Abstract 3962: Development of selection-free HER4-mutated CRISPR/Cas9 knock-ins in cutaneous melanoma cell lines

Abstract Background: Cutaneous melanoma showed the highest HER4 genomic alteration frequency (17.34%) across 33 different cancer types (cBioPortal). HER4 is a tyrosine kinase receptor belonging to the human epidermal growth factor receptor (HER/ErbB) family. The role of HER4 in cancer has not been fully studied, potentially due to its complex biology (four isoforms, different stability/tissue-specific expression patterns, and oncogenic/tumor suppressor roles). Using computational analysis, we identified two potentially oncogenic HER4 mutations in melanoma (HER4 R106C and R711C). We then developed CRISPR/Cas9-modified selection-free melanoma cell lines for interrogating the role of these mutations as biomarkers of response to the oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER). Methods: Two melanoma cell lines (WM115, SKMEL24) were genome-edited by using a selection-free CRISPR/Cas9 approach. Single guide RNAs (sgRNAs) were designed by using the CRISPRon (v1.0) web tool, and Homology Directed Repair (HDR) single-stranded OligoDeoxyriboNucleotides (ssODNs), containing the HER4 mutations, were designed with the SnapGene Software (v6.1.1). The RNP complex (Cas9 and sgRNAs) and the HDR_ssODNs, for each HER4 mutation, were delivered into the melanoma cells through electroporation. Upon validation of the CRISPR/Cas9-efficiency via PCR and Sanger Sequencing, the CRISPR/Cas9-modified cells were single-cell sorted via FACS, by using two singlets gating (FSC-A/FSC-H and SSC-A/SSC-H) and a live/dead (propidium iodide) gating, to perform single-cell cloning. Colonies were then amplified and screened via PCR and Sanger sequencing to isolate pure selection-free HER4 CRISPR/Cas9 knock-ins clones of melanoma cells. Results: The WM115 and SKMEL24 melanoma cell lines were found to be wild-type for the HER4 receptor in the Depmap Portal (mutation 22Q2, 2022 data release 2). R106C and R711C CRISPR/Cas9 knock-ins were created in the melanoma cell lines three days after the electroporation of the CRISPR/Cas9 complex. The efficiency of the HER4 R106C CRISPR/Cas9 knock-in was 22% and 7% in the SKMEL24 and WM115 melanoma cells, respectively, with a respective total indel percentage of 76% (SKMEL24) and 55% (WM115). The HER4 R711C CRISPR/Cas9 knock-in efficiency was 15% in the SKMEL24 (with 26% total indels) and 1% in the WM115 (with 33% total indels). Single-cell colonies of the HER4 CRISPR/Cas9-mediated knock-ins (HER4 R106C and R711C), were formed at four days (SKMEL24) and at one week (WM115) after single-cell sorting. Conclusions: CRISPR/Cas9 technology can be utilised to introduce single-point mutations in HER4 to melanoma cell lines. In vitro functional characterisation of the isolated HER4 CRISPR/Cas9-edited melanoma cell lines will be performed in order to understand the activating potential of HER4 R106C and R711C and their sensitivity to NER. Citation Format: Marta Valenti, Yonglun Luo, Neil T. Conlon, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Development of selection-free HER4-mutated CRISPR/Cas9 knock-ins in cutaneous melanoma cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3962.