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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

Interaction with both CBP/p300 and DNA is required in the E2A-PBX1-imposed differentiation blockade.(A) Immunophenotypic analysis of FLPs transduced with retroviruses conferring expression of E2A-PBX1 or the indicated amino acid-substituted mutants 14 days after transduction. Cells expressing unmodified E2A-PBX1, identified based upon GFP expression, fail to achieve B-lymphoid-committed, CD45R+/CD19+ status whereas this effect is lost consequent to the L20A or N51S amino acid substitutions that impair CBP/p300 recruitment or DNA binding, respectively. The ability of the L20A mutant protein to block B-lymphoid differentiation is partially restored by the A400L substitution, which increases the affinity of AD2 for CBP/p300. (B) Immunoblot of whole cell lysates from HEK293T cells transiently transfected with the indicated retroviral backbone plasmids. GFP serves as a control for transfection efficiency and gel loading. Expression of the L20A/A400L substituted E2A-PBX1 double-mutant at a level equivalent to that of un-modified E2A-PBX1 is documented elsewhere (22).
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pone.0130495.g003: Interaction with both CBP/p300 and DNA is required in the E2A-PBX1-imposed differentiation blockade.(A) Immunophenotypic analysis of FLPs transduced with retroviruses conferring expression of E2A-PBX1 or the indicated amino acid-substituted mutants 14 days after transduction. Cells expressing unmodified E2A-PBX1, identified based upon GFP expression, fail to achieve B-lymphoid-committed, CD45R+/CD19+ status whereas this effect is lost consequent to the L20A or N51S amino acid substitutions that impair CBP/p300 recruitment or DNA binding, respectively. The ability of the L20A mutant protein to block B-lymphoid differentiation is partially restored by the A400L substitution, which increases the affinity of AD2 for CBP/p300. (B) Immunoblot of whole cell lysates from HEK293T cells transiently transfected with the indicated retroviral backbone plasmids. GFP serves as a control for transfection efficiency and gel loading. Expression of the L20A/A400L substituted E2A-PBX1 double-mutant at a level equivalent to that of un-modified E2A-PBX1 is documented elsewhere (22).

Mentions: E2A-PBX1 induces proliferation in fibroblasts in culture or T-progenitor lymphoma in transgenic mice through a mechanism that does not require DNA binding mediated by the PBX1 homeodomain [3;19]. B-lymphoid commitment in the FLP model requires the E2a gene products [20]. These considerations raise the possibility that blocked B-lymphoid differentiation in our system could be attributable to dominant-negative effects on wild-type E2a gene products, perhaps involving squelching through sequestration of transcriptional co-activators by E2A-PBX1. The L20A amino acid substitution within activation domain 1 (AD1) of E2A-PBX1 markedly impairs binding to the transcriptional co-activator and histone acetyltransferase CBP/p300 whereas the N51S substitution in the PBX1 homeodomain disrupts DNA binding [5;21]. Either of these substitutions completely abrogated the differentiation blockade imposed by E2A-PBX1 in the FLP-based B-lymphopoiesis model (Fig 3A). The loss of the differentiation block was not due to sub-threshold transgene expression as both the L20A and N51S mutants are expressed at levels comparable to un-modified E2A-PBX1 (Fig 3B). These results, which were obtained in at least two independent experiments, support a model in which E2A-PBX1 blocks B-lymphoid differentiation through a mechanism that requires the recruitment of CBP/p300 to genomic loci bound by the PBX1 homeodomain.


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)

Interaction with both CBP/p300 and DNA is required in the E2A-PBX1-imposed differentiation blockade.(A) Immunophenotypic analysis of FLPs transduced with retroviruses conferring expression of E2A-PBX1 or the indicated amino acid-substituted mutants 14 days after transduction. Cells expressing unmodified E2A-PBX1, identified based upon GFP expression, fail to achieve B-lymphoid-committed, CD45R+/CD19+ status whereas this effect is lost consequent to the L20A or N51S amino acid substitutions that impair CBP/p300 recruitment or DNA binding, respectively. The ability of the L20A mutant protein to block B-lymphoid differentiation is partially restored by the A400L substitution, which increases the affinity of AD2 for CBP/p300. (B) Immunoblot of whole cell lysates from HEK293T cells transiently transfected with the indicated retroviral backbone plasmids. GFP serves as a control for transfection efficiency and gel loading. Expression of the L20A/A400L substituted E2A-PBX1 double-mutant at a level equivalent to that of un-modified E2A-PBX1 is documented elsewhere (22).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476730&req=5

pone.0130495.g003: Interaction with both CBP/p300 and DNA is required in the E2A-PBX1-imposed differentiation blockade.(A) Immunophenotypic analysis of FLPs transduced with retroviruses conferring expression of E2A-PBX1 or the indicated amino acid-substituted mutants 14 days after transduction. Cells expressing unmodified E2A-PBX1, identified based upon GFP expression, fail to achieve B-lymphoid-committed, CD45R+/CD19+ status whereas this effect is lost consequent to the L20A or N51S amino acid substitutions that impair CBP/p300 recruitment or DNA binding, respectively. The ability of the L20A mutant protein to block B-lymphoid differentiation is partially restored by the A400L substitution, which increases the affinity of AD2 for CBP/p300. (B) Immunoblot of whole cell lysates from HEK293T cells transiently transfected with the indicated retroviral backbone plasmids. GFP serves as a control for transfection efficiency and gel loading. Expression of the L20A/A400L substituted E2A-PBX1 double-mutant at a level equivalent to that of un-modified E2A-PBX1 is documented elsewhere (22).
Mentions: E2A-PBX1 induces proliferation in fibroblasts in culture or T-progenitor lymphoma in transgenic mice through a mechanism that does not require DNA binding mediated by the PBX1 homeodomain [3;19]. B-lymphoid commitment in the FLP model requires the E2a gene products [20]. These considerations raise the possibility that blocked B-lymphoid differentiation in our system could be attributable to dominant-negative effects on wild-type E2a gene products, perhaps involving squelching through sequestration of transcriptional co-activators by E2A-PBX1. The L20A amino acid substitution within activation domain 1 (AD1) of E2A-PBX1 markedly impairs binding to the transcriptional co-activator and histone acetyltransferase CBP/p300 whereas the N51S substitution in the PBX1 homeodomain disrupts DNA binding [5;21]. Either of these substitutions completely abrogated the differentiation blockade imposed by E2A-PBX1 in the FLP-based B-lymphopoiesis model (Fig 3A). The loss of the differentiation block was not due to sub-threshold transgene expression as both the L20A and N51S mutants are expressed at levels comparable to un-modified E2A-PBX1 (Fig 3B). These results, which were obtained in at least two independent experiments, support a model in which E2A-PBX1 blocks B-lymphoid differentiation through a mechanism that requires the recruitment of CBP/p300 to genomic loci bound by the PBX1 homeodomain.

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