Targeting ATG4B Inhibits The Growth Of T-ALL Cells

Objective:This thesis aims to explore the effects of targeting ATG4B on T cell acute lymphoblastic leukemia(T-ALL)cells and its underlying mechanisms.Methods:(1)The expression of several autophagy related genes in leukemia cells from T-ALL patients compared with the normal control cells was measured,and the autophagy activities of leukemia cells from T-ALL patients and normal control cells were detected with monodansylcadaverine(MDC)staining.(2)The effects of ATG4B silencing on autophagy,growth,colony-forming cells(CFC)production and apoptosis of both Jurkat and MOLT-4 cells were studied.The effect of ATG4B silencing on the leukemogenesis ability of Jurkat cells was investigated as well.(3)The effects of ATG4B silencing on the proliferation of CD3~+and CD34~+cells from normal bone marrow(NBM)were studied.(4)The effects of LV-320(an allosteric inhibitor against ATG4B)on autophagy,growth,CFC production and apoptosis of Jurkat cells in vitro and in vivo were investigated.(5)The cellular and molecular mechanisms of apoptosis induced by targeting ATG4B were studied with western blots and fluorescence staining analyses.Results:(1)The expressions of ATG4A,ATG4B,ATG4C,ATG4D and ATG5 in CD3~+cells from T-ALL patients were significantly higher than those in normal bone marrow CD3~+cells.In addition,the relative expression of ATG4B(normalized to that of BECLIN-1)was higher than the other autophagy-related genes.MDC staining analysis showed that T-ALL cells had higher autophagy activities than normal control cells.(2)Silencing ATG4B inhibited autophagy flux,growth,CFC production,and induced apoptosis in both Jurkat and MOLT-4 cells;moreover,silencing ATG4B attenuated the leukemogenesis ability of Jurkat cells in immunodeficient mice.(3)Silencing of ATG4B did not affect the growth of NBM CD3~+cells and the CFC production of NBM CD34~+cells.(4)LV-320 suppressed autophagy flux and inhibited the growth of Jurkat cells in vitro and in vivo,while sparing normal control cells.(5)Both ATG4B silencing and LV-320treatment induced caspase dependent apoptosis.LV-320 treatment led to the depolarization of mitochondrial membrane,the reduction of active mitochondria and the accumulation of reactive oxygen species(ROS).Conclusion:Autophagy is highly activated in T-ALL cells and several autophagy related genes are up-regulated in T-ALL cells compared with normal control cells,including ATG4B.Both genetical and pharmacological inhibition of ATG4B reduce autophagy flux.These operations inhibit the growth of T-ALL cells in vitro and in vivo,while sparing NBM CD3~+cells and CD34~+cells.The defective mitochondrial and ROS accumulation upon ATG4B targeting trigger caspase dependent apoptosis of T-ALL cells.Overall,these data demonstrate that ATG4B is a novel biomarker and therapeutic target of T-ALL cells.