This CRISPR/Cas9-edited polyclonal knockout cell population is derived from NCI-H1975 human lung adenocarcinoma cells, featuring targeted disruption of the HERPUD1 gene. As a heterogeneous mixture of edited cells, the polyclonal format preserves the host genetic background while avoiding clonal selection artifacts, enabling robust assessment of HERPUD1 loss-of-function effects. The knockout model is designed for direct use in functional studies examining endoplasmic reticulum (ER) stress adaptation, protein quality control, and the unfolded protein response (UPR) in a defined oncogenic setting.
NCI-H1975 cells, isolated from the pleural effusion of a non-small cell lung cancer patient, carry the EGFR L858R activating mutation and the T790M resistance mutation. This dual-mutant adenocarcinoma model is extensively utilized to investigate EGFR-driven oncogenic signaling and acquired resistance to tyrosine kinase inhibitors. The cells?? inherent dependency on proteostatic mechanisms to cope with oncogenic stress makes them particularly suitable for studying the consequences of impaired ER-associated degradation (ERAD).
HERPUD1 is a UPR target gene transcriptionally induced by ATF6 and spliced XBP1 under ER stress. It functions as an organizing scaffold for the ERAD machinery, binding the VCP/p97 ATPase, Derlin-1, and the HRD1 E3 ligase to promote retrotranslocation and proteasomal degradation of misfolded ER proteins. HERPUD1 also regulates ER calcium homeostasis and modulates apoptosis by engaging Bcl-2 family factors, ultimately influencing CHOP-mediated cell death. Additionally, it interacts with presenilins and amyloid precursor protein, connecting proteostasis networks across multiple disease contexts.
Disrupting HERPUD1 in NCI-H1975 cells is expected to cripple ERAD, leading to persistent ER stress and sensitization to apoptosis, particularly upon further UPR stimulation by agents like tunicamycin. In this EGFR-mutant background, loss of HERPUD1 may alter oncogenic signaling dynamics and drug sensitivity, offering a model to explore synthetic vulnerabilities or adaptive responses that contribute to therapeutic resistance. The knockout population is thus a powerful tool to dissect the interplay between the UPR and mutant EGFR networks.
Researchers can employ this model for mechanistic studies of ER stress tolerance using western blotting of UPR markers (BiP, CHOP, spliced XBP1), RT-qPCR for XBP1 splicing and CHOP expression, and cell viability assays with ER stress inducers. Interaction assays such as co-immunoprecipitation with VCP/p97 and immunofluorescence for calnexin or ubiquitin puncta can validate ERAD disruption. Functional readouts including proteasome activity and Annexin V apoptosis assays enable screening of agents that synergize with ER stress or circumvent drug resistance. Applications include elucidating crosstalk between UPR and EGFR signaling and evaluating HERPUD1 as a therapeutic target in NSCLC. For further technical details, contact Ascent Research.
