Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation

Abstract

Cancer stem cells, which share many common properties and regulatory machineries with normal stem cells, have recently been proposed to be responsible for tumorigenesis and to contribute to cancer resistance. The main challenges in cancer biology are to identify cancer stem cells and to define the molecular events required for transforming normal cells to cancer stem cells. Here we show that Pten deletion in mouse haematopoietic stem cells leads to a myeloproliferative disorder, followed by acute T-lymphoblastic leukaemia (T-ALL). Self-renewable leukaemia stem cells (LSCs) are enriched in the c-Kit(mid)CD3(+)Lin(-) compartment, where unphosphorylated beta-catenin is significantly increased. Conditional ablation of one allele of the beta-catenin gene substantially decreases the incidence and delays the occurrence of T-ALL caused by Pten loss, indicating that activation of the beta-catenin pathway may contribute to the formation or expansion of the LSC population. Moreover, a recurring chromosomal translocation, T(14;15), results in aberrant overexpression of the c-myc oncogene in c-Kit(mid)CD3(+)Lin(-) LSCs and CD3(+) leukaemic blasts, recapitulating a subset of human T-ALL. No alterations in Notch1 signalling are detected in this model, suggesting that Pten inactivation and c-myc overexpression may substitute functionally for Notch1 abnormalities, leading to T-ALL development. Our study indicates that multiple genetic or molecular alterations contribute cooperatively to LSC transformation.

Figure 1. VEC-Cre-mediated Pten loss leads to MPD and leukaemogenesis

a, Survival curve for mouse littermate pairs. Inset: representative spleens from mice at postnatal day 60 (P60). Mut, mutant; WT, wild type. b, Progressive alterations in PB: 30 littermate pairs (W, wild-type; M, mutant) per time point. Bars show the data range; points show averages. WBC, white blood cells. c, Identification of blast population by CD45/SSC plot in BM and spleen. The blasts were sorted for Giemsa–Wright staining, PCR genotyping and lineage analyses. Δex5, exon-5-deleted Pten allele.

Figure 2. Self-renewing LSCs are enriched in the c-Kit^midCD3⁺ compartment

a, LSC identification. The experimental design is illustrated in Supplementary Fig. 3. Top: illustration of the three subpopulations that were sorted from BM of five independent leukaemic donor mice (cell fractions are denoted on each FACS plot). Bottom: summary of independent transplantation experiments with sorted and serially diluted cells. +, leukaemia development; −, leukaemia-free for more than 100 days; n.a., viable cells after sorting were not enough for transplantation. b, LSCs are self-renewing and lead to accelerated leukaemogenesis during serial transplantations. Top: illustration of the experimental procedure. Bottom: summary of the results. Red lines in the lower chart represent the means of leukaemia latencies.

Figure 3. β-Catenin activation in LSCs and its role in leukaemogenesis

a, Accumulation of unphosphorylated β-catenin in blasts. Cytospin slides with thymic cells were stained with the monoclonal antibody 8E4, which recognizes unphosphorylated β-catenin. Top: representative fluorescent images. CP, chronic phase; BC, blast crisis. Scale bars, 25 μm. Bottom: representative confocal images. DAPI, 4′,6-diamidino-2-phenylindole. Original magnification ×100. b, Marked increase in unphosphorylated β-catenin levels in the LSC and blast populations. BM cells were pooled from two blast-crisis or WT littermates and were lineage (Lin)-depleted before FACS analysis. c, Decreased and delayed leukaemogenesis after ablation of one allele of Ctnnb1. Kaplan–Meier survival curves with Logbank statistical analysis (denoted on the curves) summarize leukaemia development in transplantation experiments. Blue line, Pten^loxP/loxP;Ctnnb1^loxP/+;VEC-Cre⁺; green line, Pten^loxP/loxP;VEC-Cre⁺.

Figure 4. The recurring translocation T(14;15) involves the Tcra/Tcrd cluster and the c-myc gene and results in aberrant overexpression of c-myc in LSCs and T-ALL blasts

a, Detection of T(14;15) by SKY analysis in metaphases prepared directly from BM of colcemid-treated WT or mutant mice. b, Breakpoint (BP) identification by two-colour FISH analysis. The results with green-labelled and red-labelled BAC probes are illustrated in the lower images and explained in the upper diagram. c, A schematic mapping of the T(14;15) translocation. BP, breakpoint. mChr, mouse chromosome. d, Recurring T(14;15) in primary T-ALL mutants and transplants with chronic-phase BM (BMT), splenic (SpT) or LSC cells was analysed by metaphase SKY (mSKY), metaphase FISH (mFISH) and interphase FISH (iFISH). Values shown are percentages. e, c-myc messenger RNA overexpression (means ± s.d.) in both primary blast-crisis (BC) mice and leukaemia transplants was detected by RT–PCR and analysed by Student's t-test. CP, chronic phase; C_t control, WT littermates (for primary mice) or an unrelated SCID mouse (for WT and leukaemic transplants). f, Overexpression of c-myc only in CD3⁺ splenic cells of T-ALL (n = 3). The percentage with c-myc overexpression in the CD3⁻ or CD3⁺ compartment is denoted in parentheses. Grey, control (a mixture of WT and T-ALL cells); blue, WT; red, T-ALL.