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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

Forced expression of E2A-PBX in FLPs deregulates hematopoietic genes.(A) Hierarchical cluster analysis performed on expression data from gene expression microarrays indicates that the closest relationship to E2A-PBX1-expressing FLPs is with pro-B-cells that were differentiated from FLPs in vitro. (B) Confirmation by qRT-PCR of differential expression of genes identified initially using expression microarrays in E2A-PBX-expressing versus pro-B-cells derived from vector-infected FLPs. Data are from 2 independent samples. Error bars indicate one standard deviation; p-values are less than 0.005 for each comparison.
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pone.0130495.g006: Forced expression of E2A-PBX in FLPs deregulates hematopoietic genes.(A) Hierarchical cluster analysis performed on expression data from gene expression microarrays indicates that the closest relationship to E2A-PBX1-expressing FLPs is with pro-B-cells that were differentiated from FLPs in vitro. (B) Confirmation by qRT-PCR of differential expression of genes identified initially using expression microarrays in E2A-PBX-expressing versus pro-B-cells derived from vector-infected FLPs. Data are from 2 independent samples. Error bars indicate one standard deviation; p-values are less than 0.005 for each comparison.

Mentions: Gene expression microarray analysis was used to further elucidate the effects of E2A-PBX1 on hematopoiesis. RNA from biological replicates of E2A-PBX1-transduced FLPs and vector-transduced, FLP-derived, CD19+ pro-B-cells was evaluated for differential gene expression. The immunophenotype of the E2A-PBX1-tranduced cells used for gene expression analysis was equivalent to that shown in Fig 2A for day 25. RNA prepared from primary, uncultured lin- FLPs and primary CD11b+ myeloid bone marrow cells was evaluated for comparison with the cultured cells. Hierarchical cluster analysis based on gene expression profiles of all of the probes that remained after initial data filtering (n = 23,457) showed that the FLP-derived pro-B-cells and the FLP-derived E2A-PBX1-expressing cells clustered together (Fig 6A). This is consistent with the common origin of these cell types from lin- FLPs and with their history of having been maintained in tissue culture for several passages. Further analysis of the data identified 3,203 probes representing 2,235 genes that showed at least a four-fold difference in expression between E2A-PBX1-expressing cells and FLP-derived pro-B-cells; these are listed in S2 Table. In S3 Table, these genes are organized according to their expression in lin- or myeloid cells. For example, abundant expression of Mycn, Il6, Bcl2, Flt3 and Hoxa9 was exclusive to E2A-PBX1-expressing FLPs, whereas abundant Rora, Meis1, Gata 2 and Gata3 was observed in both E2A-PBX1-expressing and lin- FLPs. Differential up-regulation of Rora, Meis1, Myc, Mycn, Gata3, Il6 and Kit in E2A-PBX1-expressing relative to FLP-derived pro-B-cells was confirmed by qRT-PCR (Fig 6B). Enrichment analysis for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) [24]. The top 20 KEGG pathways over-represented by genes up- or down-regulated downstream of E2A-PBX1 are listed in Tables 1 and 2, respectively; the full lists of pathways and the probe ID numbers of the genes associated with each pathway are provided in S4 and S5 Tables. Genes up-regulated in E2A-PBX1-expressing cells and over-represented from the "Cytokine-cytokine receptor interaction" pathway included many genes encoding cytokines or cytokine receptors, such as Csf1r, Flt1, Flt3, Met, Hgf, Pdgfc, Vegfc, while genes over-represented from the "Hematopoietic cell lineage" pathway included genes characteristically expressed in myeloid cells and progenitors, such as Cd14, Cd33, Cd36, Gp1ba and Gp1bb. Genes down-regulated in E2A-PBX1-expressing cells and over-represented from the "Pathways in cancer" pathway included known tumor suppressor genes, such as Cdkn1b, Pten and Foxo1; genes over-represented from the "B cell receptor signaling pathway" included those encoding signaling molecules and transcription factors such as Cd19, Cd79a, Cd79b, Blnk, Nfkb1a and Tcf3. These patterns of gene expression are consistent with failure of E2A-PBX1-expressing FLPs to commit to the B-lymphoid lineage, their retention of myeloid potential and their proliferative and leukemogenic properties.


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)

Forced expression of E2A-PBX in FLPs deregulates hematopoietic genes.(A) Hierarchical cluster analysis performed on expression data from gene expression microarrays indicates that the closest relationship to E2A-PBX1-expressing FLPs is with pro-B-cells that were differentiated from FLPs in vitro. (B) Confirmation by qRT-PCR of differential expression of genes identified initially using expression microarrays in E2A-PBX-expressing versus pro-B-cells derived from vector-infected FLPs. Data are from 2 independent samples. Error bars indicate one standard deviation; p-values are less than 0.005 for each comparison.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130495.g006: Forced expression of E2A-PBX in FLPs deregulates hematopoietic genes.(A) Hierarchical cluster analysis performed on expression data from gene expression microarrays indicates that the closest relationship to E2A-PBX1-expressing FLPs is with pro-B-cells that were differentiated from FLPs in vitro. (B) Confirmation by qRT-PCR of differential expression of genes identified initially using expression microarrays in E2A-PBX-expressing versus pro-B-cells derived from vector-infected FLPs. Data are from 2 independent samples. Error bars indicate one standard deviation; p-values are less than 0.005 for each comparison.
Mentions: Gene expression microarray analysis was used to further elucidate the effects of E2A-PBX1 on hematopoiesis. RNA from biological replicates of E2A-PBX1-transduced FLPs and vector-transduced, FLP-derived, CD19+ pro-B-cells was evaluated for differential gene expression. The immunophenotype of the E2A-PBX1-tranduced cells used for gene expression analysis was equivalent to that shown in Fig 2A for day 25. RNA prepared from primary, uncultured lin- FLPs and primary CD11b+ myeloid bone marrow cells was evaluated for comparison with the cultured cells. Hierarchical cluster analysis based on gene expression profiles of all of the probes that remained after initial data filtering (n = 23,457) showed that the FLP-derived pro-B-cells and the FLP-derived E2A-PBX1-expressing cells clustered together (Fig 6A). This is consistent with the common origin of these cell types from lin- FLPs and with their history of having been maintained in tissue culture for several passages. Further analysis of the data identified 3,203 probes representing 2,235 genes that showed at least a four-fold difference in expression between E2A-PBX1-expressing cells and FLP-derived pro-B-cells; these are listed in S2 Table. In S3 Table, these genes are organized according to their expression in lin- or myeloid cells. For example, abundant expression of Mycn, Il6, Bcl2, Flt3 and Hoxa9 was exclusive to E2A-PBX1-expressing FLPs, whereas abundant Rora, Meis1, Gata 2 and Gata3 was observed in both E2A-PBX1-expressing and lin- FLPs. Differential up-regulation of Rora, Meis1, Myc, Mycn, Gata3, Il6 and Kit in E2A-PBX1-expressing relative to FLP-derived pro-B-cells was confirmed by qRT-PCR (Fig 6B). Enrichment analysis for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) [24]. The top 20 KEGG pathways over-represented by genes up- or down-regulated downstream of E2A-PBX1 are listed in Tables 1 and 2, respectively; the full lists of pathways and the probe ID numbers of the genes associated with each pathway are provided in S4 and S5 Tables. Genes up-regulated in E2A-PBX1-expressing cells and over-represented from the "Cytokine-cytokine receptor interaction" pathway included many genes encoding cytokines or cytokine receptors, such as Csf1r, Flt1, Flt3, Met, Hgf, Pdgfc, Vegfc, while genes over-represented from the "Hematopoietic cell lineage" pathway included genes characteristically expressed in myeloid cells and progenitors, such as Cd14, Cd33, Cd36, Gp1ba and Gp1bb. Genes down-regulated in E2A-PBX1-expressing cells and over-represented from the "Pathways in cancer" pathway included known tumor suppressor genes, such as Cdkn1b, Pten and Foxo1; genes over-represented from the "B cell receptor signaling pathway" included those encoding signaling molecules and transcription factors such as Cd19, Cd79a, Cd79b, Blnk, Nfkb1a and Tcf3. These patterns of gene expression are consistent with failure of E2A-PBX1-expressing FLPs to commit to the B-lymphoid lineage, their retention of myeloid potential and their proliferative and leukemogenic properties.

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