Novel transketolase inhibitor oroxylin A suppresses the non-oxidative pentose phosphate pathway and hepatocellular carcinoma tumour growth in mice and patient-derived organoids.

Transketolase (TKT), a key rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), provides more than 85% of the ribose required for de novo nucleotide biosynthesis and promotes the development of hepatocellular carcinoma (HCC). Pharmacologic inhibition of TKT could impede HCC development and enhance treatment efficacy. However, no safe and effective TKT inhibitor has been approved. An online two-dimensional TKT protein immobilised biochromatographic system was established for high-throughput screening of TKT ligands. Oroxylin A was found to specifically bind TKT. Drug affinity responsive target stability, cellular thermal shift assay, surface plasmon resonance, molecular docking, competitive displacement assay, and site mutation were performed to identify the binding of oroxylin A with TKT. Antitumour effects of oroxylin A were evaluated in vitro, in human xenograft mice, diethylnitrosamine (DEN)-induced HCC mice, and patient-derived organoids (PDOs). Metabolomic analysis was applied to detect the enzyme activity. Transcriptome profiling was conducted to illustrate the anti-HCC mechanism of oroxylin A. TKT knocking-down HCC cell lines and PDOs were established to evaluate the role of TKT in oroxylin A-induced HCC suppression. By targeting TKT, oroxylin A stabilised the protein to proteases and temperature extremes, decreased its activity and expression, resulted in accumulation of non-oxidative PPP substrates, and activated p53 signalling. In addition, oroxylin A suppressed cell proliferation, induced apoptosis and cell-cycle arrest, and inhibited the growth of human xenograft tumours and DEN-induced HCC in mice. Crucially, TKT depletion exerted identical effects to oroxylin A, and the promising inhibitor also exhibited excellent therapeutic efficacy against clinically relevant HCC PDOs. These results uncover a unique role for oroxylin A in TKT inhibition, which directly targets TKT and suppresses the non-oxidative PPP. Our findings will facilitate the development of small-molecule inhibitors of TKT and novel therapeutics for HCC.

Jia D ，Liu C ，Zhu Z ，Cao Y ，Wen W ，Hong Z ，Liu Y ，Liu E ，Chen L ，Chen C ，Gu Y ，Jiao B ，Chai Y ，Wang HY ，Fu J ，Chen X ... -  《Clinical and Translational Medicine》

Transketolase (TKT) activity and nuclear localization promote hepatocellular carcinoma in a metabolic and a non-metabolic manner.

Qin Z ，Xiang C ，Zhong F ，Liu Y ，Dong Q ，Li K ，Shi W ，Ding C ，Qin L ，He F ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

Transketolase Deficiency Protects the Liver from DNA Damage by Increasing Levels of Ribose 5-Phosphate and Nucleotides.

De novo nucleotide biosynthesis is essential for maintaining cellular nucleotide pools, the suppression of which leads to genome instability. The metabolic enzyme transketolase (TKT) in the nonoxidative branch of the pentose phosphate pathway (PPP) regulates ribose 5-phosphate (R5P) levels and de novo nucleotide biosynthesis. TKT is required for maintaining cell proliferation in human liver cancer cell lines, yet the role of TKT in liver injury and cancer initiation remains to be elucidated. In this study, we generated a liver-specific TKT knockout mouse strain by crossing TKTflox/flox mice with albumin-Cre mice. Loss of TKT in hepatocytes protected the liver from diethylnitrosamine (DEN)-induced DNA damage without altering DEN metabolism. DEN treatment of TKT-null liver increased levels of R5P and promoted de novo nucleotide synthesis. More importantly, supplementation of dNTPs in primary hepatocytes alleviated DEN-induced DNA damage, cell death, inflammatory response, and cell proliferation. Furthermore, DEN and high-fat diet (HFD)-induced liver carcinogenesis was reduced in TKTflox/floxAlb-Cre mice compared with control littermates. Mechanistically, loss of TKT in the liver increased apoptosis, reduced cell proliferation, decreased TNFα, IL6, and STAT3 levels, and alleviated DEN/HFD-induced hepatic steatosis and fibrosis. Together, our data identify a key role for TKT in promoting genome instability during liver injury and tumor initiation. SIGNIFICANCE: These findings identify transketolase as a novel metabolic target to maintain genome stability and reduce liver carcinogenesis.

Li M ，Lu Y ，Li Y ，Tong L ，Gu XC ，Meng J ，Zhu Y ，Wu L ，Feng M ，Tian N ，Zhang P ，Xu T ，Lin SH ，Tong X ... -  《-》

HMGA1 promotes the progression of esophageal squamous cell carcinoma by elevating TKT-mediated upregulation of pentose phosphate pathway.

Esophageal squamous cell carcinoma (ESCC) possesses a poor prognosis and treatment outcome. Dysregulated metabolism contributes to unrestricted growth of multiple cancers. However, abnormal metabolism, such as highly activated pentose phosphate pathway (PPP) in the progression of ESCC remains largely unknown. Herein, we report that high-mobility group AT-hook 1 (HMGA1), a structural transcriptional factor involved in chromatin remodeling, promoted the development of ESCC by upregulating the PPP. We found that HMGA1 was highly expressed in ESCC. Elevated HMGA1 promoted the malignant phenotype of ESCC cells. Conditional knockout of HMGA1 markedly reduced 4-nitroquinoline-1-oxide (4NQO)-induced esophageal tumorigenesis in mice. Through the metabolomic analysis and the validation assay, we found that HMGA1 upregulated the non-oxidative PPP. With the transcriptome sequencing, we identified that HMGA1 upregulated the expression of transketolase (TKT), which catalyzes the reversible reaction in non-oxidative PPP to exchange metabolites with glycolytic pathway. HMGA1 knockdown suppressed the PPP by downregulating TKT, resulting in the reduction of nucleotides in ESCC cells. Overexpression of HMGA1 upregulated PPP and promoted the survival of ESCC cells by activating TKT. We further characterized that HMGA1 promoted the transcription of TKT by interacting with and enhancing the binding of transcription factor SP1 to the promoter of TKT. Therapeutics targeting TKT with an inhibitor, oxythiamine, reduced HMGA1-induced ESCC cell proliferation and tumor growth. Together, in this study, we identified a new role of HMGA1 in ESCCs by upregulating TKT-mediated activation of PPP. Our results provided a new insight into the role of HMGA1/TKT/PPP in ESCC tumorigenesis and targeted therapy.

Liu MJ ，Zhao Y ，Li QT ，Lei XY ，He KY ，Guo JR ，Yang JY ，Yan ZH ，Wu DH ，Zhang L ，Jian YP ，Xu ZX ... -  《-》

The nuclear translocation of transketolase inhibits the farnesoid receptor expression by promoting the binding of HDAC3 to FXR promoter in hepatocellular carcinoma cell lines.

Li M ，Zhang X ，Lu Y ，Meng S ，Quan H ，Hou P ，Tong P ，Chai D ，Gao X ，Zheng J ，Tong X ，Bai J ... -  《Cell Death & Disease》