Dacomitinib

Ba/F3 cells (20,000 cells/well) expressing ERBB4 variants were plated on 96-well plates in RPMI1640 medium containing 10 ng/mL NRG-1. Vector control cells were plated in RPMI1640 medium containing 5% WEHI conditioned medium. The cells were incubated in the presence or absence of 0.0005–10 µmol/L erlotinib (Santa Cruz Biotechnology), afatinib (Boehringer Ingelheim), neratinib (SantaCruz Biotechnology), dacomitinib (Cayman Chemicals), poziotinib (Selleck Chemicals), ibrutinib (Selleck Chemicals), or lapatinib (Santa Cruz Biotechnology), for 72 hours before measuring the viability of cells with MTT assay (described above). Sigmoidal dose–response curves were fitted using asymmetric five-parameter non-linear regression in GraphPad Prism 9 and are graphically displayed with GraphPad Prism 9. The IC₅₀ values are calculated with the help of the R package “drc” (34 (link)) by using quadruplicate measurements from the MTT assay for the indicated concentrations from 3–5 individual experiments and fitting four-parametric log-logistic models (LL.4, LL2.4; R). The unpaired t test (Welch two-sample t test) was used to compare IC₅₀ values.

¹³C₅-labeled Gln (catalog no. CLM-1822-H), alpha-¹⁵N Gln (catalog no. NLM-1016-PK), ¹³C₆-labeled glucose (catalog no. CLM-1396-PK), ¹⁵N-labeled alanine (catalog no. NLM-454) and ¹³C_x,¹⁵N_x-labeled amino acid standard mix (catalog no. MSK-A2) were acquired from Cambridge Isotope Laboratories. N-acetyl-l-cysteine (catalog no. A7250) dimethylsulfoxide (DMSO), methoxyamine hydrochloride and MTBSTFA + 1% t-BDMSC and DON (catalog no. D2141) were obtained from Sigma-Aldrich. Trametinib (catalog no. 16292) and dacomitinib (pan-RTK inhibitor, catalog no.9001879) were obtained from Cayman Chemical.