Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia

Flowcytometric analysis confirming multilineage engraftment.

<p>(A) Representative flowcytometric results of EV- or MLL-AF10-transduced human hematopoietic cells. The human CD45⁺ GFP⁺ cells were analyzed for their lineage distributions to B cells (CD19⁺), T cells (CD3⁺), and myeloid cells (CD33⁺). (B) Multilineage differentiation of MLL-AF10-transduced cells. The data shows cells gated on the CD45⁺GFP⁺ cell population. The graph represents the mean ± SD of the frequencies of CD33⁺ myeloid cells, CD19⁺ B cells, and CD3⁺ T cells in the BM (upper) and spleens (lower) of mice engrafted with EV-transduced (n = 8) or MLL-AF10-transduced (n = 6) CD34⁺ HSCs. No difference in the graft composition between the EV- and MLL-AF10-expressing CD34⁺ HSCs was found. Similar results were obtained in 3 independent experiments.</p

Cooperation of MLL-AF10 with activated K-ras induced acute monoblastic leukemia.

<p>(A) Kaplan-Meier survival analysis of mice receiving transplants of human HSCs transfected with EV (n = 8), K-ras^G12V (n = 12), MLL-AF10 (n = 6), or MLL-AF10 plus K-ras^G12V (n = 6) vectors. (B) GFP and Venus expression in peripheral blood cells at the indicated weeks after transplantation with human HSCs co-transfected with the MLL-AF10 and K-ras^G12V genes. (C) May-Giemsa staining of the peripheral blood of mice engrafted with human HSCs co-transfected with the MLL-AF10 and K-ras^G12V genes. Morphologic leukemia cells were found in the peripheral blood of these mice 50 days after transplantation. (D) Splenomegaly in the MLL-AF10/K-ras^G12V mice. Spleens from mice engrafted with EV-transduced HSCs (left) and MLL-AF10/K-ras^G12V co-transduced HSCs (right) are shown. The graph shows the mean ± SD of the spleen weights from mice receiving transplants of EV-transduced HSCs (n = 6) or of MLL-AF10/K-ras^G12V co-transduced HSCs (n = 6). ** represents p<0.01.</p

Enforced expression of MLL-AF10 augmented multilineage hematopoiesis, but was insufficient to induce leukemogenesis in vivo.

<p>(A) Representative RT-PCR results confirming the long-term expression of the MLL-AF10 transcript in the BM cells of mice 25 weeks after transplantation (lane 1; water, lane 2; cells from a mouse in the EV-transfused group, lane 3; cells from a mouse in the MLL-AF10-transfused group, and lane 4; positive control (MLL-AF10 plasmid)). (B) Flowcytometric analysis of the frequency of GFP⁺ cells. The indicated vector (EV, left or MLL-AF10, right)-transduced human CD34⁺ cells, whose <i>in vitro</i> GFP expression is shown in the upper panels (Before) of the flowcytometric analysis, were transplanted into NOG mice. Twenty-five weeks later, the GFP-expressing cells gated on human CD45⁺ hematopoietic cells in the BM was measured (lower panels of the FACS profiles). The data shown are representative of 3 independent experiments. The graphs show the frequency of GFP⁺ cells in human CD34⁺ cells just before transplantation (Before) and the mean ± SD of the frequency of GFP⁺ cells in the BM and spleen of mice receiving transplants of EV-transduced HSCs (n = 8) or of MLL-AF10-transduced HSCs (n = 6) 25 weeks after transplantation, in one representative experiment of three. Similar results were obtained in the 3 independent experiments.</p

Immunophenotype and clonality of the MLL-AF10/K-ras-induced leukemia.

<p>(A) Frequencies of GFP⁺/Venus⁺ cells or human CD45⁺ cells in the BM, spleen, and liver at 8 weeks after transplantation with human HSCs co-transfected with the MLL-AF10 and K-ras^G12V genes were examined by flowcytometric analysis. The flowcytometry data shown are representative of 6 to 8 mice per group in one representative experiment of two (left). The average of %frequencies of the GFP⁺ and Venus⁺ cells in whole cells in the indicated organs is shown with the standard deviation (right, upper; n = 6). The absolute cell number of human CD45⁺ cells in the indicated organs is shown with the standard deviation (right, lower; n = 6). (B) Representative RT-PCR results confirming the stable, long-term expression of the MLL-AF10 and Flag-K-ras^G12V transcripts in human hematopoietic cells in the BM of mice 8 weeks after transplantation. (C) Lineage distribution of the GFP⁺ and Venus⁺ cells in the BM of a mouse engrafted with HSCs expressing MLL-AF10 and activated K-ras. (D) Southern blot analysis of DNA prepared from the human blood cells in the spleen of mice receiving transplants of MLL-AF10/K-ras^G12V co-transduced HSCs. Independent leukemia samples derived from two mice (lane 1; mouse 1 and lane 2; mouse 2) were examined. DNA was digested with Bgl II and probed with an EGFP probe. M: marker.</p

Co-transduction of activated K-ras and MLL-AF10 into CD34⁺HSCs.

<p>(A) Schematic structure of the MLL-AF10-GFP and Flag-K-ras^G12V-Venus vectors. (B) Infectious efficiency of the MLL-AF10-GFP and Flag-K-ras^G12V-Venus co-transfection. The data and the summary shown in the flowcytometric analysis is representative of the transduced CD34⁺ HSCs in 2 experiments.</p