The KLHL22 Knockout HCT 116 Polyclonal Cells product offers a CRISPR/Cas9-edited polyclonal knockout population of HCT 116 colorectal carcinoma cells with disrupted KLHL22 gene. This pool provides a heterogeneous loss-of-function model suitable for robust functional analysis without clonal selection. The polyclonal format captures diverse editing events, reflecting genetic variability akin to tumor heterogeneity and facilitating wide-ranging studies of KLHL22-dependent biology.
HCT 116 cells originate from a human colorectal adenocarcinoma and are characterized by a KRAS G13D mutation and MLH1 deficiency, resulting in microsatellite instability (MSI-high). This well-defined genetic context is widely employed to model colorectal cancer signaling, drug responses, and DNA repair deficiencies, making it a valuable platform for interrogating oncogenic pathways. The line’s rapid growth and defined mutations render it an established workhorse for signaling studies and drug screening.
KLHL22 serves as a substrate-specific adaptor for the CUL3-RBX1 E3 ubiquitin ligase, mediating DEPTOR ubiquitination and proteasomal degradation upon amino acid stimulation. This relieves mTORC1 inhibition, allowing phosphorylation of S6K and 4E-BP1 to drive anabolic signaling. KLHL22 integrates nutrient status with mTOR and PI3K-Akt pathways, interfacing with the amino acid-sensing GATOR1 complex (NPRL2, NPRL3, DEPDC5). KLHL22 activity is governed by Cullin3 neddylation, CAND1 competition, and growth factor signals. Consequently, knockout leads to DEPTOR accumulation and mTORC1 suppression.
In the HCT 116 background, KLHL22 ablation offers a unique model to study how mTORC1 deregulation intersects with oncogenic KRAS G13D and MSI-high status. DEPTOR accumulation may counteract KRAS-driven proliferation, while altered autophagy and metabolic reprogramming can be assessed. This model is particularly suited to explore synthetic lethality, sensitivity to mTOR inhibitors (e.g., rapamycin), and proteasome-targeting therapies, given the colorectal cancer microenvironment.
Applications include Western blotting for KLHL22, phospho-S6K, phospho-4E-BP1, and DEPTOR; RT-qPCR for KLHL22 mRNA; and cell proliferation assays to assess growth phenotypes. Autophagy flux analysis via LC3 turnover, mTORC1 activity measurements, and proteasomal degradation assays with MG132 further elucidate pathway dynamics. Immunofluorescence-based mTOR localization and high-content screening for Cullin3 ligase modulators are also amenable. This polyclonal knockout pool provides a cost-effective, reproducible tool for colorectal cancer and mTOR signaling research. For further information or technical support, please contact Ascent Research.
