The TEL-AML1 leukemia fusion gene dysregulates the TGF-β pathway in early B lineage progenitor cells (original) (raw)
Induced expression of TEL-AML1 in BaF3 cells. The GeneSwitch system (20) is based on an autoregulatory feedback loop that involves the binding of a GAL4 regulatory fusion protein, pSwitch, to GAL4 upstream activating sequences in both the promoter controlling expression of the GAL4 regulatory protein and that controlling expression of TEL-AML1. The expression of TEL-AML1 is itself controlled by the presence or absence of the agonist mifepristone, which brings about a conformational change in the pSwitch regulatory protein and its subsequent activation (Supplemental Methods and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI36428DS1). We established inducible TEL-AML1 in murine BaF3 cells, a putative pro-B cell line (21). Thirty double-positive stable clones expressing the regulatory protein that also showed inducible expression of TEL-AML1 by Western blot and staining via the V5 terminal tag were selected and expanded in liquid culture. Since we observed some degree of cell death in both inducible control and TEL-AML1–expressing clones at the recommended concentrations of mifepristone, we further titrated positive clones against decreasing concentrations of agonist to determine the lowest optimal conditions for protein expression without pleiotropic effects (data not shown). All subsequent experiments were performed with 12.5 pM mifepristone and 1.5 × 104 cells/ml for 3 days (unless otherwise stated). After incubation with mifepristone, predominant nuclear speckled staining was observed by confocal microscopy using an antibody against the V5 tag in all TEL-AML1–expressing cells but not control cells (Figure 1, A and B, and Supplemental Figure 2). The results from one representative clone (i.e., 1/27) are shown in Figure 1B and from another, in Supplemental Figure 3. In a number of different clones, expression of TEL-AML1 was observed by Western blot analysis in as little as 4 hours (Figure 1C and data not shown).
Induction of TEL-AML1 protein expression in BaF3 cells. (A) Uninduced (left) and mifepristone-induced control cells (right) stained with both DAPI and an antibody against the TEL-AML1 V5 tag (original magnification, ×100). (B) Uninduced (left, DAPI and anti–V5 tag stained) and mifepristone-induced expression of TEL-AML1 (right, anti–V5 tag alone) (original magnification, ×100). The inset (×100) shows a confocal microscopy cross section. For DAPI staining, see Supplemental Figure 2E. (C) Western blot analysis over time of mifepristone-induced control and TEL-AML1–expressing BaF3 cell whole-cell protein extracts, blotted with anti-AML1 antibody. A loading control (IFN regulatory factor 2 [IRF2]) is shown below.
BaF3 cells expressing TEL-AML1 retain IL-3 dependence, grow more slowly than controls, but are less sensitive to the antiproliferative effects of TGF-β. We next evaluated the growth profile of TEL-AML1–negative and –positive BaF3 cells in standard culture conditions by determining viable cell numbers (Figure 2). For this purpose, 2 TEL-AML1–expressing clones were used (with essentially identical results), and here we show the data for one of them. No difference in growth was detected with inducible control clones grown in the presence of mifepristone (Figure 2A), while expression of the fusion gene caused a slowing of cell growth (Figure 2B). We have also observed, consistently, a slower proliferation rate in several murine B cell progenitor cell lines following transfection with retroviral TEL-AML1 constructs (C. Champseix, S. Tsuzuki, T. Enver, and M. Greaves, unpublished observations). BaF3 cells expressing TEL-AML1 retained dependence on IL-3 for viability and proliferation (Supplemental Figure 4B).
Effects of TEL-AML1 expression and TGF-β on growth rates and cell cycle of BaF3 cells. (A) Typical growth curve of control cells grown in the presence or absence of mifepristone inducer (Ind) and/or TGF-β (TGF) at 10 ng/ml. (B) Typical growth curve of clone 1/27 cells (inducible for TEL-AML1) grown in the presence or absence of inducer and/or TGF-β at 10 ng/ml. (C) Cell cycle analysis (BrdU) of inducible control and inducible TEL-AML1 cells grown in the presence or absence of inducer and/or TGF-β.
Next, we investigated whether TEL-AML1 expression, despite its negative impact on cell proliferation rate, could, nevertheless, provide some advantage in the context of growth inhibitors or apoptotic stimuli. We first tested the response of the cells to the addition of IFN-γ or TNF, IL-3 deprivation, or serum starvation, but saw no differences due to TEL-AML1 expression (Supplemental Figure 4B and data not shown). There was, however, a consistently different response of TEL-AML1–positive cells to the growth inhibitor TGF-β1 (TGF-β) at 10 ng/ml. As in previous studies (22), parental BaF3 cells were sensitive to the antiproliferative effects of TGF-β over a dose range of 1–100 ng/ml (see Methods and Supplemental Figure 4A), as were inducible control cells (Figure 2A), but TEL-AML1–positive BaF3 cells showed marked resistance to its effects, as evidenced by the finding that TGF-β did not further reduce their slower rate of proliferation (Figure 2B). The net result is that in the presence of TGF-β, TEL-AML1–positive cells proliferate at approximately the same rate as normal, equivalent cells.
In further examination of the inhibitory effects of TGF-β, we pulsed cells with BrdU for 30 minutes and analyzed the cell cycle of this synchronized population of cells after 10 hours of culture in the presence or absence of TGF-β (Figure 2C). TGF-β affected the cell cycle of TEL-AML1–negative cells, reducing the proportion of cells in S plus G2 relative to M phase (from 49% to 22%). In the absence of the mifepristone inducer, cycling of the TEL-AML1 clone was also reduced by TGF-β (51% to 32%). However, when TEL-AML1 expression was induced, the more slowly cycling cells were again resistant to further proliferative inhibition by TGF-β; i.e., there was no reduction in the proportion of cells in S plus G2 relative to M phase (Figure 2C; 21% vs. 24%). These data suggested that in the presence of TGF-β, cells expressing TEL-AML1, although proliferating slowly, might acquire at least proliferative parity if not a selective advantage.
We next performed a coculture experiment with a range of percentages of TEL-AML1–negative and –positive cells in the presence or absence of TGF-β and monitored the percentage of TEL-AML1–positive cells. Figure 3 provides data from one of 5 independent experiments that gave consistent results. After 6 days in the absence of TGF-β, there was a large relative increase in the percentage of TEL-AML1–negative cells, in accordance with their faster growth. However, in the same in vitro culture conditions in the presence of TGF-β, 10-fold more TEL-AML1–positive cells were consistently present, maintaining proliferative parity (Figure 3).
Competition assay of a mix of TEL-AML1–expressing and –nonexpressing BaF3 cells grown in the presence or absence of TGF-β. A mixture of TEL-AML1–expressing and –nonexpressing cells (84%:16%) was grown in the presence or absence of TGF-β, and the cell growth and TEL-AML1 expression profile were analyzed over time. Growth of TEL-AML1–expressing cells but not control cells is sustained in the presence of TGF-β. Flow cytometry analysis: the vertical axis represents fluorescence activity (for TEL-AML1 expression using an antibody against the V5 tag); the horizontal axis represents light scatter (cell size). In 4 repeat experiments, the starting percentage of TEL-AML1–positive cells was around 80%, and their persistence in the presence of TGF-β was consistent.
The increase in endogenous p27KIP1 transcription in response to TGF-β is blocked in the presence of TEL-AML1. In the presence of TEL-AML1 alone, we saw a relative increase in transcript levels of the cyclin-dependent kinase (Cdk) inhibitor proteins p27KIP1 (CDKN1B) and p21 (CDKN1A) (Figure 4A and Supplemental Figure 5), which control G1 phase progression and S phase entry (23). This upregulation is concomitant with the change in cell cycle. However, it has also been shown that TGF-β induces G1 arrest in B cells via upregulation of p27KIP1 protein (23). We therefore investigated next whether the resistance of TEL-AML1–positive cells to TGF-β–induced growth inhibition was associated with an impact on p27KIP1 expression. Real-time quantitative PCR (Q-PCR) analysis of p27KIP1 expression levels was performed in TEL-AML1–positive and –negative cells treated with TGF-β for different periods of time (Figure 4B) and in the absence of TGF-β (Supplemental Figure 5). As anticipated, inducible control cells in the presence of TGF-β showed a progressive increase in p27KIP1 expression. Conversely, in the presence of TGF-β, TEL-AML1–positive cells exhibited minimal further increase in p27KIP1 expression. In some experiments (as in Figure 4B), there was a transient increase in p27KIP1 in the presence of TGF-β and TEL-AML1, but this was not consistently seen.
TEL-AML1 activates expression of p21 but blocks the TGF-β–mediated activation of p27 and inhibits the TGF-β response of a target gene promoter. (A) Q-PCR analysis showing activation of p21 (CDKN1A) by TEL-AML1 in the absence of TGF-β. Control cells and cells inducible for TEL-AML1 were incubated for 3 days in the absence (–) or presence of inducer. cDNA was subjected to Q-PCR and normalized to GAPDH, to which relative expression of p21 is shown. Error bars represent the SD of an experiment performed in triplicate and repeated 3 times. (B) Q-PCR analysis showing a block in expression of TGF-β–induced p27KIP1 in the presence of TEL-AML1. cDNA was prepared from a TGF-β time-course analysis of both control and TEL-AML1–inducible cells in the presence or absence of the TEL-AML1–inducing agent (ind). Cells inducible for TEL-AML1 but not actually induced are indicated by parentheses, i.e., (TEL-AML1). Experiments were repeated 3 times. (C) Inhibition of the TGF-β–responsive IgA promoter by TEL-AML1 in a luciferase reporter assay. Activation of the IgA promoter was assayed by its transient transfection into control cells and cells inducible for TEL-AML1. Cells inducible for TEL-AML1 but not actually induced are indicated by parentheses. Growth was continued in the presence of TGF-β, either alone or after addition of the TEL-AML1–inducing agent. Error bars represent SD from 3 independent experiments. (D) TEL-AML1 associates with Smad3. Cell lysates from control and TEL-AML1–expressing cells were immunoprecipitated with anti-Smad3 antibody and half the IP subjected to Western blot analysis with an antibody against the runt homology domain (RHD) of AML1. Lane 1, uninduced cells; lane 2, TEL-AML1–induced; lane 3, TEL-AML1–induced + TGF; lane 4, control cells + TGF.
The expression of TEL-AML1 markedly represses the response of the mouse Igα promoter to TGF-β. To determine whether TEL-AML1 expression interfered only with the antiproliferative activity of TGF-β or whether its overall signaling was affected, we tested the response to TGF-β of one of the known target gene promoters. Using transient transduction reporter assays with a construct that contained the mouse Igα promoter (positively regulated by TGF-β; ref. 24), we observed that expression of TEL-AML1 markedly repressed the response of this promoter to TGF-β (Figure 4C).
Smad2/3 signaling in the presence of TEL-AML1. To gain some insight into the possible mechanism underlying the lack of response of TEL-AML1–positive cells to TGF-β, we analyzed the TGF-β pathway of these cells. First we investigated the early steps of the pathway: TGF-β binding to TGF-β II receptor and activation of the receptor complex by Smad2/3 phosphorylation in the presence or absence of a potent and specific inhibitor of TGF-β (SB-431542) (25, 26). By Western blot analysis with anti–p-Smad2/3, we showed that the phosphorylation of endogenous Smad2/3 protein, in response to TGF-β, was unaffected in TEL-AML1–positive cells, inferring that disruption to the TGF-β pathway by TEL-AML1 was subsequent to Smad2/3 activation (Supplemental Figure 6 and data not shown). Furthermore, using Q-PCR array analysis, we also analyzed the expression of a focused panel of genes known to be related to bone morphogenetic protein–mediated TGF-β signal transduction and assessed their status in the presence or absence of TEL-AML1. No changes in the levels of TGF-β II receptor gene expression were observed (data not shown), again consistent with downstream disruption of the pathway.
It has been reported that other leukemic fusion proteins that incorporate AML1, such as AML1/EVI-1 and AML1/ETO, associate with Smad3 (27, 28) and do so via the DNA binding Runt domain (27), or EVI1 in the case of AML1-EVI1 (28). Since this domain is retained in the TEL-AML1 fusion, we investigated whether TEL-AML1 was able to associate with Smad3. Smad3 immunoprecipitation from lysates of TEL-AML1–positive and –negative cells, grown for 48 hours in the presence or absence of TGF-β, confirmed association of TEL-AML1 with Smad3 (Figure 4D). We next used the CAGA12 promoter reporter gene to assess the response to TGF-β in the absence of a binding site for (TEL-)AML1. The CAGA12 promoter contains 12 tandem copies of the DNA binding site for Smad3 alone linked to luciferase (25) but no binding site for AML1. The results indicated reduced activation of the CAGA12 promoter by TGF-β in the presence of TEL-AML1, which cannot bind to this promoter (Supplemental Figure 7A). Furthermore, using EMSA with protein extracts isolated from REH t(12;21) cells and a DNA probe that contains the AMLU and AMLD binding sites as well as SMADA and SMADB binding sites (29), we show that TEL-AML1 is able to bind to its cognate binding site in the presence of Smad3 (Supplemental Figure 7B). The combined data suggest that the repressive effect seen at this TGF-β–responsive promoter in the presence of TEL-AML1 operates through interaction with Smad3 but may not require full DNA binding of the fusion protein itself. Similarly, the corepressor mSin3A is known to regulate diverse signaling pathways in both normal and malignant cell growth by forming large multiprotein repressor complexes (15, 16). In cells induced to express TEL-AML1, immunoprecipitation with antibody against mSin3a confirmed its interaction with the fusion protein (data not shown).
Functional impact of TEL-AML1 expression in progenitor cells from transgenic mice. As the lineage affiliation of BaF3 is not unambiguous, we analyzed the effect of TEL-AML1 protein expression in an alternative murine model system: BM-derived B lineage progenitor cells from mice transgenic for TEL-AML1. As expression of the fusion gene was regulated by the IgH enhancer, we anticipated that TEL-AML1 protein would be selectively expressed in the B cell lineage and, possibly, at lower levels in earlier progenitors, in which the enhancer element might be accessible to transcription factors and therefore active (30). Two transgenic lines expressing Eμ TEL-AML1 were generated. In more than 100 mice followed for 18 months, no leukemias developed, commensurate with the view that TEL-AML1 is in itself insufficient to cause overt leukemia (9, 31).
We cloned BM colony-forming cells from mice in the presence of Flt-3 ligand, IL-7, and SCF. Two morphological types of colonies were generated in both wild-type and TEL-AML1 mice (Figure 5A). These distinctive colonies were qualitatively distinguished, and we refer to them as “spread” (or diffuse) and “tight” (or dense). TEL-AML1 mice consistently showed higher numbers of spread colonies and fewer tight colonies compared with wild-type mice (Figure 5B). Replating assays revealed that spread colonies could form both spread and tight secondary colonies, but replated tight colonies only produced tight colonies. This suggests that spread CFCs are developmentally less differentiated than tight colonies, which is compatible with their phenotypes (see below). We next assessed the impact of TGF-β on progenitor CFCs with and without TEL-AML1 expression by selectively picking spread versus tight colonies and replating them in the presence of TGF-β. We observed that both spread and tight TEL-AML1–positive colonies were consistently less sensitive to the inhibitory effects of TGF-β than were wild-type colonies. Reduced sensitivity to TGF-β was reflected in colony numbers (Figure 5C and Supplemental Figure 8; data from 5 experiments). Additionally, residual colonies in TGF-β–treated TEL-AML1–positive cell were usually larger than in wild-type populations (Supplemental Figure 9).
Impact of TEL-AML1 expression on B progenitor CFC numbers and their inhibition by TGF-β. (A) Typical first-round colony morphology in a B progenitor cell colony assay. Colonies (×40) were grown in the presence of IL-7 with additional SCF and Flt-3 ligand, picked after 9 days growth, spread on glass slides, and stained with Giemsa for morphology (×100). (B) First-plate phenotype. Average colony numbers from first plating of BM cells isolated from wild-type and TEL-AML1 transgenic lines. Cells (7 × 105) were plated in methylcellulose for 9 days under B cell growth conditions with additional SCF and Flt-3 ligand. Error bars represent SD from 5 independent experiments. (C) Average resistance to TGF-β. Effect of TGF-β on second-round colonies picked from first-round tight colonies (which yield only tight colonies) and spread colonies (which yield both spread and tight colonies). Average resistance was calculated as the number of colonies in the presence of TGF-β divided by the total number of colonies in its absence in the same experiment. Error bars represent SD from 5 independent experiments.
Analysis of cells retrieved from these colonies according to morphology, immunophenotype (Figure 5A and Figure 6), and gene expression (Q-PCR) (Supplemental Figure 10) revealed that the spread colonies were a mixed population of lymphoid and myeloid cells, whereas cells in the tight colonies were all lymphoid. Wild-type and TEL-AML1–positive tight colonies showed a similar pre-B cell phenotype, being B220-, CD19-, and BP1-positive but negative for Flt-3, Sca-1, and CD11b (Figure 6). In contrast, the spread colonies were positive for Flt-3, Sca-1, and c-Kit, indicative of a more immature, progenitor-like phenotype. In line with the morphological variety of cells within the spread colonies, we also observed distinct and separate populations of cells either positive or negative for CD11b (Figure 6). These were flow sorted for future analysis. The CD11b-negative cells derived from TEL-AML1 transgenic mice were exclusively lymphoid in morphology and expressed RAG1, CD79a, and TEL-AML1 (Supplemental Figure 10). The CD11b-positive cells had myeloid morphology, expressed low levels of CD79a but not RAG1, and had low or negligible levels of TEL-AML1 and most closely resemble the pre-pro-B cells of Hardy’s fraction A (32). These data indicate that the spread colonies are derived from a common lympho-myeloid progenitor with (in the assay used) B lineage progeny. We also observed a substantial increase in the number of c-Kit+Sca-1+ cells in spread colonies in the presence of TEL-AML1 expression, which is in keeping with previous findings in BM transplantation models with TEL-AML1 (6, 8) and indicative of a selective impact on both multipotent (lympho-myeloid) and very early B cell progenitor cells.
Colony phenotypes from Eμ TEL-AML1–transgenic lines and wild-type controls. Analysis of cell immunophenotype by flow cytometry. Tight and spread colonies isolated from wild-type and TEL-AML1–transgenic lines were compared by surface phenotype using monoclonal antibodies against B220, CD19, Flt-3, BP1 (63), c-Kit, Sca-1, IL-7R, and CD11b, as well as isotype controls. The percentage of cells within each quadrant is shown in gray, and the percentage within a specific gate is shown in red.
TEL-AML1 expression provides selective advantage to candidate “preleukemic” stem cells in the presence of TGF-β. Human cord blood CD34+ cells transduced with TEL-AML1 via a lentiviral vector produce a preleukemic-like phenotype in NOD/SCID mice (13). This is dependent upon the expansion of a putative pre-LSC stem cell population with the immunophenotype CD34+CD38–CD19+ (13). We have assessed whether abrogation of TGF-β sensitivity by TEL-AML1 protein in murine models can be replicated in a system that more approximates clinical preleukemia in patients and, in particular, whether the candidate stem cell can be selected. Candidate pre-LSCs were generated in vitro by culture of TEL-AML1–transduced CD34+CD38–CD19– cord blood cells on MS-5 stroma (13). From an initial average inoculum of 93.3 TEL-AML1–expressing (GFP-positive) cells (SD 15.3, SEM 8.82, range 80–110), 5,146.7 CD34+CD38–CD19+ (candidate pre-LSCs) were generated (SD 147, SEM 85.1, range 4,980–5,260). These cells were then replated on MS-5 stroma in the presence of TGF-β. After an additional 3 weeks, they had proliferated to give rise to 19,667 pre-LSCs (SD 4,400.4, SEM 2,540, range 15,300–24,100). Thus, in the presence of TGF-β, there is an absolute increase in the number of TEL-AML1–expressing pre-LSCs of approximately 4-fold (average 3.82, range 3.1- to 4.6-fold). Interestingly, this net increase in pre-LSCs that occurs in the presence of TGF-β was accompanied by an absolute decrease in cell number of more mature TEL-AML1–expressing and wild-type cells, and this was not seen in the absence of TGF-β. The combination of increased numbers of pre-LSCs and decreased numbers of mature cells resulted in the 12-fold proportionate increase in pre-LSCs presented in Figure 7.
TGF-β “selects” candidate human pre-LSCs. Cord blood stem cell encoded (CD34+CD38–) populations were transduced with lentiviral TEL-AML1 and plated on MS-5 stroma for 3 weeks to generate pre-LSCs (CD34+CD38–CD19+) (13). Cells were replated on MS-5 with or without TGF-β. (A) Cell populations without TGF-β. (B) Cell populations with TGF-β. The percentage of cells in each particular gate or quadrant is shown from 1 experiment but was replicated and consistent in 2 separate experiments (i.e., 3 in total).