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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

Ectopic expression of Hoxa9, but not Bmi1, in fetal liver progenitors antagonizes B-lymphoid differentiation.(A) Analysis by qRT-PCR of Hoxa9 and Bmi1 gene expression in E2A-PBX1- versus vector-infected FLPs. Values for each gene are expressed relative to expression in vector-infected cells. (B) Immunophenotype of FLPs transduced with Hoxa9- versus Bmi1-expressing retroviruses. Lin- FLPs were transduced with retroviruses expressing Bmi1 or Hoxa9 and then cultured under B-lymphoid conditions. Cells were analyzed by flow cytometry on day 14 post-infection.
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pone.0130495.g007: Ectopic expression of Hoxa9, but not Bmi1, in fetal liver progenitors antagonizes B-lymphoid differentiation.(A) Analysis by qRT-PCR of Hoxa9 and Bmi1 gene expression in E2A-PBX1- versus vector-infected FLPs. Values for each gene are expressed relative to expression in vector-infected cells. (B) Immunophenotype of FLPs transduced with Hoxa9- versus Bmi1-expressing retroviruses. Lin- FLPs were transduced with retroviruses expressing Bmi1 or Hoxa9 and then cultured under B-lymphoid conditions. Cells were analyzed by flow cytometry on day 14 post-infection.

Mentions: Of the differentially regulated genes uncovered by expression analysis, Hoxa9 is an especially attractive candidate as a mediator of E2A-PBX1-induced hematopoietic effects in our experiments. Hoxa9 is expressed abundantly in HSCs and early progenitors and subsequently down-regulated in more mature progenitors [25]. Abundant expression of HOXA9 in clinical samples of acute myelogenous leukemia is associated with adverse clinical outcomes [26]. Enforced expression of Hoxa9 in murine hematopoietic progenitors antagonizes lymphopoiesis and, in collaboration with Meis1, induces myeloproliferative neoplasia [27–29]. Our microarray analysis indicated 40-fold higher expression of Hoxa9 in E2A-PBX1-expressing FLPs relative to vector-infected pro-B-cells (S2 Table) whereas qRT-PCR applied to independent cell preparations indicated 134-fold higher expression (Fig 7A). Accordingly, FLPs were transduced with a Hoxa9 retrovirus and grown under B-lymphopoietic conditions in order to investigate whether enforced expression of Hoxa9 could replicate the phenotype produced by E2A-PBX1. Flow cytometry carried out 14 days after transduction demonstrated that Hoxa9 blocked B-lymphoid commitment in what appeared to be a dose-dependent manner; whereas GFP-dim cells that presumably produced Hoxa9 less abundantly succeeded in achieving CD19+, CD11b- pro-B-cell status, GFP-bright cells expressing more abundant Hoxa9 instead manifested a CD19-, CD11b+ phenotype suggestive of myeloid differentiation (Fig 7B). The PcG protein Bmi1 normally silences the B-lymphopoietic transcription factor genes Ebf1 and Pax5 in early, lineage-uncommitted progenitors and has been implicated as a mediator of E2A-PBX1-driven oncogenesis [11;30]. However, the Bmi1 transcript was not over-expressed in E2A-PBX1-expressing FLPs and retrovirus-enforced expression of Bmi1 failed to block B-lymphoid commitment (Fig 7A and 7B). These results support the notion that induction of Hoxa9, but not Bmi1, is involved in mediating E2A-PBX1-dependent antagonism of B-lymphopoiesis in our experimental model.


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)

Ectopic expression of Hoxa9, but not Bmi1, in fetal liver progenitors antagonizes B-lymphoid differentiation.(A) Analysis by qRT-PCR of Hoxa9 and Bmi1 gene expression in E2A-PBX1- versus vector-infected FLPs. Values for each gene are expressed relative to expression in vector-infected cells. (B) Immunophenotype of FLPs transduced with Hoxa9- versus Bmi1-expressing retroviruses. Lin- FLPs were transduced with retroviruses expressing Bmi1 or Hoxa9 and then cultured under B-lymphoid conditions. Cells were analyzed by flow cytometry on day 14 post-infection.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476730&req=5

pone.0130495.g007: Ectopic expression of Hoxa9, but not Bmi1, in fetal liver progenitors antagonizes B-lymphoid differentiation.(A) Analysis by qRT-PCR of Hoxa9 and Bmi1 gene expression in E2A-PBX1- versus vector-infected FLPs. Values for each gene are expressed relative to expression in vector-infected cells. (B) Immunophenotype of FLPs transduced with Hoxa9- versus Bmi1-expressing retroviruses. Lin- FLPs were transduced with retroviruses expressing Bmi1 or Hoxa9 and then cultured under B-lymphoid conditions. Cells were analyzed by flow cytometry on day 14 post-infection.
Mentions: Of the differentially regulated genes uncovered by expression analysis, Hoxa9 is an especially attractive candidate as a mediator of E2A-PBX1-induced hematopoietic effects in our experiments. Hoxa9 is expressed abundantly in HSCs and early progenitors and subsequently down-regulated in more mature progenitors [25]. Abundant expression of HOXA9 in clinical samples of acute myelogenous leukemia is associated with adverse clinical outcomes [26]. Enforced expression of Hoxa9 in murine hematopoietic progenitors antagonizes lymphopoiesis and, in collaboration with Meis1, induces myeloproliferative neoplasia [27–29]. Our microarray analysis indicated 40-fold higher expression of Hoxa9 in E2A-PBX1-expressing FLPs relative to vector-infected pro-B-cells (S2 Table) whereas qRT-PCR applied to independent cell preparations indicated 134-fold higher expression (Fig 7A). Accordingly, FLPs were transduced with a Hoxa9 retrovirus and grown under B-lymphopoietic conditions in order to investigate whether enforced expression of Hoxa9 could replicate the phenotype produced by E2A-PBX1. Flow cytometry carried out 14 days after transduction demonstrated that Hoxa9 blocked B-lymphoid commitment in what appeared to be a dose-dependent manner; whereas GFP-dim cells that presumably produced Hoxa9 less abundantly succeeded in achieving CD19+, CD11b- pro-B-cell status, GFP-bright cells expressing more abundant Hoxa9 instead manifested a CD19-, CD11b+ phenotype suggestive of myeloid differentiation (Fig 7B). The PcG protein Bmi1 normally silences the B-lymphopoietic transcription factor genes Ebf1 and Pax5 in early, lineage-uncommitted progenitors and has been implicated as a mediator of E2A-PBX1-driven oncogenesis [11;30]. However, the Bmi1 transcript was not over-expressed in E2A-PBX1-expressing FLPs and retrovirus-enforced expression of Bmi1 failed to block B-lymphoid commitment (Fig 7A and 7B). These results support the notion that induction of Hoxa9, but not Bmi1, is involved in mediating E2A-PBX1-dependent antagonism of B-lymphopoiesis in our experimental model.

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