The Role Of SUMOylation In Acute Leukemia And The Role Of PRDM5 In Acute Myeloid Leukemia

Part I The role of SUMOylation in acute leukemiaObjective:There have been more and more research reported the role of SUMOylation,a kind of posttranslational modification that similar to ubiquitination in tumor’initiation and progression.These studies mainly focused on the function of SUMOylation in signaling pathway related proteins or oncoproteins.However,the role of enzymes involving in SUMOylation in the initiation and maintenance of acute leukemia remains unclear.Ubc9,the only E2 conjugating enzyme,plays a key function of regulating SUMOylation and may be an important biomarker for the diagnosis and prognosis of acute leukemia.Previously,we generated Ubc9^fl/+;Vav-cre mice and our previous study demonstrated that Ubc9haploinsufficiency mildly affected the steay-state hematopoiesis and accelerated the progression of MLL-AF9 induced AML.Here,we will investigate the function of Ubc9haploinsufficiency in competitive hematopoiesis and acute leukemia（MLL-ENL/ICN1overexpression leukemia models）generation and maintenance and investigate the effects of Ubc9 knockdown in human acute leukemia cell lines.Methods:We crossed Ubc9^fl/fl mice with Vav-cre mice and obtained Ubc9^fl/+;Vav-cre mice.For competitive repopulation assay,BM cells（CD45.2）from control group and experimental group mice,together with equal competitor BM cells（CD45.1）were transplanted into lethally irradiated（9.5 Gy）WT（CD45.1）recipient mice.Flow cytometry analysis was conducted to detect the reconstruction ability of CD45.2 cells in peripheral blood and measured the ratio of CD45.2+lineage positive cells and hematopoietic stem progenitor cells in BM.Then we performed the lentivirus conduction to c-Kit+/Lin-cells and generated the AML and T-ALL mouse model.We compared the survival of leukemia mice and used flow cytometry to analysis the infiltration,proportion,number,cell cycle,apoptosis and differentiation of leukemia cells in these two groups.In addition,Using CRISPR-Cas9technology,we knockdown Ubc9 in human acute leukemia cell lines,and investigated the role of Ubc9 in cell proliferation,apoptosis and cell cycle.Results:Compared with WT control,Ubc9 haploinsufficiency mice showed significantly lower level of donor chimerism in recipients’peripheral blood and the HSC appeared impared reconstitution ability and higher rate of apoptosis.Furthermore,Ubc9 haploinsufficiency accelerated transformation of MLL-ENL leukemia and promoted the progression of leukemia.Flow cytometry analysis showed that Ubc9 haploinsufficiency leukemia cells were characterized with blocked differentiation,decreased apoptosis,accelerated cell cycle and enhanced colony formation ability.Consistent with MLL-ENL leukemia model,The ICN1T-ALL mouse model also showed that Ubc9 haploinsufficiency promoted the occurrence and progression of T-ALL leukemia.Besides,the Ubc9 knockdown promoted the growth and accelerated cell cycle except apoptosis in human acute leukemia lines.Conclusion:In competitive hematopoiesis,Ubc9 haploinsufficiency impaired the ability of HSPC to rebuild hematopoiesis and affected the differentiation of HSPC into lineage positive cells.In addition,Ubc9 haploinsufficiency accelerated the transformation,progression of MLL-ENL/ICN1 leukemia cells and promoted the proliferation of human acute leukemia cells.Part Ⅱ The role of PRDM5 in acute myeloid leukemiaObjective: Acute myeloid leukemia is a group of highly heterogeneous hematopoietic malignant clonal diseases characterized with low survival,high recurrence and chemotherapy resistance.Abnormal expression of proto-oncogenes and mutation or deletion of tumor suppressor genes plays an important role in the development of AML.Many studies have shown that the expression of PRDM5 is lowly in variety of solid tumor tissues or cells and PRDM5 exhibits as a tumor suppressor gene,but the role of PRDM5 in hematopoietic malignant,especially in AML,is still unknown.In this section,we mainly focus on the effect of PRDM5 overexpression in acute myeloid leukemia cells.Methods: We used the public databases to analyze the expression of PRDM5 in prediction of AML patients’ survival.Then,we constructed the PRDM5 overexpressing lentiviral vector and infected AML cell lines OCI-AML3 and U937 cells（CT and OE groups）.The expression of PRDM5 was validated at m RNA and protein level.Viable cells counting,celltrace violet assay,colony formation,Ki67 and apoptosis analysis and Transwell assay were used to measure the ability of proliferation and migration in AML cells after PRDM5 overexpression.Subsequently,the in vivo impact of tumorigenic capability was investigated by subcutaneous inoculation of PRDM5 overexpressed tumor cells in nude mice.Results: A high level of PRDM5 in AML was associated with poor prognosis and survival.PRDM5 overexpression promoted OCI-AML3 and U937 cell proliferation,colony formation capacity,migration without affecting apoptosis in vitro and enhanced oncogenic capacity in vivo.PRDM5-mediated progression of OCI-AML3 and U937 cells is partially regulated through JNK pathway activation and c-Myc downregulation.The JNK pathway inhibitor SP600125 could effectively inhibit the growth,migration of PRDM5 overexpressing AML cells in vitro and reduce the growth ability in vivo.Conclusion:The high expression of PRDM5 is positively correlated with poor prognosis and survival of AML patients.Overexpression of PRDM5 in OCI-AML3 and U937 cells promotes the growth of cells in vitro/vivo and enhanced the migration ability in vitro by activating the JNK pathway.