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. 2014 May 8;9(5):e96711.
doi: 10.1371/journal.pone.0096711. eCollection 2014.

The Deficiency of Tumor Suppressor prep1 Accelerates the Onset of meis1- hoxa9 Leukemogenesis

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Free PMC article

The Deficiency of Tumor Suppressor prep1 Accelerates the Onset of meis1- hoxa9 Leukemogenesis

Leila Dardaei et al. PLoS One. .
Free PMC article

Abstract

Prep1 and Meis1 ortholog TALE transcription factors have opposing roles in tumorigenesis: Meis1 serves as an oncogene, Prep1 as a tumor suppressor. We now report that, Meis1 overexpression in primary Prep1-deficient (Prep1i/i) embryonic hematopoietic cells increases self-renewal potential of cells in vitro but not in vivo, whereas leukemia is instead obtained when Meis1 is combined with another oncogene, HoxA9. Prep1i/i Meis1-HoxA9-generated leukemic cells are less differentiated and grow more aggressively after the second passage in the mouse. These data indicate that Prep1 represents a barrier to the transforming activity of Meis1 in vitro, but its absence is not sufficient to induce early leukemogenesis. On the other hand, the Prep1i/i background appears to favor the insurgence of mutations that cause a more aggressive Meis1-HoxA9-generated leukemia. Indeed, the Prep1i/i leukemic cells upregulate the Polycomb protein Bmi-1 and expectedly down-regulate the Ink4a/Arf locus products. Finally, an important feature contributed by the Prep1i/i background is the post-transcriptional increase in Meis1 protein level.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Prep1 absence accelerates Meis1-HoxA9 induced myeloid transformation and leukemogenesis.
(A) Overview of the experimental strategy. FL cells taken from 14.5 WT or Prep1i/i embryos were transduced with Meis1 (B) or HoxA9 (C) or co-transduced with both oncogenes (D) and serially plated in methylcellulose cultures every 7day. Results are shown as colony numbers per 5×104 plated cells for each round of culture. (E) Survival curves are shown for cohorts of mice transplanted with 1×106 of Prep1i/i FL cells transduced with either Meis1 or Meis1-HoxA9. Five mice for each group were used. (F) Survival curves are shown for cohorts of mice serially transplanted with the indicated numbers of WT or Prep1i/i FL cells co-transduced with Meis1 and HoxA9. Five mice per group were used. In the 1st and 2nd Bone marrow transplantation (BMT), the difference between the survival of the mice transplanted with either WT or Prep1i/i transduced cells was not significant (n.s.). However, in the 3rd and 4th BMTs the p-values of mice survival transplanted with either WT or Prep1i/i transduced cells were significant (p = 0.001 and p = 0.007, respectively).
Figure 2
Figure 2. Meis1 expression is higher in Prep1i/i leukemic cells compared to WT.
(A) FACS plot represent the Meis1-GFP-positive cells percent in WT and Prep1i/i leukemic BM cells (left side). Bar graph shows the summary of Meis1-GFP-positive Prep1i/i and WT BM cells taken from serially transplanted leukemic mice (right side). Error bars indicate SEM. (B) FACS plots represent the Mac-1 and Gr-1 positive cells on GFP-positive cell populations from WT and Prep1i/i leukemic BM. (C) Representative FACS plots of Meis1-GFP expression in Prep1i/i and WT leukemic BM cells co-transduced with Meis1-GFP and HoxA9. (D) Meis1-GFP mean fluorescence intensity of Prep1i/i and WT BM cells taken from serially transplanted leukemic mice is shown as a graph. *p-value <0.05. Error bars indicate SEM. (E) 30 µg of total lysates of leukemic Prep1i/i and WT BM cells was analyzed by immunoblotting using anti-FLAG antibody. Vinculin was used as loading control. Densitometric analysis was performed using ImageJ64. (F) Bar graph shows Meis1 transcript level measured by real-time PCR in Prep1i/i and WT leukemic bone marrow cells from serially transplanted mice. Error bars show SD.
Figure 3
Figure 3. In vitro characterization of Prep1i/i and WT leukemic cells.
(A) FACS plots show c-Kit expression on GFP-positive cells in leukemic WT or Prep1i/i BM cells (right side). Bar graph represents the percent of c-Kit positive cells on GFP-positive population on BM cells from different BMTs (left side). Error bars indicate SD. *p-value <0.001. (B) FACS profiles show c-Kit and Gr-1 expressions on BM from mice with AML induced by transplantation of Prep1i/i or WT cells overexpressing Meis1-HoxA9 (left side). Bar graph shows the percent of cells co-expressing c-Kit and GR-1 on BM from serially transplanted mice. Error bars represent SD. *p-value <0.001. The number of the mice analyzed for each group is shown on the graph.
Figure 4
Figure 4. Prep1-deficient cells show altered cell cycle activity in Meis1-HoxA9 leukemia.
(A) Cell cycle profile of WT or Prep1i/i leukemic BM cells (right side) performed by flow cytometry. Bar graph summarizes data from cell cycle analysis of leukemic BM cells coming from primary, secondary and tertiary transplanted mice. Error bars indicate the SD. *p-value <0.01. The number of the mice analyzed for each group is shown on the graph. (B) mRNA expression level of the indicated transcripts in leukemic BM cells coming from seven mice transplanted with either WT or Prep1i/i Meis1-HoxA9-transduced cells. Levels were normalized to the GAPDH expression. Error bars indicate SD. *p-value <0.001 (C) Immunoblot represents the Bmi-1 protein level in the total lysate of Meis1-HoxA9-transduced leukemic WT or Prep1i/i BM cells. Vinculin was used as loading control.

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Grant support

Leila Dardaei was supported by FUV (Fondazione Umberto Veronesi) fellowship. This work was supported by grants from AIRC (Italian Association for Cancer Research), Cariplo, and the Italian Ministry of Health to FB. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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