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Retrovirus-Mediated Expression of E2A-PBX1 Blocks Lymphoid Fate but Permits Retention of Myeloid Potential in Early Hematopoietic Progenitors.

Woodcroft MW, Nanan K, Thompson P, Tyryshkin K, Smith SP, Slany RK, LeBrun DP - PLoS ONE (2015)

Bottom Line: We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself.However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis.Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada.

ABSTRACT
The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

No MeSH data available.


Related in: MedlinePlus

E2A-PBX1 induces cell cycle arrest and apoptosis in committed B-lymphoid progenitors.(A) FLP-derived pro-B-cells were infected with GFP-expressing retroviral vectors and the proportion of GFP+ cells was followed over time by flow cytometry. The graph shows the proportion of GFP+ cells relative to the value determined on day 3 after each transduction. (B) FLP-derived pro-B-cells versus lin- FLPs were infected with a retroviral vector that co-expresses GFP and E2A-PBX1 and then the mean fluorescence intensity (MFI) amongst GFP+ cells was followed over time by flow cytometry. (C) 3PER12 cells, pre-B1-derived cells that stably express EPΔ623ER, an engineered, estrogen-regulatable variant of E2A-PBX1, were induced with 10 μM estradiol (E2) versus ethanol alone (vehicle) and the accumulation of viable, Trypan blue-excluding cells over the next 72 hours was determined. (D) Flow cytometry-based cell cycle analysis of 3PER12 cells after 72 hours of induction with E2 versus vehicle. (E) Flow cytometry-based detection of apoptosis in 3PER12 cells. Cells in early apoptosis (annexin V+, propidium iodide-) or late apoptosis or death (annexin V+, propidium iodide+) are more numerous after E2 induction. Cells treated with dexamethasone (Dex) serve as a positive control.
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pone.0130495.g008: E2A-PBX1 induces cell cycle arrest and apoptosis in committed B-lymphoid progenitors.(A) FLP-derived pro-B-cells were infected with GFP-expressing retroviral vectors and the proportion of GFP+ cells was followed over time by flow cytometry. The graph shows the proportion of GFP+ cells relative to the value determined on day 3 after each transduction. (B) FLP-derived pro-B-cells versus lin- FLPs were infected with a retroviral vector that co-expresses GFP and E2A-PBX1 and then the mean fluorescence intensity (MFI) amongst GFP+ cells was followed over time by flow cytometry. (C) 3PER12 cells, pre-B1-derived cells that stably express EPΔ623ER, an engineered, estrogen-regulatable variant of E2A-PBX1, were induced with 10 μM estradiol (E2) versus ethanol alone (vehicle) and the accumulation of viable, Trypan blue-excluding cells over the next 72 hours was determined. (D) Flow cytometry-based cell cycle analysis of 3PER12 cells after 72 hours of induction with E2 versus vehicle. (E) Flow cytometry-based detection of apoptosis in 3PER12 cells. Cells in early apoptosis (annexin V+, propidium iodide-) or late apoptosis or death (annexin V+, propidium iodide+) are more numerous after E2 induction. Cells treated with dexamethasone (Dex) serve as a positive control.

Mentions: In clinical samples of t(1;19)-positive ALL, the presence of random nucleotides at the fusion point between E2A- and PBX1-derived exons and the tight clustering of breakpoints in the E2A locus suggest that the translocation may be mediated by the recombinase machinery that normally mediates immunoglobulin gene rearrangements [31]. Thus, the pre-B-cell phenotype typically observed in cases of t(1;19)-associated ALL could reflect occurrence of the leukemia-initiating translocation event in a committed B-lymphoid progenitor. In an attempt to model this leukemogenic event, FLP-derived pro-B-cells were transduced with an E2A-PBX1-expressing retroviral vector and then monitored for GFP prevalence by flow cytometry (Fig 8A). The prevalence of E2A-PBX1/GFP-expressing cells declined over time in culture, indicating that E2A-PBX1 exerts an anti-proliferative effect; this experiment was carried out twice with equivalent results (data not shown). Next, mean fluorescence intensity (MFI) for GFP was followed over time in FLP-derived pro-B-cells versus lin- FLPs after transduction with a E2A-PBX1/GFP-expressing retrovirus (Fig 8B). In lin- FLPs the MFI increased over time, consistent with the results shown in Fig 2A and suggesting that clones expressing abundant E2A-PBX1 derive a proliferative advantage. In contrast, the MFI amongst pro-B-cells declined over time, implying selection against clones expressing higher levels of E2A-PBX1. Equivalent results were obtained in multiple experiments. In order to investigate the mechanism by which E2A-PBX1 exerts its anti-proliferative effects we developed a system in which the function of E2A-PBX1 could be regulated in committed murine B-lymphoid progenitors. "Pre-B-1" cells from the bone marrow of adult mice were isolated, expanded ex vivo according to an established protocol [8], and rendered stromal cell- and IL-7-independent by stable, retrovirus-mediated expression of p210BCR-ABL. These cells were then transduced with a retrovirus conferring expression of EPΔ623ER, an estrogen-dependent derivative of E2A-PBX1 [32]; the resulting CD19+ cell line was denoted 3PER12. 3PER12 cells cultured for 72 hours in the presence of estradiol (E2) appeared small and refractile, suggesting the occurrence of widespread apoptosis, whereas control cells treated with vehicle alone, or the parent cell line treated with E2, appeared unaffected (S2 Fig and data not shown). Counts of viable cells demonstrated markedly reduced proliferation over 72 hours (Fig 8C) and cell cycle analysis determined that the majority of cells treated with E2 were arrested in G1 (Fig 8D). In comparison with vehicle-treated controls, 3PER12 cells treated with E2 displayed a marked increase in apoptosis (Fig 8E). These results indicate that, whereas E2A-PBX1 is demonstrably oncogenic when introduced into uncommitted murine hematopoietic cells, when expressed in committed B-lymphoid progenitors it induces cell cycle arrest and apoptosis.


Retrovirus-Mediated Expression of E2A-PBX1 Blocks Lymphoid Fate but Permits Retention of Myeloid Potential in Early Hematopoietic Progenitors.

Woodcroft MW, Nanan K, Thompson P, Tyryshkin K, Smith SP, Slany RK, LeBrun DP - PLoS ONE (2015)

E2A-PBX1 induces cell cycle arrest and apoptosis in committed B-lymphoid progenitors.(A) FLP-derived pro-B-cells were infected with GFP-expressing retroviral vectors and the proportion of GFP+ cells was followed over time by flow cytometry. The graph shows the proportion of GFP+ cells relative to the value determined on day 3 after each transduction. (B) FLP-derived pro-B-cells versus lin- FLPs were infected with a retroviral vector that co-expresses GFP and E2A-PBX1 and then the mean fluorescence intensity (MFI) amongst GFP+ cells was followed over time by flow cytometry. (C) 3PER12 cells, pre-B1-derived cells that stably express EPΔ623ER, an engineered, estrogen-regulatable variant of E2A-PBX1, were induced with 10 μM estradiol (E2) versus ethanol alone (vehicle) and the accumulation of viable, Trypan blue-excluding cells over the next 72 hours was determined. (D) Flow cytometry-based cell cycle analysis of 3PER12 cells after 72 hours of induction with E2 versus vehicle. (E) Flow cytometry-based detection of apoptosis in 3PER12 cells. Cells in early apoptosis (annexin V+, propidium iodide-) or late apoptosis or death (annexin V+, propidium iodide+) are more numerous after E2 induction. Cells treated with dexamethasone (Dex) serve as a positive control.
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pone.0130495.g008: E2A-PBX1 induces cell cycle arrest and apoptosis in committed B-lymphoid progenitors.(A) FLP-derived pro-B-cells were infected with GFP-expressing retroviral vectors and the proportion of GFP+ cells was followed over time by flow cytometry. The graph shows the proportion of GFP+ cells relative to the value determined on day 3 after each transduction. (B) FLP-derived pro-B-cells versus lin- FLPs were infected with a retroviral vector that co-expresses GFP and E2A-PBX1 and then the mean fluorescence intensity (MFI) amongst GFP+ cells was followed over time by flow cytometry. (C) 3PER12 cells, pre-B1-derived cells that stably express EPΔ623ER, an engineered, estrogen-regulatable variant of E2A-PBX1, were induced with 10 μM estradiol (E2) versus ethanol alone (vehicle) and the accumulation of viable, Trypan blue-excluding cells over the next 72 hours was determined. (D) Flow cytometry-based cell cycle analysis of 3PER12 cells after 72 hours of induction with E2 versus vehicle. (E) Flow cytometry-based detection of apoptosis in 3PER12 cells. Cells in early apoptosis (annexin V+, propidium iodide-) or late apoptosis or death (annexin V+, propidium iodide+) are more numerous after E2 induction. Cells treated with dexamethasone (Dex) serve as a positive control.
Mentions: In clinical samples of t(1;19)-positive ALL, the presence of random nucleotides at the fusion point between E2A- and PBX1-derived exons and the tight clustering of breakpoints in the E2A locus suggest that the translocation may be mediated by the recombinase machinery that normally mediates immunoglobulin gene rearrangements [31]. Thus, the pre-B-cell phenotype typically observed in cases of t(1;19)-associated ALL could reflect occurrence of the leukemia-initiating translocation event in a committed B-lymphoid progenitor. In an attempt to model this leukemogenic event, FLP-derived pro-B-cells were transduced with an E2A-PBX1-expressing retroviral vector and then monitored for GFP prevalence by flow cytometry (Fig 8A). The prevalence of E2A-PBX1/GFP-expressing cells declined over time in culture, indicating that E2A-PBX1 exerts an anti-proliferative effect; this experiment was carried out twice with equivalent results (data not shown). Next, mean fluorescence intensity (MFI) for GFP was followed over time in FLP-derived pro-B-cells versus lin- FLPs after transduction with a E2A-PBX1/GFP-expressing retrovirus (Fig 8B). In lin- FLPs the MFI increased over time, consistent with the results shown in Fig 2A and suggesting that clones expressing abundant E2A-PBX1 derive a proliferative advantage. In contrast, the MFI amongst pro-B-cells declined over time, implying selection against clones expressing higher levels of E2A-PBX1. Equivalent results were obtained in multiple experiments. In order to investigate the mechanism by which E2A-PBX1 exerts its anti-proliferative effects we developed a system in which the function of E2A-PBX1 could be regulated in committed murine B-lymphoid progenitors. "Pre-B-1" cells from the bone marrow of adult mice were isolated, expanded ex vivo according to an established protocol [8], and rendered stromal cell- and IL-7-independent by stable, retrovirus-mediated expression of p210BCR-ABL. These cells were then transduced with a retrovirus conferring expression of EPΔ623ER, an estrogen-dependent derivative of E2A-PBX1 [32]; the resulting CD19+ cell line was denoted 3PER12. 3PER12 cells cultured for 72 hours in the presence of estradiol (E2) appeared small and refractile, suggesting the occurrence of widespread apoptosis, whereas control cells treated with vehicle alone, or the parent cell line treated with E2, appeared unaffected (S2 Fig and data not shown). Counts of viable cells demonstrated markedly reduced proliferation over 72 hours (Fig 8C) and cell cycle analysis determined that the majority of cells treated with E2 were arrested in G1 (Fig 8D). In comparison with vehicle-treated controls, 3PER12 cells treated with E2 displayed a marked increase in apoptosis (Fig 8E). These results indicate that, whereas E2A-PBX1 is demonstrably oncogenic when introduced into uncommitted murine hematopoietic cells, when expressed in committed B-lymphoid progenitors it induces cell cycle arrest and apoptosis.

Bottom Line: We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself.However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis.Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada.

ABSTRACT
The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

No MeSH data available.


Related in: MedlinePlus