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

<div><p>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, <i>Hoxa9</i>, 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.</p></div

KEGG pathways overrepresented by genes up-regulated downstream of E2A-PBX1.

<p>KEGG pathways overrepresented by genes up-regulated downstream of E2A-PBX1.</p

Ectopic expression of <i>Hoxa9</i>, but not <i>Bmi1</i>, in fetal liver progenitors antagonizes B-lymphoid differentiation.

<p>(A) Analysis by qRT-PCR of <i>Hoxa9</i> and <i>Bmi1</i> 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 <i>Hoxa9</i>- versus <i>Bmi1</i>-expressing retroviruses. Lin^- FLPs were transduced with retroviruses expressing <i>Bmi1</i> or <i>Hoxa9</i> and then cultured under B-lymphoid conditions. Cells were analyzed by flow cytometry on day 14 post-infection.</p

E2A-PBX1-expressing bone marrow cells reconstitute early myeloid but not B-lymphoid development in transplanted mice.

<p>Primary murine bone marrow cells were transduced with E2A-PBX1-expressing or control retroviral vectors and injected into the tail vein of lethally-irradiated, syngeneic recipient mice. Mice were sacrificed 3 weeks later. Immunophenotypic analysis of (A) bone marrow, (B) spleen, and (C) thymus are shown from representative animals. Events were gated so as to restrict all of these analyses to the GFP⁺ cell population.</p

Forced expression of E2A-PBX in FLPs deregulates hematopoietic genes.

<p>(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 <i>in vitro</i>. (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.</p

FLPs expressing E2A-PBX1 remain amenable to induction of myeloid differentiation <i>in vivo</i> or <i>in vitro</i>.

<p>E2A-PBX1-transduced FLPs were injected into the tail veins of lethally-irradiated mice. Immunophenotypic analysis was performed on cells obtained from the bone marrow (A) or spleen (B) 3 weeks later. (C) E2A-PBX1 expressing FLPs were transferred from medium containing IL-7 and SCF to medium containing GM-CSF instead and analyzed by flow cytometry 7 days later.</p

B-lymphopoiesis is blocked at a point prior to the CLP stage in FLPs expressing E2A-PBX1.

<p>(A) Immunoblotting indicates markedly reduced or absent expression of the B-lymphopoietic transcription factors Ebf1 and Pax5 in FLPs expressing E2A-PBX1 relative to those infected with the empty retroviral vector. The apparent weak signal for Pax5 in the E2A-PBX1 lane may represent spillover between lanes. Anti-γ-tubulin serves as a loading control. (B) ChIP-qPCR results obtained from chromatin prepared from control, vector-infected FLPs indicate prevalence of the activating H3K4me3 mark relative to the silencing H3K27me3 mark at the <i>Ebf1</i> and <i>Pax5</i> promoters. Error bars represent 95% confidence limits. (C) Results from qRT-PCR indicating marked reduction or absence of the Ebf1 and Pax5 transcripts in FLPs expressing E2A-PBX1 relative to control cells. The asterisk indicates failure to observe a PCR product in either of the 2 independent samples. Error bars indicate one standard deviation. (D) ChIP-qPCR results obtained from chromatin prepared from FLPs expressing E2A-PBX1 indicate prevalence of the silencing H3K27me3 mark relative to the activating H3K4me3 mark at the <i>Ebf1</i> and <i>Pax5</i> promoters.</p

Interaction with both CBP/p300 and DNA is required in the E2A-PBX1-imposed differentiation blockade.

<p>(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).</p

E2A-PBX1 blocks B-lymphoid differentiation <i>in vitro</i>.

<p>(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.</p

E2A-PBX1 induces cell cycle arrest and apoptosis in committed B-lymphoid progenitors.

<p>(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.</p