Abstract 5570: Characterizing a CRISPR/Cas9-mediated deletion in the extracellular domain of HER4 in a model of cutaneous melanoma

Abstract Introduction: HER4, a tyrosine kinase receptor belonging to the Human Epidermal Growth Factor/HER/ErbB-family, has a complex biology (four isoforms (JMa-CYT1/2 and JMb-CYT1/2) and tissue specific oncogenic or tumor suppressor roles). Cutaneous melanoma (CM) showed the highest HER4 genomic alteration frequency (17.34%) across 33 cancer types (cBioPortal). To investigate the potential role of HER4 in CM, the SKMEL24 CM cell line was CRISPR/Cas9-edited to contain HER4 with a 30bp deletion (DEL) in the extracellular domain. The HER4 30bp DEL was modelled in silico and the SKMEL24 HER4 30bp DEL cell line assessed for HER-family protein expression levels, HER4 isoform RNA levels, and HER4 receptor activation upon neuregulin-1 (NRG-1) stimulation. Moreover, cellular morphology, cellular proliferation and response to the Tyrosine Kinase Inhibitor (TKI) neratinib was assessed. Material and Methods: All studies compared the wild-type (WT) and the CRISPR/Cas9-edited HER4 30bp DEL SKMEL24 cell line. For in silico HER4 3D crystal structure modelling, input files for GROMACS 2020.7 molecular dynamics simulations were generated using CHARMM-GUI. The CHARMM36m force field was used for parameterization of protein chains. The GROMACS in-built tools were used to calculate heavy atom Root Mean Square Deviation (RMSD). HER-family protein levels (EGFR, HER2, HER3, HER4) and their phosphorylated forms (pEGFR Y1173, pHER2 Y1221, pHER3 Y1197, pHER4 Y984/Y1284) were investigated via Western blot. RNA was extracted from cells 24h after seeding using TRIzol, with qRT-PCR used to examine HER4 isoform expression. NRG-1 stimulation was performed in complete growth medium with HER4 receptor activation determined by Western blot. Cellular morphology and proliferation were monitored using the IncuCyte Live Cell Analysis Instrument (Sartorius). Acid phosphatase proliferation assays were used to assess the anti-proliferative effects of neratinib. Results: In silico modelling of the HER4 30bp DEL showed reduced HER4 receptor stability compared to WT (RMSD = 6.465 Å and 6.631 Å, respectively). No changes in expression or activation of EGFR, HER2, or HER3 were detected in the SKMEL24 HER4 30bp DEL cell line. Full-length (180kDa) HER4 expression was decreased and cleaved HER4 expression (80kDa) and activation (pHER4 Y984) were increased in the HER4 30bp DEL cell line. NRG-1 treatment did not activate HER4 but activated EGFR, HER2 and HER3 in the HER4 30bp DEL cell line. The HER4 30bp DEL cell line also displayed a more stellate phenotype, reduced proliferation over 3 days in culture, and an increased sensitivity to neratinib (IC50 = 0.17 ± 0.04 µM) compared to the WT (IC50 = 0.26 ± 0.04 µM). Conclusion: These findings suggest HER4 may play a role in cellular morphology, proliferation and response to treatment. Further studies are needed to clarify the specific role of HER4 in CM. Citation Format: Marta Valenti, Shubhangi Kandwal, Yonglun Luo, Georg F. Bischof, Darren Fayne, John Crown, Denis M. Collins. Characterizing a CRISPR/Cas9-mediated deletion in the extracellular domain of HER4 in a model of cutaneous melanoma [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 5570.