The BCKDK Knockout HCT 116 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout population derived from the HCT 116 human colorectal carcinoma cell line, carrying targeted disruption of the BCKDK gene. This mixed population provides a heterogeneous loss-of-function model for studying BCKDK-dependent processes without clonal selection.
HCT 116 is a widely used colorectal adenocarcinoma model with a KRAS G13D mutation, microsatellite stability, wild-type TP53, and a near-diploid karyotype. Its epithelial origin and intact key signaling pathways make it suitable for investigating metabolic regulation in cancer.
BCKDK encodes branched-chain ketoacid dehydrogenase kinase, which phosphorylates the E1?? subunit (BCKDHA) of the branched-chain ??-ketoacid dehydrogenase complex (BCKDC), thereby inactivating the complex and limiting branched-chain amino acid (BCAA) catabolism. BCKDK activity is regulated by insulin signaling, cellular energy status, and branched-chain ketoacids such as ??-ketoisocaproic acid, and it requires ATP as a phosphate donor. BCKDK functions within a multienzyme complex that includes BCKDHA, BCKDHB, dihydrolipoyl transacylase (DBT), and dihydrolipoyl dehydrogenase (DLD), with its action controlling the flux through the BCAA degradation pathway.
In the HCT 116 background, BCKDK knockout promotes constitutive BCKDC activation, enhancing BCAA degradation and reducing intracellular BCAA levels. Given the KRAS-driven anabolic metabolism in these cells, altered BCAA homeostasis may modulate mTORC1 and insulin signaling, thereby affecting proliferation, survival, and metabolic reprogramming. This polyclonal knockout model enables dissection of BCKDK??s role in oncogenic metabolism and assessment of metabolic vulnerabilities in colorectal cancer.
Applications include studying BCAA metabolism in colorectal cancer, interrogating mTORC1 signaling, and exploring metabolic adaptations in KRAS-mutant tumors. Representative assays encompass Western blot analysis of BCKDK and phosphorylated BCKDHA, LC-MS?Cbased BCAA quantification, mTORC1 pathway assessment via p-S6K1 and p-4EBP1, viability/proliferation assays, metabolite profiling, and oxygen consumption rate measurement. Additionally, this model can be used in xenograft studies to evaluate the impact of BCKDK loss on tumor growth and response to metabolic inhibitors. It also supports research into BCKDK-linked neurological disorders. For inquiries, contact Ascent Research.
