PRPS2 Facilitates the Tumorigenesis of Diffuse Large B-Cell Lymphoma By Promoting Fatty Acid β-Oxidation

Phosphoribosyl pyrophosphate synthase 2 (PRPS2), a key enzyme in pentose phosphate pathway (PPP), catalyzes the conversion of ribulose-5-phosphate to adenosine triphosphate to produce phosphoribosyl pyrophosphate. The metabolites of the PPP are critical raw materials for the anabolism of fatty acids and nucleic acids in cells. Herein, we sought to unpack the specific regulatory mechanism underlying PRPS2's modulation of metabolism reprogramming in diffuse large B-cell lymphoma (DLBCL), expecting to propose novel diagnostic approaches and personalized therapeutic strategies.

We first performed untargeted metabolomics on peripheral blood specimens from 60 newly diagnosed DLBCL patients and 60 healthy volunteers with informed consent and identified the PPP was significantly enriched in DLBCL patients. The transcriptome sequencing data of DLBCL patients in the GEO database (GSE56315) further verified above results. Survival analysis suggested that high expression of PRPS2 a key gene in the PPP, was significantly associated with poor prognosis in DLBCL patients. We next observed the upregulation of PRPS2 mRNA and protein levels in DLBCL cells, which was further confirmed in a cohort of newly diagnosed DLBCL patients.

To further explore the biological functions of PRPS2, knockdown and overexpression models of PRPS2 were performed. PRPS2 knockdown significantly impaired cell proliferation and induced cell cycle arrest in G0/G1 phase. Moreover, PRPS2 knockdown markedly triggered cell apoptosis and dysregulation of apoptotic proteins. On the contrary, PRPS2 overexpression resulted in reduced apoptosis. To validation of PRPS2's biological function in vivo, xenograft DLBCL mice model was established and we found PRPS2 knockdown significantly reduced DLBCL tumor growth in mice and decreased Ki67 expression. Animal experiments were performed in accordance with the principles of the Institutional Animal Care.

To evaluate the potential roles of PRPS2 in DLBCL, assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) and unique molecular identifier-RNA sequencing (UMI RNA-seq) were performed on cells with stable PRPS2 knockdown. GSEA enrichment analysis showed significant enrichment in fatty acid metabolic pathway. Furthermore, PRPS2 expression was found to be positively related to triglyceride and cholesterol levels in peripheral blood serum of DLBCL patients by correlation analysis. Absolute quantitative lipidomics revealed reduced triglyceride level in PRPS2 knockdown cells, further confirming abnormalities in fatty acid metabolism. And we experimentally verified that PRPS2 knockdown significantly reduced intracellular triglyceride and lipid droplet contents. Moreover, orlistat, a fatty acid synthesis inhibitor, inhibited DLBCL cell proliferation in a dose- and time-dependent manner. PRPS2 knockdown increased orlistat’s inhibition of cell proliferation, while orlistat treatment counteracted PRPS2 overexpression’s effect on cell proliferation. The above results support that PRPS2-mediated lipid accumulation promotes DLBCL cell growth.

Next, we explore the underlying mechanism by which PRPS2 regulates fatty acid metabolism. Combining ATAC-seq data with UMI RNA-seq data, we observed increased transcription of ACOX1 in the promoter region after PRPS2 knockdown. In PRPS2 knockdown cells, ACOX1 mRNA and protein expression elevated, as well as an increase in the rate of fatty acid β-oxidation. Further investigation was carried out to determine how PRPS2 regulates ACOX1 expression. According to hTFtarget data, PU.1 had the highest peak value in the promoter region when searching for transcription factors that regulate ACOX1 expression. Evidence was provided by chromatin immunoprecipitation (ChIP)-qPCR confirming the enhanced binding of PU.1 to the ACOX1 promoter region after PRPS2 knockdown. Correlation analysis revealed that the mRNA expression level of PU.1 was negatively correlated with that of PRPS2. Our study confirmed that PRPS2 knockdown decreased PU.1 expression and increased PU.1 phosphorylation, which could promote DNA binding and cofactor recruitment.

Our study demonstrates that the suppression of PRPS2 could inhibit the development and progression of DLBCL by regulating the PU.1/ACOX1 axis to reprogram fatty acid metabolism, which provides a promising therapeutic insight into DLBCL treatment.