Suppression of leukemia development caused by PTEN loss

Abstract

Multiple genetic or molecular alterations are known to be associated with cancer stem cell formation and cancer development. Targeting such alterations, therefore, may lead to cancer prevention. By crossing our previously established phosphatase and tensin homolog (Pten)-null acute T-lymphoblastic leukemia (T-ALL) model onto the recombination-activating gene 1(-/-) background, we show that the lack of variable, diversity and joining [V(D)J] recombination completely abolishes the Tcrα/δ-c-myc translocation and T-ALL development, regardless of β-catenin activation. We identify mammalian target of rapamycin (mTOR) as a regulator of β-selection. Rapamycin, an mTOR-specific inhibitor, alters nutrient sensing and blocks T-cell differentiation from CD4(-)CD8(-) to CD4(+)CD8(+), the stage where the Tcrα/δ-c-myc translocation occurs. Long-term rapamycin treatment of preleukemic Pten-null mice prevents Tcrα/δ-c-myc translocation and leukemia stem cell (LSC) formation, and it halts T-ALL development. However, rapamycin alone fails to inhibit mTOR signaling in the c-Kit(mid)CD3(+)Lin(-) population enriched for LSCs and eliminate these cells. Our results support the idea that preventing LSC formation and selectively targeting LSCs are promising approaches for antileukemia therapies.

Fig. 1.

Rag1 deletion completely abolishes leukemia development caused by PTEN loss. (A) No T-ALL or other leukemia was detected in the bone marrow (BM) and thymus of P45–60 Pten;Rag1 null mice compared with Pten null T-ALL mice (n = 15 from 12 independent experiments). (B) Two-color interphase FISH analysis of thymocytes was performed with BAC clone probes (8): green-labeled RG-331N4 and RP23-357G5 on chromosome 14 and red-labeled RP24-194H23 and RP23-55P19 on chromosome 15. Fusion signals are indicated with white arrows. (C) c-myc was not overexpressed in Pten;Rag1 null CD4⁺CD8⁺ thymocytes (P60; percentage ± SD, n = 3 from three independent experiments). (D) Two million Pten;Rag1 null cells failed to develop leukemia in transplanted recipients in contrast to P30–45 Pten null preleukemic cells (representative; 3–5 mo, n = 7 from three separate experiments).

Fig. 2.

PTEN loss enables Rag1 null thymocytes to bypass β-selection. (A) The mass difference of P30 and P60 Rag1 null and Pten;Rag1 null thymuses was compared by Student t test analysis (n = 4–9 per group; P values are indicated as **P ≤ 0.01 or *P < 0.05). (B) Pten;Rag1 null thymocytes differentiate into DP cells at P30–45 (n = 3–6 per group from five independent experiments; error bars represent SD). (C) FACS-Gal analysis determined that only Pten;Rag1 null thymocytes could bypass β-selection (8, 25). LacZ⁺ cells (green dots) and LacZ⁻ cells (red dots) in the same samples are overlaid. (D) Pten loss restored cell size (FSC) and nutrient potentials (CD98 and CD71) of Rag1 null DN3 cells at P30–45 (percentage ± SD, n = 4; Fig. S4).

Fig. 3.

Rapamycin restores the β-selection checkpoint in Pten;Rag1 null mice. (A) Rapamycin dramatically reduced total thymocytes in Pten;Rag1 null mice after 14-d treatment (n = 4–5 per group from four independent experiments; P values of t test analysis are indicated as **P ≤ 0.01 or *P < 0.05). (B) Fourteen-day rapamycin treatment restored the β-selection checkpoint in P30–45 Pten;Rag1 null mice (n = 4 per group from four independent experiments; representative FACS plots are in Fig. S4). Error bars represent SD. (C) Rapamycin reduced nutrient potentials (CD98 and CD71) of Pten;Rag1 null DN3 thymocytes after 14-d treatment (percentage ± SD, n = 3; Fig. S5).

Fig. 4.

Rapamycin blocks T-cell differentiation to DP thymocytes and thereby, suppresses T-ALL development. (A) Rapamycin dramatically decreased DP cells, which are susceptible to the Tcrα rearrangement and Tcrα/δ-c-myc translocation, and thereby, reduced the chance of translocation and LSC development (n = 4–5 per group from three independent experiments). P values of Student t test analysis are indicated as **P ≤ 0.01 or *P < 0.05, and error bars represent SD (Fig. S5). (B) Long-term rapamycin treatment on preleukemic Pten null mice suppressed T-ALL development, as shown by the Kaplan–Meier survival curves (Left) and a CD45-SSC flow cytometrianalysis of WT and preleukemic mutant mice treated with rapamycin for 80 d (Right). (C) Leukemia occurs in some Pten null mice treated as in B after rapamycin withdrawal. Leukemia mice analyzed for c-myc overexpression (Right) are highlighted with red stars on the Kaplan–Meier survival curves (Left; Fig. S7).

Fig. 5.

Pten null LSCs are resistant to rapamycin treatment. (A) Rapamycin treatment reduced P-S6 levels (as measured by the percentage of P-S6⁺ cells and P-S6 fluorescence median) in c-Kit⁻CD3⁺Lin⁻ T-ALL blasts but not in c-Kit^midCD3⁺Lin⁻ LSCs (three independent experiments with 6,929–49,342 LSCs in placebo-treated samples and 474–64,808 LSCs in rapamycin-treated ones). P-S6 fluorescence medians are normalized to those for CD3⁻ cells as fold changes in corresponding experiments. (B) Rapamycin treatment results in the increased ratio of LSCs to leukemia blasts (three independent experiments). (C) Schematic illustration of the roles of RAG1 and rapamycin in blocking Tcrα/δ-c-myc translocation, LSC formation, T-cell differentiation, and leukemia development. P values of Student t test analysis are indicated as **P ≤ 0.01 or *P < 0.05, and error bars represent SD.