DNA replication in bone marrow (BM) progenitor cell populations is severely impaired in E2f1^−/−E2f2^−/− (DKO) mice, but not in E2f1^+/−E2f2^+/− mice (phenotypically wild type; henceforth called E2f1⁺2⁺), leading to severe hematopoietic deficiencies [7]. We tested the effects of E2f1 and E2f2 mutation on the expansion and leukemogenicity of cells expressing the p190 Bcr-Abl fusion oncogene, which is preferentially associated with ALL in humans [5]. For most experiments, we limited transduction with mouse stem cell viruses (MSCVs) encoding Bcr-Abl and/or green fluorescent protein (GFP) to only a small percentage of progenitors to more accurately reflect the rare occurrence of oncogenic initiating events in a background of unmutated cells, allowing for competition between Bcr-Abl-positive and -negative progenitor cells. The transduced cells were then transplanted into wild-type irradiated recipient mice, and competitive reconstitution was analyzed (see Figure 1 for the experimental design).

At low transduction efficiencies (less than 5% of transplanted cells expressing Bcr-Abl), we found that Bcr-Abl expression clearly provides a substantial advantage for DKO, but not E2F1⁺2⁺ progenitors, as reflected by contributions to peripheral blood cells at 3 wk post-BM transplantation (BMT) (Figure 2A and 2B). The recipients of Bcr-Abl-transduced DKO cells had dramatically increased percentages of B cells and myeloid cells expressing Bcr-Abl. Increased contributions of Bcr-Abl-expressing cells in the DKO relative to E2F1⁺2⁺ background were also observed at high infection efficiency, although a modest (but for many recipients, transient) advantage of Bcr-Abl expression was evident among E2F1⁺2⁺ cells in terms of contributions to mature blood cells (Figure S1). As a control, we transduced BM progenitors with vector expressing GFP only, and in numerous experiments we found that E2f1/E2f2 loss did not noticeably affect the ability to transduce stem and progenitor populations, as similar short-term and long-term reconstitution of all lineages as determined by GFP expression was observed in recipients of vector-transduced E2f1⁺2⁺ and DKO BM progenitors (Figures 2A, 2C, and unpublished data).

In order to directly determine contributions to hematopoietic progenitor populations, we analyzed BM from the same set of mice for the expression of GFP (vector or Bcr-Abl) in particular progenitor pools. The lineage negative (Lin⁻) population in the BM expresses low levels of lineage-specific markers, and is thus enriched for progenitor cells. The Lin⁻ Flk2⁻ Sca1⁺ population is enriched for HSC but will also include short-term progenitors [18]. Of note, while Sca1 is expressed on only about 25% of HSC in BALB/c mice, the Lin⁻ Sca1⁺ population is substantially enriched for long-term HSC activity in these mice [19]. We observed a dramatic selective advantage for Bcr-Abl expression in the Lin⁻ Flk2⁻ Sca1⁺ population in the DKO, but not the E2f1⁺2⁺, background (Figure 2C, upper right quadrants), correlating with contributions to peripheral blood cells. Of note, a transient advantage within more committed progenitors (such as Lin⁻ Sca1⁻ cells; Figure 2C, lower right quadrants) is often evident for Bcr-Abl expression in E2f1⁺2⁺ cells. As we find that c-Kit is down-regulated on Lin⁻ Sca1⁺ cells post-BMT (unpublished data), we have been unable to further examine Bcr-Abl expression among highly defined HSC. In summary, Bcr-Abl expression provides a dramatic competitive advantage to progenitor cells in the replication-impaired DKO background, but not in the replication-competent E2f1⁺2⁺ background.

The competitive expansion of Bcr-Abl-expressing cells relative to untransduced cells in the DKO background could be the result of overcoming cell cycle defects observed in DKO progenitors. To test this possibility, we assayed the effect of Bcr-Abl expression on S-phase progression rates in DKO progenitor cells using a bromodeoxyuridine (BrdU) pulse-chase method [20]. Two weeks post-BMT, at which point no signs of leukemia development were evident, mice were injected with BrdU and then sacrificed after 2 h. BrdU injection essentially provides a short pulse of the label, since BrdU half-life in vivo is very short [21]. Following flow cytometric isolation of GFP⁺ Lin⁻ or GFP⁻ Lin⁻ progenitors from the spleen, the average DNA content of BrdU-positive cells was assayed by propidium iodide (PI) intensity profiling. The rates of S phase progression were determined as the shift of the average PI intensity from the middle of S phase toward the G2 peak using formulas described by Begg et al. [20]. Although the calculated progression rates do not provide accurate absolute values for S phase length, since the presumption that the average PI intensity lies in the middle of S phase is not true for DKO cells (which accumulate in early S phase [7]), this method can still accurately analyze the changes of the rates caused by Bcr-Abl expression. Significantly, while S phase progression is substantially slowed in DKO progenitor cells, as previously reported [7], the expression of Bcr-Abl in these cells essentially restored S phase progression to control progression rates (Figure 3). In contrast, the expression of Bcr-Abl had no noticeable effects on S phase progression in wild-type progenitors. In addition, we previously showed that a fraction of DKO BM progenitors appear arrested in S phase, exhibiting S phase DNA content but no detectable BrdU incorporation [7]. As shown in Figure 3A, Bcr-Abl expression in DKO progenitors prevents the accumulation of this S phase-arrested population.

Thus, the competitive advantage provided by Bcr-Abl expression to DKO progenitors appears to derive from restored cell cycle progression. Furthermore, as we find that the expression of Bcl2 does not provide an advantage to DKO progenitors relative to wild-type progenitors (Figure S2), the ability of Bcr-Abl to block apoptosis does not appear to underlie the selective advantage conferred by Bcr-Abl expression in the DKO background, although decreased apoptosis could be a contributing factor.

As leukemogenesis appears to require initiating mutations in stem or progenitor cell populations, we addressed whether the greater competitive advantage provided to Bcr-Abl-expressing DKO progenitors translates to increased tumor incidence in transplanted mice. Strikingly, all recipients of Bcr-Abl-transduced DKO progenitors developed rapid pre-B ALL-like disease using both low and high initial transduction efficiencies (Figures 4A and 4B), preceded by rapid increases of peripheral GFP⁺ B220⁺ cells (Figures 2A and S1). In contrast, only about half of the recipients of E2f1⁺2⁺ cells transduced at high efficiency, and none of the recipients at low transduction efficiency, developed leukemia (also pre-B ALL; Figure 4). For leukemias arising in both genetic backgrounds, leukemic infiltrates were evident in spleen, liver, and lung, and peripheral blood GFP⁺ cells expressed low B220, exhibiting a large, immature morphology with pale-staining nuclei (Figure 4C and unpublished data). Leukemia cells in the spleen and BM were mostly negative for the Lin markers GR-1, Ter119, CD4, and CD3 (unpublished data), expressing from undetectable to low B220 levels depending on the leukemias (Figure 4C; examples shown express little to no B220). Interestingly, we consistently observed a greater fraction of Sca1⁺CD34⁺ cells in DKO relative to E2f1⁺2⁺ leukemias (Figure 4C), consistent with the early advantage provided by Bcr-Abl expression in DKO progenitor-enriched populations (Figure 2C).

While leukemogenesis correlates with the ability of Bcr-Abl-expressing cells to competitively expand within progenitor pools, based on increased leukemia rates alone we cannot distinguish whether or not cell autonomous effects of E2f1/E2f2 loss, such as accelerated mutagenesis or decreased apoptosis, are the major factors underlying increased leukemia rates. To directly address this issue, we included E2f1⁺2⁺ untransduced competitor cells with Bcr-Abl-transduced DKO progenitors; amazingly, these competitors were able to almost completely eliminate leukemogenesis (Figures 4A and 4B). E2f1⁺2⁺ competitors also substantially reversed the selective advantage conferred by Bcr-Abl expression in the DKO background as reflected in peripheral blood cells (Figures 2A, 2B, and S1) and within stem cell-enriched populations (Figure 2C). Similarly, transplantation of Bcr-Abl-transduced DKO progenitors into sublethally irradiated recipients resulted in the rebound of host (wild-type) hematopoiesis and complete prevention of leukemias (unpublished data). Thus, enhanced leukemogenesis is not primarily due to cell autonomous effects of E2f1 and E2f2 loss, but most likely results from the poor ability of DKO progenitor cells to compete with Bcr-Abl-expressing DKO progenitors.

Importantly, these results suggest that the failure of many recipients of Bcr-Abl-transduced E2f1⁺2⁺ (particularly at low transduction efficiency) to develop leukemia was not due to our inability to introduce Bcr-Abl into the appropriate progenitor cells, but to the inability of Bcr-Abl⁺ progenitors to be maintained in the face of replication competent competitors. Finally, while it remains formally possible that DKO stem/progenitor cells home poorly to BM niches and that Bcr-Abl restores homing to DKO progenitors, the fact that a low percentage of cotransplanted E2f1⁺2⁺ BM can reverse the selective expansion of Bcr-Abl⁺ DKO progenitors and leukemogenesis (Figures 4 and S1) strongly argues against a role for differential homing in genotype-dependent leukemia rates.

In summary, a DKO progenitor pool promotes Bcr-Abl-dependent leukemogenesis due to the inability of untransduced DKO progenitors to provide effective competition. The ability of Bcr-Abl to restore S phase progression to replication impaired DKO progenitors most likely underlies the competitive advantage provided by Bcr-Abl specifically within the DKO background.

In order to further explore the generality of the selection for Bcr-Abl-expressing cells under conditions of impaired DNA replication as well as to examine Bcr-Abl-mediated leukemogenesis in a more clinically relevant context, we asked how the suppression of proliferation within hematopoietic progenitor pools during treatment of mice with HU, an inhibitor of ribonucleotide reductase, affects Bcr-Abl-dependent competition within wild-type BM. HU has been used for over 40 y as a cytostatic drug [22]. HU-induced dNTP depletion results in replication stalling and the activation of checkpoint pathways [23]. We initially asked whether HU treatment creates a poorly competitive environment that favors the expansion of Bcr-Abl-expressing blood progenitors ex vivo. As shown in Figure 5A, HU differentially affected Bcr-Abl- and vector-expressing progenitors, allowing Bcr-Abl⁺ cells to competitively expand. In fact, while the expansion of vector-expressing progenitors was almost completely inhibited by all concentrations of HU used, Bcr-Abl-expressing progenitors continued expanding at doses ranging from 5 to 25 μM (Figure 5A, lower panels). Higher doses of 50 and 100 μM HU did effectively inhibit the expansion of Bcr-Abl⁺ progenitors, indicating that the advantage provided by Bcr-Abl expression during HU treatment is relative but not absolute. While Bcr-Abl expression alone provided a modest advantage to wild-type progenitors in culture, 5 to 25 μM HU treatment conferred an even greater advantage to Bcr-Abl-expressing cells (Figure 5A, upper panel). Furthermore, as determined by GFP expression levels, HU selects for cells expressing higher levels of Bcr-Abl (Figure 5B). Notably, increased Bcr-Abl expression is commonly observed in patients during CML progression from chronic phase to blast crisis [24].

Mice were transplanted with MSCV-p190 Bcr-Abl-transduced BALB/c BM progenitor cells, and half of the recipients were switched to water containing HU (20 mg/kg/d; similar to doses used clinically for cytoreduction [22]) at 2 wk post-BMT. Strikingly, by 5 and 8 d after switching recipient mice to HU, a substantial competitive advantage for Bcr-Abl⁺ cells within the progenitor-enriched Lin⁻ Sca1⁺ population was evident, while a similar selection for Bcr-Abl expression was not observed without HU treatment (Figure 6A). Note that about two-thirds of the progenitor-enriched Lin⁻ Sca1⁺ cells expressed Bcr-Abl in HU-treated mice, while very few Bcr-Abl⁺ Lin⁻ Sca1⁺ cells were detected in untreated mice, despite the fact that these mice were transplanted with the same pool of transduced BM progenitors. The rapid competitive advantage provided by Bcr-Abl specifically following HU treatment in vivo, together with the almost immediate selection for Bcr-Abl expression by HU ex vivo, argues that HU-induced mutagenesis does not account for the effects of HU on the competitive expansion of Bcr-Abl⁺ progenitors.

To understand why Bcr-Abl provides such an advantage specifically following HU treatment, we analyzed cell cycle progression in Bcr-Abl-expressing progenitors in vivo using the same method used for Figure 3. While HU decreased the percentage of cells undergoing DNA synthesis and increased the length of S phase, Bcr-Abl expression resulted in more normal S phase progression in leukocytic progenitors in HU-treated recipients (Figure 6B). This result provides a mechanistic insight into why HU treatment improves the competitive expansion of Bcr-Abl-expressing progenitors in vivo and ex vivo: While HU impedes the proliferation of competing progenitors, Bcr-Abl overcomes this block. In contrast, Bcr-Abl expression does not appear to affect DNA replication in untreated progenitors, and thus Bcr-Abl expression does not provide an apparent competitive edge without HU.

We addressed whether HU treatment of mice with Bcr-Abl⁺ progenitors promotes leukemogenesis in vivo. For each experiment, aliquots from a single pool of MSCV-Bcr-Abl (or vector)-transduced BALB/c BM stem cells were transplanted into multiple syngeneic recipients. For all recipients, ∼3% of peripheral B cells were GFP⁺ at 2 wk post-BMT, at which point half of the mice were switched to water containing HU. By treating with HU starting 2 wk post-BMT, potential effects of HU on retroviral infection efficiency or homing to BM niches are avoided. Strikingly, all of the HU-treated mice developed a pre-B ALL-like disease within 2 mo (Figure 7A) preceded by increases in the percentages of Bcr-Abl⁺ cells in the B220⁺ subset (unpublished data). In contrast, mock-treated mice that received Bcr-Abl-transduced stem cells developed leukemia with much lower penetrance (3 of 8) and longer latency periods. HU treatment also enhanced leukemogenesis induced by p210 Bcr-Abl, although interestingly, these mice all succumbed to CML-like disease (Figure 7B). Thus, in our model system, p190 Bcr-Abl causes B-ALL and p210 causes CML, thus closely modeling the human diseases caused by the respective translocation products. Given that HU treatment promotes the expansion of Bcr-Abl⁺ progenitors within 5 d (Figure 6A), it is improbable that HU-promoted mutagenesis alone accounts for more rapid disease, although increased mutation accumulation may be a contributing factor. Furthermore, as HU similarly promotes leukemogenesis in analogous experiments using donor BM from Rag2^−/− mice, in which case mature T and B cells were nearly undetectable in recipients (Figure S3), HU-mediated increases in Bcr-Abl-dependent leukemogenesis does not appear to result from immunosuppression.

Leukemic p190 Bcr-Abl recipient mice exhibited massive increases in pre-B-like large B220⁺ CD43⁺ cells, high leukocyte blast counts in peripheral blood, and splenomegaly (Figures 7C and S4). Pathological characterization of spleens and other tissues determined that the lymphoproliferative diseases arising in all HU and the three non-HU-treated mice were large cell leukemias, with frequent involvement of liver, lung, and spleen (unpublished data). Spleens contained a mixture of leukemic cells, with most exhibiting lymphoid characteristics and a subset exhibiting a myeloid phenotype (unpublished data). For most HU (relative to non-HU)-treated mice, the predominant GFP⁺ (i.e., Bcr-Abl⁺) population in the BM and spleen contained a more substantial subpopulation of cells expressing the progenitor markers c-Kit and CD34 (Figure 7D). We transplanted spleen or BM cells (10³–10⁵) from morbid leukemic HU- and non-HU-treated Bcr-Abl BMT recipients into BALB/c recipients, and most recipients for both groups developed leukemia, underscoring the malignant nature of the leukemias (Figure S5). Thus, by providing a competitive advantage to Bcr-Abl⁺ progenitor cells (as shown ex vivo and in vivo), HU treatment promotes Bcr-Abl-mediated ALL and CML.

While our experiments provide strong evidence that backgrounds of impaired DNA replication enhance the competitive expansion of Bcr-Abl-expressing cells, we wanted to test if this phenomenon can be applicable to other oncogenic events. The p53 tumor suppressor gene is mutated in over half of human tumors, and mutation of p53 has been shown to disrupt checkpoints that prevent cell cycle progression in the face of various cellular stresses [25]. Mutation of p53 in human cancers frequently results in the expression of a dominant negative mutant p53 protein (DNp53) that forms inactive multimers with wild-type p53. Given the known ability of DNp53 to alleviate checkpoint-dependent cell cycle arrests, we asked whether the expression of DNp53 (175H) could provide a competitive advantage for E2f1/E2f2 mutant progenitor expansion. In addition, we engineered a retrovirus to express the oligomerization domain of p53 (DDp53), which also inhibits p53 activity [26]. Strikingly, MSCV-mediated expression of DNp53 or DDp53 in hematopoietic progenitors from DKO, but not wild-type, mice resulted in a dramatic selective advantage for these cells after only 2 wk post-BMT (Figure 8A). Again, the percentages of peripheral blood cells expressing vector were very similar for wild-type and DKO cells. Thus, the inhibition of p53 provides a competitive advantage during hematopoiesis in DKO, but not wild-type, BM.

We used genetic disruption of p53 [27] as an alternative to retroviral DNp53 expression. We bred the p53 mutation into the E2f1/E2f2 mutant and BALB/c backgrounds together with a ubiquitously expressed GFP transgene [28]. In competitive BMT experiments using wild-type BM in competition with p53^−/−GFP⁺ BM (93:7 ratio), p53^−/−GFP⁺ BM contributions to hematopoiesis were similar to its percentage within the original BM transplanted (Figure 8B), indicating that p53 mutation does not provide an immediate competitive advantage during hematopoiesis. In contrast, DKO/p53^−/−GFP⁺ BM progenitors exhibited significant competitive expansion relative to DKO progenitors, and this competitive expansion was completely abrogated by the inclusion of E2f1⁺2⁺ competitors (Figure 8B; compare second and third columns). Thus, as in the Bcr-Abl experiments shown in Figure 2, the increased expansion of DKO/p53^−/− cells in a DKO background relative to p53^−/− cells in a wild-type background is not due to cell autonomous contributions of E2f1/E2f2 loss, but instead to the poor competition provided by DKO hematopoiesis.

The generation of the E2f1 and E2f2 mutant mice (backcrossed seven times into BALB/c) has been previously described [52,53]. BALB/c and p53^−/− mice were purchased from Jackson Labs (Bar Harbor, Maine, United States). GFP transgenic mice were the generous gift of Dr. Brian Schaefer, and were backcrossed with p53 mutation into the BALB/c background. The University of Colorado Health Sciences Center Animal Care and Use Committee approved all mouse experiments.

The retroviral MSCV-iresGFP vectors expressing either p190 or p210 Bcr-Abl were provided by Drs. Zhonghan Dai and Warren Pear. MSCV-Bcl2-iresGFP was provided by Dr. Scott Lowe. To construct similar retroviral vectors expressing DNp53 and DDp53, the p53Arg175His cDNA fragment from plasmid pCMV-NEO pC53–175 (provided by Dr. Bert Vogelstein) and DDp53 cDNA from pLXSNp53DD (provided by Dr. Xiao-Fan Wang) were cloned into the MSCV-iresGFP vector.

MSCV viruses were prepared by transient transfection of Phoenix-E cells together with pCL-Eco, and titered on fibroblasts using GFP as the marker, so that similar multiplicities of infection for oncogene and vector viruses were used. Donor DKO, E2f1⁺2⁺, or wild-type mice were injected intraperitoneally with 100 mg/kg 5-fluorouracil (Sigma, St. Louis, Missouri, United States) 3 d prior to BM harvest. Harvested donor BM cells were enriched for c-Kit⁺ cells by MACS (Miltenyi Biotec, Bad Gladbach, Germany) and then cultured overnight in IMDM plus 15% defined fetal bovine serum (HyClone, South Logan, Utah, United States) and 0.1% bovine serum antigen (Sigma), with the cytokines 100 ng/ml human stem cell factor (gift from Dr. Chris Hogan), 100 ng/ml human IL-11 (PeproTech, Rocky Hill, New Jersey, United States),1.5 ng/ml mouse IL-3 (PeproTech), and 100 ng/ml hIL-6 (PeproTech). Cells were transduced with MSCV-containing supernatants (∼1/5 volume) supplemented with 6 μg/ml Polybrene (Sigma) for 6 h. Cells were then transplanted intravenously into lethally (1,200 Rads for E2F experiments) or sublethally (600 Rads for HU experiments) irradiated BALB/c mice. A subset of the transduced cells was further cultured for 2 d, and the percentage of GFP⁺ cells was determined by flow cytometry in order to determine the initial infection efficiency. Ex vivo culturing of Bcr-Abl-transduced cells for Figure 5 was performed under the same culture conditions.

Single-cell suspensions were washed in PBS containing 5% FBS (FBS-PBS). About 10⁶ cells were stained in 20 μl of antibody solution (1:100 dilution of each antibody) for 30 min at room temperature. Cells were washed once with 1 ml of FBS-PBS and resuspended in 400 μl of PBS for flow cytometric analysis. The following PharMingen (San Diego, California, United States) antibodies against mouse were used: phycoerythrin (PE)-linked anti-B220, PE-anti-Ter119, PE-anti-GR-1, PE-anti-CD3, PE-anti-CD4, PE-anti-Flk2/Flt3, and PE-anti-CD43; and allophycocyanin (APC)-linked anti-B220, APC-anti-Thy1.2, and APC-anti-CD117; and biotin-linked anti-GR-1, biotin-anti-CD34, biotin-anti-IgM, and PE-cyanine (PE-Cy7)-linked anti-biotin. APC-anti-Sca1 antibodies were purchased from eBioscience (San Diego, California, United States). Fluorescence was detected with a Cytomics FC 500 (Beckman Coulter, Allendale, New Jersey, United States) cytometer. For BrdU incorporation analyses, mice were injected intraperitoneally with BrdU (1 mg per 25 g of body weight; Roche, Indianapolis, Indiana). After 2 h, pooled spleen and BM cells were purified using a MoFlo (Cytomation, Fort Collins, Colorado, United States) cell sorter for GFP⁺ or GFP⁻ subsets of progenitors: B220⁺ plus GR-1⁺ (B-cell and myeloid lineages) for Figure 3 or Lin⁻ for Figure 6. Immunofluorescence, flow cytometric analyses, and calculations of the length of S phase were performed as described previously [7,20].

Mice were monitored for disease development, as judged by increasing percentages of GFP⁺ cells with blast morphology in peripheral blood of transplanted animals, as well as symptoms, such as abnormal gait and labored breathing. Moribund animals were sacrificed and examined for tumors. Touch preps and tissue sections were obtained from spleen and liver.

The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers of the genes discussed in this paper are Bcr-Abl (X06418), E2f1 (L21973), E2f2 (AK087452), and p53 (P04637).