Investigation of Lipid Metabolism Dysregulation in Chronic Lymphocytic Leukemia and the Role of ENPP2 in Tumorigenesis of Chronic Lymphocytic Leukemia

Introduction

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world, and although the advent of novel targeted drugs has greatly improved the prognosis of CLL patients, CLL remains incurable at present. Improving the risk stratification of CLL patients and finding new CLL therapeutic targets are urgent issues to be addressed.

Methods

We performed untargeted metabolomic analysis using LC-MS on sera from CLL patients and healthy controls (HC). Statistical analysis was used to screen for differential metabolites (DMs). Differentially expressed genes (DEGs) were also screened for enrichment pathway enrichment relying on transcriptome data analysis through the GEO database. Finally, DMs and DEGs were analyzed together to explore CLL metabolic alteration pathways. In this study, we targeted the gene ENPP2. Peripheral blood samples from 82 CLL patients were collected at the Department of Hematology in Shandong Provincial Hospital. Expressions levels of ENPP2 in CLL patients were determined by quantitative RT-PCR. In addition, cell viability and apoptosis were assessed by cell counting kit-8 and annexin V-PE/7AAD staining.

Results

A total of 52 differential metabolites were annotated, 40 of them were up-regulated and 12 were down-regulated. There were significant differences in sphingolipids (SP), glycerolipids (GL), glycerophospholipids (GP), and fatty acids (FA) levels in CLL patients compared to health controls. Fifty-two differential metabolites between samples from CLL patients and HC were analyzed by pathway analysis using metaboanalyst v5.0. KEGG enrichment showed that differential metabolites in CLL patients and HC were mainly annotated and enriched in human diseases, metabolism, and organismal systems (Figure 1A).

Furthermore, we obtained differential expression information from the GEO database for CLL patients with HC. A total of 539 DEGs were screened, of which 279 were up-regulated and 260 were down-regulated. A total of 257 KEGG pathways were enriched. In the current work, a joint analysis of the two histological data was performed to validate each other. A total of 20 KEGG pathways were repeatedly obtained from metabolomics and transcriptomics data-based pathway analysis, including pathways in cancer, insulin resistance, long-term depression, regulation of lipolysis in adipocytes metabolic pathways, glycerophospholipid metabolism, glycerolipid metabolism, sphingolipid metabolism, arachidonic acid metabolism, ether lipid metabolism, inositol phosphate metabolism, choline metabolism in cancer, phospholipase D signaling pathway. A PPI network was constructed for the identified significantly different genes, from which we also screened for the gene ENPP2, a secreted lysophospholipase D, that we desired to investigate further (Figure 1B). We validated the difference in ENPP2 expression between CLL patients and healthy donors in the GEO database and its guiding effect on CLL prognosis. The results showed that elevated expression of ENPP2 in CLL patients and relatively high expression in CLL patients implied a worse prognosis.

In addition, to validate the role of ENPP2 in CLL, we did the further investigation in CLL patients in our own center. Aberrantly increased expression of ENPP2 were detected in CLL primary cells and cell line (MEC1) at mRNA compared with normal B cells. To investigate the biological processes of ENPP2 involving in CLL progression, functional assays were performed. The survival rate of CLL cells treated with ENPP2 inhibitor PF-8380 was significantly decreased, while apoptosis was increased. In addition, we performed a combination of ibrutinib with PF8380, which showed a further decrease in cell viability and showed better anti-CLL effects.

Conclusion

Taken together, the present work provides evidence that the identified metabolic biomarkers can be used for CLL diagnosis and screening. The combined analysis of metabolomics and transcriptomics data provides a comprehensive understanding of the metabolic pathways in CLL. Besides, this study was the first investigation on the role of ENPP2 in CLL. ENPP2 expression was increased in CLL cells, and the ENPP2 inhibitor PF-8380 inhibited cell proliferation and induced apoptosis, with therapeutic potential.

No relevant conflicts of interest to declare.