The IMPACT Knockout NCI-H1975 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population with disrupted IMPACT gene in the NCI-H1975 human lung adenocarcinoma line. This polyclonal format provides a heterogeneous pool of cells carrying diverse gene-editing events, making it well-suited for pooled functional genomics studies of the integrated stress response in EGFR-mutant non-small cell lung cancer.
The NCI-H1975 cell line was derived from a female patient with non-small cell lung carcinoma and harbors the EGFR L858R and T790M mutations, which drive constitutive signaling and mediate resistance to first- and second-generation EGFR tyrosine kinase inhibitors. As a widely accepted model for acquired resistance to third-generation inhibitors such as osimertinib, these cells are instrumental for exploring mechanisms of drug tolerance and for preclinical therapeutic testing in EGFR-mutant lung adenocarcinoma.
IMPACT encodes an evolutionarily conserved protein that functions as a negative regulator of the GCN2-dependent integrated stress response (ISR). Mechanistically, IMPACT binds the ribosome-associated scaffold GCN1, thereby inhibiting GCN1-mediated activation of the kinase GCN2. This interaction prevents GCN2 from phosphorylating eIF2??, a translation initiation factor, which in turn suppresses the translation of ATF4 mRNA and downstream expression of the pro-apoptotic transcription factor CHOP. The IMPACT?CGCN1?CGCN2 axis is modulated by inputs from amino acid sufficiency, mTORC1 activity, and the transcription factor ATF4, embedding it within a network that controls protein synthesis and cell fate under nutrient stress.
In EGFR-mutant NCI-H1975 cells, the ISR contributes to cell survival under the metabolic stresses imposed by rapid proliferation and drug exposure. IMPACT-mediated inhibition of GCN2 may provide a protective mechanism that raises the threshold for stress-induced apoptosis. Disruption of IMPACT in this background thus creates a sensitized system where loss of restraint on GCN2 signaling allows for dissection of how sustained ISR activation impacts EGFR TKI susceptibility, stress granule dynamics, and translational reprogramming in lung adenocarcinoma.
This polyclonal knockout model enables detailed investigation using standard assays such as immunoblotting for phospho-eIF2?? and eIF2??, RT-qPCR for ATF4 and CHOP, and cell viability measurements under amino acid deprivation. Additional applications include co-immunoprecipitation to verify IMPACT?CGCN1 complex disruption, puromycin incorporation for global translation monitoring, immunofluorescence for stress granule formation, and GCN2 kinase activity assays. The cells are also suitable for synthetic lethal screens with GCN2 inhibitors and for evaluating ISR-targeted compounds in combination with EGFR inhibitors. For technical inquiries, please contact Ascent Research.
