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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 blocks B-lymphoid differentiation in vitro.(A) Immunophenotype of E2A-PBX1-expressing FLPs after 14 days (top and middle rows) and 25 days (bottom row) of propagation in medium containing IL-7 and SCF. (B) Differential effects of SCF deprivation on E2A-PBX1-expressing FLPs. The bar graph shows the number of cells that were deprived of SCF expressed as a percentage of those that were maintained in SCF-containing medium. Data are from 2 independent experiments. Error bars represent one standard deviation. (C) Enumeration of culture-initiating cells by limiting dilution assays in 96-well plates. The slope of the solid line represents the log-active cell fraction and the dotted lines represent the 95% confidence intervals. Data points with no negative response are represented by down-pointing triangles. The calculated prevalence of culture-initiating cells is indicated on the plot.
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pone.0130495.g002: E2A-PBX1 blocks B-lymphoid differentiation in vitro.(A) Immunophenotype of E2A-PBX1-expressing FLPs after 14 days (top and middle rows) and 25 days (bottom row) of propagation in medium containing IL-7 and SCF. (B) Differential effects of SCF deprivation on E2A-PBX1-expressing FLPs. The bar graph shows the number of cells that were deprived of SCF expressed as a percentage of those that were maintained in SCF-containing medium. Data are from 2 independent experiments. Error bars represent one standard deviation. (C) Enumeration of culture-initiating cells by limiting dilution assays in 96-well plates. The slope of the solid line represents the log-active cell fraction and the dotted lines represent the 95% confidence intervals. Data points with no negative response are represented by down-pointing triangles. The calculated prevalence of culture-initiating cells is indicated on the plot.

Mentions: In order to determine hematopoietic lineage fate, lineage-specific inductive signals must act upon multipotent progenitors that are competent to respond to them [17]. Therefore, the competency of E2A-PBX1-expressing, lineage-uncommitted progenitors to respond to environmental signals that normally induce B-lymphoid differentiation was investigated using an established, more experimentally tractable in vitro system. Lin- hematopoietic progenitors harvested from day 12 to 14 fetal livers differentiate to committed pro-B-cells (also called “pre-B1 cells”) after 9 to 14 days of co-culture with OP9 stromal cells in the presence of IL-7 and SCF [18]. Whereas the immunophenotype of control cells confirmed their CD45R+/CD19+, B-lymphoid-committed, status (Fig 2A), most E2A-PBX1-transduced cells displayed a relatively immature, lineage-ambiguous phenotype characterized by the absence of CD45R or CD19, persistent expression of abundant CD117 and Sca-1, and variable expression of IL-7R and CD11b. Initially, minor populations of CD45R+/CD19+ (24%) and CD45R+/CD19- (11%) cells were observed. While the CD45R+/CD19+ population was lost with continued culture (compare the CD45R/CD19 plots at 14 and 25 days), the CD45R+/CD19- population was retained. The presence of these cells could reflect slight leakiness of the differentiation block or the presence at the time of retroviral transduction of a small number of lin- progenitors with a bias towards the B-lymphoid fate. Equivalent results were obtained in two experiments. Despite its obvious effect on hematopoietic differentiation, expression of E2A-PBX1 did not affect growth kinetics during the first 14 days after retroviral transduction (S1 Fig). However, the ability of GFP+, E2A-PBX1-transduced cells to out-compete GFP- cells by 25 days after transduction (Fig 2A) suggests the outgrowth of more proliferative subpopulations of cells many of which express E2A-PBX1 more abundantly. This is further supported by the right-ward shift in GFP signal intensity observed by day 25 for E2A-PBX1-transduced cells (Fig 2A). However, given the persistent expression of CD117 in E2A-PBX1-transduced FLPs, we considered whether the immature, E2A-PBX1-transduced cells were dependent on SCF, the ligand for CD117. Lin- FLPs were deprived of SCF 5 days after transduction and the cumulative number of GFP+ cells was determined 9 days later. E2A-PBX1-transduced FLPs suffered a greater impairment of proliferation upon SCF deprivation than did control cells, indicating greater dependency on this cytokine (Fig 2B). Limiting dilution in 96-well plates and the web-based ELDA tool were used to determine the prevalence of culture-initiating cells. Whereas culture-initiating cells were present at a frequency of 1/12.6 cells among vector-infected FLPs, the frequency among E2A-PBX1-infected FLPs was nine-fold higher, at 1/1.4 (95% CI 1/17.5-1/9.06 versus 1/2.13-1/0.954, respectively, p < 0.001); Fig 2C) [12]. Equivalent results were obtained in three replicate experiments. The presence of features characteristic of early hematopoietic progenitors, including an immature immunophenotype, persistent differential responsiveness to SCF and a higher prevalence of culture-initiating cells in FLPs forced to express E2A-PBX1, supports the idea that E2A-PBX1 antagonizes aspects of hematopoietic maturation and enforces persistence of features characteristic of hematopoietic stem cells (HSCs) or early progenitors.


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 blocks B-lymphoid differentiation in vitro.(A) Immunophenotype of E2A-PBX1-expressing FLPs after 14 days (top and middle rows) and 25 days (bottom row) of propagation in medium containing IL-7 and SCF. (B) Differential effects of SCF deprivation on E2A-PBX1-expressing FLPs. The bar graph shows the number of cells that were deprived of SCF expressed as a percentage of those that were maintained in SCF-containing medium. Data are from 2 independent experiments. Error bars represent one standard deviation. (C) Enumeration of culture-initiating cells by limiting dilution assays in 96-well plates. The slope of the solid line represents the log-active cell fraction and the dotted lines represent the 95% confidence intervals. Data points with no negative response are represented by down-pointing triangles. The calculated prevalence of culture-initiating cells is indicated on the plot.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130495.g002: E2A-PBX1 blocks B-lymphoid differentiation in vitro.(A) Immunophenotype of E2A-PBX1-expressing FLPs after 14 days (top and middle rows) and 25 days (bottom row) of propagation in medium containing IL-7 and SCF. (B) Differential effects of SCF deprivation on E2A-PBX1-expressing FLPs. The bar graph shows the number of cells that were deprived of SCF expressed as a percentage of those that were maintained in SCF-containing medium. Data are from 2 independent experiments. Error bars represent one standard deviation. (C) Enumeration of culture-initiating cells by limiting dilution assays in 96-well plates. The slope of the solid line represents the log-active cell fraction and the dotted lines represent the 95% confidence intervals. Data points with no negative response are represented by down-pointing triangles. The calculated prevalence of culture-initiating cells is indicated on the plot.
Mentions: In order to determine hematopoietic lineage fate, lineage-specific inductive signals must act upon multipotent progenitors that are competent to respond to them [17]. Therefore, the competency of E2A-PBX1-expressing, lineage-uncommitted progenitors to respond to environmental signals that normally induce B-lymphoid differentiation was investigated using an established, more experimentally tractable in vitro system. Lin- hematopoietic progenitors harvested from day 12 to 14 fetal livers differentiate to committed pro-B-cells (also called “pre-B1 cells”) after 9 to 14 days of co-culture with OP9 stromal cells in the presence of IL-7 and SCF [18]. Whereas the immunophenotype of control cells confirmed their CD45R+/CD19+, B-lymphoid-committed, status (Fig 2A), most E2A-PBX1-transduced cells displayed a relatively immature, lineage-ambiguous phenotype characterized by the absence of CD45R or CD19, persistent expression of abundant CD117 and Sca-1, and variable expression of IL-7R and CD11b. Initially, minor populations of CD45R+/CD19+ (24%) and CD45R+/CD19- (11%) cells were observed. While the CD45R+/CD19+ population was lost with continued culture (compare the CD45R/CD19 plots at 14 and 25 days), the CD45R+/CD19- population was retained. The presence of these cells could reflect slight leakiness of the differentiation block or the presence at the time of retroviral transduction of a small number of lin- progenitors with a bias towards the B-lymphoid fate. Equivalent results were obtained in two experiments. Despite its obvious effect on hematopoietic differentiation, expression of E2A-PBX1 did not affect growth kinetics during the first 14 days after retroviral transduction (S1 Fig). However, the ability of GFP+, E2A-PBX1-transduced cells to out-compete GFP- cells by 25 days after transduction (Fig 2A) suggests the outgrowth of more proliferative subpopulations of cells many of which express E2A-PBX1 more abundantly. This is further supported by the right-ward shift in GFP signal intensity observed by day 25 for E2A-PBX1-transduced cells (Fig 2A). However, given the persistent expression of CD117 in E2A-PBX1-transduced FLPs, we considered whether the immature, E2A-PBX1-transduced cells were dependent on SCF, the ligand for CD117. Lin- FLPs were deprived of SCF 5 days after transduction and the cumulative number of GFP+ cells was determined 9 days later. E2A-PBX1-transduced FLPs suffered a greater impairment of proliferation upon SCF deprivation than did control cells, indicating greater dependency on this cytokine (Fig 2B). Limiting dilution in 96-well plates and the web-based ELDA tool were used to determine the prevalence of culture-initiating cells. Whereas culture-initiating cells were present at a frequency of 1/12.6 cells among vector-infected FLPs, the frequency among E2A-PBX1-infected FLPs was nine-fold higher, at 1/1.4 (95% CI 1/17.5-1/9.06 versus 1/2.13-1/0.954, respectively, p < 0.001); Fig 2C) [12]. Equivalent results were obtained in three replicate experiments. The presence of features characteristic of early hematopoietic progenitors, including an immature immunophenotype, persistent differential responsiveness to SCF and a higher prevalence of culture-initiating cells in FLPs forced to express E2A-PBX1, supports the idea that E2A-PBX1 antagonizes aspects of hematopoietic maturation and enforces persistence of features characteristic of hematopoietic stem cells (HSCs) or early progenitors.

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