Hoxa9 and Hoxa10 induce CML myeloid blast crisis development through activation of Myb expression

Abstract

Mechanisms underlying the progression of Chronic Myeloid Leukemia (CML) from chronic phase to myeloid blast crisis are poorly understood. Our previous studies have suggested that overexpression of SETBP1 can drive this progression by conferring unlimited self-renewal capability to granulocyte macrophage progenitors (GMPs). Here we show that overexpression of Hoxa9 or Hoxa10, both transcriptional targets of Setbp1, is also sufficient to induce self-renewal of primary myeloid progenitors, causing their immortalization in culture. More importantly, both are able to cooperate with BCR/ABL to consistently induce transformation of mouse GMPs and development of aggressive leukemias resembling CML myeloid blast crisis, suggesting that either gene can drive CML progression by promoting the self-renewal of GMPs. We further identify Myb as a common critical target for Hoxa9 and Hoxa10 in inducing self-renewal of myeloid progenitors as Myb knockdown significantly reduced colony-forming potential of myeloid progenitors immortalized by the expression of either gene. Interestingly, Myb is also capable of immortalizing primary myeloid progenitors in culture and cooperating with BCR/ABL to induce leukemic transformation of mouse GMPs. Significantly increased levels of MYB transcript also were detected in all human CML blast crisis samples examined over chronic phase samples, further suggesting the possibility that MYB overexpression may play a prevalent role in driving human CML myeloid blast crisis development. In summary, our results identify overexpression of HOXA9, HOXA10, and MYB as critical drivers of CML progression, and suggest MYB as a key therapeutic target for inhibiting the self-renewal of leukemia-initiating cells in CML myeloid blast crisis patients.

Keywords: Hoxa10; Hoxa9; Myb; blast crisis; chronic myeloid leukemia.

Figure 1. Constitutive expression of Hoxa9 or Hoxa10 alone is capable of inducing immortalization of myeloid progenitor cells

Representative Wright-Giemsa staining and FACS analysis of indicated marker expression of cells immortalized by transduction with retroviruses expressing either Hoxa9 (A and B) or Hoxa10 (C and D) after passaging in liquid media containing SCF and IL-3 for 2 months.

Figure 2. Hoxa9 cooperates with BCR/ABL to induce development of CML myeloid blast crisis

(A). Survival curves of lethally-irradiated B6-Ly5.2 mice receiving GMPs transduced with MSCV-Hoxa9-PGK-Neo virus alone, MSCV-BCR/ABL-IRES-GFP virus alone, the combination of both viruses, or 1 × 10⁶ spleen cells from primary leukemic mice. (B). Representative cytospin analysis of bone marrow and spleen cells from leukemic mice. (C). Representative FACS analysis of GFP and CD45.2 double positive leukemia cells from the bone marrow of leukemic mice using the indicated antibodies. Numbers represent the percentages of gated events. (D). H&E staining of spleen and liver tissue sections showing leukemic infiltration in two Hoxa9+BCR/ABL leukemic mice in comparison to a healthy control mouse.

Figure 3. Hoxa10 cooperates with BCR/ABL to induce development of CML myeloid blast crisis

(A). Survival curves of lethally-irradiated B6-Ly5.2 mice receiving GMPs transduced with MSCV-Hoxa10-PGK-Puro virus alone, MSCV-BCR/ABL-IRES-GFP virus alone, the combination of both viruses, or 1 × 10⁶ spleen cells from primary leukemic mice. (B). Representative cytospin analysis of bone marrow and spleen cells from leukemic mice. (C). Representative FACS analysis of GFP and CD45.2 double positive leukemia cells from the bone marrow of leukemic mice using the indicated antibodies. Numbers represent the percentages of gated events. (D). H&E staining of spleen and liver tissue sections showing leukemic infiltration in two Hoxa10+BCR/ABL leukemic mice in comparison to a healthy control mouse.

Figure 4. Myb is a critical mediator of Hoxa9/Hoxa10-induced self-renewal of myeloid progenitors

(A). Upper panels, colony-forming potential of myeloid progenitors immortalized by Hoxa9 (left) and Hoxa10 (right) plated 48hrs after infection with lentiviral shRNA targeting Myb (Myb-sh1, Myb-sh5) or control shRNA (NC-sh1). Lower panel, Western blotting analysis of Myb protein at 72hrs after infection in the same cells corresponding to the upper panel. (B). Cytospin analyses of Hoxa9 (top) and Hoxa10-immortalized cells (lower panels) 72hrs after infection with the indicated lentiviral shRNAs. (C). Real-time PCR analysis of Lyz2 and Cd11b mRNA levels in either Hoxa9 or Hoxa10-immortalized myeloid progenitor cells 72hrs after infection with the indicated lentiviral shRNAs. Relative expression levels were calculated by normalizing to β-Actin mRNA levels in the same sample and also in cells infected by NC-sh1. (D). Real-time PCR analysis of Myb mRNA levels in mouse 5-FU treated BM progenitor cells 72hrs after infection with the indicated retrovirus. Non-transduced cells were eliminated by selection with 2 ug/ml of puromycin added at 48 hrs after infection. Relative expression levels were calculated by normalizing to β-Actin mRNA levels in the same sample and also in cells infected by empty virus. The mean and SD of each relative expression level is shown. (E). Representative cytospin analysis of Myb-immortalized cells. ^*, P <0.05; ^**, P < 0.01; ^***, P < 0.001; ^****, P < 0.0001 (two-tailed Student's t test).

Figure 5. Myb is capable of cooperating with BCR/ABL to induce development of CML myeloid blast crisis

(A). Survival curves of lethally-irradiated C57BL6-Ly5.2 mice receiving GMPs transduced with MSCV-Myb-IRES-GFP virus alone, MSCV-BCR/ABL-IRES-GFP virus alone, the combination of both viruses, or 1 × 10⁶ spleen cells from primary leukemic mice. (B). Representative cytospin analysis of bone marrow and spleen cells from leukemic mice. (C). Representative FACS analysis of GFP and CD45.2 double positive leukemia cells from the bone marrow of leukemic mice using the indicated antibodies. Numbers represent the percentages of gated events. (D). H&E staining of spleen and liver tissue sections showing leukemic infiltration in two Myb+BCR/ABL leukemic mice in comparison to a healthy control mouse.

Figure 6. Increased expression of MYB in CML blast crisis patients

Real-time RT-PCR analysis of MYB mRNA levels in total RNA isolated from whole bone marrow of healthy volunteers (NL) and CML chronic phase (CP), advanced phase (AP), and blast crisis phase (BC) patients. Relative expression levels were calculated by normalizing to BCR mRNA levels in the same sample. ^****, P < 0.0001 (two-tailed Student's t test).