1035 - ENPP2 mediates lipid metabolism and tumorigenesis in chronic lymphocytic leukemia through AMPK signaling pathway

Introduction
Disorders of lipid metabolism are critical factors in the progression of chronic lymphocytic leukemia (CLL). Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), an adipocyte-derived lysophospholipase D, is closely correlated with obesity and disorders of glucolipid metabolism in obese individuals. ENPP2 has been described to be engaged in the progression of several solid neoplasms, but its functional role in CLL remains unclear. Hence, the aim of this study was to investigate the functional mechanism and clinical significance of ENPP2 regulation in CLL.

Methods
In this study, peripheral blood samples of 82 de novo patients diagnosed with CLL were collected at the Department of Hematology in Shandong Provincial Hospital. The mRNA expressions levels of ENPP2 in CLL patients were determined by quantitative RT-PCR. Plasmids were utilized to knockdown and overexpression ENPP2 expression in MEC1 and EHEB CLL cells. ENPP2 targeted inhibitor PF-8380 was used to inhibit ENPP2 impression. RNA-sequencing and functional enrichment analysis were performed. In addition, cell viability and apoptosis were assessed by cell counting kit-8 and annexin V-PE/7AAD staining. Bodipy staining was used to explore lipid deposition in CLL cells.

Results
The mRNA expression level of ENPP2 was examined in 82 CLL primary samples from Shandong Provincial Hospital CLL (SPHCLL) database. RT-PCR confirmed the significant upregulation of ENPP2 in CLL patients compared to normal B cells (p < 0.01; Figure 1A). To validate the altered pattern of ENPP2 expression in CLL patients, we further investigated and found ENPP2 over-experssion in CLL patients in two independent microarrays (p < 0.0001; Figure 1B). Kaplan-Meier curves showed higher ENPP2 expression patients were associated with adverse overall survival (HR=2.33, p < 0.043; Figure 1C).

To investigate the biological processes of ENPP2 involving in CLL progression, functional assays were performed. CLL cells with ENPP2 knockdown resulted in slow proliferation, G0/G1 phase cell cycle arrest, increased apoptosis and lipid accumulation in CLL cells (Figure 1D-F). In contrast, upregulation of ENPP2 produced the opposite effect (Figure 1G-I). Similarly, treated with ENPP2 targeted inhibitor PF-8380 exhibited attenuated cell proliferation, increased apoptosis and induced G0/G1 phase arrest. Additionally, the expression of cyclin-related proteins, including C-myc, Cyclin D1, CDK4, P21, and P27 was reduced by treated. The expression of pro-apoptotic proteins Bax was increased, while the expression of anti-apoptotic protein Bcl-2 was reduced (Figure 2A-B).

To futher explore the mechanism of ENPP2 regulation in the tumorigenesis of CLL, RNA-sequencing was implemented between the MEC-1 cells transfected ShControl and ShENPP2#2 respectively. We identified that ENPP2 was concentrated in pathways linked to metabolisms, such as the TCA cycle, AMPK signaling pathway and glycerophospholipid metabolism (Figure 2C), through analysis of Kyoto encyclopedia of genes and genomes (KEGG). Gene ontology (GO) analysis implicated that ENPP2 was closely associated with metabolic processes, cellular processes, biological regulation (Figure 2D). Accordantly, knockdown of ENPP2 and treated with PF-8380 reduced intracellular lipid deposition. In contrast, intracellular lipid deposition was increased after overexpression of ENPP2. Western blot suggested that ENPP2 was involved in the regulation of lipid metabolism in slow lymphoid cells through participation in the AMPK/SREBP1/FAS signaling pathway (Figure 2E).

Conclusion
Taken together, the present study was the first investigation on the role of ENPP2 in the tumorigenesis of CLL. ENPP2 expression was aberrantly increased in CLL cells, and affected cell lipid metabolism through the AMPK pathway. ENPP2 targeted inhibitor PF-8380 inhibited CLL cell proliferation and induced apoptosis, highlighting potent therapeutic potential.

Keywords: Chronic lymphocytic leukemia; ENPP2, Lipid metabolism