Disrupted differentiation and oncogenic transformation of lymphoid progenitors in E2A-HLF transgenic mice

Abstract

The hepatic leukemia factor (HLF) gene codes for a basic region-leucine zipper (bZIP) protein that is disrupted by chromosomal translocations in a subset of pediatric acute lymphoblastic leukemias. HLF undergoes fusions with the E2A gene, resulting in chimeric E2a-Hlf proteins containing the E2a transactivation domains and the Hlf bZIP DNA binding and dimerization motifs. To investigate the in vivo role of this chimeric bZIP protein in oncogenic transformation, its expression was directed to the lymphoid compartments of transgenic mice. Within the thymus, E2a-Hlf induced profound hypoplasia, premature involution, and progressive accumulation of a T-lineage precursor population arrested at an early stage of maturation. In the spleen, mature T cells were present but in reduced numbers, and they lacked expression of the transgene, suggesting further that E2a-Hlf expression was incompatible with T-cell differentiation. In contrast, mature splenic B cells expressed E2a-Hlf but at lower levels and without apparent adverse or beneficial effects on their survival. Approximately 60% of E2A-HLF mice developed lymphoid malignancies with a mean latency of 10 months. Tumors were monoclonal, consistent with a requirement for secondary genetic events, and displayed phenotypes of either mid-thymocytes or, rarely, B-cell progenitors. We conclude that E2a-Hlf disrupts the differentiation of T-lymphoid progenitors in vivo, leading to profound postnatal thymic depletion and rendering B- and T-cell progenitors susceptible to malignant transformation.

FIG. 1

Structure and expression of the E2A-HLF transgene. (A) Schematic illustration of the E2A-HLF transgene construct consisting of the E2A-HLF^HAL-01 cDNA driven by an Eμ enhancer and SV40 early promoter. (B) Expression of E2a-Hlf protein in bone marrow (BM), thymocytes (Thy.), and spleens (Spl.) of transgenic mice at 16 weeks of age as determined by Western blot analysis using an anti-E2a monoclonal antibody. Nontransgenic control mice displayed no E2a-Hlf expression. Gel loadings were standardized based on total protein content (lower panel). wt, wild type.

FIG. 2

Thymic hypoplasia and rapid involution in E2A-HLF mice. (A) Total cell numbers were determined on viable cell suspensions of thymuses (left) from transgenic and nontransgenic (NTG) littermates at the indicated ages. Numbers of splenocytes (right) expressing B220 or CD3 were determined by FACS at 4 weeks of age. Results represent the average and standard deviations of determinations from four to six transgenic or two to four nontransgenic animals at each time point. (B) Spleen cells (16 weeks of age) were cultured at a density of 10⁶ cells/ml in normal tissue culture medium without addition of cytokines or mitogens. The number of viable cells was determined by microscopy using a hemocytometer and trypan blue exclusion. Data are means and standard deviations of triplicate determinations from three animals in each cohort (open boxes, E2A-HLF mice; filled boxes, nontransgenic mice). The data for EμSV-bcl-2-22 (filled circles) are from reference . FACS analysis of splenocytes prior to explantation showed a modestly skewed B/T ratio in E2A-HLF mice. Numbers indicate percentages of cells in each phenotypic subset.

FIG. 3

FACS analysis of premalignant thymocytes from E2A-HLF mice and their nontransgenic (NTG) littermates. Two-color contour plots show expression of CD4 and CD8 in thymocytes from mice at different ages (indicated at the top). Progressive loss of CD4⁺8⁺ DP and then SP thymocytes is observed in E2A-HLF mice. Numbers indicate percentages of cells in each phenotypic subset.

FIG. 4

Expansion of a primitive thymocyte population in E2A-HLF transgenic mice. (A) Two-color contour plots of CD4 and CD3 expression in CD4⁺ gated thymocytes or splenocytes from mice at different ages show accumulation of a CD3⁻4^lo population in thymuses of transgenic but not normal mice. A similar population is not detected in the spleen at 24 weeks. (B) Two-color contour plots demonstrating that CD3⁻4^lo gated thymocytes express low levels of c-Kit and CD25. CD3⁺8⁺ thymocytes from same transgenic animal served as a negative control.

FIG. 5

Expression of E2a-Hlf within various lymphoid populations purified by flow sorting from E2A-HLF mice at 6 and 24 weeks of age. Identities and sources of the cell populations are indicated above the gel lanes. Western blot analysis was performed with an anti-E2a monoclonal antibody (24). Extracts from equal numbers of cells were loaded per gel lane. The control lane contains extract from transiently transfected cells expressing wild-type (wt) and chimeric E2a proteins of human origin. Wild-type E2a proteins were not detected in most lanes due to minimal cross-reactivity of the YAE antibody with mouse E2a proteins (24) at the low amounts of protein available for analysis.

FIG. 6

Tumor development in E2A-HLF mice. The mortality for a cohort (n = 28 animals) of E2A-HLF mice is shown over the 27-month observation period. Deaths were the result of malignant lymphoma confirmed by histologic examination. No deaths from lymphoma were observed in a comparable cohort of normal littermates.

FIG. 7

Histologic and molecular features of tumors arising in E2A-HLF transgenic mice. (A and B) Tissue sections were stained with hematoxylin and eosin for thymic tumor and liver infiltrated by lymphoma, respectively (magnification, ×36). (C and D) Immunohistochemical analyses showing CD3 and B220 expression in two different thymic and nodal tumors, respectively. (E) Southern blot analysis of TCR gene rearrangements in lymphomas. Dash indicates migration of the germ line band. Lanes: C, germ line control; 1 to 3, 5, and 6, T-lineage tumors; 4, B-lineage tumor. (F) Western blot analysis showing expression of E2a-Hlf in lymphomas (lanes 1 to 5, T lineage; lane 6, B lineage).

FIG. 8

Schematic illustration of thymocyte maturational arrest induced by E2A transgenes. The profile of thymocyte differentiation is based on the data of Moore and Zlotnik (34). Vertical bars denote stages of maturational arrest in E2A-HLF and E2A-PBX1 mice, respectively. Temporal expression profiles for selected surface antigens are shown below. TN, triple negative.