The HACL2 Knockout NCI-H1975 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal knockout cell population designed for loss-of-function analysis of the HACL2 gene in a human lung adenocarcinoma background. This polyclonal pool contains a mixture of cells with targeted gene disruptions at the HACL2 locus, allowing researchers to study the functional consequences of impaired peroxisomal alpha-oxidation while avoiding the clonal selection biases often inherent to single-cell-derived knockouts.
The host cell line, NCI-H1975, is an adherent epithelial line derived from a lung adenocarcinoma of a female non-smoker. It harbors an activating EGFR exon 19 deletion and a PIK3CA mutation, while KRAS remains wild-type, making it a widely used model for investigating oncogenic signaling and metabolic rewiring in non-small cell lung cancer (NSCLC). The line possesses functional peroxisomes, providing a relevant cellular context for examining the metabolic roles of peroxisomal enzymes such as HACL2.
HACL2 encodes a peroxisomal 2-hydroxyacyl-CoA lyase that cleaves 2-hydroxyacyl-CoAs, particularly 2-hydroxyphytanoyl-CoA, into fatty aldehydes and formyl-CoA during alpha-oxidation of phytanic acid. This reaction requires peroxisomal import via interaction with PEX5. HACL2 is regulated by PPAR?? agonists and fatty acids, and it functions downstream of phytanoyl-CoA hydroxylase (PHYH). The resulting fatty aldehydes are oxidized by ALDH3A2, while formyl-CoA enters ether lipid and lipid mediator synthesis through formyl-CoA transferase. Consequently, HACL2 is a pivotal enzyme integrating peroxisomal lipid catabolism with cellular signaling.
In the NCI-H1975 background, ablation of HACL2 provides a powerful system to interrogate how peroxisomal alpha-oxidation influences the metabolic phenotype of lung adenocarcinoma cells. The concomitant EGFR and PIK3CA mutations??both drivers of growth and metabolism??allow dissection of crosstalk between oncogenic pathways and peroxisomal function. Loss of HACL2 is expected to cause accumulation of phytanic acid intermediates, potentially reshaping cellular bioenergetics, altering lipid mediator profiles, and modifying responses to oxidative stress. The polyclonal nature of the knockout pool offers a more representative cellular phenotype, reducing artifacts from single-cell adaptations and enabling robust assessment of metabolic dependencies in NSCLC.
These polyclonal knockout cells support diverse assays: western blotting and RT-qPCR confirm HACL2 disruption, phytanic acid accumulation and peroxisomal staining assess pathway blockade, and lipidomics reveal altered lipid profiles. Metabolic flux analysis, cell viability assays, and drug screening delineate functional consequences and therapeutic vulnerabilities. Applications include cancer metabolism, peroxisomal biology, functional genomics, and drug discovery. For further technical details, contact Ascent Research.
