Poziotinib

The interleukin‐3–dependent murine pro–B‐cell line Ba/F3 was provided by Riken Bio Resource Center. Cells were cultured in RPMI 1640 medium (Wako) with 10% FBS (Sigma‐Aldrich). EGFR‐TKIs (afatinib, poziotinib, and osimertinib) were purchased from Selleck Chemicals. Tarloxotinib and tarloxotinib‐E were provided by Rain Therapeutics, Inc. Mycoplasma contamination was checked routinely using the TaKaRa PCR Mycoplasma Detection Set (Takara).
Each EGFR exon 20 mutation was introduced into the Ba/F3 cells by retroviral vectors as previously described.¹³ We generated A763insFQEA, D770insSVD, and H773insNPH mutations from full‐length complementary DNA that encoded the wild‐type EGFR gene (Addgene), using the Prime STAR Mutagenesis Basal Kit (Takara) with specific primers (Table S1). Full‐length complementary DNA that encoded V769insASV and H773insH was purchased from Addgene. We also used Ba/F3 cells with secondary mutations, plus either EGFR exon 19 deletion or L858R point mutation, which our previous study showed to be potential factors in resistance to frontline osimertinib.¹³

We individually validated 8 compounds in same concentration (20 μM). Here, we exposed the drug for 16 h either at 32 or 37°C, and evaluated by flow cytometry as previously described. In a 96-well plate 20,000 of either 293 or 293+SP1-MHI cells were seeded per well in a total volume of 200 μL of media. After cells had attached to the wells (the morning after), cells were exposed to 20 μM of each drug, controls were exposed to the same quantity of DMSO as drug, for 16 h and harvested for flow cytometry analysis. Flow cytometry analysis was conducted as previously described. Drugs that were tested for further validation after initial screen were: Orantinib (Selleckchem, S1470), Dasatinib (Selleckchem, S7782), Poziotinib (Selleckchem, S7358), Tamoxifen Mylan (Mylan AB*), Erythromycin (Stragen Pharma GmbH*), Naloxone (B. Braun*), Entacapone (Orion Corporation*) and Cinacalcet (Ratiopharm GmbH*). Drugs labeled with “*” were pharmaceutical grade medications gifted from the Pharmacy at Landspítali University Hospital. Analysis was performed as described above.