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

Thermal denaturation profile of CHOP measured using DSC.

<p>Top panel: Normalized DSC scan of CHOP in 5 mM phosphate buffer (pH 7.0) in the absence (solid triangle) or presence (open circle) of 80 µM l-cysteine. Middle panel: Two-state fitting (dashed line) of normalized DSC scan of CHOP in presence of 80 µM l-cysteine. Bottom panel: Normalized DSC scan of CHOP in presence of 4 mM l-cysteine. The heating rate was 45°C per hour. Approximately 10 µM CHOP was used for each run.</p

Thermal denaturation of CHOP measured using far-UV CD.

<p>Top panel shows a graph depicting the changes in [θ]₁₉₀ and [θ]₂₂₂ with at increasing temperatures. Far-UV CD spectra of CHOP were recorded at increasing temperatures (20°C–80°C) on an OLIS RMS CD spectrophotometer with a Pt100 thermal probe fitted right under the cuvette. Temperatures in the cuvette were controlled using a Julabo F30-C circulating water bath. A total of 8 scans were conducted at each temperature and averaged, with 10 minutes of equilibration time between each experiment. Bottom panel depicts a phase diagram showing the dependence of [θ]₁₉₀ vs. [θ]₂₂₂ on temperature. Note that the linear regression is representative of an all-or-none transition.</p

<i>In silico</i> disorder analysis of CHOP using various prediction programs (IVLXT, VSL2, VL3, IUPred, TopIDP, Foldindex and PONDR-FIT).

<p>(A) Distribution of predicted disorderedness per-residue. (B) Cumulative distribution function (CDF) analysis of CHOP. (C) Charge-hydropathy analysis of CHOP (grey) in comparison to natively folded proteins (closed circles) and natively unfolded proteins (open circles).</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

Two-dimensional ¹H-¹⁵N-HSQC spectra of CHOP in the absence (background) and presence of 2 mM (inset A) and 8 mM (inset B) l-cysteine at 4°C.

<p>Circles identify backbone ¹H-¹⁵N resonances affected by l-cysteine while arrows identify a region comprising side chain amide resonances affected l-cysteine.</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

Phase diagram showing the dependence of [θ]₁₉₀ vs. [θ]₂₂₂ on increasing l-cysteine concentrations.

<p>Data was adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034680#pone-0034680-g002" target="_blank">Figure 2</a>. Note the two-state linear regression, which is representative of consecutive conformational transitions in CHOP.</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

CD-based secondary structure measurements of CHOP.^a

a<p>Measurements of CHOP were performed in 10 mM phosphate buffer (pH 7.0, 25°C), in the presence of increasing concentrations of l-cysteine. Calculations were performed using CDNN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034680#pone.0034680-Uversky11" target="_blank">[108]</a>.</p