Abstract 247: Investigating the impact of HER4 mutations on intracellular signalling pathways

Abstract Background: HER4 is the least studied member of the HER/ErbB receptor tyrosine kinase family that also includes EGFR, HER2 and HER3. HER4 is frequently mutated in skin, lung and gastro-intestinal tumours but there are limited data on the impact of HER4 point mutations (MUTs) on intracellular signalling pathways. This study identifies 8 HER4 point MUTs (2 extracellular domain (ECD) and 6 intracellular domain (ICD)) and utilises plasmid-based overexpression to assess the impact of the HER4 point MUTs on intracellular signalling pathways in HEK293 cells. Methods: 8 HER4 point MUTs were identified using publicly available Catalogue Of Somatic Mutations in Cancer (COSMIC) data and Provean/SIFT analysis. 4 HER4 MUTs were deemed deleterious/damaging and 4 MUTs were classified as neutral or tolerated. MUT1 (p.E202D) and 2 (p.P224R) are ECD, MUT3-8 (p.T1036I, p.Y1242C, p.H1245N, p.S1246R, p.L1247M, p.H1255N) are ICD. Mutant primers were designed and constructed (Sigma Aldrich). Site-directed mutagenesis (QuikChange Lightning kit) was carried out using the pRK5 HER4 wild-type (WT) plasmid as template creating 8 HER4 MUT plasmids. Sequences were validated (SequiServe). HEK293 cells were transfected (Fugene) with either empty vector, GFP control, WT HER4 or the HER4 MUT plasmids. Transfection was confirmed by Western blot. Plasmid transfected HEK293 protein lysates were analysed in biological triplicate by reverse phase protein array (RPPA) to determine the levels of 50 total proteins and 34 phospho-proteins (n= 84 targets). Increases or decreases in protein levels were determined as significant (p<0.05, Student’s t test) vs appropriate control, WT HER4 vs. empty vector or MUT HER4 vs WT HER4. Results: HEK293 cells express WT EGFR and negligible levels of HER3 and HER4. Total EGFR levels were unchanged in response to WT HER4 transfection but EGFR phosphorylation at Y992 (p=0.01), and Y1068 (p<0.001) increased 15-fold and 2-fold, respectively. Total and phosphorylated Akt (S473 and T308) and MAPK-ERK1/2 (T202/Y204) were not impacted by WT HER4 transfection. Overexpression of WT HER4 resulted in increased levels of phosphorylated p38 MAPK (T180/Y182) (p=0.04), Src (Y416) (p<0.001) and STAT3 (Y705) (p<0.001). The 8 HER4 MUTs assessed had no impact on the activity or total expression of EGFR, Akt and Stat3. MUT2 decreased phosphorylated MAPK-ERK1/2 (p=0.01) and total p38 MAPK levels (p=0.04). MUT4 decreased phosphorylated MAPK-ERK1/2 levels (p<0.001). MUT6 decreased total p38 MAPK levels (p=0.03) and increased phosphorylated Src levels (p=0.01). MUT7 decreased levels of total MAPK-ERK1/2 (p=0.03) and total p38 MAPK (p<0.001). Conclusions: WT HER4 activates EGFR and downstream signaling through p38 MAPK, Src and STAT3. The HER MUTs investigated affected MAPK-ERK1/2, p38 MAPK and Src signaling pathways, suggesting phenotypic impact that supports a role in tumorigenesis. Citation Format: Denis M. Collins, Marta Valenti, Johanna Gaubatz, Birgit Bossenmaier, Bryan Hennessy, John Crown. Investigating the impact of HER4 mutations on intracellular signalling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 247.