American Society of Hematology

Figure 7.

$Overexpression of Bach2 suppresses the growth of MZPs, transformed lymphoma and leukemia cells, and B-myeloid conversion. (A) Scheme of 5′ Bach2 locus including exon 1 and the upstream regulatory region. Predicted binding sites of Notch pathway component MYC and NF-κB/RelB are indicated with short colored dashes below the scheme. Column diagrams show ChIP-qPCR data demonstrating binding of Myc and RelB to the Bach2 locus in TriB spleen B cells. (B) Outlines of Bach2 overexpression experiments. (C) Enforced expression of Bach2 inhibits growth of TriB MZPs. Cells were retrovirally transfected with Bach2 expression vector (white column) or empty vector used as control (black), fractionated by flow sorting, and cocultured with OP9 stromal cells in the presence of SCF, Flt3l, interleukin-7 (IL-7), and IL-4 (10 ng/mL each). The experiment was performed in 3 biological replicates. The cell numbers were calculated by total cell numbers × 45.2% (to exclude cocultured stromal cells; data not shown). (D) Experiment was performed as described in panel C, except the MZP/OP9 coculture was supplemented with SCF, Flt3L, and IL-3 (10 ng/mL each). Flow cytometry revealed the Bach2-dependent shift from Mac1+ myeloid cells to CD19+ B cells. (E) Experiment was performed as described in panel C except instead of coculturing in vitro, cells were transferred to lethally irradiated and BM-rescued CD45.1 recipient mice (n = 4). (F) qPCR analysis of Bach2, Cebpα, and Spic expression in TriB MZPs cocultured with OP9 cells. MZPs overexpressing Bach2 (white) are compared with MZPs containing normal levels of Bach2 (vector, black; n = 3). (G) Growth inhibition of flow-sorted TriB lymphoma cells containing elevated levels of Bach2. Cells were retrovirally transduced with Bach2 or empty vector and cocultured with OP9 stromal cells in the presence of SCF, Flt3l, IL-7, and IL-4 (10 ng/mL each). Cell numbers were calculated by multiplying the total cell numbers × CD45.2 percentage (n = 3). (H) Sorted inducible Bach2-transduced converted leukemia cells (Bswitch) were cultured in RPMI with 15% fetal bovine serum (FBS) in the presence of SCF, Flt3L, and IL3 (10 ng/ml each) with or without 4-OHT and cells were counted 3 days later (n = 3). (I) BrdU (3 hours, 10 μM, in vitro) incorporation assay indicating suppressed proliferative activity of Bach2-expressing converted leukemia cells (n = 3). *P < .05, **P < .01.$

Overexpression of Bach2 suppresses the growth of MZPs, transformed lymphoma and leukemia cells, and B-myeloid conversion. (A) Scheme of 5′ Bach2 locus including exon 1 and the upstream regulatory region. Predicted binding sites of Notch pathway component MYC and NF-κB/RelB are indicated with short colored dashes below the scheme. Column diagrams show ChIP-qPCR data demonstrating binding of Myc and RelB to the Bach2 locus in TriB spleen B cells. (B) Outlines of Bach2 overexpression experiments. (C) Enforced expression of Bach2 inhibits growth of TriB MZPs. Cells were retrovirally transfected with Bach2 expression vector (white column) or empty vector used as control (black), fractionated by flow sorting, and cocultured with OP9 stromal cells in the presence of SCF, Flt3l, interleukin-7 (IL-7), and IL-4 (10 ng/mL each). The experiment was performed in 3 biological replicates. The cell numbers were calculated by total cell numbers × 45.2% (to exclude cocultured stromal cells; data not shown). (D) Experiment was performed as described in panel C, except the MZP/OP9 coculture was supplemented with SCF, Flt3L, and IL-3 (10 ng/mL each). Flow cytometry revealed the Bach2-dependent shift from Mac1⁺ myeloid cells to CD19⁺ B cells. (E) Experiment was performed as described in panel C except instead of coculturing in vitro, cells were transferred to lethally irradiated and BM-rescued CD45.1 recipient mice (n = 4). (F) qPCR analysis of Bach2, Cebpα, and Spic expression in TriB MZPs cocultured with OP9 cells. MZPs overexpressing Bach2 (white) are compared with MZPs containing normal levels of Bach2 (vector, black; n = 3). (G) Growth inhibition of flow-sorted TriB lymphoma cells containing elevated levels of Bach2. Cells were retrovirally transduced with Bach2 or empty vector and cocultured with OP9 stromal cells in the presence of SCF, Flt3l, IL-7, and IL-4 (10 ng/mL each). Cell numbers were calculated by multiplying the total cell numbers × CD45.2 percentage (n = 3). (H) Sorted inducible Bach2-transduced converted leukemia cells (Bswitch) were cultured in RPMI with 15% fetal bovine serum (FBS) in the presence of SCF, Flt3L, and IL3 (10 ng/ml each) with or without 4-OHT and cells were counted 3 days later (n = 3). (I) BrdU (3 hours, 10 μM, in vitro) incorporation assay indicating suppressed proliferative activity of Bach2-expressing converted leukemia cells (n = 3). *P < .05, **P < .01.

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