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A. Sharma et al.
   Supplementary Figure S4E and F). In support of the GSEA analysis, a strong downregulation of the anti-apoptotic Bcl2 gene family, Mcl1 and Survivin was shown by qRT- PCR (Figure 2C). MLL-AF9/Mn1null cells showed low lev- els of the hematopoietic stem/progenitor cell marker c- Kit/CD117 but high expression of the mature myeloid cell marker Mac1/CD11b and Gr-1/Ly-6G in Mn1 null cells compared to MLL-AF9/Mn1wt cells (Figure 2D and Online Supplementary Figure S6A). This observation was also sup- ported by morphological analysis, as Mn1 null cells had a lower nuclear-cytoplasmic ratio and were more granulated (Figure 2E), and gene sets were enriched for myeloid and lymphoid cell differentiation (Online Supplementary Tables S4 and S5, and Online Supplementary Figures S4B and S6B and C). MLL-AF9 oncogene expression was found to be unaltered or increased upon loss of Mn1 in MLL- AF9/Mn1null cells when compared with MLL- AF9/Mn1wt cells, excluding the possibility that downreg- ulation of the oncogene is responsible for the anti- leukemic effects of MN1 loss (Online Supplementary Figure S7A-C). Hence, MN1 deletion suppresses cell cycle, pro- motes apoptosis, and induces differentiation in MLL posi- tive leukemia cells.
MLL-AF9-induced leukemogenesis requires Meningioma 1
To further investigate the role of Mn1 deletion in MLL- AF9 leukemogenesis in vivo, we transplanted MLL- AF9/Mn1wt and MLL-AF9/Mn1null cells in syngeneic mice and monitored onset of leukemia and survival. Mice transplanted with MLL-AF9/Mn1null cells had <1% engraftment in all ten mice, whereas mice trans- planted with MLL-AF9/Mn1wt cells showed >90% engraftment at week 4 (Figure 3A). At eight weeks, engraftment in MLL-AF9/Mn1null was below 2% (except for one mouse with 88% MLL-AF9 expressing cells, which consequently died), while 8 of 10 MLL- AF9/Mn1wt mice died from leukemia and the two sur- viving mice showed >90% engraftment (Figure 3A). Correspondingly, white blood cell (WBC) counts were significantly lower in MLL-AF9/Mn1null than in MLL- AF9/Mn1wt mice (Figure 3B). Loss of Mn1 significantly prolonged survival as compared to Mn1wt mice (Figure 3C). At death, mice transplanted with MLL-AF9/Mn1 null cells had significantly lower spleen weight (Figure 3D). Since loss of Mn1 in MLL-AF9 cells did not show a potent engraftment ability, and to exclude the possibility of homing defects, we transplanted equal numbers of MLL-AF9 (Mn1wt/null) cells intravenously in sub-lethal- ly irradiated mice. Mice were sacrificed after 8 and 24 hours and MLL-AF9/Mn1null cells showed better hom- ing in bone marrow than mice transplanted with MLL- AF9/Mn1wt cells, suggesting that differences in homing could not account for the differences in survival (Figure 3E). In addition, to rule out the presence of any immuno- logical effects against MLL-AF9/Mn1null cells, we trans- planted MLL-AF9 (Mn1wt or null) cells in NSG mice (lacking cellular and humoral immunity) and monitored leukemia onset and survival. Similar to syngeneic mouse transplantation studies, MLL-AF9/Mn1null mice did not develop leukemia, while their MLLAF9/Mn1wt counter- part quickly died from leukemia (Online Supplementary Figures S8A and B and S9A-H for blood counts). Thus, MLL-AF9 positive murine leukemia requires MN1 expression to induce leukemia in vivo.
MLL-AF9 and MLL-AF4 rearranged human leukemias also depend on Meningioma 1
Besides syngeneic MLL-AF9 transplantation studies, we also assessed the role of MN1 deletion in MLL-AF4 (MV- 4-11) and MLL-AF9 (THP-1) leukemias in vivo. Equal num- bers of THP-1 and MV-4-11 (MN1wt or MN1null) cells were transplanted intravenously in NSG mice, and the onset of leukemia and survival were assessed. MN1null cells showed lower engraftment of transplanted cells in peripheral blood at four weeks and improved blood counts compared to their wild-type counterparts (Figure 4A and B and Online Supplementary Figures S10 and S11). MN1 deletion significantly prolonged survival of mice transplanted with THP-1/MN1null and MV-4-11/MN1null clones (Figure 4C and D). At death, engraftment in bone marrow and spleen weight were also found lower in mice transplanted with MN1 deletion clones of THP-1 and MV- 4-11 (Figure 4E and F and Online Supplementary Figure S12A and B). In addition, we also evaluated the tumor-forming ability of THP-1/MN1null and MV-4-11/MN1null clones by subcutaneous transplantation in NOD-SCID mice. Tumor volumes were monitored every five days starting 15 days after transplantation. Deletion of MN1 led to sig- nificantly reduced tumor volumes for 2 of 3 MN1null clones as compared to wild-type MN1 human leukemic cell lines (Online Supplementary Figure S13A and B). Our in vivo transplantation studies suggest that loss of MN1 critically affects leukemia proliferation in human MLL- rearranged AML.
Meningioma 1 overexpression restores leukemogenicity in MLL-AF9/Mn1null cells
In an attempt to rescue the deleterious effects caused by Mn1 deletion, we over-expressed control (MIY) vector or MN1 in MLL-AF9/Mn1 null cells and characterized its properties in vitro and in vivo. MLL-AF9/Mn1null cells transduced with MN1 overcame the reduced proliferative capacity of MLL-AF9/Mn1null cells similarly to MLL- AF9/Mn1wt cells (Figure 5A). Similarly, MLL- AF9/Mn1null cells with MN1 expression restored the reduced colony-forming potential of MLL-AF9/Mn1null cells akin to MLL-AF9/Mn1wt cells (Figure 5B and C). We also studied the leukemogenic potential of MLL- AF9/Mn1null cells rescued by MN1 expression in compar- ison with MLL-AF9 (Mn1wt or null) cells transduced with vector control. As previously mentioned in our manu- script, MLL-AF9/Mn1null cells do not possess the ability to engraft in mice. However, MLL-AF9/Mn1null cells with ectopic MN1 expression engrafted in mice and induced a leukemic phenotype with short survival similar to MLL- AF9/Mn1wt mice (Figure 5D and E and Online Supplementary Figure S14). Hence, deleterious effects caused by loss of MN1 expression can be rescued by restoring the expression of MN1.
Meningioma 1 maintains expression of the distal HOXA cluster and MEIS1
MLL-AF9-mediated leukemogenesis is primarily medi- ated by upregulation of the Hox/Meis1 gene cluster and their target genes.33,35 DOT1L methylates histone H3 on lysine 79 (H3K79me2), which has been associated with MLL-AF9 binding and expression of Hoxa cluster genes and Meis1 in normal hematopoietic progenitors and MLL- r leukemias.25 Gene expression analysis by qRT-PCR in MLL-AF9/Mn1null and Mn1wt cells showed that Hoxa3
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