Ectopic <i>TLX1</i> Expression Accelerates Malignancies in Mice Deficient in <i>DNA-PK</i>

<div><p>The noncluster homeobox gene <i>HOX11/TLX1</i> (<i>TLX1</i>) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell acute lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of <i>TLX1</i> in T cells. The oncogenic potential of <i>TLX1</i> was demonstrated in <i>IgHμ-TLX1^Tg</i> mice which develop mature B cell lymphoma after a long latency period, suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed <i>IgHμ-TLX1^Tg</i> mice with mice deficient in the DNA repair enzyme DNA-PK (<i>Prkdc^Scid/Scid</i> mice). <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice developed T-ALL and acute myeloid leukemia (AML) with reduced latency relative to control <i>Prkdc^Scid/Scid</i> mice. Further analysis of thymi from premalignant mice revealed greater thymic cellularity concomitant with increased thymocyte proliferation and decreased apoptotic index. Moreover, premalignant and malignant thymocytes exhibited impaired spindle checkpoint function, in association with aneuploid karyotypes. Gene expression profiling of premalignant <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> thymocytes revealed dysregulated expression of cell cycle, apoptotic and mitotic spindle checkpoint genes in double negative 2 (DN2) and DN3 stage thymocytes. Collectively, these findings reveal a novel synergy between TLX1 and impaired DNA repair pathway in leukemogenesis.</p></div

<i>TLX1</i> accelerates T-ALL and AML in <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>Cohorts of mice were monitored for signs of disease for 25 months. A diagnosis of AML or T-ALL was made based on histological examination of bone marrow, spleen and thymic tissues. Survival of <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice are indicated by blue or red lines, respectively. (A) Disease free survival of <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice during the observation period, (p<0.0001). (B) T-ALL free survival of <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice during the 25 month observation period, (p<0.003). (C) AML free survival of <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice during the 25 month observation period, (p<0.0001). (D) Median survival of the complete <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> mouse cohorts developing T-ALL and/or AML. The column labeled as T-ALL/AML corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089649#pone-0089649-g001" target="_blank">Figure 1A</a> and shows the median survival of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> mice from cohorts developing either T-ALL or AML. The columns labeled T-ALL and AML correspond to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089649#pone-0089649-g001" target="_blank">Figure 1B and 1C</a> and indicate median survival of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> or <i>Prkdc^Scid/Scid</i> mice from cohorts developing T-ALL or AML respectively. (E) A one-year Kaplan-Meier survival probability estimate of the complete cohort of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> mice developing T-ALL or AML.</p

Heat map of the top ranking differentially expressed genes in flow sorted premalignant thymocytes.

<p>(A) Venn diagrams depicting TLX1-associated up-regulated and down-regulated genes in DN1, DN2 and DN3 fractions. (B–D) Gene expression heat map of the top ranking differentially expressed genes in DN1 (B), DN2 (C) and DN3 (D) flow-sorted thymocytes from <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> mice. For each heat map, the first four columns (<i>TLX1</i>⁺) show <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> thymocyte-derived samples, and the last four columns (<i>TLX1</i>^-) represent samples derived from thymocytes of <i>Prkdc^Scid/Scid</i> mice.</p

List of primers.

<p>List of primers.</p

Aberrant checkpoint regulation in thymocytes of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>(A) Cell cycle analysis showing the percentage of thymocytes in S and G2/M as determined by PI staining for DNA content. Percentages of cells in S and G2/M are shown above the histograms. (B) BrdU labeling to assess bypass of the G2/M cell cycle checkpoint in <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> thymocyte cultures. Thymocytes were treated with colchicine to induce mitotic arrest then exposed to BrdU to assess cell cycling. BrdU incorporation was detected by BrdU immunolabeling and nulcei were revealed by DAPI staining. White arrows on the merged images indicate cycling thymocytes. The histogram depicts the mean percentages of BrdU-positive cells assessed by scoring 20 random fields. Statistically significant differences (p<0.05) are indicated by asterisks.</p

Immunophenotype of <i>TLX1</i>-induced T-ALL.

<p>Immunophenotype distribution showing heterogeneous expression of CD44, CD25, CD4 and CD8 in <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> T-ALL. Representative flow diagrams showing heterogeneous expression of CD4 and CD8 in <i>TLX1</i>-initiated leukemia are presented.</p

<i>TLX1-</i>induced T-ALL in <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>(A) Hematoxylin and eosin staining of tissues isolated from premalignant <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice diagnosed with T-ALL. Magnification x40 (overview) and x100 (insert). Scale bars, 10 µm. (B) Immunohistochemical analysis of thymus and spleen from a premalignant <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mouse and a moribund <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mouse stained with an anti-Thy1.2 antibody. Magnification x20. Scale bars, 10 µm. (C) Cells from thymi, spleens and bone marrow of premalignant and moribund <i>IgHµ</i>-<i>TLX1^TgPrkdc^Scid/Scid</i> mice were examined for cell surface expression of CD44, CD25, CD4, CD8, CD3, TCRαβ, TCRγδ and Thy1.2 (for T cells) and Gr-1 and Mac-1 (for myeloid cells).</p

Validation of differential gene expression in premalignant thymocytes <i>of Prkdc^Scid/Scid</i> and <i>IgHμ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>qRT-PCR analysis of selected genes whose protein products are involved in chromosome segregation (<i>Chek1, Aurka</i> and <i>Bub1</i>), cell cycle progression (<i>Cyclin A, Cyclin B1, Anapc5, c-myc</i> and <i>c-myb</i>) and apoptosis (<i>Birc5, Brca1</i>). Samples are presented as pairs with the first bar representing the level of expression of cells isolated from <i>Prkdc^Scid/Scid</i> mice and the second from <i>IgHµ-TLX1</i>^Tg<i>Prkdc^Scid/Scid</i> mice. Data were normalized relative to <i>β-actin</i>. Red bars: DN1; brown: DN2; green: DN3 cells; black; total unsorted thymocytes. Error bars represent SD. Statistical testing was performed using the student’s T-test. Statistically significant differences are indicated by asterisks (* depicts p<0.05, ** depicts p<0.01, *** depicts p<0.001).</p

qRT-PCR analysis of thymic tumors derived from <i>Prkdc^Scid/Scid</i> and <i>IgHμ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>qRT-PCR analysis of expression of <i>Bcl11b</i>, <i>Pten</i> and <i>Notch1</i> in tumors isolated from ten <i>Prkdc^Scid/Scid</i> and ten <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice. Data were normalized relative to <i>β-actin</i>. Each bar represents one tumor sample.</p

Expression of <i>TLX1</i> in <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> premalignant thymocytes increases cell viability and provides a proliferative advantage.

<p>(A) Absolute cell numbers of thymocytes isolated from 20, six week old <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and 20 <i>Prkdc^Scid/Scid</i> littermates (p<0.0001). (B) Thymocytes were flow sorted based on expression of CD44 and CD25 and absolute numbers of DN thymocytes were calculated using percentages obtained after flow sorting (p<0.0001). (C) Thymocytes from three, six week old <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> littermates were stained with Annexin V and PI than assessed by flow cytometric analysis for cell viability. The lower left quadrant of each panel contains viable cells, the upper right quadrant contains dead cells and the lower right quadrant contains early apoptotic cells. The percentage of cells in each quadrant is indicated. (D) Percentages of viable, apoptotic and dead thymocytes in thymi of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> littermates, as determined by flow cytometry with Annexin V and PI staining. Error bars represent SD. (E) Histogram showing premalignant thymocytes obtained from <i>Prkdc^Scid/Scid</i> and <i>IgHµ</i>-<i>TLX1^TgPrkdc^Scid/Scid</i> mice, 2 and 7 hours after intraperitoneal injection with 10 µM BrdU. (F) The percentages of proliferating cells in thymi of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> littermates as determined by BrdU and PI staining. Data represent means of triplicate measurements with error bars to represent ± SD (p<0.0001). Statistically significant differences between compared samples are indicated by asterisks.</p