Fig. 1. MAPK1 mutations found in Asian HNSCC are drivers for growth.

a Table showing HNSCC cases with somatic MAPK1 mutations in the US-TCGA-HNSCC Provisional cohort (N = 512 tumors) and the Asian HK-HNSCC cohort (N = 105 tumors). b Mapping of mutation sites of the MAPK1 gene based on the pan-cancer data from TCGA (refs ^9,10) and the COSMIC database (ref. ¹¹). Each mutational event is represented by one triangular symbol. Color annotation of various cancer types are shown at the bottom. c Conserved regions of the MAPK1 (ERK2) proteins across species around amino acid positions p.D321 and p.R135 are shown. The amino acid residues of the KIM-docking site are indicated by red arrows. d The X-ray crystallography structure of the human MAPK1 (ERK2) protein (locked with the ATP competitive inhibitor 5-Iodotubercidin and the allosteric inhibitor peptide-type ERK2 inhibitor; PDB ID: 5AX3 (ref. ¹³); MMDB ID: 136379 (ref. ¹⁴). Amino acid residues R135, D321, and E322 are highlighted in red, blue, and green, respectively. Residue R135 is 9.0 Å away from E322 and 11.3 Å away from D321. The peptide sequence of the KIM domain is highlighted and labeled in yellow. e The same X-ray crystallography structure of MAPK1 protein showing the peptide sequence of the ATP-binding domain highlighted in yellow, and the ATP molecule shown in gray color. f Driver activity assay, by MTT assay, of FaDu cells that ectopically expressed MAPK1-WT, MAPK1p.D321N, and MAPK1p.R135K mutants. Cells were seeded on a 48-well plate at a density of 1.2 × 10⁴ cell/well with DMEM and 5% FBS. MTT assay were conducted at 96 h after seeding. A cumulative graph of three independent repeats is shown (total N ≥ 14 wells). Driver activity was normalized against MAPK1-WT. The MAPK1p.D321N is a driver for FaDu cell growth (P < 0.0001; 88.65% ± 1.262 SEM), while the MAPK1p.R135K moderately suppresses cell growth (P < 0.0001; 122.3% ± 4.060 SEM). g Western blotting showing the level of p-EGFR(Y1173), t-EGFR, p-MAPK, and t-MAPK in FaDu cells expressing MAPK1-WT, MAPK1p.R135K, and MAPK1p.D321N mutants, respectively. The p-EGFR and total EGFR levels were normalized to actin, and shown as bar graphs. Three independent repeats were performed and all repeats showed similar trends.