The Role Of DBC1 In Pathogenesis Of MLL Rearranged Acute Myeloid Leukemia

Objective:MLL rearranged leukemia is characterized by chromosomal rearrangements at11q23.According to WHO classification of tumors of hematopoietic and lymphoid tissues in 2016,it is divided into MLL-rearranged acute myeloid leukemia（MLL-r AML）,acute leukemia with mixed lineage and MLL-rearranged acute lymphoblastic leukemia（MLL-r ALL）.The disease progresses rapidly and the prognosis is poor.The research on the regulatory mechanism of DOT1L protein stability,which plays an important role in the initiation and development of MLL rearranged leukemia,not only helps to deepen the understanding of the pathogenesis of MLL rearranged leukemia,but also has important theoretical and practical significance for the targeted treatment of MLL rearranged leukemia.This research mainly involves in the effect of DBC1 on the proliferation of MLL-r AML and its related molecular mechanism,so as to provide theoretical and experimental basis for the development of drugs targeting the stability of DOT1L protein.Methods:The effect of knockdown of DBC1 on the proliferation of MLL-r AML cell line was observed in vitro.MLL-r AML cell lines（THP-1 and MV4-11）and non-MLL leukemia cell lines（KG1a and NB4）with DBC1 knockdown were established by lentivirus-mediated RNA interference.Furthermore,the effect of DBC1 knockdown on the apoptosis and cell cycle distribution of were investigated in MLL-r AML cells.The effect of Dbc1 deletion on the disease progression of MLL-r AML mice model induced by MLL-AF9 fusion protein was studied in vivo.With retroviral technology,we transduced MLL-AF9 fusion gene into bone marrow hematopoietic stem/progenitor cells of Dbc1 knockout mice to establish MLL-r AML mouse models in Dbc1^fl/fl and Dbc1^-/-mice and investigated the effects of Dbc1 deletion on the initiation and development of MLL-r AML disease in mice,including survival time,tumor load in peripheral blood,leukemia infiltration in liver and spleen and so on.Meanwhile,the effect of Dbc1 deletion on the normal hematopoiesis of mice was studied in the noncompetitive transplantation experiments.Detection of the degree of allogeneic chimerism and the frequency of mature blood cells and hematopoietic stem/progenitor cells in recipient mice was performed to evaluate whether the deletion of Dbc1 affects the hematopoietic reconstruction.At the molecular level,we explored the molecular mechanism of DBC1affecting the progression of MLL-r AML,which was explored by RNA interference and overexpression of related genes combined with immunocoprecipitation。Results:In vitro,downregulation of DBC1 promoted the proliferation of MLL-r AML cell lines（THP-1 and MV4-11）,rather than non-MLL rearranged AML cell lines（KG1a and NB4）;Furthermore,downregulation of DBC1 increased the proportion of S-phase in the MLL-r AML cell lines,but had no significant effect on the apoptosis in these cell lines.In vivo,on the one hand,the deletion of Dbc1 increased the proportion of leukemia cells in the peripheral blood of mice,promoted the infiltration of leukemia cells in the liver and spleen,and shortened the survival time of MLL-AF9 leukemia mice.On the other hand,Dbc1 deletion had no significant effect on the hematopoietic chimerism and the proportion of terminal differentiated and hematopoietic stem/progenitor cells during the hematopoietic reconstitution.Mechanically,we investigated that the increased proliferation rates in the MLL-r AML cells with DBC1 knock down were mediated by the upregulation of DOT1L protein.Furthermore,DBC1 promoted the degradation of DOT1L via the ubiquitin-proteasome pathway.And then the upregulation of DOT1L,resulting from the increased level of acetylation at K358 of DOT1L in the DBC1 downregualtion cells,was dependent on CBP and coutributed to the increased proliferation.;At last,DBC1,which interacted directly with CBP,competed with the CBP-binding protein DOT1L to bind CBP.Conclusion:In this study,we preliminarily confirmed that DBC1 competed with histone H3K79 methyltransferase DOT1L to bind histone acetyltransferase CBP,which mediates DOT1L K358 acetylation,and promoted the ubiquitination degradation of DOT1L,therefore deletion or knockdown of DBC1 could accelarate the progress of MLL-r AML in vivo and in vitro.