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

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

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

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

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

Pathological phenotypes of the leukemia.

<p>(A) Hematoxylin and eosin staining showing the infiltration of leukemic cells in the indicated organs of mice engrafted with HSCs expressing the MLL-AF10 and K-ras^G12V genes compared to control mice. (B) Immunostaining by a human CD45 mAb in the BM, spleen, and liver in mice engrafted with HSCs expressing the MLL-AF10 and K-ras^G12V genes.</p

Structure and <i>in vitro</i> analysis of foamy virus vectors.

<p>(<b>A</b>) Structure of the foamy virus vectors. The UCOE631 promoter sequence from the human HNRPA2B1-CBX3 locus and transgenes were inserted into the FV vector. (<b>B</b>) Cell-surface expression of γc on ED40515(–) cells transduced with the FV-IL2RG vector. (<b>C</b>) STAT5 phosphorylation upon IL-2 stimulation. ED40515(–) cells, an ED40515(–)-derived transfectant with a γc gene, EDγ cells, and ED40515(–) cells transduced with the indicated vectors were stimulated with IL-2 for 30 min, and STAT5 phosphorylation in each cell line was detected by a STAT5 phosphospecific mAb.</p

Profile of provirus integration in transduced cells.

<p>(<b>A</b>) Position of FV and RV integration sites. The percentage of all integration sites within 15 kb of transcriptional start sites, within genes that contain putative microRNA genes, and within 30 kb of oncogenes is shown for FV vector- or RV vector-treated cells. *p<0.05, χ²-test. (<b>B</b>) A 100-kb window centered on TSS in the RefSeq database is shown. Relative frequencies of FV and RV vector integrations in each interval were calculated by dividing the percentage of integration b the indicated interval length.</p

Reconstitution of T and B cells.

<p>(<b>A</b>) Cell-surface expression of γc on peripheral CD8⁺ T cells in γc-KO mice treated with FV-IL2RG-treated HSCs. The upper and lower panels show isotype-control and γc-specific stainings, respectively. (<b>B</b>) The absolute numbers of CD4⁺ T, CD8⁺ T, sIgM⁺ B, and NK cells in the spleen of γc-KO mice treated with FV-EGFP and FV-IL2RG (n = 4 in each group). <i>N.D.</i>, not detectable. (<b>C</b>) Serum IgM, IgG, and IgA in FV-IL2RG-treated mice. Serum levels of IgM, IgG, and IgA were measured by ELISA. Results shown are the mean ± SD of the stimulation index from 4 mice in each group. *p<0.05 and **p<0.01, Student t-test.</p