CRISPR/Cas9-Mediated HKDC1 Depletion to Assess Ferroptosis and Lenvatinib Responses in Hepatocellular Carcinoma.

Hexokinase domain-containing protein 1 (HKDC1) has been implicated in hepatocellular carcinoma (HCC) progression, but its relationship to regulated cell death remains incompletely defined. This article presents an in vitro workflow that combines public cohort analysis, paired clinical tissues, CRISPR/Cas9-mediated HKDC1 depletion, cell growth assays, lenvatinib treatment, pharmacological rescue, and biochemical measurements of glutathione (GSH) and malondialdehyde (MDA). HKDC1 expression was higher in HCC than in non-tumor liver tissue and was associated with poorer overall and disease-free survival in public datasets. HKDC1 depletion reduced growth and colony formation in Hep3B and SNU449 cells. HKDC1 depletion also increased the response to lenvatinib and ML-162. Independent targeting with sg-HKDC1-2 reproduced the ML-162-associated viability phenotype, and ferrostatin-1 restored viability after ML-162 exposure in SNU449 cells. These results support an association between HKDC1 depletion and increased susceptibility to regulated cell-death stress in HCC cells. Because direct lipid-reactive oxygen species, GPX4/SLC7A11 activity, glucose flux, and in vivo efficacy were not measured, the downstream mechanism and therapeutic relevance require further validation.