Establishment Of Humanized Mouse Model Of MLL Rearranged Leukemias And Lineage Switch Determination

Background and objective:MLL gene rearrangement is one of the most common chromosomal abnormalitis in childhood and adult acute leukemias.With the relatively specific biological and clinical characteristics,MLL rearranged leukemias is a unique group of acute leukemias.However,the etiology,pathogenesis and lineage switch determination are not yet clear.In order to establish humanized mouse model of MLL rearranged acute leukemias,we transduced the CD34+ cells derived from human fetal liver(h FL)or adult bone marrow(a BM)by MLL-AF9 retrovirus and injected to NSG and NSG/SGM3 mice irradiated at 1.2 Gy,respectively.We followed these mice and compared the differences of leukemias induced in these mice.The role of environment(the presence of cytokine)and the target cells(h FLCs and a BMCs)in the determination of lineage was another aim of the current study.Methods:1.We transduced MLL-AF9 retrovirus into CD34+ stem cells in vitro and transplanted the cells into sublethally irradiated NSG and NSG/SGM3 mice to establish the humanized mouse model of MLL rearranged leukemias.2.Morphology and flow cytometry were used to detect the type of leukemia cells.3.Serially transferred MLL-AF9 h FLCs-NSG/SGM3 AML cells to NSG mice with or without IL-3 plasmid hydrodynamiclly injection to confirm the role of IL-3 in the development and lineage determination of MLL-AF9 leukemia.4.Southern blotting and IGH rearrangement were used to detect the colonality of MLL-AF9 leukemias.Results:1.Human fetal liver derived CD34+ cells expressing MLL-AF9 gave rise to acute lymphoid leukemia in NSG miceWe transduced MLL-AF9 retrovirus into human fetal liver CD34+ stem cells(hereafter referred to MLL-AF9 h FLCs)in vitro and transplanted intravenously the cells into sublethally irradiated NSG mice(hereafter referred to MLL-AF9 h FLCs-NSG mice).The MLL-AF9 h FLCs-NSG mice developed leukemia and the median survival time of was 183.0(179.0-192.0)days after transplantation.These phenotypic,morphological and diffuse organ infiltration characteristics of the leukemia cells in the model are fully compatible with the human acute lymphoid leukemia with MLL-AF9 rearrangement.To detect the leukemia initiating potential,we did adaptive transfer to NSG mice sequentially with different number of leukemic cells from primary MLL-AF9 h FLCs-NSG mice.Secondly adaptive transfer mice indicated that even less than 500 leukemic cells in NSG mice were sufficient to give rise to ALL.2.Human fetal liver derived CD34+ stem cells expressing MLL-AF9 produced acute myeloid leukemia in NSG/SGM3 miceWe injected intravenously the MLL-AF9 transduced human fetal liver CD34+ cells(same as the cells injected to NSG mice of Result 1)into sublethally irradiated NSG/SGM3 mice(hereafter referred to MLL-AF9 h FLCs-NSG/SGM3 mice).The MLL-AF9 h FLCs-NSG/SGM3 mice developed leukemia and the median survival time of was 34.0(34.0-36.0)days after transplantation.These phenotypic,morphological and diffuse organ infiltration characteristics of the human leukemia cells in these MLL-AF9 h FLCs-NSG/SGM3 mice were consistent with the human acute monoblastic leukemias,which is relatively common in MLL-rearranged AML.We also performed adaptive transfer to NSG/SGM3 mice sequentially with different number of leukemic cells from primary MLL-AF9 h FLCs-NSG/SGM3 mice.Secondly adaptive transfer mice indicated that more than 1×105 leukemic cells in NSG/SGM3 mice might give rise to AML.3.Adult BM derived CD34+ cells expressing MLL-AF9 could not give rise to leukemia in NSG miceWe transduced MLL-AF9 retrovirus into adult bone marrow CD34+ stem cells(hereafter referred to MLL-AF9 a BMCs)in vitro and performed intra-bone marrow transplantation into sublethally irradiated NSG mice(hereafter referred to MLL-AF9 a BMCs-NSG).MLL-AF9 a BMCs-NSG mice were healthy until 50 weeks after transplantation.The engraftment of human cells was low and GFP positive cells were undetectable in peripheral blood during serially monitor every 3 to 4 weeks.However,we detected the human CD45+GFP-cells in bone marrow at 21 weeks after transplantation.Postmortem analysis showed normal size of spleen and liver.Flow cytometry confirmed there were not GFP+ cells in spleen,bone marrow and peripheral blood.Thus,NSG mice transplanted with MLL-AF9 transduced adult BM cells did not develop leukemia.4.Adult bone marrow derived CD34+ stem cells expressing MLL-AF9 produced acute myeloid leukemia in NSG/SGM3 miceWe injected the MLL-AF9 transduced adult bone marrow derived CD34+ cells(same as the cells injected to NSG mice of Result 3)into sublethally irradiated NSG/SGM3 mice(hereafter referred to MLL-AF9 a BMCs-NSG/SGM3).The MLL-AF9 a BMCs-NSG/SGM3 mice developed AML and the median survival time after transplantation was 99.0(84.0-113.0)days.The phenotype is similar to the AML derived from NSG/SGM3 mice received the fetal liver CD34+ cells transduced with MLL-AF9..We also performed adaptive transfer to NSG/SGM3 mice sequentially with different number of leukemic cells from primary MLL-AF9 a BMCs-NSG/SGM3 mice.Secondly adaptive transfer mice injected with more than 5×106 cells in NSG/SGM3 mice might give rise to AML.5.Injection of IL-3 plasmid strongly accelerated the development of AML in NSG miceMLL-AF9 h FLCs NSG/SGM3 AML express human IL-3 receptor(hu CD123),but not human GM-CSF receptor or SCF receptor.MLL-AF9 h FLCs NSG/SGM3 AML were injected into NSG mice with or without IL-3 plasmid hydrodynamic injection.5/5 NSG mice with IL-3 plasmid hydrodynamic injection developed leukemia,3/5 died of leukemia in 37 weeks after transplantation.No leukemic cells were detected in PB of NSG mice without IL-3 plasmid hydrodynamic injection before 37 weeks after transplantation,while leukemic cells were detectable in BM in 4/5 mice.6.ALL cells appeared in IL-3 plasmid-injected NSG mice that received primary MLL-AF9 h FLCs NSG/SGM3 AML cellsWe injected the MLL-AF9 h FLCs NSG/SGM3 AML cells into NSG mice with IL-3 plasmid.3/5 mice developed AML,which is similar to primary MLL-AF9 h FLCs NSG/SGM3 AML.We detected ALL(CD19+CD33-GFP+)cells(named 2°MLL-AF9 h FLCs MLL-CD19+)and AML(CD19-CD33+GFP+)cells(named 2°MLL-AF9 h FLCs MLL-CD33+)at the same time in 2/5 mice.Southern blotting were detected the same bands in parimary MLL-AF9 h FLCs NSG/SGM3 AML?2°MLL-AF9 AML ? 2°MLL-AF9 h FLCs MLL-CD19+ and 2°MLL-AF9 h FLCs MLL-CD33+ cells.IGH rearrangement were positive in 2°MLL-AF9 h FLCs MLL-CD19+ cells,but negative in primary MLL-AF9 h FLCs NSG/SGM3 AML?2°MLL-AF9 AML and 2°MLL-AF9 h FLCs MLL-CD33+ cells.Conclusions:1.Human fetal liver derived CD34+ stem cells transduced with MLL-AF9 retrovirus were able to establish MLL-AF9 ALL humanized model in NSG mice.2.Human fetal liver and adult bone marrow derived CD34+ stem cells transduced with MLL-AF9 retrovirus were able to establish MLL-AF9 AML humanized model in NSG/SGM3 mice.3.Human cytokines(e.g.,IL-3)promote survival/engraftment and migration from BM to PB of MLL-AF9 AML cells.4.The intrinsic properties of the cell of origin(fetal liver or adult bone marrow),in addition to extrinsic cues(cytokines),dictate lineage of the MLL-AF9 leukemia cells.