Growth-arrest Specific 2 Promotes BCR-ABL Mediated Malignant Transformation

Objective:The fusion gene BCR-ABL is believed to induce human chronic myeloid leukemia(CML).This study aims to elucidate the role and underlying mechanism of growth-arrest specific2(GAS2)in BCR-ABL mediated malignant transformation in a mice model.Methods:(1)GAS2 and its dominant negative form(GAS2DN)were delivered into BaF3 or BaF3/BCR-ABL cells with lentiviral vectors,and the effects of GAS2 and GAS2DN on the proliferation and colony-forming cell(CFC)production were assessed.(2)The variously treated BaF3 cells were injected into lethally irradiated mice through tail vein,including control,GAS2 or GAS2DN alone,BCR-ABL alone and GAS2 or GAS2DN with BCR-ABL.The mice were monitored closely to exam the formation of mice leukemia.(3)Western blot and qRT-PCR were used to detect the protein and mRNA expression of isocitrate dehydrogenase 2(IDH2)upon GAS2 or GAS2DN expression in MEG-01,Ba F3/BCR-ABL and K562 cells.(4)Two independent shRNAs against IDH2 were delivered with lentiviral vector to MEG-01 cells,and the effect of IDH2 silencing on the growth and CFC ability were studied.(5)CD34~+cells from healthy donors and CML patients in chronic phase(CP)and blast crisis(BC)were collected and the expression of GAS2 and IDH2 were assessed with qRT-PCR as well.Results:(1)GAS2 inhibited Calpain activity,while its overexpression didn't affect the proliferation or CFC production of neither Ba F3 nor Ba F3/BCR-ABL cells.In contrast,GAS2DN elevated Calpain activity.GAS2DN significantly suppressed the growth and CFC production of BaF3/BCR-ABL cells,while it didn't affect BaF3 cells.(2)GAS2 overexpression was not able to induce leukemia in this experimental setting,however,it significantly accelerated BCR-ABL induced leukemia(P=0.0001).In contrast,GAS2DN significantly delayed BCR-ABL induced leukemia(P=0.0006);in addition,GAS2DN significantly reduced the weights of spleen and liver in the diseased mice.We also showed that in a cohort of Chinese patients,GAS2 expression was significantly increased along disease progression.(3)GAS2DN reduced the protein expression of IDH2,while had no effect on the mRNA expression of IDH2.Similar results were obtained with GAS2 or GAS2DN expressed BaF3/BCR-ABL cells.Moreover,we showed that GAS2DN mediated IDH2 reduction required Calpain2 in K562 cells.(4)IDH2 transcript was expressed significantly higher in CML CD34~+than their normal control cells.IDH2 silencing inhibited the growth and CFC production of MEG-01 cells.Conclusion:GAS2 promoted the malignant transformation ability of BCR-ABL,while which was attenuated by GAS2DN;GAS2 modulated the expression of IDH2posttranscriptionally and requiring activity of Calpain2;IDH2 was aberrantly expressed in CML CD34~+cells compared with normal control cells,and the silence of IDH2 in CML cells led to growth inhibition and reduced CFC production.