Metabolic dysfunction-associated steatotic liver disease (MASLD), including its severe form MASH (metabolic dysfunction associated steatohepatitis), is the most common chronic liver condition, with a global prevalence of 30%. MASH is characterized by lipid accumulation, inflammation and frequently fibrosis, and is associated with the development of life-threatening diseases, including liver cancer.
MASLD is characterized by defective lipid metabolism, with increased fatty acid uptake and de novo lipogenesis being the major hallmarks, causing hepatic steatosis and inflammation, key events that precede and contribute to fibrosis and are important for disease progression. Hence, tackling early metabolic defects (i.e., inhibiting hepatic steatosis) is likely to prevent the development of MASH and liver cancer.
To systematically uncover novel genes required for hepatic lipid accumulation, we performed five independent, pooled genome-wide screens in HepG2 liver cells using the human GeCKO v2 CRISPR library. Cells were treated with/without excess fatty acid (FA) to mimic chronic lipid overload, followed by Bodipy lipid staining and fluorescence-activated cell sorting. In Control cells, we collected the highest 10% of cells to select mutants that retained high levels of lipid due to defects in lipid processing (‘negative regulators’), while in the FA-treated cells, we collected the lowest 10% of cells, thereby screening for cells bearing mutations to genes necessary for FA-induced lipid accumulation (‘positive regulators’).
Using single gRNA knockout combined with functional assessment of lipid metabolism, we validated the top 36 candidates, and identified C1orf35 (Chromosome 1 open reading frame 35) as a novel regulator of hepatic lipid metabolism and contributor to MASLD.
Deletion of C1orf35 in HepG2 cells reduces lipid accumulation by >80% by suppressing lipogenesis and fatty acid uptake and enhancing triglyceride secretion. Mechanistically, C1orf35 acts as a transcriptional regulator of key lipid metabolism enzymes, including SREBP1, FASN and CD36. Using liver-targeted lipid nanoparticles (LNP) to deliver sgRNA targeting C1orf35 to mice expressing Cas9 in hepatocytes, we further show reduced lipogenesis, increased triglyceride secretion, and an overall 45% reduction in hepatocyte triglyceride content following C1orf35 deletion. In addition, C1orf35 deletion sensitizes liver cancer cells to the anti-cancer drugs etoposide and sorafenib/lenvatinib, inhibits cell migration and invasion in vitro, and suppresses tumorigenicity in the NSG xenograft model.
Taken together, these data highlight that targeting C1orf35 may be a feasible therapeutic option for MASLD and liver cancer, and, in combination with traditional cancer therapy, may reduce disease burden and improve long-term prognosis.