‐CD56 IgG, PC5‐conjugated anti‐CD8 IgG, PC7‐conjugated anti‐CD3 IgG, and each isotype control IgG were purchased from Beckman Coulter (Brea, CA). Fluorescein isothiocyanate (FITC)‐labeled polyclonal rabbit IgG antibodies for phosphorylated proteins, including pJAK1 (Y1034, #3238R), pJAK2 (Y1007, #2485R), pSTAT1 (Y701, #1657R), pSTAT3 (Y705, #1658R), pERK (T202/Y204, #1646R), pJNK (T183/Y185, #1640R), pp38 (T180/Y182, #2210R), pAKT (Y315, #5193R), and isotype control (#0295P) were purchased from Bioss (Wobun, MA, USA).