Oligomerization Of Oncoproteins In Leukemogenesis And Target Therapy

This dissertation comprises two parts: I)Study on the mechanism of oridonin in treating t(8;21)acute myeloid leukemia(AML),which shows that oligomerization of AML1-ETO is disrupted by oridonin-induced?AML1-ETO.II)Study on the pathogenesis and arsenic-targeted therapy of acute promyelocytic leukemia(APL),which shows that PML-RING tetramerization is essential for the function of PML and PML-RAR? and response to arsenic.The study of pathogenesis and targeted therapy on the two subtypes of leukemia,indicates that oligomerization of oncoproteins is essential for their function and could be used as target for treatment.These findings allow us to establish a solid foundation for understanding leukemogenesis and developing more effective treatment strategy.PART ?STUDY ON THE MECHANISM OF ORIDONIN IN TREATING T(8;21)ACUTE MYELOID LEUKEMIA: OLIGOMERIZATION OF AML1-ETO IS DISRUPTED BY ORIDONIN-INDUCED ?AML1-ETOThe t(8;21)(q22;q22)translocation is the most frequent chromosomaltranslocation in AML(12–20%),which results in the fusion gene of AML1-ETO.It has been well established that AML1-ETO protein plays a critical role in the pathogenesis of t(8;21)leukemia through its oligomerization and aberrant transcription regulation of target genes.Nearly60% of patients with this type of AML fail to achieve long-term disease-free survival.Our previous studies demonstrated that oridonin selectively induced apoptosis of t(8;21)leukemia cells through a truncated fragment?AML1-ETO by caspase-3-dependent cleavage.However the molecular mechanism of oridonin treatment as well as function of ?AML1-ETO remain unclear.Here,we show that oridonin interacts with the sulfhydryl groups of glutathione and thioredoxin/thioredoxin reductase to increase intracellular reactive oxygen species,which in turn activates caspase-3 in t(8;21)cells.Moreover,oridonin binds AML1-ETO,directing the enzymatic act cleavage at aspartic acid 188 via caspase-3 to generate ?AML1-ETO and preventing the protein from further proteolysis.?AML1-ETO interacts with AML1-ETO through NHR2 domain,abrogates oligomerization of AML1-ETO,and interfers with the trans-regulatory functions of remaining AML1-ETO oncoprotein,thus acting as a tumor suppressor that mediates the anti-leukemia effect of oridonin.Furthermore,oridonin inhibits the activity of c-Kit+ leukemia-initiating cells and prolongs the survival of AML1-ETO9 a leukemia mouse model.Therefore,oridonin,which can target AML1-ETO,is a potential lead compound for molecular target-based therapy of leukemia.Meanwhile,we find that ETO is colocalized with PML,and ETO may participate in the assembly and function of PML nulear body.PART ?STUDY ON THE PATHOGENESIS AND ARSENIC-TARGETED THERAPY OF APL: PML-RING TETRAMERIZATION IS ESSENTIAL FOR THE FUNCTION AND ARSENIC RESPONSE OF PML AND PML-RARAAPL,that used to be a highy fatal acute leukemia,is now highly curable.The frequency of APL is 10-15% in all AML cases,while over 95% of APL patients harbor t(15;17)(q22;q21)translocation which results in PML-RAR?oncoprotein.PML-RAR? is able to form homodimers or oligomers and recruit co-repressor proteins including histone deacetylase and DNA methylating enzymes,therefore repressing the transcription of target genes responsible for granulocytic differentiation and contributing to APL development.Arsenic,a potential agent for APL,is able to bind directly to PML/PML-RAR?,and induce their oligomerization and then facilitate subsequent sumoylation/ubiquitination and proteasomal degradation.PML is critical component of sub-nuclear structures,which are thereby called PML nuclear bodies(NBs).Oligomerization of PML constitutes the basis for structure and function of PML NB.PML-RAR? can also form heterodimers with PML to disrupt PML NB assembly.For a better understanding of the mechenism of arsenic treatment effect at molecular level,we carried out the exploration and analysis of structural protein of PML/PML-RAR?.We have solved the PML-RING domain structure and found it to be a highly symmetrical ring-shape tetramer via two highly conserved amino acid interfaces.The tetramerization of PML-RING is essential for the function of PML and PML-RAR?.Disruption of the interfaces inhibits PML oligomerization,abolishes PML NB biogenesis and sumoylation,impaires PML-RAR? transformation and their response to arsenic,and may even influences APL pathogenisis.