The HM13 Knockout NCI-H1975 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population designed for the disruption of the HM13 gene in the human NCI-H1975 lung adenocarcinoma cell line. This loss-of-function model provides a genetically defined background for investigating intramembrane proteolysis and its downstream effects on immune signaling pathways. As a polyclonal product, it preserves the cellular heterogeneity inherent to CRISPR/Cas9-mediated gene disruption, enabling robust functional studies without the selection bias associated with monoclonal isolates.
The host NCI-H1975 line is an adherent epithelial cell line derived from the pleural effusion of a 43-year-old female with lung adenocarcinoma. It harbors activating EGFR L858R and T790M mutations, along with a TP53 mutation, making it a clinically relevant model for EGFR-mutant non-small cell lung cancer with acquired resistance to first-generation tyrosine kinase inhibitors. These features render the cells particularly suitable for investigating tumor-intrinsic mechanisms of immune modulation and therapy resistance.
HM13 encodes signal peptide peptidase-like 2A (SPPL2A), an intramembrane aspartyl protease resident in endosomal and lysosomal membranes. SPPL2A proteolytically processes type II transmembrane substrates, most notably the invariant chain CD74, to regulate MHC class II antigen presentation. It cleaves the CD74 N-terminal fragment, generating a soluble fragment and a membrane-bound C-terminal domain that influences transcriptional regulation. SPPL2A also processes tumor necrosis factor-alpha (TNF-??), Fas ligand (FasL), and integral membrane protein 2B (ITM2B). Its activity is modulated by NF-??B and interferon-gamma, and it functions downstream of endosomal sorting pathways. Knockout of HM13 disrupts CD74 turnover, leading to accumulation of CD74 fragments, impaired peptide loading on MHCII, and defective activation of CD4+ T cells.
In the NCI-H1975 context, loss of HM13/SPPL2A enables the study of how disrupted intramembrane proteolysis impacts anti-tumor immunity. Since aberrant MHCII expression in non-immune cells can influence tumor immune surveillance, this knockout model is instrumental for dissecting SPPL2A’s role in immune evasion mechanisms in EGFR-driven lung adenocarcinoma. It also allows exploration of cross-talk between endosomal proteolysis and oncogenic signaling, potentially uncovering vulnerabilities associated with SPPL2A deficiency.
Typical applications include western blotting for CD74 processing intermediates, flow cytometric analysis of surface MHCII and antigen presentation competence, T cell co-culture assays to assess functional T cell activation, and cytokine secretion profiling. Immunofluorescence can be used to visualize CD74 accumulation in endocytic compartments, while protease activity assays confirm loss of SPPL2A function. These cells support investigation of the tumor microenvironment’s immunopeptidome and facilitate screening for modulators of antigen presentation. For additional technical information, please contact Ascent Research.
