Gene expression profiling for circPETH overexpression and knockout

Metabolic reprogramming fuels cancer cell metastasis and remodels immunosuppressive tumor microenvironment (TME). We report here that a circRNA packaged by extracellular vesicles (EVs) from tumor-associated macrophages (TAMs) to hepatocellular carcinoma (HCC) cells, circPETH, facilitates glycolysis and metastasis of HCC cells. Mechanistically, circPETH-147aa, encoded by circPETH in m6A-driven manner, promotes the PKM2-catalyzed ALDOA-S36 phosphorylation via MEG pocket. Furthermore, circPETH-147aa impairs anti-HCC immunity by elevating the HuR-dependent SLC43A2-mRNA stability and stimulating the deficiency of methionine and leucine in cytotoxic CD8+ T cells. Importantly, we successfully identified a novel small molecule, Norathyriol, by virtual and experimental screening as an effective inhibitor targeting MEG pocket on circPETH-147aa surface. Norathyriol reverses circPETH-147aa-facilitated metabolic and metastatic phenotypes of HCC cells, improves anti-PD1 efficacy and boosts cytotoxic CD8+ T cells function. Our findings determine Norathyriol as a promising anti-HCC agent, and contribute to overcoming the resistance of advanced HCC to immune checkpoint blockades (ICBs) therapies. To gain insights into the regulatory mechanism of circPETH in terms of comprehensive gene expression, RNA-sequencing (RNA-seq) was conducted to compare the following two expression profiles: OE-circPETH versus Vector; and circPETH KO versus NTC.