The ITPK1 Knockout NCI-H1975 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the NCI-H1975 human lung adenocarcinoma cell line. These cells feature targeted disruption of the ITPK1 gene, encoding inositol-tetrakisphosphate 1-kinase, a key enzyme in inositol phosphate metabolism. The polyclonal format provides a heterogeneous pool of edited cells, each with distinct CRISPR-generated modifications, creating a robust loss-of-function model that reflects tumor genetic diversity. This product enables interrogation of ITPK1-dependent processes without residual wild-type protein, eliminating single-cell cloning while ensuring batch consistency.
The NCI-H1975 cell line is a non-small cell lung carcinoma (NSCLC) model from a lung adenocarcinoma patient. It carries the EGFR T790M mutation, which imparts resistance to EGFR TKIs and is widely used in drug resistance research. These cells retain functional PI3K-AKT and PLC signaling, facilitating oncogenic pathway studies. The adherent line supports standard proliferation, apoptosis, and drug sensitivity assays.
ITPK1 is a key kinase in inositol phosphate metabolism, phosphorylating IP3 to IP4 in a calmodulin-dependent, ATP-driven reaction. The enzyme is regulated by PKA, PKC, calcium, and EGF pathways, positioning it downstream of multiple oncogenic cascades. Its product IP4 is the precursor for IP5 and IP6, synthesized with IPMK, which regulate calcium release channels and mRNA export factors. This node connects ITPK1 to PI3K-AKT signaling, as IP4 and downstream polyphosphates promote PDK1-mediated activation of AKT, governing cell survival. ITPK1 knockout thus abolishes IP4 synthesis, reducing higher inositol phosphates and attenuating AKT signaling while disrupting calcium homeostasis.
In NCI-H1975 cells, ITPK1 knockout offers a model to explore the crosstalk between inositol phosphate metabolism and TKI resistance. Oncogenic EGFR signaling via PLC generates IP3, the direct substrate of ITPK1, linking EGFR directly to inositol phosphate flux. Disruption of ITPK1 may therefore blunt PI3K-AKT pro-survival signals that sustain the T790M-resistant phenotype, potentially resensitizing cells to EGFR inhibitors like osimertinib. Moreover, the downstream effects on mRNA export factors could alter the expression of genes involved in drug tolerance. This system permits dissection of whether IP4-dependent pathways are essential for maintaining resistance or can be targeted to overcome it.
Researchers can apply this polyclonal knockout population in diverse assays, including calcium imaging to monitor IP3-mediated calcium release, Western blotting for phospho-AKT to gauge PI3K pathway activity, and MTT viability assays to assess proliferative changes. Drug dose-response experiments with osimertinib or other TKIs quantify shifts in drug sensitivity, while RT-qPCR profiling of ITPK1 and inositol phosphate-responsive genes uncovers transcriptional adaptation. The model further supports functional genomics screens to pinpoint synthetic lethal interactions or compensatory signaling nodes. For technical assistance or project-specific inquiries, please reach out to Ascent Research.
