Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors

Abstract

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.

Systematically developing a registry of splice-site creating variants utilizing massive publicly available transcriptome sequence data

“…In an effort to conduct a detailed functional analysis of SSCVs, we established CRISPR genome editing models derived from PC-9 46,47 , a lung cancer cell line, for each SSCV (c.5048-132G>C and c.5048-132G>T). We successfully introduced the NOTCH1 deep intronic SSCV into the cell line, confirming a 129 bp exon extension (Figure 6c, Supplementary Figure 20).…”

“…NOTCH1 SSCVs (c.5048-132G>C, c.5048-132G>T) were created in the PC-9 cell lines as previously reported 46,47 . We designed sgRNA and donor templates for CRISPR-Cas9 homology directed repair (HDR) using Alt-R HDR Design Tool (Integrated DNA Technologies, IDT).…”

“…Since the transcript induced by the SSCVs is expected to elongate the same juxtamembrane region, we considered the possibility that these SSCVs could cause similar activation (Figure 6b). In an effort to conduct a detailed functional analysis of SSCVs, we established CRISPR genome editing models derived from PC-9 46,47 , a lung cancer cell line, for each SSCV (c.5048-132G>C and c.5048-132G>T). We successfully introduced the NOTCH1 deep intronic SSCV into the cell line, confirming a 129 bp exon extension (Figure 6c, Supplementary Figure 20).…”

Combined Dacomitinib and Selpercatinib Treatment for a Patient with EGFR-Mutant Non-Small Cell Lung Cancer and Acquired CCDC6-RET Fusion

“…Kobayashi et al conducted a comprehensive study on fusion genes and their role in mediating resistance to EGFR TKIs in EGFR- mutant lung cancer. 12 Specifically, they identified that only a minority of detected fusion genes, such as CCDC6-RET , are functional and capable of imparting resistance to EGFR inhibitors. In a brief review, Zhao et al reported that the co-occurrence of EGFR mutations and RET fusions in patients with EGFR -mutant NSCLC is predominantly induced by osimertinib, a third-generation EGFR-TKI.…”

Mechanistic patterns and clinical implications of oncogenic tyrosine kinase fusions in human cancers

“…1). Inspired by the recent reports indicating fusion oncogene formation as a mechanism of resistance to EGFR inhibitors 7,20,21 , we applied FACTS into the in vitro model of EGFR inhibitor resistance using PC-9 cells, harboring EGFRactivating mutation (EGFR E746-A750del) 22 .…”