The Role Of PON2 In The Pathogenesis Of B-cell Acute Lymphoblastic Leukemia (B-ALL)

[Objective]Philadelphia(Ph)chromosome-like B-ALL is a subtype of ALL without Ph-chromosome or BCR-ABL1-fusion gene,but displayed similar gene-expression profile with that of Ph⁺B-ALL.Tyrosine-kinase inhibitors significantly improves the clinical outcome of patients with either Ph⁺B-ALL or Ph-like B-ALL.However,the prognosis of these patients is worse than that of in other B-ALL patients.Paraoxonase 2(PON2)mRNA is upregulated in Ph-like B-ALL and defined as a marker for Ph-like B-ALL patients.However,the role of PON2 in the pathogenesis of B-ALL remains unclear.Our lab previously found that Pon2-deficiency substantially impaired B-ALL cell proliferation in the settings of both ex vivo and in vivo.Here,we further explore the clinical significance of PON2 in B-ALL;establish Pon2-deficiency in both mouse and human models for B-ALL to determine if PON2 involve in the pathogenesis of B-ALL via its role in glucose metabolism and the mechanism by which PON2 participates in glucose metabolism;besides,we test the potential therapeutic strategy targeting PON2.Addressing of these questions will significantly advance our understanding of the function of PON2 and its contribution during the development of B-ALL and provide the rationale for targeted therapies.[Method]1.Data mining was performed on gene expression data and clinical data from clinical trials or public database regarding B-ALL or mature B-cell malignancies to study the expression profile of PON2 and its clinical relevance;2.PON2 protein levels were examined in patient-derived B-ALL cells;3.Enriched splenic B-cells from Pon2^+/+and Pon2^-/-donor mice were transplanted into mature B-cell-deficient mouse(?MT)by tail vein injection.Following engraftment,NP-KLH mixed with adjuvant or adjuvant alone was intraperitoneally injected into recipient mice to trigger immunization.Mice were eventually sacrificed to study the role of Pon2in B-cell maturation and the formation of NP-specific B cell.4.Bone marrow from Pon2^+/+and Pon2^-/-mice were harvested and cultured in vitro and transduced with BCR-ABL1and NRAS ^G12D to create B-ALL models.Glucose uptake,total ATP,lactate production and extracellular acidification rates(ECARs)were measured for mouse BCR-ABL1 or NRAS ^G12D B-ALL cells.5.Tet-on inducible Pon2-overexpression system were established on Pon2^+/+and Pon2^-/-mouse BCR-ABL1 B-ALL cells.The impacts of Pon2expression in glucose metabolism were examined upon doxycycline treatment.6.Lentivirus-carried shRNA-mediated downregulation of Stom was performed on Pon2^+/+and Pon2^-/-mouse BCR-ABL1 B-ALL cells,and the impacts of Pon2 expression in glucose metabolism were examined.7.Electroporation-delivered and CRISPR/Cas9-mediated deletion of PON2 was performed on human B-ALL cell line(BV173),and single-cell sorting followed by cell expansion was used to isolate individual CRISPR-edited clones deficient in PON2.The impacts of PON2-deficiency on cellular and metabolic phenotype were examined.8.CRISPR/Cas9-mediated deletion of STOM was performed on PON2^+/+and PON2^-/-human B-ALL cell line.The impacts of STOM-deficiency on glucose uptake and colony formation of PON2^-/-human B-ALL cell line were examined.9.The impacts of PON2-deficiency and PON2-overexpression on protein levels of STOM and GLUT1 were examined.10.Co-immunoprecipitation(Co-IP)assay was performed in BV173 cells under endogenous-or exogenous-PON2 expression to test the interactions among PON2,STOM,and GLUT1 and to determine the impacts of PON2-deficiency on STOM-GLUT1 interaction.11.Flow cytometry were performed to examine the cell surface expression of PON2.12.The impacts of PON2-deficiency on cytotoxic effects of glucocorticoids(GCs)were examine in both mouse and human B-ALL cells.[Result]1.PON2 mRNA and protein levels were significantly higher in Ph⁺B-ALL and Ph-like B-ALL patient samples in comparison to that in patients with other B-ALL subtypes.;2.High PON2 mRNA levels(PON2 ^high)correlated with poor clinical outcome of B-ALL patients but were not associated with that of patients with mature B-cell malignancies.3.Pon2-deficiency did not impact affinity maturation of germinal center B cells and the formation of NP-specific B cells.4.Pon2-deficiency impaired glucose uptake,total ATP production,lactate generation and glycolysis efficiency.Inducible overexpression of Pon2 augmented intracellular ATP levels in Pon2^+/+BCR-ABL1 B-ALL cells.Likewise,reconstitution of Pon2 expression increased ATP levels in Pon2^-/-BCR-ABL1 B-ALL cells.5.Partial rescue of ATP production were observed upon knockdown of Stom in Pon2^-/-BCR-ABL1 mouse B-ALL cells.6.PON2 deletion impaired colony forming capacity of human B-ALL(BV173)cells and resulted in G_0/1-cell cycle arrest.7.PON2-deficiency curtailed glucose uptake and total ATP production in human B-ALL cells.8.Protein levels of GLUT1 and STOM were not impacted by genetic inactivation and activation of PON2.9.Co-IP assays confirmed that PON2 directly interacts with GLUT1 and STOM in human B-ALL cells.Genetic inactivation of PON2markedly augmented interaction between STOM and GLUT1.10.Complete restoration of glucose uptake paralleled by partial rescue of ATP production and colony forming capacity were observed upon deletion of STOM in PON2^-/-B-ALL cells.11.Cell surface localization of PON2 were confirmed in HEK293T and mouse BCR-ABL1 B-ALL cells.12.Loss of Pon2 function potentiated the effects of GCs in mouse BCR-ABL1-and NRAS ^G12D-driven B-ALL cells.Likewise,genetic ablation of PON2 in human Ph⁺B-ALL cells substantially shifted the dose-response curve for GCs treatment.[Conclusion]1.High expression of PON2 is of unique significance for prognosis of B-ALL patients.2.PON2facilitates glucose transport and ATP production and enables B-cell malignant transformation by interfering interaction between GLUT1 and STOM.3.PON2-deficiency sensitizes B-ALL cells to glucose uptake inhibitor GCs.Targeting PON2-related glucose metabolism represents a promising way to treat Ph-like B-ALL and to improve clinical outcome of relapsed/refractory B-ALL patients.